Eukaryotic motor protein
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.19.548188v1?rss=1 Authors: Chai, Y., Li, D., Gong, W., Ke, J., Tian, D., Chen, Z., Guo, A., Guo, Z., Li, W., Feng, W., Ou, G. Abstract: KIF1A, a microtubule-based motor protein responsible for axonal transport, is linked to a group of neurological disorders known as KIF1A-associated neurological disorder (KAND). Current therapeutic options for KAND are limited. Here, we introduced the clinically relevant KIF1A(R11Q) variant into the C. elegans homolog UNC-104, resulting in uncoordinated animal behaviors. Through genetic suppressor screens, we identified intragenic mutations in UNC-104's motor domain that rescued synaptic vesicle localization and coordinated movement. We showed that two suppressor mutations partially recovered motor activity in vitro by counteracting the structural defect caused by R11Q at KIF1A's nucleotide-binding pocket. We found that supplementation with fisetin, a plant flavonol, improved KIF1A(R11Q) worms' movement and morphology. Notably, our biochemical and single-molecule assays revealed that fisetin directly restored the ATPase activity and processive movement of human KIF1A(R11Q) without affecting wild-type KIF1A. These findings suggest fisetin as a potential intervention for enhancing KIF1A(R11Q) activity and alleviating associated defects in KAND. 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/2023.07.12.548706v1?rss=1 Authors: Wu, Y., Ding, C., Weinreb, A., Manning, L., Swaim, G., Yogev, S., Colon-Ramos, D., Hammarlund, M. Abstract: Mitochondria transport is crucial for mitochondria distribution in axons and is mediated by kinesin-1-based anterograde and dynein-based retrograde motor complexes. While Miro and Milton/TRAK were identified as key adaptors between mitochondria and kinesin-1, recent studies suggest the presence of additional mechanisms. In C. elegans, ric-7 is the only single gene described so far, other than kinesin-1, that is absolutely required for axonal mitochondria localization. Using CRISPR engineering in C. elegans, we find that Miro is important but is not essential for anterograde traffic, whereas it is required for retrograde traffic. Both the endogenous RIC-7 and kinesin-1 act at the leading end to transport mitochondria anterogradely. RIC-7 recruitment to mitochondria requires its N-terminal domain and partially relies on MIRO-1, whereas RIC-7 accumulation at the leading end depends on its disordered region, kinesin-1 and metaxin2. We conclude that polarized transport complexes containing kinesin-1 and RIC-7 form at the leading edge of mitochondria, and that these complexes are required for anterograde axonal transport. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
Dr. Ahmed Hamdy, CEO and Co-Founder of Vincerx, is focused on targeting a specific antigen found on cancer cells. With a unique enzyme, it is effective with solid tumors and hematological malignancies, releasing a kinesin spindle protein inhibitor that inhibits the division of cancer cells. Their first bioconjugate VIP236 targets a specific molecule expressed on several metastatic tumors. Ahmed explains, "At Vincerx, we have a very exciting pipeline that's designed to solve a lot of problems with the current treatments for cancer therapies. In today's world, cancer continues growing exponentially. And thankfully, there are a lot of treatments out there for different types of cancer. Yet, the current treatments come with quite a bit of morbidities. Throughout my career, the morbidity of medicine has been something that I've always been concerned about, especially for patients and their caregivers. At Vincerx, we have very exciting, unique types of treatments that can be paradigm-shifting from a safety perspective and efficacy perspective." "For solid tumors, we have a compound that's currently in dose escalation phase 1 trial that is designed for advanced metastatic tumors, where we target a specific antigen that is found on the cancer cells themselves. And with a unique enzyme, it cleaves what we're describing as a warhead or the substance that can kill the cancer cell, which is an optimized camptothecin. And the word optimize means it's a well-known topoisomerase inhibitor designed to allow for very rapid permeability or intake by the cancer cell, with very low pumping out." #VincerxPharma #VIP236 #VIP943 #ADCs #Bioconjugates #Cancer #SolidTumors #Biotech #PrecisionMedicine Vincerx.com Listen to the podcast here
Dr. Ahmed Hamdy, CEO and Co-Founder of Vincerx, is focused on targeting a specific antigen found on cancer cells. With a unique enzyme, it is effective with solid tumors and hematological malignancies, releasing a kinesin spindle protein inhibitor that inhibits the division of cancer cells. Their first bioconjugate VIP236 targets a specific molecule expressed on several metastatic tumors. Ahmed explains, "At Vincerx, we have a very exciting pipeline that's designed to solve a lot of problems with the current treatments for cancer therapies. In today's world, cancer continues growing exponentially. And thankfully, there are a lot of treatments out there for different types of cancer. Yet, the current treatments come with quite a bit of morbidities. Throughout my career, the morbidity of medicine has been something that I've always been concerned about, especially for patients and their caregivers. At Vincerx, we have very exciting, unique types of treatments that can be paradigm-shifting from a safety perspective and efficacy perspective." "For solid tumors, we have a compound that's currently in dose escalation phase 1 trial that is designed for advanced metastatic tumors, where we target a specific antigen that is found on the cancer cells themselves. And with a unique enzyme, it cleaves what we're describing as a warhead or the substance that can kill the cancer cell, which is an optimized camptothecin. And the word optimize means it's a well-known topoisomerase inhibitor designed to allow for very rapid permeability or intake by the cancer cell, with very low pumping out." #VincerxPharma #VIP236 #VIP943 #ADCs #Bioconjugates #Cancer #SolidTumors #Biotech #PrecisionMedicine Vincerx.com Download the transcript here
Join Hugh Ross and Jeff Zweerink as they discuss new discoveries taking place at the frontiers of science that have theological and philosophical implications, including the reality of God's existence. Lunar Catastrophes The Moon is exquisitely fine-tuned in various ways that are essential for advanced life to exist on Earth's surface. However, astronomers have determined that the Moon will reverse its outward migration from Earth and crash into our planet about 40 billion years from now. Astronomer Bradley Hansen recently demonstrated that for the majority of planet-moon systems capable of sustaining life, the moon will collide with the planet long before conditions on that planet permit the existence of advanced life. Hansen has discovered yet another habitability requirement: a large dynamically stable moon orbiting a large rocky planet. References: Consequences of Dynamically Unstable Moons in Extrasolar Systems Peering Deep Inside Cells Scientists continue to develop tools to investigate the detailed workings inside the cell. In one recent example researchers tracked the motion of individual molecules that transport resources around the cell. Advances over past attempts now demonstrate the step-by-step movements of motor protein kinesin-1 as it moves through the cell, and data gives hints that the molecule might be twisting as it traverses the intracell highways. These advances not only show the incredible design within the cell but continue to add to the overwhelming evidence that humans are truly exceptional among all the animals on Earth. References: Superresolution Microscopy Tracks a Walking Biomolecule Direct Observation of Motor Protein Stepping in Living Cells Using MINFLUX MINFLUX Dissects the Unimpeded Walking of Kinesin-1
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.01.538972v1?rss=1 Authors: Gergely, Z., Jones, M. H., Zhou, B., Cash, C., McIntosh, R., Betterton, M. Abstract: 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/2023.04.18.537280v1?rss=1 Authors: Kita, T., Chiba, K., Wang, J., Nakagawa, A., Niwa, S. Abstract: Kinesin-3 is a family of microtubule-dependent motor proteins that transport various cargos within the cell. However, the mechanism underlying kinesin-3 activations remains largely elusive. In this study, we compared the biochemical properties of two Caenorhabditis elegans kinesin-3 family proteins, KLP-6 and UNC-104. Both KLP-6 and UNC-104 are predominantly monomeric in solution. As previously shown for UNC-104, non-processive KLP-6 monomer is converted to a processive motor when artificially dimerized. We present evidence that releasing the autoinhibition is sufficient to trigger dimerization of monomeric UNC-104 at nanomolar concentrations, which results in processive movement of UNC-104 on microtubules, although it has long been thought that enrichment in the phospholipid microdomain on cargo vesicles is required for the dimerization and processive movement of UNC-104. In contrast, KLP-6 remains to be a non-processive monomer even when its autoinhibition is unlocked, suggesting a requirement of other factors for full activation. By examining the differences between KLP-6 and UNC-104, we identified a coiled-coil domain called CC2 that is required for the dimerization and processive movement of UNC-104. Our results suggest a common activation mechanism for kinesin-3 family members, while also highlighting their diversification. 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/2023.04.17.535593v1?rss=1 Authors: Aureille, J., Barnett, S., Arnal, I., Lafanechere, L., Low, B. C., Kanchanawong, P., Mogilner, A., Bershadsky, A. Abstract: Microtubules regulate cell polarity and migration by local activation of focal adhesion turnover, but the mechanism of this process is insufficiently understood. Molecular complexes containing KANK family proteins connect microtubules with the major component of focal adhesions, talin. Local optogenetic activation of KANK1-mediated links which promoted microtubule targeting to individual focal adhesion resulting in its centripetal sliding and rapid disassembly. The sliding is preceded by a local increase of traction force due to accumulation of myosin-II and actin in the proximity of the focal adhesion. Knockdown of Rho activator GEF-H1 prevented development of traction force and abolished sliding and disassembly of focal adhesion upon KANK activation. Other players participating in microtubule-driven KANK-dependent focal adhesion disassembly include kinases ROCK and PAK, as well as microtubules/focal adhesions associated proteins Kinesin-1, APC and TAT. Finally, we propose a physical model of a microtubule-driven focal adhesion disruption involving local GEF-H1/RhoA/ROCK dependent activation of contractility which is consistent with experimental data. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
While DNA may be the blueprint of life, proteins are the workhorses, says Polly Fordyce, a bioengineer, explaining how one of her favorites, kinesin, “walks” in 8-nanometer steps transporting chemical cargo through the body. More remarkable still, Fordyce says, kinesin is just one among thousands of “incredible” proteins that make life happen, as she tells host Russ Altman on this episode of Stanford Engineering's The Future of Everything podcast.
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.06.535716v1?rss=1 Authors: Montgomery, A. C., Mendoza, C. S., Garbouchian, A., Quinones, G. B., Bentley, M. Abstract: Neurons are polarized cells that require accurate membrane trafficking to maintain distinct protein complements at dendritic and axonal membranes. The Kinesin-3 family members KIF13A and KIF13B are thought to mediate dendrite-selective transport, but the mechanism by which they are recruited to polarized vesicles and the differences in the specific trafficking role of each KIF13 have not been defined. We performed live-cell imaging in cultured hippocampal neurons and found that KIF13A is a dedicated dendrite-selective kinesin. KIF13B confers two different transport modes, both dendrite- and axon-selective transport. Both KIF13s are maintained at the trans-Golgi network by interactions with the heterotetrameric adaptor protein complex AP-1. Interference with KIF13 binding to AP-1 resulted in disruptions to both dendrite- and axon- selective trafficking. We conclude that AP-1 is the molecular link between the sorting of polarized cargoes into vesicles and the recruitment of kinesins that confer polarized transport. 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/2023.02.07.527540v1?rss=1 Authors: Chew, Y. M., Cross, R. A. Abstract: Taxol is a critically important cancer drug that stabilises microtubules. We report that taxol acts differently on different metazoan tubulin isotypes. 50 nM taxol blocks catastrophe of human or zebrafish 1{beta}4 but has no effect on human 1{beta}3 microtubules. 500 nM taxol blocks catastrophe in both 1{beta}3 and 1{beta}4 microtubules but introduces kinks only into 1{beta}4 microtubules. Taxol washout relaxes the kinks, suggesting taxol expands 1{beta}4 but not 1{beta}3 lattices. Kinesin-driven microtubule gliding detects this conformational shift - 1{beta}4 microtubules glide at ~450 nm/sec in 400 nM taxol, but at ~750 nm/sec in 10 M taxol, whereas 1{beta}3 microtubules glide at ~450 nm/sec, even in 10 M taxol. Thus, taxol readily stabilises 1{beta}4 GDP-tubulin lattices and shifts them to a fast-gliding conformation, but stabilises 1{beta}3 lattices much less readily and without shifting their conformation. These isotype-specific actions of taxol may drive the switch to {beta}3 tubulin commonly seen in taxol-resistant tumours. 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/2023.02.06.526840v1?rss=1 Authors: Rivera Alvarez, J., Asselin, L., Tilly, P., Benoit, R., Batisse, C., Richert, L., Batisse, J., Morlet, B., Levet, F., Schwaller, N., Mely, Y., Ruff, M., Reymann, A.-C., GODIN, J. D. Abstract: Completion of neuronal migration is critical for brain development. Kif21b is a plus-end directed kinesin motor protein that promotes intracellular transport and controls microtubule dynamics in neurons. Here we report a physiological function of Kif21b during radial migration of projection neurons in the mouse developing cortex. In vivo analysis in mouse and live imaging on cultured slices demonstrate that Kif21b regulates the radial glia-guided locomotion of new-born neurons independently of its motility on microtubules. Unexpectedly we show that Kif21b directly binds and regulates the actin cytoskeleton both in vitro and in vivo in migratory neurons. We establish that Kif21b-mediated regulation of actin cytoskeleton dynamics influences branching and nucleokinesis during neuronal locomotion. Altogether, our results reveal atypical roles of Kif21b on the actin cytoskeleton during migration of cortical projection neurons. 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/2023.01.07.523106v1?rss=1 Authors: Gumusderelioglu, S., Resch, L., Brock, T., Undiagnosed Diseases Network,, Luxton, G. G., Tan, Q. K.-G., Hopkins, C. E., Starr, D. A. Abstract: Hereditary spastic paraplegia (HSP) is a group of degenerative neurological disorders. We identified a variant in human kinesin light chain KLC4 that is suspected to be associated with autosomal dominant HSP. How this and other variants relate to pathologies is unknown. We created a humanized C. elegans model where klc-2 was replaced with human KLC4 and assessed the extent to which hKLC4 retained function in the worm. We observed a slight decrease in motility but no nuclear migration defects in the humanized worms, suggesting that hKLC4 retains much of the function of klc-2. Five hKLC4 variants were introduced into the humanized model. The clinical variant led to early lethality with significant defects in nuclear migration when homozygous, and a weak nuclear migration defect when heterozygous, possibly correlating with the clinical finding of late onset HSP when the proband was heterozygous. Thus, we were able to establish humanized C. elegans as an animal model for HSP and use it to test the significance of five variants of uncertain significance in the human gene KLC4. 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/2022.12.27.522002v1?rss=1 Authors: Ghosh, S. K., Shah, S., Mittal, P., Kumar, D., Mittal, A. Abstract: The characteristic bi-lobed organization of the kinetochores observed during mitotic metaphase is a result of separation of the sister kinetochores into two clusters upon their stable end-on attachment to the microtubules emanating from opposite spindle poles. In contrast, during metaphase I of meiosis despite bi-orientation of the homologs, we observe that the kinetochores are linearly dispersed between the two spindle poles indicating that pole-distal and pole-proximal kinetochores are attached laterally and end-on, respectively to the microtubules. Colocalization studies of kinetochores and kinesin motors suggest that budding yeast kinesin 5, Cin8 and Kip1 perhaps localize to the end-on attached kinetochores while kinesin 8, Kip3 resides at all the kinetochores. Unlike mitosis in budding yeast, in meiosis, the outer kinetochores assemble much later after prophase I. From the findings including co-appearance of kinesin 5 and the outer kinetochore protein Ndc80 at the centromeres after prophase I and a reduction in Ndc80 level in Cin8 null mutant, we propose that kinesin motors are required for reassembly and stability of the kinetochores during early meiosis. Thus, this work reports yet another meiosis specific function of kinesin motor. 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/2022.12.22.521561v1?rss=1 Authors: Niwa, S., Chiba, K. Abstract: Kinesin-1, a motor protein composed of the kinesin heavy chain (KHC) and the kinesin light chain (KLC), is fundamental to cellular morphogenesis and function. A monoclonal antibody (mAb) called H2 recognizes the KHC in a broad range of species and is one of the most widely used mAbs in cytoskeletal motor research. Here, we generated vectors that expressed recombinant H2 in mammalian cells. We demonstrated that the recombinant H2 performed as well as the hybridoma-derived H2 in western blotting and immunofluorescence assays. The recombinant H2 could detect all three human KHC isotypes (KIF5A, KIF5B, and KIF5C) and amyotrophic lateral sclerosis (ALS)-associated KIF5A aggregates in the cell. Immunofluorescence microscopy showed that the single chain variable fragment (scFv) derived from the H2 mAb could specifically recognize KHCs in cells. In addition, we developed a chickenized anti-KHC scFv(H2), which broadens the application of H2 in immunofluorescence microscopy. Collectively, our findings validate recombinant H2 as useful for studying the function of KHCs. 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/2022.12.22.521263v1?rss=1 Authors: Albisetti, A. C., Douglas, R. L., Welch, M. D. Abstract: Trypanosoma brucei, the causative agent of African sleeping sickness, uses its flagellum for movement, cell division, and signaling. The flagellum is anchored to the cell body membrane via the flagellar attachment zone (FAZ), a complex of proteins, filaments, and microtubules that spans two membranes with elements on both flagellum and cell body sides. How FAZ components are carried into place to form this complex is poorly understood. Here, we show that the trypanosome-specific kinesin KIN-E is required for building the FAZ in bloodstream-form parasites. KIN-E is localized along the flagellum with a concentration at its distal tip. Depletion of KIN-E by RNAi rapidly inhibits flagellum attachment and leads to cell death. A detailed analysis reveals that KIN-E depletion phenotypes include failure in cytokinesis completion, kinetoplast DNA mis-segregation, and transport vesicle accumulation. Together with previously published results in procyclic form parasites, these data suggest KIN-E plays a critical role in FAZ assembly in T. brucei. 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/2022.12.01.518806v1?rss=1 Authors: Andreu-Carbo, M., Egoldt, C., Velluz, M.-C., Aumeier, C. Abstract: The properties of single microtubules within the microtubule network can be modulated through posttranslational modifications (PTMs), including acetylation within the lumen of microtubules. To access the lumen, the enzymes could either enter through the microtubule ends or at damage sites along the microtubule shaft. Here we show that the acetylation profile depends on damage sites, which can be caused by the motor protein kinesin-1. Indeed, the entry of the deacetylase HDAC6 into the microtubule lumen depends on kinesin-1-induced damage sites. In contrast, activity of the microtubule acetylase TAT1 is independent of kinesin-1 and shaft damage. On a cellular level, our results show that microtubule acetylation distributes in an exponential gradient. This gradient results from tight regulation of microtubule (de-)acetylation and scales with the size of the cells. The control of shaft damage represents a novel mechanism to regulate PTM inside the microtubule by giving access to the lumen. 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/2022.11.08.515201v1?rss=1 Authors: Seneviratne, P. B., Lidagoster, S., Valbuena-Castor, S., Lashley, K., Saha, S., Kreitzer, G. Abstract: Kinesin family motors are microtubule (MT)-stimulated ATPases known best as transporters of cellular cargoes through the cytoplasm, regulators of MT dynamics, organizers of the mitotic spindle, and for insuring equal division of DNA during mitosis. Several kinesins have also been shown to regulate transcription by interacting with transcriptional cofactors and regulators, nuclear receptors, or with specific promotor elements on DNA. We previously showed that an LxxLL nuclear receptor box motif in the kinesin-2 family motor KIF17 mediates binding to the orphan nuclear receptor estrogen related receptor alpha (ERR1) and is responsible for the suppression of ERR1-dependent transcription by KIF17. Analysis of all kinesin family proteins revealed that multiple kinesins contain this LxxLL motif, raising the question as to whether additional kinesins motors contribute to regulation of ERR1. In this study, we interrogated the effects of multiple kinesins with LxxLL motifs on ERR1-mediated transcription. We demonstrate that the kinesin-3 motor KIF1B contains two LxxLL motifs, one of which binds to ERR1. In addition, we show that expression of a KIF1B fragment containing this LxxLL motif inhibits ERR1-dependent transcription by regulating nuclear entry of ERR1. We also provide evidence that the effects of expressing the KIF1B-LxxLL fragment on ERR1 activity are mediated by a mechanism distinct from that of KIF17. Because LxxLL domains are found in many kinesins, our data suggest an expanded role for kinesins in nuclear receptor mediated transcriptional regulation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
Heyo Wiwas, es ist schon wieder Sonntag. Das kommt nicht nur für dich, sondern auch für uns überraschend. So überraschend, dass der Texteschreiber erst jetzt seine Finger zur Höchstform motivieren konnte. Im heutigen Podcast geht es um das Innerste unserer Köpfe. Das Gehirn, unser Bewusstsein und der Frage, ob wir uns manchmal ganz gerne selber belügen. Wir gehen der Sache auf den Grund und hoffen auf ein freudiges oooooohhhh aus euren Reihen. Denkt dran, die nächsten 3 Wochen machen wir Pause und sind dann im November wieder für euch am Start. Kinesin protein walking on microtubule https://www.youtube.com/watch?v=y-uuk4Pr2i8 Können wir uns selbst trauen? | 42 - Die Antwort auf fast alles | ARTE https://www.youtube.com/watch?v=jFVYxD33ECk
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.08.24.505074v1?rss=1 Authors: Nagel, M., Noss, M., Xu, J., Horn, N., Ueffing, M., Boldt, K., Schuele, R. Abstract: Neurons critically depend on regulated RNA localization and tight control of spatio-temporal gene expression to maintain their morphological and functional integrity. Mutations in the kinesin motor protein gene KIF1C cause Hereditary Spastic Paraplegia, an autosomal recessive disease leading to predominant degeneration of the long axons of central motoneurons. In this study we aimed to gain insight into the molecular function of KIF1C and understand how KIF1C dysfunction contributes to motoneuron degeneration. We used affinity proteomics in neuronally differentiated neuroblastoma cells (SH-SY5Y) to identify the protein complex associated with KIF1C in neuronal cells; candidate interactions were then validated by immunoprecipitation and mislocalization of putative KIF1C cargoes was studied by immunostainings. We found KIF1C to interact with all core components of the exon junction complex (EJC); expression of mutant KIF1C in neuronal cells leads to loss of the typical localization distally in neurites. Instead, EJC core components accumulate in the pericentrosomal region, here co-localizing with mutant KIF1C. These findings suggest KIF1C as a neuronal transporter of the EJC. Interestingly, the binding of KIF1C to the EJC is RNA-mediated, as treatment with RNAse prior to immunoprecipitation almost completely abolishes the interaction. Silica-based solid-phase extraction of UV-crosslinked RNA-protein complexes furthermore supports direct interaction of KIF1C with RNA, as recently also demonstrated for kinesin heavy chain. Taken together, our findings are consistent with a model where KIF1C transports mRNA in an EJC-bound and therefore transcriptionally silenced state along neurites, thus providing the missing link between the EJC and mRNA localization in neurons. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer
On today's ID the Future, distinguished British physician and author David Galloway explains why he's convinced that the human fetal circulatory system is irreducibly complex and therefore beyond the reach of blind gradualistic evolution to have built. In his conversation with host and fellow physician Geoffrey Simmons, Galloway also mentions some molecular machines that he's convinced are irreducibly complex and shout intelligent design. The occasion for the conversation is Galloway's new book, Design Dissected. Source
Evolutionists have no plausible theory on how something as sophisticated as kinesin could have evolved in a gradual fashion. However, when we see similar machines and operating systems in our everyday life at work or home, they are always the result of intelligent and intentional design. This episode article was written by Calvin Smith and podcast produced by Joseph Darnell out of the CMI-USA office. Become a monthly contributor at our site. You can also help out by telling your family and friends to check out the podcasts. ✍️ Links and Show Notes Original article: Incredible Kinesin! New bacteria show ‘wonder upon wonder' Germ with seven motors in one! Design in living organisms (motors: ATP synthase) Virus has powerful mini-motor to pack up its DNA Germ's miniature motor has a clutch Fantastic voyage Cosmos by Neil deGrasse Tyson Even a tiny virus has a powerful mini-motor Irreducible complexity and cul-de-sacs Molecular motors show incredible design
TWiV reviews Michael Worobey's dissection of the early COVID-19 cases in Wuhan, and the discovery that herpesviruses assimilate cellular kinesin to produce motorized virus particles. Hosts: Vincent Racaniello, Alan Dove, and Kathy Spindler Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode Early COVID-19 cases in Wuhan (Science) Herpesviruses assimilate kinesin (Nature) Letters read on TWiV 833 Timestamps by Jolene. Thanks! Weekly Picks Kathy – Power of poop Alan – Under the Sky We Make, by Kimberly Nicholas Vincent – Enzymatic amplification of beta-globing sequences Listener Picks Paula – Reasons to be Cheerful and The Night Witches by Bruce Myles Grant – George Carlin – Germs, Immune System Intro music is by Ronald Jenkees Send your virology questions and comments to twiv@microbe.tv
TWiV reviews Michael Worobey's dissection of the early COVID-19 cases in Wuhan, and the discovery that herpesviruses assimilate cellular kinesin to produce motorized virus particles. Hosts: Vincent Racaniello, Alan Dove, and Kathy Spindler Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode Early COVID-19 cases in Wuhan (Science) Herpesviruses assimilate kinesin (Nature) Letters read on TWiV 833 Timestamps by Jolene. Thanks! Weekly Picks Kathy – Power of poop Alan – Under the Sky We Make, by Kimberly Nicholas Vincent – Enzymatic amplification of beta-globing sequences Listener Picks Paula – Reasons to be Cheerful and The Night Witches by Bruce Myles Grant – George Carlin – Germs, Immune System Intro music is by Ronald Jenkees Send your virology questions and comments to twiv@microbe.tv
TIME STAMP 0:00 ช่วงแรก แนะนำตัวพี่ชิ้นและอ.ป๋วย 26:05 จารย์ป๋วยเล่าสงครามโปรตีน (AlphaFold vs Rosetta Fold) AI เจ้าไหนจะไขปริศนาทำนายโครงสร้าง 3 มิติของโปรตีนได้เจ๋งกว่ากัน 32:05 เบรกปูพื้นฐานชีววิทยาโมเลกุล DNA, RNA, Protein ให้คนไม่เคยเรียน ใครรู้อยู่แล้วข้ามช่วงนี้ไปก็ได้ 41:05 กลับมาฟังจารย์ป๋วยเล่าต่อ 1:14:15 พี่ชิ้นช่วยเสริมเรื่องที่มาของชื่อ Rosetta ตามด้วยช่วงนักชีวโมเลกุลหัวร้อนกับการตั้งชื่อโปรตีน 1:31:40 พี่ชิ้นเล่าเรื่องงานวิจัยสนุกๆ จากคอลัมน์ที่เขียน และหนังสือเล่มใหม่ เธอ ฉัน สวรรค์ นรก แขกรับเชิญตอนนี้คือ พี่ชิ้น นําชัย ชีววิวรรธน์ เจ้าของผลงานล่าสุด หนังสือ "เธอ ฉัน สวรรค์ นรก" และ อ. ป๋วย อุ่นใจ หนึ่งในผู้เขียนหนังสือ "Vaccine War สมรภูมิวัคซีนโควิด-19" วิดิโอไลฟ์สด https://www.youtube.com/watch?v=PThn7gbneEk คอลัมน์ "ทะลุกรอบ" ของอาจารย์ป๋วยตอน สงครามปัญญาประดิษฐ์ ไขปริศนาจักรกลนาโน ภาค 1 สงครามปัญญาประดิษฐ์ ไขปริศนาจักรกลนาโน ภาค 2 : การกลับมาของกูเกิลดีปมายด์ ภาพโครงสร้างสามมิติของโปรตีนอินเตอร์ลิวคิน-12 กำลังจับกับโปรตีนตัวรับของมัน ทำนายโดยโรเซ็ตต้าโฟลด์ (ภาพโดย Ian Haydon, UW Medicine Institue for Protein Design) AlphaFold - 1,2 คลิปอนิเมชั่นโมเลกุลโปรตีนเดินได้ Kinesin และ Dynein https://www.youtube.com/watch?v=y-uuk4Pr2i8 https://www.youtube.com/watch?v=-7AQVbrmzFw พี่ชิ้นเล่างานวิจัยเรื่อง เวลาทำข้อสอบไม่ควรเชื่อสัญชาตญานแรก ยกเว้นข้อที่มั่นใจจริงๆ -1,2 รูปปกเป็นภาพวาดไวรัส SARS-CoV-2 กำลังบุกเข้าเซลล์ เห็นโครงสร้างโมเลกุลต่างๆ สวยงาม ผลงานโดย David Goodsell
Vale explains how doing science often follows a winding path with unexpected, sometimes wonderful surprises, and uses his own story to illustrate his point. When Vale was a graduate student, he initially hoped to show that myosin was involved in axonal transport, but ended up discovering a new molecule which he called kinesin.
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.22.308320v1?rss=1 Authors: Lam, A. J., Rao, L., Anazawa, Y., Okada, K., Chiba, K., Niwa, S., Gennerich, A., Nowakowski, D. W., McKenney, R. J. Abstract: KIF1A, a kinesin-3 family member, plays critical roles as a long-distance cargo-transporter within neurons. Over 100 known KIF1A mutations in humans result in KIF1A Associated Neurological Disease (KAND), developmental and degenerative neurological conditions for which there is no cure. A de novo missense mutation, P305L, was recently identified in several children diagnosed with KAND, but the underlying molecular basis for the disease phenotype is unknown. Interestingly, this residue is highly conserved in kinesin-family proteins, and together with adjacent conserved residues also implicated in KAND, forms an unusual 310-helical element immediately C-terminal to loop-12 (L12, also known as the K-loop in KIF1A) in the conserved kinesin motor core. In KIF1A, the disordered K-loop contains a highly charged insertion of lysines that is thought to endow the motor with a high microtubule-association rate. Here, we characterize the molecular defects of the P305L mutation in KIF1A using genetic, biochemical, and single-molecule approaches. We find the mutation negatively impacts the velocity, run-length, and force generation of the motor. However, a much more dramatic effect is observed on the microtubule-association rate of the motor, revealing that the presence of an intact K-loop is not sufficient for its function. We hypothesize that an elusive K-loop conformation, mediated by formation of a highly conserved adjacent 310-helix that is modulated via P305, is critically important for the kinesin-microtubule interaction. Importantly, we find that the function of this proline is conserved in the canonical kinesin, KIF5, revealing a fundamental principle of the kinesin motor mechanism. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.03.281576v1?rss=1 Authors: Budaitis, B. G., Jariwala, S., Rao, L., Sept, D., Verhey, K., Gennerich, A. Abstract: The kinesin-3 motor KIF1A functions in neurons where its fast and superprocessive motility is thought to be critical for long-distance transport. However, little is known about the force-generating properties of kinesin-3 motors. Using optical tweezers, we demonstrate that KIF1A and its C. elegans homolog UNC-104 undergo force-dependent detachments at ~3 pN and then rapidly reattach to the microtubule to resume motion, resulting in a sawtooth pattern of clustered force generation events that is unique among the kinesin superfamily. Whereas UNC-104 motors stall before detaching, KIF1A motors do not. To examine the mechanism of KIF1A force generation, we introduced mutations linked to human neurodevelopmental disorders, V8M and Y89D, based on their location in structural elements required for force generation in kinesin-1. Molecular dynamics simulations predict that the V8M and Y89D mutations impair docking of the N-terminal ({beta}9) or C-terminal ({beta}10) portions of the neck linker, respectively, to the KIF1A motor domain. Indeed, both mutations dramatically impair force generation of KIF1A but not the motor's ability to rapidly reattach to the microtubule track. Homodimeric and heterodimeric mutant motors also display decreased velocities, run lengths, and landing rates and homodimeric Y89D motors exhibit a higher frequency of non-productive, diffusive events along the microtubule. In cells, cargo transport by the mutant motors is delayed. Our work demonstrates the importance of the neck linker in the force generation of kinesin-3 motors and advances our understanding of how mutations in the kinesin motor domain can manifest in disease. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.12.247544v1?rss=1 Authors: Angerani, S., Lindberg, E., Klena, N., Bleck, C. K. E., Aumeier, C., Winssinger, N. Abstract: Kinesin-1 is a processive motor protein that uses ATP-derived energy to transport a variety of intracellular cargoes toward the cell periphery. As tracks for cargo delivery, kinesin-1 uses a subset of microtubules within the dense microtubule network. It is still debated what defines the specific binding of kinesin-1 to a subset of microtubules. Therefore, the ability to visualize and monitor kinesin transport in live cells is critical to study the myriad of functions associated with cargo trafficking. Herein we report the discovery of a fluorogenic small molecule substrate for kinesin-1 that yields a precipitating dye. The activity of kinesin-1 thus leaves a fluorescent trail along its walking path and can be visualized without loss of signal due to diffusion. Kinesin-1 specific transport of cargo from the Golgi appears as trails of fluorescence over time. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.10.244491v1?rss=1 Authors: Alfieri, A., Gaska, I., Forth, S. Abstract: The proper structural organization of the microtubule-based spindle during cell division requires the collective activity of many different types of proteins. These include non-motor microtubule-associated proteins (MAPs) whose functions include crosslinking microtubules to regulate filament sliding rates and assembling microtubule arrays. One such protein is PRC1, an essential MAP that has been shown to preferentially crosslink overlapping antiparallel microtubules at the spindle midzone. PRC1 has been proposed to act as a molecular brake, but insight into the mechanism of how PRC1 molecules function cooperatively to resist motor-driven microtubule sliding and to allow for the formation of stable midzone overlaps has been lacking. Here we employ a modified microtubule gliding assay to rupture PRC1-mediated microtubule pairs using surface-bound kinesins. We discovered that PRC1 crosslinks always reduce bundled filament sliding velocities relative to single microtubule gliding rates, and do so via two distinct emergent modes of mechanical resistance to motor-driven sliding. We term these behaviors braking and coasting, where braking events exhibit substantially slowed microtubule sliding compared to coasting events. Strikingly, braking behavior requires the formation of two distinct high-density clusters of PRC1 molecules near microtubule tips. Our results suggest a cooperative mechanism for PRC1 accumulation when under mechanical load that leads to a unique state of enhanced resistance to filament sliding and provides insight into collective protein ensemble behavior in regulating the mechanics of spindle assembly. Copy rights belong to original authors. Visit the link for more info
Latter Day Radio, now podcasting from The Intersection of Faith & Freedom.
Our initial podcasts offering “evidence” or “witnesses” focus on the arguments for “intelligent design,” namely, that when you find a watch in the forest, somewhere there must be a watchmaker. Watches do not assemble themselves, and as two of our guests, Dr. John West and Dr. Ann Gauger, explain, even life at its simplest is infinitely more complex than a watch. Our guests from the Discovery Institute in Seattle construct an increasingly compelling case that things as complex as an animal–vertebrate, invertebrate, complex or simple–cannot simply arise from itself alone through the process of natural selection. Complexity cannot arise from simplicity. Perpetual motion machines are impossible in physics–the science of biology is just as strict. In this last segment, cellular biologist Dr. Ann Gauger explores the wonderful work accomplished by a tiny machine inside of our cells called kinesin. Kinesin has an odd shape — two long legs, two feet, and a stalk-like body — that is eminently suited for its work. When it receives a package to carry, it attaches itself to one of the many microtubule highways of the cell and begins to stride, one foot over the other, dragging its cargo behind it and looking for all the world like a little stick man walking down a road with a great big bundle on his back. Kinesin is the UPS delivery man of the cell — a molecular motor that can muscle its way through the jostling tangle of the cytoplasm, dragging its cargo with it. Because of its amazing tenacity kinesin is rarely dislodged, placing one foot securely after the other; by working in teams it is able to transport cellular objects as large as mitochondria. When it encounters an obstacle it can't get around, kinesin will first recruit more kinesin motors to help. Failing that, it partners with another motor traveling in the opposite direction. They rock back and forth taking turns pulling in opposite directions, like a driver caught in a snow drift, until finally they dislodge their cargo. Then the original kinesin resumes travel in its old direction. How do the proteins and organelles get from where they are made to where they are used? And how do things that need to be recycled return to the cell body? It's by means of kinesin (for outward bound travel) and another motor protein called dynein (for inward bound travel). Remarkably, dynein and kinesin cooperate, rather than compete—otherwise there would be a constant tug of war in the cell. They “know” where packages are meant to go, and which motor protein should do the job. How does kinesin know which package to carry and what road to take? Apparently there are labels that kinesin recognizes, saying “Carry me!” but no one knows what the map is or how kinesin reads it. The big question is, could such a tiny little intra-cellular UPS man like kinesin create itself? Could the chaos of the natural world accidentally create such a thing with such abilities? The rational answer is NO. And the only possible explanation, that there was an Intelligent Designer with remarkable capabilities.
Nels and Vincent reveal how a motor protein in corn causes preferential transmission of chromosomes to egg cells, leading to non-Mendelian inheritance. Hosts: Nels Elde and Vincent Racaniello Become a patron of TWiEVO Molecular Evolution and the Cellmeeting Selfishcorn chromosomes (Cell) Dawe Lab- neocentromeres and meiotic drive Image credit Letters readon TWiEVO 30 Science Picks Nels - Craypot stinkhorn mushroom (Colus pusilus) Vincent -Doubts raised over plan to release herpesvirus to wipe out carp Music on TWiEVO is performed by Trampled by Turtles Send your evolution questions and comments to twievo@microbe.tv
Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 05/06
Mon, 13 Oct 2014 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/17534/ https://edoc.ub.uni-muenchen.de/17534/1/Koesem_Sueleyman.pdf Kösem, Süleyman ddc:570, ddc:500, Fakultät für Biologie
Fakultät für Physik - Digitale Hochschulschriften der LMU - Teil 05/05
Epithelzellen, die Modell-Zelllinien dieser Dissertation, sind für die Aufteilung und Abtrennung verschiedener Kompartimente eines Organismus zuständig, indem sie sich zu Grenzflächen zusammenschliessen, welche häufig hohen physikalischen Spannungen und Kräften ausgesetzt sind. Um diese physikalischen Kräfte zu verarbeiten oder sie selbst zu produzieren, verwenden Epithelzellen, wie alle anderen Zelltypen auch, das Zytoskelett, das sich im Allgemeinen aus den Komponenten Mikrotubuli, Intermediär-Filamenten und Aktin sowie den damit korrespondierenden Motorproteinen Dynein, Kinesin sowie Myosin zusammensetzt. In dieser Dissertation wird das Zusammenspiel von Aktin und Myosin auf der apikalen Seite von Epithelzellen untersucht. Im Falle von konfluenten Zellen mit vollständig ausgebildeten Zell-Zell-Kontakten sind auf der apikalen Seite der Zellen Mikrovilli zu finden, kleine, mit Aktin-Bündeln gefüllte Ausstülpungen aus der Zelloberfläche, welche für die optimierte Nahrungsaufnahme sowie als Antennen für Signalverarbeitung zuständig sind. Im Zuge der Arbeit konnten wir feststellen, dass sich der Aktin-Myosin-Aufbau auf der apikalen Seite von Einzelzellen ohne Zell-Zell-Kontakte, sogenannten nicht-konfluenten Zellen, grundsätzlich ändert. Mittels Fluoreszenz-Mikroskopie und anderen experimentellen Methoden zeigen wir, dass zwar ähnliche Ausstülpungen auf der apikalen Oberfläche von Einzelzellen zu finden, diese jedoch häufig verlängert, gebogen, hoch-dynamisch und oft parallel zur Zellmembran orientiert sind. Wir zeigen mittels molekularbiologischer Methoden, dass ein zusätzliches, innerhalb der apikalen Zellmembran liegendes isotropes Akto-Myosin-Netzwerk für die dynamische Reorganisation der Mikrovilli-Ausstülpungen verantwortlich ist. Der Identifzierung des isotropen Akto-Myosin-Netzwerkes, welches eine der Hauptaussagen dieser Dissertation ist, wird eine detaillierte Analyse der dynamischen Netzwerkreorganisation angefügt, die mittels temporaler und örtlicher Bild-Korrelationsanalysen charakteristische Zeiten und Längen der Dynamik definiert. Des Weiteren entwickeln wir mehrere Bild- Analyseverfahren, allen voran die Methode der iterativen temporalen Bildkorellation sowie des optischen Flusses, wodurch wir eine Oszillation der Netzwerk-Reorganisationsgeschwindigkeit identifizieren und parametrisieren können. Verschiedene, auf Fluoreszenzmikroskopie und automatisierter optischer Fluss-Bildanalyse basierende Experimente geben Hinweise auf zwei mögliche Erklärungen für die identifizierten Oszillationen. Sowohl Myosin aktivitätsregulierende Proteine als auch spontan auftretende Spannungsfluktuationen im unter Zugspannung liegenden Netzwerk können mögliche Ursachen für die identifizierten Netzwerkoszillationen sein. Obwohl eine eindeutige zelluläre Funktion des apikalen Akto-Myosin-Netzwerkes im Rahmen dieser Doktorarbeit noch nicht identifiziert werden konnte, so können wir aufgrund von verschiedenen Resultaten dennoch postulieren, dass das hier identifizierte Netzwerk eine entscheidende Rolle bei der Zellmigration und Signaltransduktion einnimmt. Unabhängig davon repräsentiert das hier gefundene Netzwerk die faszinierende Möglichkeit, ein aktives, zweidimensionales Akto-Myosin-Netzwerk nicht nur in vitro, sondern in seiner natürlichen Umgebung studieren und biophysikalische Eigenschaften analysieren zu können.
Motors give a new twist to platelet activation The discoid shape of resting platelets is maintained by a peripheral ring of bundled microtubules called the marginal band. Diagouraga et al. reveal that, upon platelet activation, the motor protein dynein slides microtubules apart, inducing marginal band coiling and the conversion of platelets to a spherical shape. This biosights episode presents the paper by Diagouraga et al. from the January 20, 2014, issue of The Journal of Cell Biology and includes an interview with senior author Karin Sadoul (Institut Albert Bonniot, Grenoble, France). 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
Mon, 18 Nov 2013 10:35:12 GMT https://sakai.rutgers.edu/access/content/group/c7b3269a-bc92-488e-b4b1-0e20648073af/Podcasts/Lect
In animal cells, the nuclear lamina keeps nuclear pore complexes evenly distributed throughout the nuclear envelope. Steinberg et al. reveal that fungi, which lack nuclear laminae, prevent their nuclear pores from clustering by moving them around on cytoskeletal tracks, a process that also helps to organize fungal chromosomes and optimize nucleocytoplasmic transport. This biosights episode presents the paper by Steinberg et al. from the August 6, 2012, issue of the Journal of Cell Biology and includes an interview with senior author Gero Steinberg (University of Exeter, 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
Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 04/06
Fri, 20 Jul 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14587/ https://edoc.ub.uni-muenchen.de/14587/1/Vukajlovic_Marija.pdf Vukajlovic, Marija
Desmosomes are intercellular adhesions whose adhesive core is formed by two distinct classes of cadherin molecules – desmogleins and desmocollins. Nekrasova et al. reveal that these two cadherins are independently transported to the cell surface by two different kinesin motors. This biosights episode presents the paper by Nekrasova et al. from the December 26, 2011, issue of the Journal of Cell Biology and includes an interview with senior author Kathleen Green (Northwestern University Feinberg School of Medicine, Chicago, IL). 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
Many cellular processes, including polarization and differentiation, require the nucleus to move to a specific location within the cytoplasm. Fridolfsson and Starr reveal how the microtubule motors dynein and kinesin-1 control the bi-directional movements of nuclei in the embryonic hypoderm of C. elegans. This biosights episode presents the paper by Fridolfsson and Starr from the October 4, 2010 issue of The Journal of Cell Biology, and includes an interview with senior author Daniel Starr (UC Davis, CA). Produced by Caitlin Sedwick and Ben Short. Subscribe to biosights via iTunes or RSS View biosights archive The Rockefeller University Press biosights@rockefeller.edu
Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 03/06
Mon, 14 Dec 2009 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13811/ https://edoc.ub.uni-muenchen.de/13811/1/Huemmer_Stefan.pdf Hümmer, Stefan ddc:570, ddc:500, Fakultät für Biologie
Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 03/06
Kinesins form a large microtubule-associated motor protein super-family that can be found in every eukaryotic genome sequenced so far. Not only is the translocation of a large number of organelles, protein complexes and mRNAs carried out by them, but also the formation of the meiotic spindle and mitotic spindle integrity are strongly dependent on the kinesins. Fourteen different sub-families of kinesin have been reported. However, previous analyses were based on a relatively small number of selected kinesins (
Melanosomes are specialized pigment-producing organelles that arise from the endosomal system. A new study reveals that the clathrin adaptor AP-1 and the kinesin motor KIF13A combine to sort melanosomal cargo and position endosomes near to developing melanosomes where they deliver the cargo via direct tubular contacts. This biosights episode presents the paper by Delevoye et al. from the October 19th, 2009 issue of the Journal of Cell Biology, and includes interviews with authors Graça Raposo and Cédric Delevoye. Produced by Eun Choi and Ben Short. Subscribe to biosights via iTunes or RSS View biosights archive The Rockefeller University Press biosights@rockefeller.edu
Soft Active Materials: From Granular Rods to Flocks, Cells and Tissues
This presentation discusses the kinesins involved in microtubule depolymerization and the factors that determine the depolymerization rate.
Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 03/06
Fri, 4 Apr 2008 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/8368/ https://edoc.ub.uni-muenchen.de/8368/1/Ebbing_Bettina.pdf Ebbing, Bettina ddc:500, ddc:570, Fakultät für Biologie 0
Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 02/06
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.
Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 01/06
Thu, 22 Jul 2004 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/2619/ https://edoc.ub.uni-muenchen.de/2619/1/Hahlen_Katrin.pdf Hahlen, Katrin ddc:570, ddc:500, Fakultät für Biologie
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 02/19
Thu, 29 Apr 2004 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/2215/ https://edoc.ub.uni-muenchen.de/2215/1/Hartel_Michaela.pdf Hartel, Michaela
Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 01/06
Konventionelle Kinesine sind Mikrotubuli assoziierte Motorproteine. Sie benutzen die Energie der ATP-Hydrolyse, um gerichtete Bewegungen entlang des Zytoskeletts zu ermöglichen. Tierische konventionelle Kinesine sind aus zwei schweren Ketten und zwei leichte Ketten aufgebaut. Die niederen Organismen, wie Pilze, besitzen dagegen nur die zwei schweren Ketten. Das konventionelle Kinesin des roten Brotschimmels Neurospora crassa (NcKin) bewegt sich wie auch andere Pilzkinesine in vitro im mikroskopischen Gleittest mit Geschwindigkeiten, die etwa drei- bis fünffach höher sind (zwischen 2,0 und 2,6 µm/s), als die der tierischen Kinesine (zwischen 0,2 und 0,8 µm/s). Trotz der hohen Sequenzähnlichkeit von Tier- und Pilzkinesinen, sind spezifische Unterschiede festgestellt worden, vor allem im Halsbereich. Weil es bisher keine zufrieden stellende Erklärung der schnellen Gleitgeschwindigkeit von NcKin gibt, liegt es nahe, in diesen pilzspezifischen Sequenzbereichen die Grundlage hierfür zu vermuten. In dieser Dissertation wurde daher untersucht, welchen Einfluss die einzelnen Kinesin-Domänen auf die Motilität und den ATP-Umsatz haben. Zu diesem Zweck wurden (i) bakterielle Expressionsvektoren hergestellt, die für C-terminal verkürzte Kinesinkonstrukte kodieren. Hierbei wurden zunächst rekombinante Motoren hergestellt, die an den Domänengrenzen endeten, wie sie durch kristallografische Modelle und Sekundärstrukturvorhersagen abgeleitet worden waren. Aufgrund der Ergebnisse an diesen Proteinen wurden weitere C-terminal verkürzte Kinesine konstruiert, die eine genauere funktionelle Kartierung der Scharnierdomäne zum Ziel hatten. Mit diesen Konstrukten wurden kinetische Studien durchgeführt, um ein Gesamtbild von deren ATPase-Aktivität und Prozessivität zu bekommen. Da ähnliche Studien an dem homologen Drosophila Kinesin durchgeführt worden waren, war ein direkter Vergleich zu diesen Vertretern der Tierkinesine möglich. (ii) Um den Beitrag der einzelnen Domänen zur hohen Geschwindigkeit von NcKin zu ermitteln, wurden in einem zweiten Teil der vorliegenden Arbeit gezielt NcKin Domänen in die entsprechenden Bereiche des humanen Kinesins eingeführt, und die entstandenen Chimären auf einen Geschwindigkeitszuwachs getestet.
Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 01/06
Die vorliegende Arbeit beschäftigt sich mit den molekularen Grundlagen polaren Wachstums im phytopathogenen Basidiomycet Ustilago maydis. Zunächst wurde die zelluläre Rolle des t-SNAREs Yup1 analysiert. Ein temperatursensitiver Defekt im yup1-Gen hatte zu Störungen in der Zelltrennung und im polaren Wachstum von Sporidien geführt. Mutante Zellen bildeten dabei lange verzweigte Ketten aus verdickten Zellen. Die Lokalisation eines Yup1-GFPFusionsproteins auf beweglichen Organellen hatte zum Aufstellen eines spekulativen Modells geführt, bei dem Yup1 auf Endosomen die Fusion mit ankommenden endozytotischen Vesikeln vermittelt. Eine erstmalige Charakterisierung der Endozytose von U. maydis in dieser Arbeit zeigte, dass es sich bei den mit Yup1-GFP markierten, schnellen Organellen tatsächlich um frühe Endosomen handelte. Diese akkumulierten Zellzyklus-abhängig an Regionen aktiven Wachstums in BSDs. Die Akkumulation früher Endosomen im Apex von Hyphen war für das Spitzenwachstum erforderlich. In yup1ts-Zellen war bei restriktiver Temperatur eine gestörte Endozytose zu beobachten. Dieser Zusammenhang zwischen Zellmorphologie und polarer Sekretion einerseits und Endozytose andererseits deutete darauf hin, dass Membranrecycling über frühe Endosomen entscheidend am polaren Wachstum von U. maydis-Zellen im Speziellen und pilzlichen Hyphen im Allgemeinen mitwirkt. Mittels des Yup1-GFP-Fusionsproteins konnten die molekularen Grundlagen der beobachteten Bewegung von Endosomen untersucht werden. Es wurde gezeigt, dass sich frühe Endosomen entlang von MT bewegen. Für diese Bewegung war in erster Linie das Kinesin Kin3 verantwortlich. Dieses Molekül ist ein neues Mitglied der Unc104/KIF1-Familie von Kinesin-ähnlichen molekularen Motoren und bewegt als solches vermutlich in Richtung der plus-Enden von MT. Gelfiltrationsexperimente legten nahe, dass Kin3 in der Zelle als Monomer vorliegt. Die N-terminale Motordomäne zeigte in vitro eine MTstimulierte ATPase Aktivität. Ein Kin3-GFP-Fusionsprotein lokalisierte in schnell beweglichen Flecken, die im Bewegungsverhalten den frühen Endosomen glichen. Ein Kin3-YFP-Fusionsprotein bewegte entlang von MT und kolokalisierte zudem mit einem Yup1-CFP-Fusionsprotein auf Endosomen. Die Deletion von kin3 führte zu einer starken Reduzierung der Endosomenbewegung. Die in vivo-Untersuchung der MT-Dynamik im Δkin3-Stamm ergab, dass der Großteil der Endosomen in Akkumulationen an den minus-Enden der MT konzentriert war. Entsprechend führte die Überexpression von kin3 zu einer verstärkten Konzentration der Endosomen an den plus-Enden von MT. Die Zellform einzelner Sporidien war im kin3-Deletionsstamm nicht verändert. Allerdings trennten sich die Zellen nach der Teilung wie in der yup1ts-Mutante nicht voneinander. Dieser Trennungsdefekt und ein verändertes Knospungsmuster führten zur Bildung von großen Baum-ähnlichen Zellaggregaten. Im Hyphenstadium führte die Deletion von kin3 außerdem zu einer deutlichen Störung des polaren Wachstums. Die nach Deletion von kin3 beobachtete Restbewegung der Endosomen beruhte fast ausschließlich auf der Aktivität des zytoplasmatischen Dyneins von U. maydis. Das konventionelle Kinesin von U. maydis, Kin2, zeigte ebenfalls einen Einfluss auf die Organisation und Position endosomaler Akkumulationen, obwohl es vermutlich nicht direkt am Transport einzelner Endosomen beteiligt ist. Die präsentierten Daten zeigen, dass Endosomen MT- und Zellzyklus-abhängig organisiert sind. Die Position der BSDs korrelierte dabei mit Funktionen der Endosomen bei der Zelltrennung, in der Bestimmung des Knospungsmusters und beim polaren Wachstum. Da die MT während des Knospenwachstums unipolar ausgerichtet sind, nutzt die U. maydis-Zelle das Wechselspiel des plus-Motors Kin3 und des minus-Motors Dynein, um die Endosomen Zellzyklus-abhängig an den plus- oder minus-Enden der MT zu akkumulieren.