Podcasts about Dictyostelium

En el episodio de hoy hablamos de los dictiostélidos o mohos mucilaginosos celulares. Estas amebas sociables pasan parte de su vida en soledad, pero cuando les falta el alimento cooperan para formar una estructura multicelular, con todas las complicaciones y dramas que eso conlleva. Aquí una imagen de babosas y esporangios de Dictyiostelium discoideum en distintas fases de formación: Dictyostelium_discoideum_02.jpg (2901×1635) ------------------------------------------------- Podcast de divulgación científica en español. El objetivo es enseñar conceptos de biología en lenguaje sencillo para público no especializado y de forma breve (10-20 minutos). Empezaremos por lo básico pero iremos pasando a cosas más complejas y de actualidad con el tiempo. Este programa se realiza con el apoyo de la Fundación Laboratorio de Anatomía Animal, visítanos en ⁠http://anatolab.net/⁠. El logo es un trabajo de NiModo Creations (⁠https://www.nimodocreations.com/)⁠. Muchas gracias a Those Who Ride with Giants por permitirme usar partes de su tema: The Scent of the Old Ocean como intro y outro (⁠https://thosewhoridewithgiants.bandca⁠...)

Hugues de ThéCollège de France - Année 2023-2024Oncologie cellulaire et moléculaireColloque - Journée François Jacob : La vie sociale des microbes - Physical Properties of Single Cells and Social Behaviour in Dictyostelium DiscoideumIntervenant(s)Silvia De Monte, CNRS IBENS & Max Planck Institute for Evolutionary Biology, Plön, GermanyLes Journées François JacobLes Journées François Jacob, organisées par l'Institut de Biologie du Collège de France, rassemblent chaque année les meilleurs spécialistes français et étrangers autour d'un thème à la pointe des enjeux de la recherche en biologie.Le lauréat du prix Antoine Lacassagne, attribué chaque année par le Collège de France à un chercheur en biologie, est traditionnellement invité à recevoir son prix lors des Journées François Jacob et à y donner un séminaire en relation avec ses travaux.Ces journées sont nommées en l'honneur de François Jacob, titulaire de la chaire Génétique cellulaire du Collège de France (1964-1991), prix Nobel de physiologie ou médecine 1965 avec André Lwoff et Jacques Monod pour la découverte de la régulation génétique de la synthèse des enzymes et des virus.

How can we reconcile the evolutionary problem of cooperation? What can social amoebae tell us about the origins of multicellularity? In this episode, we talk to Joan Strassmann and David Queller, professors at Washington University in St. Louis, about the evolution of cooperation and conflict. From social insects to humans, we can find instances of individuals seemingly sacrificing fitness for the good of the group. But, truly altruistic behavior poses a problem for evolutionary biologists because it challenges the assumption that natural selection favors individuals over groups. We talk with Joan and David about their work with the social amoeba, Dictyostelium discoideum. This species is known for its remarkable developmental cycle: when there is no more to eat, the starving amoebae aggregate into a slug-like organism, which then forms a fruiting body that releases spores in hopes of dispersing to a better place. The problem, evolutionarily, is that only a fraction of the cells in the fruiting body get to live on through offspring. This facultative lifestyle and the ability to combine genetically different cells makes D. discoideum a prime study species for understanding how relatedness impacts cooperation and conflict and the possible origins of multicellular organisms. Towards the end of the episode, we also talk about Joan's new book Slow Birding: The Art and Science of Enjoying the Birds in Your Own Backyard. Cover art: Keating Shahmehri

This episode: Single-celled bacteria can act independently to create patterns and structure in their biofilm communities! Download Episode (9.6 MB, 14.0 minutes) Show notes: Microbe of the episode: Dictyostelium discoideum Skipper virus News item Takeaways Large multicellular organisms like us have interesting mechanisms for using one set of genetic instructions present in all cells to form a large, complex community of many different types of cells with different structures and functions, all working together. Single-celled microbes do not have the same requirements for genetic or structural complexity, but they do often display interesting communal patterns and behaviors. In this study, bacteria growing in colonies on agar displayed a particular mechanism of pattern formation previously seen only in eukaryotes, called segmentation clock or clock and wavefront process. In this process, the cells in the colony are all acting individually without communication with each other, but nevertheless form a repeating ring structure in the colony as it grows, possibly allowing some measure of differentiation of cells that could help the community survive various challenges.   Journal Paper: Chou K-T, Lee DD, Chiou J, Galera-Laporta L, Ly S, Garcia-Ojalvo J, Süel GM. 2022. A segmentation clock patterns cellular differentiation in a bacterial biofilm. Cell 185:145-157.e13. Email questions or comments to bacteriofiles at gmail dot com. Thanks for listening! Subscribe: Apple Podcasts, Google Podcasts, Android, or RSS. Support the show at Patreon, or check out the show at Twitter or Facebook.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.07.548115v1?rss=1 Authors: Listian, S. A., Kol, M., Ufelmann, E., Eising, S., Froehlich, F., Walter, S., Holthuis, J. C. M., Barisch, C. Abstract: Dictyostelium discoideum is a professional phagocyte frequently used as experimental model to study cellular processes underlying the recognition, engulfment and infection course of microbial pathogens. Sphingolipids are abundant components of the plasma membrane that bind cholesterol, control vital membrane properties, participate in signal transmission and serve as adhesion molecules in recognition processes relevant to immunity and infection. While the pathway of sphingolipid biosynthesis has been well characterized in plants, animals and fungi, the identity of sphingolipids produced in D. discoideum, an organism at the crossroads between uni- and multicellular life, is not known. Combining lipidomics with a bioinformatics-based cloning strategy for key sphingolipid biosynthetic enzymes, we show here that D. discoideum produces phosphoinositol-containing sphingolipids with predominantly phytoceramide backbones. Cell-free expression of candidate inositol-phosphorylceramide (IPC) synthases from D. discoideum in defined lipid environments enabled identification of an enzyme that selectively catalyses the transfer of phosphoinositol from phosphatidylinositol onto ceramide. The corresponding IPC synthase, DdIPCS1, is non-homologous to but shares multiple sequence motifs with yeast IPC and human sphingomyelin synthases and localizes to the Golgi apparatus as well as the contractile vacuole of D. discoideum. Collectively, these findings open up important opportunities for exploring a role of sphingolipids in phagocytosis and infection across major evolutionary boundaries. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.24.538041v1?rss=1 Authors: Franzkoch, R., Anand, A., Breitsprecher, L., Psathaki, O. E., Barisch, C. Abstract: The infection course of Mycobacterium tuberculosis is highly dynamic and comprises sequential stages that require damaging and crossing of several membranes to enable the translocation of the bacteria into the cytosol or their escape from the host. Many important breakthroughs such as the restriction of vacuolar and cytosolic mycobacteria by the autophagy pathway and the recruitment of sophisticated host repair machineries to the Mycobacterium-containing vacuole have been gained in the Dictyostelium discoideum/M. marinum system. Despite the availability of well-established light and advanced electron microscopy techniques in this system, a correlative approach that integrates both methodologies with almost native ultrastructural preservation is still lacking at the moment. This is most likely due to the low ability of D. discoideum to adhere to surfaces, which results in cell loss even after fixation. To address this problem, we improved the adhesion of cells and developed a straightforward and convenient workflow for 3D-correlative light and electron microscopy. This approach includes high-pressure freezing, which is an excellent technique for preserving membranes. Thus, our method allows to monitor the ultrastructural aspects of vacuole escape which is of central importance for the survival and dissemination of bacterial pathogens. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.17.537276v1?rss=1 Authors: Anand, A., Mazur, A.-C., Rosell-Arevalo, P., Franzkoch, R., Breitsprecher, L., Listian, S. A., Hüttel, S. V., Müller, D., Schäfer, D. G., Vormittag, S., Hilbi, H., Maniak, M., Gutierrez, M., Barisch, C. Abstract: Several intracellular pathogens, such as Mycobacterium tuberculosis, damage endomembranes to access the cytosol and subvert innate immune responses. The host counteracts endomembrane damage by recruiting repair machineries that retain the pathogen inside the vacuole. Here, we show that the endoplasmic reticulum (ER)-Golgi protein oxysterol binding protein (OSBP) and its Dictyostelium discoideum homologue OSBP8 are recruited to the Mycobacterium-containing vacuole (MCV) after ESX-1-dependent membrane damage. Lack of OSBP8 causes a hyperaccumulation of phosphatidylinositol-4-phosphate (PI4P) on the MCV and decreased cell viability. OSBP8-depleted cells had reduced lysosomal and degradative capabilities of their vacuoles that favoured mycobacterial growth. In agreement with a function of OSBP8 in membrane repair, human macrophages infected with M. tuberculosis recruited OSBP in an ESX-1 dependent manner. These findings identified an ER-dependent repair mechanism for restoring MCVs in which OSBP8 functions to equilibrate PI4P levels on damaged membranes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.13.536664v1?rss=1 Authors: Yi, X., Chen, P., Kang, C., Li, D., Yang, Y., Cai, H., Li, B., Wu, C. Abstract: Cell migration towards stiff substrates has been coined as durotaxis and implicated in development, wound healing and cancer, where complex interplays between immune and non-immune cells are present. Compared to the emerging mechanisms underlying the strongly adhesive mesenchymal durotaxis, little is known about whether immune cells - migrating in amoeboid mode - could follow mechanical cues. Here we develop an imaging-based confined migration device that provides stiffness gradient for cell migration. By tracking live cell trajectory and analyzing the directionality T cells and neutrophils, we observe that amoeboid cells can durotax. We further delineate the underlying mechanism to involve non-muscle myosin IIA (NMIIA) polarization towards the soft-matrix-side but may not require differential actin flow up- or down-stiffness gradient. Using the protista Dictyostelium, we further demonstrate the evolutionary conservation of amoeboid durotaxis. Finally, these experimental phenomena are theoretically captured by an active gel model capable of mechanosensing. Collectively, these results may shed new lights on immune surveillance and recently identified confined migration of cancer cells, within the tumor microenvironment or the inflamed fibrotic tissues. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.01.529730v1?rss=1 Authors: Velle, K. B., Garner, R. M., Beckford, T. K., Weeda, M., Liu, C., Kennard, A. S., Edwards, M., Fritz-Laylin, L. K. Abstract: Controlling intracellular osmolarity is essential to all cellular life. Cells that live in hypo-osmotic environments like freshwater must constantly battle water influx to avoid swelling until they burst. Many eukaryotic cells use contractile vacuoles to collect excess water from the cytosol and pump it out of the cell. Although contractile vacuoles are essential to many species, including important pathogens, the mechanisms that control their dynamics remain unclear. To identify basic principles governing contractile vacuole function, here we investigate the molecular mechanisms of two species with distinct vacuolar morphologies from different eukaryotic lineages - the discoban Naegleria gruberi, and the amoebozoan slime mold Dictyostelium discoideum. Using quantitative cell biology we find that, although these species respond differently to osmotic challenges, they both use actin for osmoregulation, as well as vacuolar-type proton pumps to fill contractile vacuoles. We also use analytical modeling to show that cytoplasmic pressure is sufficient to drive water out of contractile vacuoles in these species, similar to findings from the alveolate Paramecium multimicronucleatum. Because these three lineages diverged well over a billion years ago, we propose that this represents an ancient eukaryotic mechanism of osmoregulation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.27.530330v1?rss=1 Authors: Gentili, H. G., Pignataro, M. F., Olmos, J., Pavan, M. F., Ibanez, L. I., Santos, J., Velazquez, F. Abstract: Here we present the development of a new model system for Friedreichs Ataxia (FA) using D. discoideum (Dd). FA is a rare disease caused by disfunction of frataxin (FXN), a protein involved in Fe-S cluster assembly machinery. We firstly investigated the conservation of function between human and D. discoideum. In this work we show that DdFXN can substitute the human version in the interaction and activation of Fe-S assembly supercomplex. DdFXN can in vitro displace HsFXN in competition assays and also it can activate cysteine desulfurase activity in the context of human Fe-S assembly supercomplex. We then manage to edit fxn locus and isolated clone 8, a defective mutant with undetectable levels of frataxin. Clone 8 presents landmarks of frataxin deficiency such as decrease in Fe-S cluster dependent enzymatic functions, growth rate reduction and increase sensitivity to oxidative stress. Besides these phenotypes, shared with other FA models, clone 8 presents defects in the multicellular developmental program induced by starvation in this protist. We then assessed the rescuing capacity of DdFXN G122V, a version that mimics a human variant presented in some FA patients. While expression of DdFXN G122V rescues growth and enzymatic activities defects as well as DdFXN does, multicellular development defects were only partially rescued. This work opens the door to develop drug or treatment screenings that would help to design, and/or evaluate therapeutical strategies. Besides this biological model offers a wide range of possibilities to easily explore diverse phenotypes in FA. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.16.528874v1?rss=1 Authors: Rijal, R., Ismail, I., Jing, S., Gomer, R. H. Abstract: Dictyostelium discoideum is a soil-dwelling unicellular eukaryote that accumulates extracellular polyphosphate (polyP). At high cell densities, when the cells are about to overgrow their food supply and starve, the corresponding high extracellular concentrations of polyP allow the cells to preemptively anticipate starvation, inhibit proliferation, and prime themselves to begin development. In this report, we show that starved D. discoideum cells accumulate cell surface and extracellular polyP. Starvation reduces macropinocytosis, exocytosis, and phagocytosis, and we find that these effects require the G protein-coupled polyP receptor (GrlD) and two enzymes, Polyphosphate kinase 1 (Ppk1), which is required for synthesizing intracellular polyP, cell surface polyP, and some of the extracellular polyP, and Inositol hexakisphosphate kinase (I6kA), which is required for cell surface polyP and polyP binding to cells, and some of the extracellular polyP. PolyP reduces membrane fluidity, and we find that starvation reduces membrane fluidity, and this effect requires GrlD and Ppk1 but not I6kA. Together, these data suggest that in starved cells, extracellular polyP decreases membrane fluidity, possibly as a protective measure. In the starved cells, sensing polyP appears to decrease energy expenditure from ingestion, and decrease exocytosis, to both decrease energy expenditures and retain nutrients. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.03.522496v1?rss=1 Authors: Banerjee, T., Matsuoka, S., Biswas, D., Miao, Y., Pal, D. S., Kamimura, Y., Ueda, M., Devreotes, P. N., Iglesias, P. A. Abstract: The plasma membrane is widely regarded as the hub of the signal transduction network activities that drives numerous physiological responses, including cell polarity and migration. Yet, the symmetry breaking process in the membrane, that leads to dynamic compartmentalization of different proteins, remains poorly understood. Using multimodal live-cell imaging, here we first show that multiple endogenous and synthetic lipid-anchored proteins, despite maintaining stable tight association with the inner leaflet of the plasma membrane, were unexpectedly depleted from the membrane domains where the signaling network was spontaneously activated such as in the new protrusions as well as within the propagating ventral waves. Although their asymmetric patterns resembled those of standard peripheral "back" proteins such as PTEN, unlike the latter, these lipidated proteins did not dissociate from the membrane upon global receptor activation. Our experiments not only discounted the possibility of recurrent reversible translocation from membrane to cytosol as it occurs for weakly bound peripheral membrane proteins, but also ruled out the necessity of directed vesicular trafficking and cytoskeletal supramolecular structure-based restrictions in driving these dynamic symmetry breaking events. Selective photoconversion-based protein tracking assays suggested that these asymmetric patterns instead originate from the inherent ability of these membrane proteins to "dynamically partition" into distinct domains within the plane of the membrane. Consistently, single-molecule measurements showed that these lipid-anchored molecules have substantially dissimilar diffusion profiles in different regions of the membrane. When these profiles were incorporated into an excitable network-based stochastic reaction-diffusion model of the system, simulations revealed that our proposed "dynamic partitioning" mechanism is sufficient to give rise to familiar asymmetric propagating wave patterns. Moreover, we demonstrated that normally uniform integral and lipid-anchored membrane proteins in Dictyostelium and mammalian neutrophil cells can be induced to partition spatiotemporally to form polarized patterns, by optogenetically recruiting membrane domain-specific peptides to these proteins. Together, our results indicate dynamic partitioning as a new mechanism of plasma membrane organization, that can account for large-scale compartmentalization of a wide array of lipid-anchored and integral membrane proteins in different physiological processes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.07.519354v1?rss=1 Authors: Dowdell, A., Paschke, P., Thomason, P., Tweedy, L., Insall, R. Abstract: Negative chemotaxis, where eukaryotic cells migrate away from repellents, is important throughout biology, for example in nervous system patterning and resolution of inflammation. However, the mechanisms by which molecules repel migrating cells are unknown. Here, we use a combination of modelling and experiments with Dictyostelium cells to show that competition between different ligands that bind to the same receptor leads to effective chemorepulsion. 8-CPT-cAMP, widely described as a simple chemorepellent, is inactive on its own, and only repels cells if it interacts with the attractant cAMP. If cells degrade either competing ligand, the pattern of migration becomes more complex; cells may be repelled in one part of a gradient but attracted elsewhere, leading to populations moving in different directions in the same assay, or converging in an arbitrary place. More counterintuitively still, two chemicals can each attract cells on their own, but repel cells when combined together. We have thus identified a new mechanism that drives reverse chemotaxis, verified by mathematical models and experiments with real cells, and important anywhere several ligands compete for the same receptors. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Nels and Vincent discuss how duplication of a gene encoding a transcription factor led to evolution of a novel cell type in the slime mold Dictyostelium. Hosts: Nels Elde and Vincent Racaniello Subscribe (free): Apple Podcasts, Google Podcasts, RSS, email Become a patron of TWiEVO Links for this episode Gene duplication leads to novel cell type (Current Biol) Evolution of multicellularity (TWiEVO 11) Letters read on TWiEVO 81 Time stamps by Jolene. Thanks! Science Picks Nels – Dall-E artificial intelligence meets art Vincent – John Bonner's slime mold movies Music on TWiEVO is performed by Trampled by Turtles Send your evolution questions and comments to twievo@microbe.tv

Aujourd'hui je vous propose de découvrir dicty, l'un des êtres vivants les plus étranges au monde ! Son nom scientifique est en fait Dictyostelium discoideum mais on va l'appeler dicty* pour plus de simplicité.*Le petit nom "dicty" a été emprunté à Audrey Dussutour, qui mentionne cette incroyable créature dans son ouvrage Tout ce que vous avez toujours voulu savoir sur le blob sans jamais oser le demander, une petite pépite de vulgarisation scientifique dont je vous reparlerai très bientôt ;). See acast.com/privacy for privacy and opt-out information.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.29.273185v1?rss=1 Authors: Honda, G., Saito, N., Fujimori, T., Hashimura, H., Nakamura, M. J., Nakajima, A., Sawai, S. Abstract: In fast moving cells such as amoeba and immune cells, spatio-temporal regulation of dendritic actin filaments shapes large-scale plasma membrane protrusions. Despite the importance in migration as well as in particle and liquid ingestion, how these processes are affected by the micrometer-scale surface features is poorly understood. Here, through quantitative imaging analysis of Dictyostelium on micro-fabricated surfaces, we show that there is a distinct mode of topographically guided cell migration (phagotaxis) directed by the macropinocytic Ras/PI3K signaling patches. The topography guidance was PI3K-dependent and involved nucleation of a patch at the convex curved surface and confinement at the concave surface. Due to the topography-dependence, constitutive cup formation for liquid uptake in the axenic strain is also destined to trace large surface features. Given the fact that PI3K-dependency of phagocytosis are restricted to large particles in both Dictyostelium and immune cells, topography-dependency and the dual-use of membrane cups may be wide-spread. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.17.246082v1?rss=1 Authors: Cochet-Escartin, O., Demircigil, M., Hirose, S., Allais, B., Calvez, V., Funamoto, K., Rieu, J.-P. Abstract: It is well known that eukaryotic cells can sense oxygen (O2) and adapt their metabolism accordingly. It is less known that they can also move towards regions of higher oxygen level (aerotaxis). Using a self-generated hypoxic assay, we show that the social amoeba Dictyostelium discoideum displays a spectacular aerotactic behavior. When a cell colony is covered by a coverglass, cells quickly consume the available O2 and the ones close to the periphery move directionally outward forming a dense ring keeping a constant speed and density. To confirm that O2 is the main molecular player in this seemingly collective process, we combined two technological developments, porphyrin based O2 sensing films and microfluidic O2 gradient generators. We showed that Dictyostelium cells exhibit aerotactic and aerokinetic (increased speed at low O2) response in an extremely low range of O2 concentration (0-1.5%) indicative of a very efficient detection mechanism. The various cell behaviors under self-generated or imposed O2 gradients were modeled with a very satisfactory quantitative agreement using an in silico cellular Potts model built on experimental observations. This computational model was complemented with a parsimonious 'Go or Grow' partial differential equation (PDE) model. In both models, we found that the collective migration of a dense ring can be explained by the interplay between cell division and the modulation of aerotaxis, without the need for cell-cell communication. Explicit wave solutions of the PDE model also informed about the relative contributions of division and directed motion on the collective speed. Copy rights belong to original authors. Visit the link for more info

This episode: Slime molds have special cells that capture and kill bacteria using traps made of DNA! Download Episode (11.2 MB, 12.25 minutes) Show notes: Follow-up study from ep 255 group related to this study Link to Audiommunity episode about Neutrophil Extracellular Traps Journal Paper: Zhang X, Zhuchenko O, Kuspa A, Soldati T. 2016. Social amoebae trap and kill bacteria by casting DNA nets. Nat Commun 7:10938. Other interesting stories: Making E. coli produce propane fuel (paper) Bacteria can convert algae directly into ethanol (paper) Certain chronic virus infections could indicate your family history Viruses can transfer pigment production ability between bacteria Different sizes of bacteria impose different limitations   Post questions or comments here or email to bacteriofiles at gmail dot com. Thanks for listening! Subscribe at iTunes or Google Play, support the show at Patreon, or check out the show at Twitter or Facebook  

This episode: Some bacteria seem to cause slime mold amoebas to carry around other bacteria for food! Download Episode (12.4 MB, 13.5 minutes) Show notes: News item Journal Paper: DiSalvo S, Haselkorn TS, Bashir U, Jimenez D, Brock DA, Queller DC, Strassmann JE. 2015. Burkholderia bacteria infectiously induce the proto-farming symbiosis of Dictyostelium amoebae and food bacteria. Proc Natl Acad Sci 112:E5029–E5037. Other interesting stories: Fish oil vs. lard affect gut microbes differently Engineered mix of engineered bacteria produce controlled oscillations Mutation and selection makes better biomass-degrading enzymes Antibodies can be produced inside bacteria (paper) Fungus can be used to kill mosquitoes (paper) Post questions or comments here or email to bacteriofiles at gmail dot com. Thanks for listening! Subscribe at iTunes, check out the show at Twitter or Facebook

Min Zhao and Peter Devreotes discuss the results from a genetic screen to identify genes important for electrotaxis in the slime mold Dictyostelium discoideum.

The genus Legionella consists of environmental bacteria which are the causative agents of the severe pneumonia Legionnaires’ disease. L. longbeachae and L. pneumophila are able to replicate intracellularly in human alveolar macrophages and aquatic or soil amoebae. In order to replicate within host cells the bacteria establish a compartment derived from the endoplasmatic reticulum (ER) which is called “Legionella-containing vacuole” (LCV). A bacterial intracellular multiplication/defective in organelle transport (Icm/Dot) type IV secretion system (T4SS) is essential for the formation of this LCV. The Icm/Dot T4SS enables translocation of effector proteins into the host cell. More than 100 effector proteins are presumably translocated during an L. longbeachae infection whereas around 300 translocated effector proteins are known for L. pneumophila. During maturation the LCV communicates with vesicles from the endocytic vesicle trafficking pathway, avoids fusion with lysosomes and instead fuses with the ER. Phosphoinositides (PI) such as phosphatitdylinositol-4-phosphate (PtdIns(4)P) are enriched on the LCV which mediate the binding of Icm/Dot translocated effector proteins like SidCLpn (substrate of Icm/Dot transporter) as well as its paralogous protein SdcALpn. The 73 kDa effector SidM but not the 106 kDa SidCLpn was found in a previous phosphoinositide pulldown assay with L. pneumophila lysate to be the major PtdIns(4)P binding protein. Using L. longbeachae lysate we showed binding of the 111 kDa SidCLlo to PtdIns(4)P in a phosphoinositide pulldown. This result was confirmed by protein-lipid overlay assays using “PIP-strips”. In further analysis the P4C (PtdIns(4)P-binding of SidC) domain was identified as a 19 kDa domain of SidCLlo located in the amino acid region 609 to 782. This P4C domain was located in the same region as the 20 kDa SidCLpn_P4C domain of L. pneumophila. Both P4C domains can be used as LCV markers. This was shown with GST-tagged proteins binding to LCVs in a cell homogenate. The two P4C domains show a sequence identity of only 45% and the full-length protein of 40%. Circular dichroism measurements revealed that the secondary structure of the two proteins is similar. Moreover, isothermal titration calorimetric measurements indicated a 3.4 higher affinity of SidCLlo towards PtdIns(4)P compared with SidCLpn. In RAW 264.7 macrophages infected with L. longbeachae we showed that endogenous SidCLlo as well as heterologously produced SidCLpn is translocated to the LCV in an Icm/Dot-dependent manner. The deletion of the sidCLlo gene led to a reduced recruitment of calnexin to the LCV in infected Dictyostelium discoideum. This effect was complemented by adding plasmid-encoded SidCLlo, SidCLpn or SdcALpn. The same recruitment defect for a L. pneumophila strain lacking the sidCLpn and sdcALpn genes was complemented by the production of SidCLlo and SidCLpn as published before. Therefore, these effectors play a role for pathogen-host interactions by promoting the recruitment of ER to the LCV. L. longbeachae or L. pneumophila wild-type strains outcompeted their sidC deletion mutant in a competition assay in Acanthamoeba castellanii. However neither of the deletion mutants were impaired in their growth in single strain replication experiments. In summary despite of the small sequence identity and the higher binding affinity to PtdIns(4)P of SidCLlo compared to SidCLpn both effector proteins seem to have similar functions during an infection of Legionella. For the characterization of L. longbeachae-containing vacuoles through proteomic analysis, LCVs had to be isolated from infected D. discoideum or RAW 264.7 macrophages. Endogenous SidCLlo or heterologously produced SidCLpn were used as LCV markers for the isolation. Pathogen vacuoles harbouring L. longbeachae were isolated by immuno-affinity purification using antibodies specifically recognizing SidCLlo or SidCLpn. Future investigations aim at optimizing the LCV purification protocol for L. longbeachae to determine the proteome composition of the L. longbeachae-containing vacuole.

The cytoskeleton in most eukaryotes consists of actin filaments, intermediate filaments, microtubules and specific associated proteins. It determines the shape and the polarity of a cell and is inevitable for the coordination of cell movement. The regulation of this complex structure requires a highly organised and specialised signalling network. Ste20-like kinases and the regulator protein Mo25 (Morula protein 25) are part of this signalling network. The main objective of this work was the functional characterisation of the regulator protein Mo25 and the Ste20-like kinases Fray1, Fray2 (Frayed kinase 1/2) and DstC (Dictyostelium serine threonine kinase C) in the amoeba Dictyostelium discoideum (D. discoideum). An additional project was to map and characterise the actin and actin related genes in the genome of the fresh water foraminifer Reticulomyxa filosa (R. filosa). Mo25 is a highly conserved 40 kDa scaffolding protein with a 60% identity from amoeba to man. The disruption of the mo25 gene in D. discoideum results in very large, multinucleated cells which are unable to complete cytokinesis. Growth as well as development is severely delayed in the Mo25-minus strain. Furthermore, in phototaxis assays performed with multicellular aggregates (slugs), the Mo25-minus slugs were unable to migrate towards the light source. These findings imply that Mo25 plays an important role in cytokinesis, growth and cell polarity. We could link the Ste 20-like kinase SvkA (severin kinase), a homolog of the human Mst3, Mst4 (Mammalian Ste20-like kinase 3/4) and Ysk1/Sok1 (Yeast Sps1/Ste20-related kinase 1, Suppressor of Kinase 1) kinases to Mo25 as a binding partner. To further elucidate the interaction of Mo25 with SvkA as well as their role in cytokinesis or polarity signalling, we generated a series of GFP–Mo25 rescue constructs with distinct point mutations in protein-protein interaction surfaces and transformed these into the Mo25-minus background. The kinase domains of the Ste20-like kinases, Fray1 and Fray2 in D. discoideum are highly homologous to the catalytic domains of OSR1 (Oxidative stress response kinase 1) and SPAK (Ste20/SPS1-related proline-alanine-rich protein kinase) in humans and Frayed in fruit fly. Here, we generated the knockout clones Fray1-minus, Fray2-minus, and the double knockout Fray2Fray1-minus in D. discoideum. In developmental studies, Fray2-minus did not show an altered phenotype, whereas Fray1-minus and Fray2Fray1-minus developed slightly slower into fruiting bodies. When grown in shaking culture, Fray1-minus and Fray2Fray1-minus showed a reduced growth rate compared to Fray2-minus and the wild type. In addition, by using a GFP-Trap resin we identified a binding partner of Fray1, a yet unknown protein that we named FRIP (Fray Interacting Protein). FRIP mainly consists of a CBS (Cystathionine beta synthase) domain pair and is 30% identical to the gamma subunit of the AMPK (5‘ adenosine mono phosphate-activated protein kinase) complex in D. discoideum. The Ste20-like kinase DstC has been described to be a regulator of the actin driven process of phagocytosis. The catalytic domain of DstC is most similar to the mammalian kinase Mst2 (Mammalian Ste20-like kinase 2) and Hippo (“Hippopotamus”-like phenotype) of D. melanogaster. We could map the sorting signal that localises DstC to phagocytic cups and acidic vesicles to about 90 amino acids. Here we present an array of distinct point mutations for the identification of the exact localisation signal. R. filosa is a fresh water protist which belongs to the the group of Foraminifera within the Rhizaria. The R. filosa genome is the first foraminiferal and only the second rhizarian genome to be deciphered. In this bioinformatics project, we could identify, map and characterise four new actin genes in addition to the already known actin and about 40 genes that code for potential actin related proteins of seven different protein classes.

Mon, 17 Nov 2014 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/17681/ https://edoc.ub.uni-muenchen.de/17681/1/Sanftenberg_Linda.pdf Sanftenberg, Linda ddc:570, ddc:500, Fakultät für Biologie

The highly conserved protein actin is the building block in the cytoskeleton of eukaryotic cells and provides a structural framework known as the microfilament system.The molecular principle of actin-based amoeboid movement was so successful in evolution that it was kept nearly identically from lower (e.g. amoebae) to higher (e.g. neutrophils) eukaryotes.To understand this type of cellular movement one has first to identify and to characterize the proteins which play a major role during the dynamic rearrangement of actin. The collection of actin isoforms, of actin-variants and actin related proteins (Arps) in a given cell is known as the 'actinome' whose number of proteins can be quite different from one organism to the next. Therefore, the present work describes studies on the actinome of thesocial amoeba Dictyostelium discoideum, compares the findings with actinomes from other organisms, and discusses similarities and alterations that might have happened during evolution. D. discoideum is among the oldest organisms which exhibit actin-based amoeboid movement, the genome is completely sequenced and the system can be easily studied by molecular and biochemical approaches. The study was started using bioinformatics and the computational methods provided a global view on the D. discoideum actinome. It turned out that the D. discoideum genome conprises a total of 33 actin and 8 Arp genes, seven actin genes are putative pseudogenes. Interestingly, there are 17 distinguishable actin genes which code for identical proteins. Phylogenetic analyses helped to understand the putative duplication events during evolution. Modelling of the three-dimensional structures showed that the typical actin-fold, the ATP-binding pocket, and other functional domains are highly conserved. Homologues of the members of the D. discoideum actinome across various model organisms clearly demonstrated which amino acids in conserved domains are of special importance. All Arp subfamilies that are found in mammals are also present in D. discoideum. Two of the actin related proteins, Arp5 and Arp6, were selected for molecular and cellular studies. Using fluorescently labeled fusion proteins first data indicated that both Arps are present also in the nucleus, suggesting an involvement in chromatin reorganization.

Blebs lead the way in Dictyostelium chemotaxis Membrane blebs can help the leading edge of migrating cells protrude forwards, but the contribution of blebs to the motility of Dictyostelium cells is unclear. Zatulovskiy et al. reveal that blebs form at the front of chemotaxing Dictyostelium cells, particularly when the cells are faced with a mechanically resistant environment, and that this process is guided by a PI3-kinase-dependent signaling pathway. This biosights episode presents the paper by Zatulovskiy et al. from the March 17, 2014, issue of The Journal of Cell Biology and includes an interview with senior author Robert Kay (MRC Laboratory of Molecular Biology, Cambridge, UK). Produced by Caitlin Sedwick and Ben Short. See the associated paper in JCB for details on the funding provided to support this original research. Subscribe to biosights via iTunes or RSS View biosights archive The Rockefeller University Press biosights@rockefeller.edu

Background: The receptor for activated C-kinase 1 (RACK1) is a conserved protein belonging to the WD40 repeat family of proteins. It folds into a beta propeller with seven blades which allow interactions with many proteins. Thus it can serve as a scaffolding protein and have roles in several cellular processes. Results: We identified the product of the Dictyostelium discoideum gpbB gene as the Dictyostelium RACK1 homolog. The protein is mainly cytosolic but can also associate with cellular membranes. DdRACK1 binds to phosphoinositides (PIPs) in protein-lipid overlay and liposome-binding assays. The basis of this activity resides in a basic region located in the extended loop between blades 6 and 7 as revealed by mutational analysis. Similar to RACK1 proteins from other organisms DdRACK1 interacts with G protein subunits alpha, beta and gamma as shown by yeast two-hybrid, pull-down, and immunoprecipitation assays. Unlike the Saccharomyces cerevisiae and Cryptococcus neoformans RACK1 proteins it does not appear to take over G beta function in D. discoideum as developmental and other defects were not rescued in G beta null mutants overexpressing GFP-DdRACK1. Overexpression of GFP-tagged DdRACK1 and a mutant version (DdRACK1mut) which carried a charge-reversal mutation in the basic region in wild type cells led to changes during growth and development. Conclusion: DdRACK1 interacts with heterotrimeric G proteins and can through these interactions impact on processes specifically regulated by these proteins.

Actin is one of the most abundant proteins in eukaryotic cells and regulation of the microfilament system is crucial for a wide range of cellular functions including cell shape, cell motility, cell division and membrane dynamics. The aim of this thesis was (1) to gain a better understanding of the function of distinct actin binding domains in the regulation of the actin cytoskeleton and (2) to elucidate the role of actin variants. WH2 domains (WH2, Wiskott-Aldrich syndrome protein homology 2) are ubiquitous multifunctional regulators of actin dynamics. The protein Spire contains four central WH2 domains A-B-C-D with about 20 amino acids each and the cyclase-associated protein CAP2 contains only one WH2 domain. Under certain conditions, they can (1) nucleate actin polymerization, (2) disintegrate actin filaments and (3) sequester actin monomers. Here, the influence of selected Drosophila melanogaster Spire-WH2 and Mus musculus CAP2-WH2 domain constructs on actin dynamics was tested in vitro. To act as a filament nucleator, at least two WH2 domains are required, and nucleation of actin polymerization was only observed at substoichiometric concentrations of WH2 domains over actin. At higher concentrations, the sequestering activity of WH2 domains takes over. Preformed and purified SpireWH2-actin complexes act as extremely efficient nuclei for actin polymerization, even at superstoichiometric WH2 concentrations, under which free WH2 domains would sequester actin. All analyzed constructs, including these with only a single WH2 domain, sequester actin as well as they can disrupt filaments. This latter and most peculiar behavior of WH2 domains was observed in fluorometric, viscometric and TIRF assays. The WH2 domains seem to have such a high affinity for actin that they can forcefully sequester monomers even from filaments and filament bundles, thus breaking the whole structures. Taken together, the data clearly show that SpireWH2-actin complexes are the intermediates that account for the observed nucleating activity, whereas free WH2 domains can disrupt filaments and filament bundles within seconds, again underlining the intrinsic versatility of this regulator of actin dynamics. These data have been confirmed by crystallography in collaboration with the groups of Prof. Dr. Tad Holak and Prof. Dr. Robert Huber (Martinsried, Germany). Besides the well-studied conventional actins many organisms harbor actin variants with unknown function. The model organism Dictyostelium discoideum comprises an actinome of a total of 41 actins, actin isoforms and actin-related proteins. Among them is filactin, a highly conserved actin with an elongated N-terminus. The 105 kDa protein has a distinct domain organization and homologs of this protein are present in other Dictyosteliidae and in some pathogenic Entamoebae. Here, the functions of filactin were studied in vivo and in vitro. Immunofluorescence studies in D. discoideum localize endogenous and GFP-filactin in the cytoplasm at vesicle-like structures and in cortical regions of the cell. A most peculiar behavior is the stress-induced appearance of full length filactin in nuclear actin rods. To perform in vitro analyses recombinant filactin was expressed in Sf9 cells. Fluorescence studies with the filactin actin domain suggest that it interferes with actin polymerization by sequestering G-actin or even capping filaments. Gel filtration assays propose a tetrameric structure of full length filactin. Protein interaction studies suggest that filactin is involved in the ESCRT (endosomal sorting complexes required for transport) pathway which is responsible for multivesicular body formation. The data on filactin suggest that only the conventional actins are the backbone for the microfilamentous system whereas less related actin isoforms have highly specific and perhaps cytoskeleton-independent subcellular functions.

Background: Dictyostelium harbors several paralogous Sec7 genes that encode members of three subfamilies of the Sec7 superfamily of guanine nucleotide exchange factors. One of them is the cytohesin family represented by three members in D. discoideum, SecG, Sec7 and a further protein distinguished by several transmembrane domains. Cytohesins are characterized by a Sec7-PH tandem domain and have roles in cell adhesion and migration. Results: We study here Sec7. In vitro its PH domain bound preferentially to phosphatidylinositol 3,4-bisphosphate (PI(3,4) P-2), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P-3). When following the distribution of GFP-Sec7 in vivo we observed the protein in the cytosol and at the plasma membrane. Strikingly, when cells formed pseudopods, macropinosomes or phagosomes, GFP-Sec7 was conspicuously absent from areas of the plasma membrane which were involved in these processes. Mutant cells lacking Sec7 exhibited an impaired phagocytosis and showed significantly reduced speed and less persistence during migration. Cellular properties associated with mammalian cytohesins like cell-cell and cell-substratum adhesion were not altered. Proteins with roles in membrane trafficking and signal transduction have been identified as putative interaction partners consistent with the data obtained from mutant analysis. Conclusions: Sec7 is a cytosolic component and is associated with the plasma membrane in a pattern distinctly different from the accumulation of PI(3,4,5)P-3. Mutant analysis reveals that loss of the protein affects cellular processes that involve membrane flow and the actin cytoskeleton.

Chemotaxis, the ability of cells to detect and migrate directly towards a source of a chemically active agent, is the result of a sophisticated interplay of proteins within a complex regulatory network. However, partially redundant pathways that simultaneously mediate chemotaxis and dynamic protein distributions complicate the experimental identication of distinct signaling cascades and their inuence on chemotactic migration. Yet, increasingly precise generation and rapid modication of chemotactic stimuliin microuidic devices promise further insight into the basic principles of cellular feedback signaling. I developed a Chemotactic Gradient Generator (CGG) for the exposure of living cells to chemotactic gradient elds with alternating gradient direction based on a double T-junction microuidic chamber. A large extension of the concentration gradients enables the parallel exposure of several dozens of cells to identical chemotactic stimuli, allowing for a reliable quantitative analysis of the chemotactic migration behavior. Two pressure pumps and a syringe pump facilitate accurate control of the inow velocities at the individual ow chamber inlets, pivotal for precise manipulation of the chemotactic stimuli. The CGG combines homogeneous gradients over a width of up to 300 µm and rapid alterations of gradient direction with switching frequencies up to 0.7 Hz. Fast gradient switching in our experimental design facilitates cell stimulation at the intrinsic time scales of their chemotactic response as demonstrated by a gradual increase in the switching frequency of the gradient direction. We eventually observe a "chemotactically trapped" state of Dictyostelium discoideum (D. discoideum) cells at a switching rate of 0.01 Hz. Here, gradient switching proves too fast for the cells to respond to the altered gradient direction by migration. In contrast, we observe oscillatory runs at switching frequencies of less than 0.02 Hz. We distinguish between re-polymerizing cells that exhibit an internal re-organization of the actin cortex in response to chemotactic stimulation and stably polarized cells that gradually adjust their leading edge when the gradient is switched. To experimentally characterize both response types, we record cell shape and the intracellular distribution of actin polymerization activity. Cell shape is readily described by the eccentricity of the cell and to record F-actin polymerization dynamics we introduce a fluorescence distribution moment (FDM). Accurate description of the migratory response behavior facilitates a quantitative analysis of the inuence of both the experimental boundary conditions such as gradient shape, ongoing starvation of the cells, and in particular the inuence of distinct signaling cascades on chemotactic migration. Here, we demonstrate this ability of the GCC by inhibition of PI3-Kinase with LY 294002. PI3-Kinase initiates the formation of fresh pseudopods in the direction of the chemotactic gradient and therefore is one of the key signaling pathways mediating the chemotactic response. In shallow gradients and with ongoing starvation of the cells, we find a decreased ratio of re-polymerizing cells, pointing towards a diminished influence of PI3-Kinase. After inhibition of PI3-Kinase, cell re-polymerization in response to a switch in gradient direction is hindered at 5h of starvation, whereas at 7h of starvation evidence is found that chemotactic migration is more efficient. We observe the astonishing result that in dependency of the boundary conditions of the experiment inhibition of PI3-Kinase promotes an effective chemotactic response. Thus, the CGG for the rst time facilitates a quantitative analysis of the starvation time dependent effect of PI3-Kinase inhibition on D. discoideum chemotaxis.

Wed, 1 Dec 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13338/ https://edoc.ub.uni-muenchen.de/13338/1/Kastner_Peter.pdf Kastner, Peter Michael ddc:570, ddc:500, Fakultät für Biologi

Fri, 22 Jan 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/11082/ https://edoc.ub.uni-muenchen.de/11082/1/Batsios_Petros.pdf Batsios, Petros

Filamin and Cortexillin are F-actin crosslinking proteins in Dictyostelium discoideum allowing actin filaments to form three-dimensional networks. GAPA, an IQGAP related protein, is required for cytokinesis and localizes to the cleavage furrow during cytokinesis. Here we describe a novel interaction with Filamin which is required for cytokinesis and regulation of the F-actin content. The interaction occurs through the actin binding domain of Filamin and the GRD domain of GAPA. A similar interaction takes place with Cortexillin I. We further report that Filamin associates with Rac1a implying that filamin might act as a scaffold for small GTPases. Filamin and activated Rac associate with GAPA to regulate actin remodelling. Overexpression of filamin and GAPA in the various strains suggests that GAPA regulates the actin cytoskeleton through interaction with Filamin and that it controls cytokinesis through association with Filamin and Cortexillin.

Dictyostelium, an amoeboid motile cell, harbors several paralogous Sec7 genes that encode members of three distinct subfamilies of the Sec7 superfamily of Guanine nucleotide exchange factors. Among them are proteins of the GBF/BIG family present in all eukaryotes. The third subfamily represented with three members in D. discoideum is the cytohesin family that has been thought to be metazoan specific. Cytohesins are characterized by a Sec7 PH tandem domain and have roles in cell adhesion and migration. Dictyostelium SecG exhibits highest homologies to the cytohesins. It harbors at its amino terminus several ankyrin repeats that are followed by the Sec7 PH tandem domain. Mutants lacking SecG show reduced cell-substratum adhesion whereas cell-cell adhesion that is important for development is not affected. Accordingly, multicellular development proceeds normally in the mutant. During chemotaxis secG(-) cells elongate and migrate in a directed fashion towards cAMP, however speed is moderately reduced. The data indicate that SecG is a relevant factor for cell-substrate adhesion and reveal the basic function of a cytohesin in a lower eukaryote.

The contractile vacuole is an osmoregulatory organelle in protozoans that expels excess water from the cytosol through a complex sequence of membrane movements. A new study identifies a myosin motor protein that orchestrates these movements in Dictyostelia. This biosights episode presents the paper by Jung et al. from the August 24th, 2009 issue of the Journal of Cell Biology, and includes an interview with senior author John Hammer III. Produced by Eun Choi and Ben Short. Subscribe to biosights via iTunes or RSS View biosights archive The Rockefeller University Press biosights@rockefeller.edu

Dictyostelium cells migrate in an orderly head-to-tail arrangement. They do this by leaving a trail of vesicles (thought to contain chemoattractant) for their fellow cells to follow. This biosights episode presents a paper by Kriebel et al. in the Journal of Cell Biology, and includes excerpts from an interview with senior author Carole Parent. Produced by Justin Paul and Ruth Williams.   Subscribe to biosights via iTunes or RSS View biosights archive The Rockefeller University Press biosights@rockefeller.edu

The goal of this project was the characterisation of a new guanine nucleotide exchange factor (GEF protein) for Rac GTPases in Dictyostelium discoideum. GEF proteins function as stimulating proteins of Rac GTPases which are of central importance in the regulation of actin-involving processes. Actin is a major component of the cytoskeleton, constituting a dynamic network of filamentous structures which provide the basis for elementary tasks of growth, differentiation, cell movement and cell division. The ameba D. discoideum is a haploid unicellular eukaryote. It serves as a model organism to study basic actin-involving processes in higher organisms. The D. discoideum genome has been fully sequenced recently and comprises a wide range of regulatory components for the actin cytoskeleton. The gene of a novel GEF protein with a coding sequence of 3597 bp was studied in detail. The gene was cloned and the sequence verified. The protein sequence of 1198 amino acids comprises three Calponin homology domains (CH) and one Dbl homology/Pleckstrin homology tandem domain which is a typical sequence feature of GEF proteins. On the basis of this domain architecture the protein was named 'Trix' in short for 'triple Calponin exchange factor'. The CH domains were classified as type 1 and type 1 CH domain (type 3 - type 3 - type 1). This resembles a novel combination of CH domains in a Rho GEF protein. A recombinant fragment carrying the three CH domains was shown to bind and bundle actin filaments which is not explained by the functions that have been described for CH domains so far. In vivo Trix is localised in the area of the actin-rich cell cortex as well as on the membranes of late endocytic vesicles. This suggests a regulatory role for Trix in the assembly or the disassembly of the actin coat that is associated with endocytic vesicles during the late stages of endocytosis. It was shown that Trix is mainly expressed during the vegetative stadium of D. discoideum which would be consistent with the increased endocytosis during growth. An association of Rac GTPases with late endocytic vesicles in D. discoideum was not described so far. Trix displayed no GDP/MANT-GDP exchange activity with the Rac GTPases Rac1a, RacC and RacE, hence the protein could not be allocated to a Rac GTPase signaling pathway. Some of the interactions between exchange factors and their respective Rac GTPases are of a very specific nature. Thus a potential activity of Trix might be directed against a Rac GTPase that has not yet been tested. The activation of Trix might also depend on further uncharacterised regulatory components. Finally, the results of the in vitro assays might differ from the in vivo situation as it has been demonstrated for other Rho GEFs. The Trix gene was disrupted in AX2 wildtype cells by a gene replacement approach. This allowed detailed characterisation of the protein's function in vivo. Trix is not an essential protein. There were no significant differences in the expression of important marker proteins, in phototaxis, chemotaxis, phagocytosis, cytokinesis, and growth of Trix- mutants. The mutant cells showed subtle changes in the organisation of the actin system as well as a slight delay during the developmental cycle. The most severe phenotypic deviation displayed by Trix- mutants consisted in a marked reduction of exocytosis. This provides further evidence for a regulatory function of the protein in exocytosis. The data suggest that Trix plays a role in the organisation of actin-involving processes in D. discoideum, especially in the regulation of the late endocytic cycle. Trix could not be allocated to a specific Rac GTPase signaling pathway and definite structural or dynamic tasks on the basis of the protein’s actin-binding and actin-bundling properties. The subtle phenotypic alterations in Trix- mutants might be due to a general redundancy in the functions of D. discoideum Rho GEF proteins.

Actin belongs to the most abundant proteins in eukaryotic cells which harbor usually many conventional actin isoforms as well as actin-related proteins (Arps). To get an overview over the sometimes confusing multitude of actins and Arps, we analyzed the Dictyostelium discoideum actinome in detail and compared it with the genomes from other model organisms. The D. discoideum actinome comprises 41 actins and actin-related proteins. The genome contains 17 actin genes which most likely arose from consecutive gene duplications, are all active, in some cases developmentally regulated and coding for identical proteins (Act8-group). According to published data, the actin fraction in a D. discoideum cell consists of more than 95% of these Act8-type proteins. The other 16 actin isoforms contain a conventional actin motif profile as well but differ in their protein sequences. Seven actin genes are potential pseudogenes. A homology search of the human genome using the most typical D. discoideum actin (Act8) as query sequence finds the major actin isoforms such as cytoplasmic beta-actin as best hit. This suggests that the Act8-group represents a nearly perfect actin throughout evolution. Interestingly, limited data from D. fasciculatum, a more ancient member among the social amoebae, show different relationships between conventional actins. The Act8-type isoform is most conserved throughout evolution. Modeling of the putative structures suggests that the majority of the actin-related proteins is functionally unrelated to canonical actin. The data suggest that the other actin variants are not necessary for the cytoskeleton itself but rather regulators of its dynamical features or subunits in larger protein complexes.

The cellular slime mold Dictyostelium discoideum contains a total number of 13 kinesins. Two of them, kinesins Kif3 and Kif5, represent the Kinesin-1 family (formerly conventional kinesins) in D. discoideum whose members are dimeric molecular motors that move as single molecules micrometer-long distances on microtubules by using the energy from ATP hydrolysis. In this study constructs of both kinesins were expressed in E. coli, purified, and tested in biochemical assays. A GFP-fusion protein of Kif3 revealed an overall cytoplasmic localization with accumulations that could not be assigned to a specific cellular structure or vesicle. Using immunofluorescence staining an association with the endoplasmic reticulum or mitochondria was ruled out. Full-length and truncated Kif3 motors were active in gliding and ATPase assays. They showed a strong dependence on ionic strength. Like the full-length motor, the truncated Kif3-592 motor (amino acids 1-592; comprising motor domain, neck and partial stalk) reached its maximum speed of around 2.0 µms-1 at a potassium acetate concentration of 200 mM. The velocity from the microtubule-gliding assay was confirmed using kinesin labeled with Q-Dots. The shortened Kif3-342 motor (amino acids 1-342; comprising motor domain, partial neck) and the Kif3-592 construct showed an ATP turnover comparable to the fungal Nkin motor. Kif3-full-length displayed less activity in ATPase assays, possibly resulting from tail-motor inhibition. Results from the duty ratio calculations and single-molecule gliding assays indicated that Kif3 is a processive enzyme. Overall, D. discoideum’s Kif3 revealed a closer similarity to fungal rather than animal kinesins. The truncated motor Kif5-476 (amino acids 1-476; comprising motor domain, neck and partial stalk) turned out to bind microtubules, but was immotile in gliding assays. Still, this construct, as well as the shorter variant Kif5-353 (amino acids 1-353; comprising motor domain), showed activity in ATPase assays, indicating that a significant portion of the isolated protein was active. Unlike Kif3, the Kif5 motor protein was sensitive to potassium-acetate concentrations exceeding 25 mM and lost its capability to bind microtubules with increasing ionic strength. D. discoideum knockout strains showed no apparent phenotype under standard culture conditions or during development. Merely a reduced growth speed was observed in submerged cultures of kif5-null cells. A GFP-Kif5 construct showed a strong accumulation in the cell’s peripheries, in agreement with previous reports. Microtubule recovery experiments after nocodazole treatment did not reveal any significant differences between wild type and knockout strains, arguing against an influence of Kif5 on microtubule organization.

The major goal of the project was the investigation of proteins that regulate dynamic rearrangements of the actin cytoskeleton in Dictyostelium discoideum amoebae. Among the proteins studied in detail were (i) D. discoideum vasodilator stimulated phosphoprotein DdVASP and a new profilin isoform as putative regulators of filopodia formation, and (ii) the Ste20-like kinases Krs1 and Severin-Kinase as members of signalling cascades towards the actin cytoskeleton. Filopodia are bundles of actin filaments projecting from the cell surface. They are found on a variety of cell types and are needed among others, for cell adhesion, sensory and exploratory functions. Filopodia are frequently found associated with sheet-like arrays of actin filaments called lamellipodia and membrane ruffles. The function of the VASP homolog from D. discoideum in filopodia formation was studied using molecular, biochemical and cell biological approaches. The protein sequence of DdVASP shares a significant homology to the members from other species. The protein harbours two Ena/VASP homology domains EVH1 and EVH2 separated by a polyproline stretch. The EVH2 domain is characterised by a G-actin binding site (GAB), an F-actin binding site (FAB) and a tetramerisation domain. As a tetramer the DdVASP protein can nucleate actin polymerization and bundle actin filaments. The in vitro nucleating activity of DdVASP is salt dependent and its nucleating activity is completely abolished at 100 mM salt. However, the F-actin bundling activity as determined by the low speed sedimentation assay was not disturbed. The ability of DdVASP to influence the binding of capping protein to the growing ends of the actin filaments was tested through elongation of capped F-actin seeds and by depolymerization of capped filaments upon dilution below the critical concentration of the barbed ends. Results from both sets of experiments showed that DdVASP cannot remove capping protein from the barbed ends. The D. discoideum formin dDia2, which was previously reported to be essential for filopodia formation could elongate the capped F-actin seeds. In vitro biochemical data led to the conclusion that the bundling activity of DdVASP is the essential in vivo function to stabilise actin filaments in emerging filopodia. To test this hypothesis, a DdVASP null mutant was isolated. As expected the mutant failed to produce any filopodia. Expression of wild type DdVASP, but not DdVASPFAB, rescued the phenotype suggesting the importance of the bundling activity of DdVASP in filopodia formation. To confirm that the data obtained with DdVASP were not species specific, key biochemical functions of HsVASP were also tested. The results indicated that VASPs are functionally well conserved throughout evolution. During this study, a third profilin isoform, profilin III, was further characterised. Specific interaction between profilin III and DdVASP was discovered. Profilin III shares a limited homology at the amino acid level with the other two and well studied profilins. Polyclonal antibodies that recognise only the profilin III isoform showed that in wild type cells profilin III represents less than 1% of all profilins. This suggests a distinct role for profilin III, because a low protein concentration argues against an actin sequestering function. Immunolocalisation studies showed profilin III in filopodia and enriched at their tips. Cells lacking the profilin III protein show defects in cell motility during chemotaxis. The second part of the project dealt with the characterisation of two D. discoideum Germinal Centre Kinases (GCK). The catalytic domain of Krs1 was found to be highly homologous to the catalytic region of human MST1 and MST2 from the GCK-II subfamily. The regulatory region harbours the putative inhibitory domain (aa 330-379) and a possible multimerization (SARAH) domain (aa 412-458) described for GCKs in higher organisms. This SARAH region spans about 50 amino acid residues, is located at the extreme carboxyl terminus and most likely forms an  - helical coiled-coil motif. GFP-Krs1 overexpressing wild type cells showed an enrichment of the kinase in the cell cortex, and motility of these cells during aggregation was reduced. Krs1 knockout strains exhibited only subtle differences to wild type cells. Severin kinase is encoded by the gene svkA, and phylogenetic analysis groups it into subfamily GCK-III, along with human MST3, MST4 and YSK/SOK-1. Immunoblot analysis with polyclonal antibodies showed an uniform expression level throughout development. Gene disruption of svkA resulted in cells that had problems to divide both in submerged or in shaking cultures. Though the motility and chemotaxis of these cells remain unaltered compared to the wild type cells, the movement of the multicellular slugs is disturbed. In addition, development was delayed and the mutant formed aberrant fruiting bodies.

Auf der Suche nach bislang unbekannten Proteinen des Centrosoms von Dictyostelium wurde zunächst auf der Ebene des Dictyostelium-Genomprojekts, basierend auf Ähnlichkeiten zu bekannten centrosomalen Proteinen anderer Spezies, nach möglichen Kandidaten gesucht. Zu den ca. 120 wahrscheinlich centrosomalen Proteinen in Tieren konnten hier nur 38 Homologe gefunden werden. Allerdings besteht das Dictyostelium-Centrosom wahrscheinlich aus ähnlich vielen verschiedenen Proteinen, sodass mit dieser Methode ein Großteil unentdeckt blieb. In einem Proteomics-Ansatz mit verschiedenen Auftrennungsmethoden wurde das Dictyostelium-Centrosom systematisch untersucht. Hierfür wurde zunächst ein Verfahren erarbeitet, Centrosomen in hinreichender Reinheit für massenspektrometrische Analysen zu präparieren. Am Ende der Bemühungen wurden 33 neue mögliche centrosomale Proteine gefunden, von denen bereits drei bestätigt werden konnten. Parallel wurde im Dictyostelium-System die Krankheit Lissenzephalie untersucht, eine Migrationsstörung von Neuronen bei der Gehirnentwicklung, bei der Centrosom-assozierte Proteine eine wichtige Rolle spielen. Zellmotilität und Entwicklung sind in Dictyostelium besonders gut zu beobachten, außerdem existieren hier Homologe zu den miteinander interagierenden Proteinen LIS1 und DCX, deren Mutationen beim Menschen Lissenzephalie auslösen. Mit DdDCX wurde ein Homologes (29 % Identität) zum humanen DCX gefunden und unter Einsatz von Fusionsproteinen und eines Antikörpers umfangreich charakterisiert. DdDCX bindet an Mikrotubuli und wird hauptsächlich in der Aggregationsphase exprimiert. Die generierte Nullmutante zeigte jedoch keinen Phänotyp. Das centrosomale Protein DdLIS1 hat zahlreiche wichtige Dynein-assoziierte Funkionen in vegetativ wachsenden Zellen. Hier konnte in durch gezielte Mutationen gezeigt werden, dass DdLIS1 eine Rolle bei der Entwicklung spielt, auch wenn es selbst nicht entwicklungsreguliert ist. Eine klare Aussage wurde erst durch die Generierung einer Doppelmutante möglich: Bei dieser ist die Aggregation in der Entwicklung gestört, also die Phase, in der wie bei Neuronen Zellbewegung und die Kommunikation zwischen den Zellen besonders wichtig sind. Da gezeigt werden konnte, dass Mikrotubuli dafür nicht essentiell sind, sind Spekulationen über gestörte Mikrotubuli-Dynamik als Ursache für die Migrationsstörung in Dictyostelium nicht haltbar. Mögliche Erklärungen bieten dagegen die nachgewiesene Interaktion mit Aktin oder die Beteiligung von LIS1 an der Regulation von PAF, einem intrazellulären Botenstoff, der auch in Neuronen eine Rolle spielt.

Rap1 is a ubiquitous Ras-like guanine-nucleotide-binding protein that is involved in a variety of signal-transduction processes especially during cytoskeletal rearrangements. Rap1 is regulated by guanine-nucleotide- exchange factors (GEFs) and GTPase-activating proteins (GAPs) which increase the slow intrinsic GTPase activity by many orders of magnitude and allow tight regulation of signaling. In this study a new Dictyostelium RapGAP1 gene was cloned and characterized. RapGAP1 was discovered by screening the sequences of the D. discoideum database. The Dictyostelium RapGAP1 gene encodes a protein with 1212 amino acids protein which shows at the C-terminal region 53% sequence similarity to human RapGAP. RapGAP1 mRNA was present during all stages of D. discoideum development with a strong upregulation at 9 hours of development. Furthermore, to investigate the role of RapGAP1 in cellular processes RapGAP1 null cells where generated by inserting a gene replacement construct into the endogenous gene. RapGAP1 minus mutants did not show any significant phenotypic abnormalities except that there was a slight delay in development. This delay by about three hours was confirmed by testing the expression of developmentally regulated genes like csA, a cell adhesion protein, and MUD1, a prespore-specific cell surface antigen. However, the mutant was able to complete normal developmental and to form fruiting bodies containing mature spores. Studies on cell motility showed that RapGAP1 null cells moved faster than AX2 wild type cells. This finding suggests that RapGAP1 belongs to a signal transduction chain which ultimately leads to changes in cytoskeletal dynamics.

Die Organisation und Dynamik des Mikrotubuli-Cytoskeletts wird von großen Proteinkomplexen an den plus- und minus-Enden der Mikrotubuli reguliert. Am minus-Ende befindet sich das Centrosom, das als Mikrotubuli-organisierendes Zentrum dient. Am plus-Ende der Mikrotubuli findet sich ein Komplex von Proteinen, der die Dynamik der Mikrotubuli reguliert sowie ihre Verankerung am Zellcortex vermittelt. DdCP224 ist ein centrosomales und Mikrotubuli-assoziiertes Protein bei Dictyostelium discoideum, das zur ubiquitären XMAP215-Familie gehört und eine wichtige Rolle bei der Dynamik des Centrosoms und des Mikrotubuli-Cytoskeletts spielt. Ziel dieser Arbeit war die Charakterisierung zuvor unbekannter Dictyostelium-Proteine, die mit DdCP224 bei diesen dynamischen Vorgängen zusammenwirken. Mit DdEB1, DdMoe1 und DdLIS1 konnten drei neue Mikrotubuli-assoziierte Proteine bei Dictyostelium identifiziert und charakterisiert werden. Alle drei Proteine konnten gleichzeitig auch als echte centrosomale Bestandteile nachgewiesen werden, da ihre Lokalisation am Centrosom unabhängig von Mikrotubuli ist. DdEB1 ist aufgrund seines Molekulargewichts, das größte Mitglied der ubiquitären EB1-Proteinfamilie. DdEB1 zeigte eine cytosolische Interaktion mit DdCP224 und Dynein. Am Beispiel von DdEB1 und DdCP224 konnte in dieser Arbeit nicht nur erstmals die Interaktion von Proteinen aus der EB1- und XMAP215-Familie, sondern auch ihre lange vermutete Colokalisation an Mikrotubuli-plus-Enden nachgewiesen werden. Mit Hilfe der Expression von GFP-DdEB1-Deletionsmutanten konnte gezeigt werden, dass die DdEB1 Bindung an Mikrotubuli von einer Homo-Oligomerisierung des Proteins abhängt, die durch eine „coiled-coil“-Domäne vermittelt wird. DdEB1-Nullmutanten zeigen in erster Linie mitotische Defekte, d.h. Störungen der Centrosomenduplikation, Spindelbildung und Chromosomensegregation. Die mikroskopische Analyse lebender Zellen ergab, dass DdEB1 für die Spindelbildung, nicht aber für die Spindelelongation oder die Mikrotubuli/Zellcortex-Interaktion benötigt wird. Bei der Suche nach möglichen DdEB1-Interaktoren wurde mit DdMoe1 das Dictyostelium-Homologe von Schizosaccharomyces pombe Moe1 isoliert, das dort ein Interaktionspartner des entsprechenden EB1-Proteins ist. Eine solche Interaktion ist den durchgeführten Untersuchungen zufolge bei Dictyostelium jedoch unwahrscheinlich. Dafür konnte hier zum ersten mal ein Moe1-homologes Protein als echte Centrosomenkomponente identifiziert werden und die Mikrotubuli-Bindung eines solche Proteins in vivo nachgewiesen werden. Wie EB1 ist auch das humane LIS1-Protein ein Mikrotubuli-plus-End und Dynein-assoziiertes Protein. Mutationen in diesem Gen führen zu einer schweren Entwicklungsstörung des Gehirns (Lissenzephalie), aufgrund eines neuronalen Migrationsdefekts. Dictyostelium LIS1 (DdLIS1) bindet nicht nur an Dynein, sondern auch an DdCP224, womit auch erstmals die Interaktion mit einem Protein der XMAP215-Familie nachgewiesen werden konnte. DdLIS1 spielt gemeinsam mit Dynein eine Rolle bei der Mikrotubuli/Zellcortex Verankerung, was in DdLIS1-Überexpressionsmutanten deutlich wurde. Die Überexpression von DdLIS1 führte außerdem zur Centrosomenamplifikation, Defekten bei der Organisation der Mitosespindel, schweren Cytokinesedefekten und einer drastisch eingeschränkten Zellmotilität. Letztere steht im Einklang mit dramatischen Veränderungen der Aktindynamik, bei der charakteristische wandernde Aktin-Polymerisationswellen am Zellcortex auftreten. Da derselbe Aktin-Phänotyp auch durch Behandlung von Kontrollzellen mit der F-Aktin depolymerisierenden Droge Latrunculin A simuliert werden konnte wurde angenommen, dass die DdLIS1-Überexpression den Aktin-Gehalt beeinflusst. Tatsächlich konnte in mikroskopischen und biochemischen Nachweisen bestätigt werden, dass die Überexpression von DdLIS1 den F-Aktin Gehalt der Zellen vermindert. Das Mikrotubuli-assoziierte Protein DdLIS1 ist also ein mögliches Bindeglied zwischen dem Mikrotubuli- und Aktin-Cytoskelettsystem.

Die vorliegende Arbeit hatte zum Ziel, (i) die regulatorische Funktion bestimmter integraler Membranproteine im Dictyostelium Zytoskelett und (ii) die biochemische Interaktion einer Kinase eines infektiösen Bakteriums mit Aktin aus der Wirtszelle genauer zu analysieren. (i) Die ubiquitären Mitglieder der CD36/LIMPII-Familie sind integrale Membranproteine, die als Lipidrezeptoren und Zelladhäsionsproteine in der Plasmamembran oder - mit bisher unbekannter Funktion - in Membranen endosomaler Vesikel vorkommen. In Dictyostelium discoideum führte die Inaktivierung eines lysosomalen Membranproteins aus dieser Gruppe zur Suppression des Phänotyps einer Profilin-minus Mutante. Im Zuge der vollständigen Sequenzierung des D. discoideum-Genoms konnte festgestellt werden, daß es neben diesem DdLmpA noch die beiden weiteren homologen Proteine DdLmpB und DdLmpC gibt. Da der Mechanismus der Suppression des Profilin-minus Phänotyps ungeklärt ist, wurden die beiden Isoformen im Rahmen der vorliegenden Arbeit genauer charakterisiert. Sowohl für DdLmpB wie auch für DdLmpC konnte die familientypische Membran-Topologie einer Haarnadelstruktur nachgewiesen werden. Dabei weist die zentrale, lumenale Domäne beider Proteine zahlreiche Glykosylierungen auf. Durch Immunofluoreszenz und Saccharosegradienten wurde die Lokalisation der drei Isoformen an endolysosomalen Vesikeln nachgewiesen. Es stellte sich dabei heraus, daß die drei DdLmp-Proteine in unterschiedlichen Vesikelpopulationen auftraten. Auch “pulse-chase“-Experimente mit TRITC-Dextran und nachfolgender Markierung der Vesikel mit DdLmp-spezifischen Antikörpern ergaben unterscheidbare Zeitmuster für die Rekrutierung der Membranproteine in Vesikeln. Die für DdLmpA oft beobachtete Kolokalisation mit Makropinosomen konnte z.B. für DdLmpB und DdLmpC nur selten festgestellt werden. Nach zahlreichen Versuchen und der Konstruktion von verschiedenen Vektoren konnte am Ende der praktischen Arbeiten eine DdLmpB-minus Mutante im Wildtyp-Hintergrund isoliert werden. (ii) Im zweiten Teil der Arbeit wurde die in der Literatur beschriebene Interaktion zwischen Aktin und der Kinase YopO, die durch Yersinia enterocolitica als Effektorprotein in die Wirtszelle transloziert wird, biochemisch genauer untersucht. Es konnte festgestellt werden, daß G-Aktin und nicht F-Aktin für die Aktiverung der YopO-Kinase verantwortlich ist. Dabei tritt Nichtmuskel-Aktin im Vergleich zum Muskel-Aktin als ein deutlich besserer Aktivator von YopO auf. Obwohl die aktivierte Kinase in vivo das Aktin-Zytoskelett beeinflußt, ist Aktin offensichtlich kein Substrat von YopO. Mittels Fluoreszenzspektroskopie konnte gezeigt werden, daß sowohl die native Kinase YopO als auch das durch Punktmutation inaktivierte YopO K269A die Polymerisierungskinetik von Aktin behindern. Für eine mutmaßliche Aktin-Binderegion von 20 Aminosäuren aus dem C-terminalen Ende konnte hingegen kein Effekt beobachtet werden. Der Einfluß von aktinbindenden Proteinen, aktinmodifizierenden Substanzen und YopO-bindenden GTPasen auf die Aktivierung der Kinase durch Aktin deutet darauf hin, dass die Aktivität der Kinase in der Wirtszelle nicht nur durch Aktin alleine, sondern auch durch weitere Zytoskelett-Komponenten reguliert wird.

In this study a new Dictyostelium STE20-like protein kinase DST2 (Dictyostelium STE20-like kinase 2) was cloned and characterised. STE20 (Sterile 20) kinase was first identified in yeast as a pheromone-induced serine/threonine protein kinase that acts upstream of a MAP kinase cascade. Based on the domain structure, DST2 belongs to the GCK subfamily of STE20-like protein kinases, which include the mammalian STE20-like kinases (MST1/2/3), oxidant stress response kinase SOK-1, and DST1 in Dictyostelium discoideum which phosphorylates severin, a gelsolin-like F-actin fragmenting protein. DST2 was discovered by screening of the D. discoideum cDNA project database using DST1 as query. To confirm the existence of the DST2 gene and its expression, Southern, Northern and Western analyses of DST2 were carried out. It revealed that DST2 may have two copies in the Dictyostelium genome and that DST2 was expressed during all stages of D. discoideum development. In vitro kinase assays with bacterially expressed fusion protein of full length DST2 (aa461), the catalytic domain (aa287) and the regulatory domain (aa174) showed that autophosphorylation of DST2 occurson the regulatory domain and phosphorylates severin in the presence of a Mn2+ or Mg2+. Purified catalytic domain of PKA phosphorylated the regulatory domain of DST2 and caused an increase in the basal autophosphorylation activity of DST2, suggesting that PKA may be a potential upstream kinase of DST2 through the phosphorylation of its regulatory domain. To understand the function of the non-catalytic domain of DST2, three C-terminal truncation constructs (aa1-421, aa1-368 and aa1-326) were used in comparison to full length DST2 in in vitro kinase assays. Deletion of C-terminal regions revealed an inhibitory region amino acids 326-461 of DST2. Gel filtration chromatography showed that DST2 was eluted in a broad peak ranging from approximately 63 kDa to 400 kDa, suggesting that DST2 may exist in vivo as a monomer as well as a high molecular weight complex. The influence of phosphorylated and unphosphorylated severin on F-actin solutions was investigated using falling-ball viscometry and fluorescence spectroscopy. It turned out that phosphorylation by DST2 inhibits the F-actin fragmenting activity of severin, suggesting that DST2 may be directly involved in actin-cytoskeleton rearrangements.

The cyclase-associated protein (CAP) from Dictyostelium discoideum was studied in detail regarding its structure and function relationships. The second part of the thesis describes the characterization of the novel actin-related protein filactin from D. discoideum. CAP homologs are multifunctional proteins: they are involved in signal dependent changes in the actin cytoskeleton, in vesicle transport and cell development. The binding of monomeric actin through the C-terminal domain represents a common feature of all CAPs. From four highly conserved regions in this domain the verprolin homology region of the D. discoideum CAP was analyzed in this work. Loss of this region led to a clear decrease, but not suppression of the actin-sequestering activity. In agreement to these data, stable complexes of the modified CAP-C with G-actin could be identified in chemical crosslinking experiments, and it could be shown that CAP-C is able to dimerize. Considering the high conservation of cyclase-associated proteins and their importance for cell biological processes it is remarkable that no structure of this protein has been elucidated so far. Therefore, in the frame of this thesis the structure of the membrane-associated N-terminal domain of D. discoideum CAP was to be determined in cooperation with the group of T. Holak at the MPI f. Biochemistry (Martinsried). For this purpose numerous constructs from the N-terminal domain had to be cloned and expressed, to be purified and examined for their stability and threedimensional folding. It turned out that the stable core of the aminoterminal domain covers the amino acids 51-226. The structure was determined by nuclear magnetic resonance spectroscopy and X-ray crystallography. Six antiparallel a-helices are connected by loop elements and form a cylindrical core domain which can form a dimer in the crystal, dimerization occurs through Mg2+ ions. The clarification of the N-terminal structure of D. discoideum CAP will now simplify the research on its interaction with phospholipids and CAP binding proteins The second part of this work dealt with the characterization of filactin. The unique 105 kDa protein contains two filamin homologous regions in its N-terminal, and a clearly actin-related domain within the C-terminal part. While in resting cells the endogenous filactin shows cytoplasmic distribution and interaction with protein aggregates as well, the GFP construct of the actin-related domain displays an actin-like behavior during cell movement or phagocytosis. A stimulus-induced colocalization of actin and filactin was observed in experiments with chemotactically stimulated cells. The alignment of its C-terminal amino acid sequence with the structure of muscle actin predicts a globular, actin-related structure containing all residues that are important for ATP/ADP binding.

Three functionally important centrosomal proteins from Dictyostelium were identified, cloned and characterized. The work focuses on the Dictyostelium-homologue of centrin and two proteins belonging to soluble gamma-tubulin complexes in other organisms.

In the past 25 years many techniques have been developed to characterize cell adhesion and to quantify adhesion forces. Atomic force microscopy (AFM) has been used to measure forces in the pico-newton range, an experimental technique known as force spectroscopy. We modified such an AFM to measure adhesion forces between live cells or between cells and surfaces. This strategy required functionalizing the surface of the sensors for immobilizing the cell. We used Dictyostelium discoideum cells which respond to starvation by surface expression of the adhesion molecule csA and consequent aggregation to measure the adhesion force of a single csA-csA bond. Relevant experimental parameters include the duration of contact between the interacting surfaces, the force against which this contact is maintained, the number and specificity of interacting adhesion molecules and the constituents of the medium in which the interaction occurs. This technology also permits the measurement of the viscoelastic properties of single cells or cell layers. Copyright (C) 2002 S, Karger AG, Basel.

A large number of actin-binding proteins regulates the dynamics of the actin cytoskeleton. Here we report the identification and characterization of two new proteins in Dictyostelium discoideum: the novel cytoskeleton protein villidin and a third profilin isoform. One goal of the project was to analyze the features of the villidin sequence in detail and to investigate the protein by molecular, biochemical and cell biological approaches. Villidin has a calculated molecular mass of 190,000 Da. Based on the domain structure of the protein, villidin can be assigned to the gelsolin/villin-family as well as to the WD-repeat family. In principal, the group of the WD-repeat proteins includes regulatory proteins which are involved in signal transduction and other important cellular processes. The N-terminus of villidin harbours between four and eight of the specific WD-repeats and forms probably a β-propeller structure. The WD-domain is followed by an intervening domain of 400 amino acids that leads to the second characteristic villidin domain at the C-terminus. This part of the sequence exhibits a similarity to villin, though the first of the six villin domains is absent in villidin. However the typical headpiece is present. Villidin mRNA and protein are expressed in low amounts during growth and early aggregation, they are increased during development and reach highest levels at the tipped aggregate stage. The protein is present in the cytosol as well as in the cytoskeletal and the membrane fraction. These biochemical results are in agreement with the immunofluorescence data. The endogenous villidin is homogeneously distributed throughout the cytosol and localizes at vesicular structures. Colocalization experiments lead to the assumption that these structures might belong to a still unknown population of vesicles. GFP fusion proteins with the villin homology domains show a similar distribution, whereas GFP fusions of the N-terminal part encompassing the WD-repeats are present in F-actin rich regions at the plasma membrane and on internal membranes during motility, pinocytosis and phagocytosis. Mutants lacking villidin do not show an aberrant phenotype during growth and development but are defective in motility and phototaxis in the multicellular slug stage. Based on this defect and the multidomain structure of the protein, villidin might be involved in signal transduction processes leading to phototactic movement. The second part of the project dealt with a new gene which codes for a third profilin isoform in D. discoideum. The well-known Dictyostelium profilin isoforms I and II are able to interact with G-actin, PIP2 and poly-L-prolin. As in the case of profilin I and II, profilin III is encoded by a single gene. In contrast to profilin I and II, the transcription of profilin III is not developmentally regulated. All three isoforms show the typical limited homology at the amino acid level. The recombinant Profilin III protein cosediments with poly-L-prolin, inhibits the actin-polymerisation and the PIP2 interaction of profilin III competes with the G-actin affinity. The low expression of Profilin III mRNA in growing and developing cells suggests a distinct role of profilin III because a low protein concentration argues against an actin sequestering function.

Zyklisches 3’:5’-Adenosinmonophosphat (cAMP) ist sowohl als extrazellulärer Lockstoff als auch als intrazelluläres Signalmolekül von entscheidender Bedeutung in der Entwicklung von Dictyostelium discoideum. In der vorliegenden Arbeit wird gezeigt, dass dieser sekundäre Botenstoff auch eine zentrale Rolle in der hyperosmotischen Stressantwort von D. discoideum spielt. Wildtypzellen reagieren auf hyperosmotischen Stress mit einem transienten Anstieg der intrazellulären cAMP-Konzentration, der von dem Hybridhistidinkinase-Homolog DokA abhängig ist. DokA ist essentiell für das Überleben von D. discoideum-Zellen unter hyperosmotischen Bedingungen. Die Osmosensitivität von dokA--Zellen beruht auf dem gestörten cAMPMetabolismus und kann durch die transiente Zugabe eines membrangängigen cAMP-Analogs weitgehend aufgehoben werden. Der Einfluss von DokA auf den intrazellulären cAMP-Spiegel zeigt sich auch in ungeschockten Zellen: Während die basale cAMP-Konzentration in dokA-- Zellen reduziert ist, weisen Zellen, die die Regulator-Domäne von DokA überexprimieren, einen erhöhten cAMP-Spiegel auf. Basierend auf diesen Daten wurde ein Modell der Regulation des intrazellulären cAMP-Spiegels durch DokA entwickelt. Die Regulator-Domäne von DokA wirkt hierbei als Phosphatase des Histidin-Phosphotransferproteins RdeA, welches durch Phosphorylierung die Phosphodiesterase RegA aktiviert. Durch die Phosphatase-Aktivität von DokA wird somit der über den RdeA/RegAPhosphorelay gesteuerte intrazelluläre cAMP-Abbau inhibiert. Die in vitro-Dephosphorylierung von RdeA durch die Regulator-Domäne von DokA konnte ebenso nachgewiesen werden wie die verringerte Phosphodiesterase-Aktivität bei homologer Überexpression des DokA-Regulators. Die Effekte dieser Überexpression auf cAMP-Haushalt und Entwicklung sind DokA-spezifisch und von dem konservierten Aspartylrest D1567 der Regulator-Domäne abhängig. Bei Untersuchungen zu den Effektoren der hyperosmotischen Stressantwort in D. discoideum wurden Veränderungen in Aufbau und Zusammensetzung von Membran und Cytoskelett beobachtet. Dabei konnten sieben Proteine identifiziert werden, die eine deutliche Translokation bei hyperosmotischem Stress erfahren. Eine Übersicht stellt die osmoregulatorischen Signalwege in D. discoideum dar und vergleicht die Rolle von 2-Komponenten-Systemen in der Osmoregulation bei Eukaryoten.

During the slug stage of cellular slime mold Dictyostelium discoideum, up to 105 cells coordinate their movement and migrate as a single organism. Slugs have a cylindrical shape with tip and tail; their morphological polarity corresponds to the polarity of migration. A large body of results suggest that cyclic AMP-mediated cell-cell signaling is the mechanism coordinating multicellular movement. Waves of cyclic AMP generated at the anterior tip propagate towards the tail and induce the chemotactic movement of cells toward the tip. Slugs exhibit highly sensitive environmental reactions: phototaxis, chemotaxis and thermotaxis. Although many studies have investigated how Dictyostelium slugs move toward a light source, the mechanism of phototaxis is still unclear. It has been known that slugs turn towards the light at the anterior end. In addition, previous research identified mutations and drug treatments that interfere with phototaxis but the strategy for analyzing phototaxis has been limited to low resolution both temporarily and spatially. In this thesis methods have been developed to analyze phototactic behavior on two different scales, the slug level and cellular level. The analyses revealed dynamic features of slug behavior during phototaxis which have not been previously described. Following light irradiation slugs moved with approximately 50% higher speed; they showed prominent serpentine movement of their tip as if they were scanning and correcting migration direction; they elongated and decreased the diameter of their body; and their tip remained lifted off the substrate for long periods. The analysis of cell movement during phototactic turning showed that the cell movement pattern was unlike any predicted from earlier hypotheses. Some cells in the anterior zone moved away from the light source across the slug, thus increasing the volume on the “dark” side (“asymmetric cell accumulation”) and bending the anterior zone like a lever-arm toward the light source. Furthermore, it was discovered that light irradiation enhances secretion of cyclic AMP from the slug and that light interferes with cyclic AMP cell-cell signaling during other multicellular stages as well. A model for phototaxis has been proposed based on these results. Laterally irradiated light is focused on the distal side of the slug by a lens effect and locally induces cyclic AMP release. Some cells accumulate chemotactically on the side away from the light source and cause a bending of the anterior zone towards the light source. Since cell movement within the slug is organized by cyclic AMP waves, light induced cyclic AMP release interferes with the endogenous signaling pattern. The consequence is an overall change in the shape and the behavior of slug. The mechanism by which light induces the release of cyclic AMP from slug cells may involve a histidine kinase phosphorelay pathway, since such a pathway is known to be functional in Dictyostelium and is used for environmental responses in many other organisms.

Methoden der multidimensionalen konfokalen Mikroskopie wurden für die in vivo Beobachtung dynamischer Prozesse in der Amöbe Dictyostelium discoideum verwendet. Um diese Vorgänge sichtbar zu machen, wurde Grün Fluoreszierendes Protein (GFP) als Reporter von Genexpression und als Fusionsprotein eingesetzt. Mit Hilfe von 4D-Mikroskopie und Mehrkanal-Detektion konnten dabei in vivo die Zellsortierung in vielzelligen Aggregaten und die intrazelluläre Lokalisierung des Zytoskelett-Proteins Severin visualisiert und analysiert werden. Es wurde eine Methode entwickelt, spektral unterschiedliche GFP-Varianten im Konfokalmikroskop simultan zu detektieren und verläßlich zu trennen. Aufgrund der Anregungswellenlänge und der Verwendung nur eines Aufnahmevorgangs zur Erfassung beider GFP-Varianten eignet sich dieser Ansatz zur schonenden Beobachtung lebender Zellen auch über längere Zeiträume. Diese Eigenschaften ermöglichen darüber hinaus die Verwendung dieser Konfiguration bei den wiederholten Aufnahmen einer 4D-Messung. In D. discoideum-Slugs wurde die Lokalisierung von Prestalk-Zellen (pstA- und pst0-Zellen) während der Migration und der Spitzenneubildung mit GFP als Reporter für spezifische Genexpression untersucht. In migrierenden Slugs fallen Prestalk-Zellen aus dem pstA-Bereich der Spitze in den Prespore-Bereich zurück. Dieses Zurückfallen in der zentralen Längsachse läßt sich durch die Organisation der Spitzenregion durch cAMP-Spiralwellen mit einem Zentrum in der Längsachse erklären. Prestalk-Zellen gelangen über diesen Weg aus der Spitze in den Prespore-Bereich. An der Neubildung von Spitzen im Prespore-Bereich sind ehemalige Prestalk-Zellen gemeinsam mit den Anterior-like-Zellen des Prespore-Bereichs maßgeblich beteiligt. Diese Zelltypen bewegen sich an die Slugoberseite und bilden dort rotierende Zentren, aus denen sich neue Spitzen bilden. Das Verhalten dieser Zellgruppen und die 4DTrajektorien einzelner Zellen deuten auf eine Organisation dieser Zentren durch cAMPSignale. Darüber hinaus konnte gezeigt werden, daß es innerhalb dieser Zellen Gruppen gibt, die zu unterschiedlichen Zeiten in neue Spitzen rekrutiert werden. PstA-Zellen sind in neuen Spitzen wieder im vordersten Bereich lokalisiert, obwohl sie aus dem Prespore-Bereich kommen, während pst0-Zellen im gesamten Prestalk-Bereich zu finden sind. Artspezifische Zellsortierungsprozesse gemeinsam vorkommender Dictyostelium-Arten wurden mittels der Markierung von Zellen der beteiligten Arten untersucht. Es konnte gezeigt werden, daß die Sortierung von D. discoideum/D. mucoroides-Mischungen schon in den Aggregationsströmen anfängt und im Mound abgeschlossen wird. Die Trennung erfolgt über Unterschiede in der Zelladhäsion. Erheblich beschleunigt wird dieser Sortierungsvorgang durch die gerichtete chemotaktische Bewegung beider Arten auf dieselben Signale, die bei den sich in den Zellströmen schneller bewegenden D. mucoroides-Zellen zu einer Anreicherung im Moundzentrum führt. Somit sind in vivo zwei Faktoren gemeinsam für die artspezifische Sortierung von D. discoideum und D. mucoroides verantwortlich. In Mischungen von D. discoideum und Polysphondylium spec. geschieht die Aussortierung über die Nutzung unterschiedlicher Botenstoffe für die Chemotaxis, so daß die Arten getrennt aggregieren. Es konnte gezeigt werden, daß diese getrennte Aggregation sehr spezifisch ist und es dennoch zu einer Interaktion zwischen D. discoideum und fortgeschrittenen Polysphondylium-Aggregaten kommt, da in Polysphondylium in späten Aggregationsphasen cAMP für die interzelluläre Kommunikation verwendet wird. In dieser Phase wird von Polysphondylium sowohl cAMP als auch Glorin sekretiert. Severin ist ein mit Gelsolin verwandtes 40 kDa Protein, das F-Aktin-Filamente fragmentiert und an das (+)-Ende des entstehenden Fragments bindet. Mit Hilfe eines GFP-Fusionsproteins wurde die intrazelluläre Lokalisierung dieses Proteins in vivo während einer Vielzahl zellulärer Aktivitäten untersucht. Severin ist in den aktiven Bereichen des F-Aktin Zellkortex angereichert. Obwohl die Morphologie von D. discoideum-Zellen in den verschiedenen Phasen der Entwicklung sehr unterschiedlich ist, ist die Lokalisierung von Severin in allen Stadien primär von der Bildung neuer Zellfortsätze trennbar. Die Anreicherung von Severin ist hingegen mit der Bewegung der Zelle in diese neu gebildeten Fortsätze assoziiert. Erstmals wurde im Rahmen dieser Arbeit der Effekt von cAMP-Signalen auf das Zytoskelett einzelner Zellen in Aggregationsströmen ausführlich dargestellt.

We describe the application of a novel image processing method, which allows quantitative analysis of cell and tissue movement in a series of digitized video images. The result is a vector velocity field showing average direction and velocity of movement for every pixel in the frame. We apply this method to the analysis of cell movement during different stages of the Dictyostelium developmental cycle. We analysed time-lapse video recordings of cell movement in single cells, mounds and slugs. The program can correctly assess the speed and direction of movement of either unlabelled or labelled cells in a time series of video images depending on the illumination conditions. Our analysis of cell movement during multicellular development shows that the entire morphogenesis of Dictyostelium is characterized by rotational cell movement. The analysis of cell and tissue movement by the velocity field method should be applicable to the analysis of morphogenetic processes in other systems such as gastrulation and neurulation in vertebrate embryos.

There are cells acattered in the rear, prespore region of the Dictyostelium slug that share many of the properties of the prestalk cells and that are therefore called anterior-like cells (ALCs). By placing the gene encoding a cell surface protein under the control of an ALC-specific promoter and immunologically labeling the living cells, we analyze the movement of ALCs within the slug. There is a posterior to anterior cellular flow, and the ALCs change their movement pattern as they enter the prestalk zone. Prestalk cells are periodically shed from the migrating slug. They must be replaced if the correct ratio of prestalk to prespore cells is to be maintained, and we present evidence for the trans-differentiation of prespore into prestalk cells, with ALCs functioning as intermediates in the transition. The slug has, therefore, a surprisingly dynamic structure, both with respect to cellular differentiation and cell movement.

Fri, 1 Jan 1993 12:00:00 +0100 http://epub.ub.uni-muenchen.de/5968/ http://epub.ub.uni-muenchen.de/5968/1/5968.pdf Siegert, Florian; Weijer, Cornelis J. Siegert, Florian und Weijer, Cornelis J. (1993): The role of periodic signals in the morphogenesis of Dictyostelium discoideum. In: Rensing, Ludger (Hrsg.), Oscillations and morphogenesis. Dekker: New York, pp. 133-152. Geowissensc

Cells in Dictyostelium slugs follow well-defined patterns of motion. We found that the chemotactic cell response is controlled by a scroll wave of messenger concentration in the highly excitable prestalk zone of the slug that decays in the less-excitable prespore region into planar wave fronts. This phenomenon is investigated by numerical solutions of partial differential equations that couple local nonlinear kinetics and diffusive transport of the chemotactic signal. In the interface of both regions a complex twisted scroll wave is formed that reduces the wave frequency in the prespore zone. The spatio-temporal dynamics of waves and filaments are followed over 33 periods of rotation. These results yield an explanation of collective self-organized cell motion in a multicellular organism.

To test the hypothesis that periodic signals and chemotaxis direct later morphogenesis in Dictyostelium discoideum, we investigated cell behavior and cell movement in slugs. Trails of neutral red-stained prestalk and anterior-like cells were recorded by high-resolution digital image processing. Neutral red-stained anterior-like cells in the prespore zone of slugs move straight forward in the direction of slug migration and, furthermore, show coherent periodic cell movement. In contrast, cells in the prestalk zone move along completely different trajectories. Prestalk cells move perpendicular to the direction of slug migration; that is, they rotate around the tip. The cell movement data show that the chemotactic signal in the slug propagates as a three-dimensional scroll wave in the prestalk zone and as a planar wave in the prespore zone. The different behavior of prestalk and prespore cells is most likely caused by a difference in the oscillatory properties of the two cell types. We provide evidence that the slug stage of Dictyostelium behaves like an excitable system and that a (twisted) scroll wave organizes slug formation and migration.

We have studied optical density wave propagation during aggregation of the cellular slime mould Dictyostelium discoideum in a quantitative manner by digital image analysis. The waves are mostly single ended spiral waves starting from an aggregation center. We can measure a variety of parameters such as oscillation frequency, wave propagation velocity and wave shape. This allows the construction of dispersion curves under a variety of experimental conditions. During later development where the optical density waves are no longer visible we have started to measure movement of fluorescently labelled cells. Our main conclusions from these measurements are that the cells continue to move chemotactically to periodic signlas both in aggregates and in slugs. There is a dramatic difference in the movement pattern of prestalk and prespore cells: Prestalk cells move perpendicular to the long axis of the slug, they are most likely organized by a scroll wave. Prespore cells seem to move almost perpendicular to the prestalk cells, in the direction of the tip. This behaviour is explained on the basis of different relay properties of prespore and prestalk cells.

Waves of chemotactic movement during the early phase of aggregation in Dictyostelium discoideum were analyzed by digital image processing in a manner that immediately shows the following parameters: wave propagation velocity, period length, wave amplitude und wave shape. We have characterized the aggregation of AX-2 and the streamer F mutant NP 377 in terms of these parameters and investigated the influence of caffeine and ammonia. It was found that during normal aggregation oscillation frequency increases while at the same time wave propagation velocity decreases. Caffeine, a known inhibitor of cyclic AMP relay, reduces oscillation frequency and wave propagation velocity in a dose-dependent manner but most notably leads to the appearance of bimodal (harmonic) oscillations. These bimodal waves are also found in streamer F mutants without caffeine during early aggregation. The effect of caffeine is interpreted as an increase in the average chemotactic deadaptation time due to elevated cyclic GMP levels after a cyclic AMP stimulus. This increased deadaptation time results in some cells responding to every chemotactic signal, while others respond only to every second signal, leading to mixed population behavior and hence biphasic optical density waves. Ammonia has no significant influence on oscillation frequency and wave propagation velocity but shows a clear increase in the amplitude of the optical density waves. This may indicate a more vigorous chemotactic response by individual cells or a better synchronization of the responding cell populations due to shortened chemotactic deadaptation times.

The relationship between the cell cycle phase of vegetative amoebae and prestalk and prespore differentiation in the slug stage were investigated in the slime mould Dictyostelium discoideum. Cells were synchronized by release from the stationary phase. Samples were taken at various times during the course of a synchronous cell doubling, fluorescently labelled and mixed with cells of random cell cycle phase from exponentially growing cultures. The fate of the fluorescently labelled cells was recorded at the slug stage. Cells early in the cycle exhibit strong prestalk sorting; cells taken later in the cycle exhibit strong prespore sorting. The period of prestalk sorting occurs immediately following mitosis and lasts about 1 h in a cell cycle of about 7 h duration. Accompanying the altered sorting behaviour is a marked change in the prestalk-prespore proportions in slugs formed from synchronized populations of cells. Cells synchronized early in the cycle form slugs with 55 % prespore cells; cells synchronized late in the cycle form slugs with 90% prespore. The results are discussed in terms of models for the formation of the prestalk-prespore pattern in slugs.

Sun, 1 Jan 1984 12:00:00 +0100 http://epub.ub.uni-muenchen.de/3334/ http://epub.ub.uni-muenchen.de/3334/1/061.pdf Weijer, Cornelis J.; Duschl, Gertrud; David, Charles N. Weijer, Cornelis J.; Duschl, Gertrud und David, Charles N. (1984): A REVISION OF THE DICTYOSTELIUM DISCOIDEUM CELL CYCLE. In: Journal of Cell Science, Vol. 70, Nr. 1: pp. 111-131. Biologie

Sun, 1 Jan 1984 12:00:00 +0100 http://epub.ub.uni-muenchen.de/3419/ http://epub.ub.uni-muenchen.de/3419/1/3419.pdf MacWilliams, H.; David, Charles N. MacWilliams, H. und David, Charles N. (1984): Pattern formation in Dictyostelium. In: Losick, Richard (Hrsg.), Microbial Development. Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, pp. 255-274. Biologie

A pattern of two tissue types exists in the Dictyostelium slug. Contained within the posterior tissue are anterior-like cells which comprise about 10% of the developing cell mass. For more than 72 hr of slug migration the proportion of these cells is closely regulated. They are randomly distributed along the anterior-posterior axis but about twice as many are localized in the ventral portion of the slug posterior than in the dorsal portion. As the slug begins to form a fruiting body, the anterior-like cells sort out into two groups. One group moves toward the anterior region and one toward the prebasal disc region. In the mature fruiting body the anterior-like cells remain as undifferentiated amoebae at the apex and base of the sorus. Removal of anterior tissue from a slug initiates two events. (1) Some of the anterior-like cells, probably guided by chemotaxis to cyclic AMP, sort out from the posterior tissue. (2) Some prespore cells redifferentiate into anterior-like cells. These events result in the regeneration of a new anterior-posterior pattern after 2 hr and the reestablishment of the original proportions of each cell type by about 8 hr. Furthermore, while the anterior-like cells which lie in slug posteriors remain as amoebae in fruiting bodies, the anterior-like cells which form the anteriors of regenerated slugs subsequently become stalk cells. Thus, it appears that for a cell to differentiate as a stalk cell, it must first be exposed to some form of signal which is present in both the anterior and prebasal disc regions.

Formation of the prestalk-prespore pattern in Dictyostelium was investigated in slugs and submerged clumps of cells. Prestalk and prespore cells were identified by staining with vital dyes, which are shown to be stable cell markers. Dissociated slug cells reaggregate and form slugs that contain a prestalk-prespore pattern indistinguishable from the original pattern. The pattern forms by sorting out of stained prestalk cells from unstained prespore cells. Sorting also occurs in clumps of dissociated slug cells submerged in liquid or agar. A pattern arises in 2 h in which a central core of stained cells is surrounded by a periphery of unstained cells. Sorting appears to be due to differential chemotaxis of stained and unstained cells to cAMP since exogenous cAMP (>10−7 M) reverses the normal direction of sorting-out such that stained cells sort to the periphery of the clumps. Isolated portions of slugs regenerate a new prestalk-prespore pattern. Posterior isolates regenerate a pattern within 2 h due to sorting of a population of vitally stained ‘anterior-like’ cells present in posteriors. Anterior-like cells do not sort in intact slugs due to the influence of a diffusible inhibitor secreted by the anterior region. During posterior regeneration this signal is absent and anterior-like cells rapidly acquire the ability to sort. Anterior isolates regenerate a staining pattern more slowly than posterior isolates by a process that requires conversion of stained prestalk cells to unstained prespore cells. The results suggest that pattern formation in Dictyostelium consists of two processes: establishment of appropriate proportions of two cell types and establishment of the pattern itself by a mechanism of sorting-out.

We have investigated the formation of the prestalk-prespore pattern in Dictyottelium discoideum. Pattern formation occurs in clumps of Dictyostelium cells embedded in agar under a 100% oxygen atmosphere. Agar embedding allows us to control spatially the environment surrounding the cell clumps. Our results suggest that the ambient oxygen concentration plays a role in controlling the size of the multicellular mass. Further, oxygen gradients established across clumps embedded in agar or held in holes in a plastic barrier cause orientation of the prestalk-prespore pattern such that the anterior prestalk region forms at the highest end of the gradient. The results also indicate that developing cells have the ability to migrate up a gradient of oxygen.

Mon, 1 Jan 1979 12:00:00 +0100 http://epub.ub.uni-muenchen.de/3332/ http://epub.ub.uni-muenchen.de/3332/1/056.pdf Sternfeld, John; David, Charles N. Sternfeld, John und David, Charles N. (1979): AMMONIA PLUS ANOTHER FACTOR ARE NECESSARY FOR DIFFERENTIATION IN SUBMERGED CLUMPS OF DICTYOSTELIUM. In: Journal of Cell Science, Vol. 38: pp. 181-191.