Podcasts about arp2

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.28.550203v1?rss=1 Authors: Chen, T., Huertas Fernandez-Espartero, C., Illand, A., Tsai, C.-T., Yang, Y., Klapholz, B., Jouchet, P., Fabre, M., Rossier, O., Cui, B., Leveque-Fort, S., Brown, N. H., Giannone, G. Abstract: Morphogenesis requires building stable macromolecular structures from highly dynamic proteins. Muscles are anchored by long-lasting integrin adhesions to resist contractile force. However, the mechanisms governing integrin diffusion, immobilization, and activation within developing tissue remain elusive. Here, we show that actin polymerisation-driven membrane protrusions form nanotopographies resulting in strong adhesions in the Drosophila muscle attachment site. With super-resolution microscopy and single protein tracking, we show that integrins assemble invadosomes-like adhesive belts around Arp2/3-dependent actin protrusions, which promotes enhanced integrin molecular immobilization and confinement in diffusion traps. Actin filaments also display restricted motion and confinement, indicating strong mechanical connection with integrins. Using isolated muscle cells, we show that substrate nanotopography, instead of rigidity, drives adhesion maturation by regulating actin protrusion, integrin diffusion and immobilization. These results point to the existence of a molecular clutch in developing tissue required for the formation of stable adhesions and highlight the importance of geometrical information in cellular and tissue morphogenesis. 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/2023.07.27.550884v1?rss=1 Authors: Jury-Garfe, N., You, Y., Martinez, P., Redding-Ochoa, J., Karahan, H., Johnson, T. S., Zhang, J., Kim, J., Troncoso, J. C., Lasagna-Reeves, C. A. Abstract: Asymptomatic Alzheimer 's disease (AsymAD) describes the status of subjects with preserved cognition but with identifiable Alzheimer 's disease (AD) brain pathology (i.e. A {beta}-amyloid deposits, neuritic plaques, and neurofibrillary tangles) at autopsy. In this study, we investigated the postmortem brains of a cohort of AsymAD cases to gain insight into the underlying mechanisms of resilience to AD pathology and cognitive decline. Our results showed that AsymAD cases exhibit an enrichment of core plaques and decreased filamentous plaque accumulation, as well as an increase in microglia surrounding this last type. In AsymAD cases we found less pathological tau aggregation in dystrophic neurites compared to AD and tau seeding activity comparable to healthy control subjects. We used spatial transcriptomics to further characterize the plaque niche and found autophagy, endocytosis, and phagocytosis within the top upregulated pathways in the AsymAD plaque niche, but not in AD. Furthermore, we found ARP2, an actin-based motility protein crucial to initiate the formation of new actin filaments, increased within microglia in the proximity of amyloid plaques in AsymAD. Our findings support that the amyloid-plaque microenvironment in AsymAD cases is characterized by microglia with highly efficient actin-based cell motility mechanisms and decreased tau seeding compared to AD. These two mechanisms can potentially provide protection against the toxic cascade initiated by A {beta} that preserves brain health and slows down the progression of AD pathology. 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/2023.07.10.548473v1?rss=1 Authors: Singh, S. B., Rajput, S. S., Sharma, A., Ananthanarayanan, V., Nandi, A., Patil, S. P. V., majumdar, a., Subramanyam, D. Abstract: Protein aggregation is a common underlying feature of neurodegenerative disorders. Cells expressing neurodegeneration associated mutant proteins show altered uptake of ligands, suggestive of impaired endocytosis, in a manner as yet unknown. Using live cell imaging, we show that clathrin mediated endocytosis (CME) is affected due to altered actin cytoskeletal organization in the presence of Huntingtin aggregates. Additionally, we find that cells containing Huntingtin aggregates are stiffer and less viscous than their wild type counterparts due to altered actin conformation, and not merely due to the physical presence of aggregate(s). We further demonstrate that CME and cellular viscosity can be rescued by overexpressing Hip1, Arp2/3 or transient LatrunculinA treatment. Examination of other pathogenic aggregates revealed that only a subset of these display defective CME, along with altered actin organization and increased stiffness. Together, our results point to an intimate connection between functional CME, actin organization and cellular stiffness in the context of neurodegeneration. 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/2023.04.26.538419v1?rss=1 Authors: Willet, A. H., Ren, L., Chen, J.-S., Gould, K. Abstract: Myosin-1s are monomeric actin-based motors that function at membranes. Myo1 is the single myosin-1 isoform in Schizosaccharomyces pombe that works redundantly with Wsp1-Vrp1 to activate the Arp2/3 complex for endocytosis. Here, we identified Ank1 as an uncharacterized cytoplasmic Myo1 binding partner. We found that in ank1{Delta} cells, Myo1 dramatically redistributed from endocytic patches to decorate the entire plasma membrane and endocytosis was defective. Biochemical analysis and structural predictions suggested that the Ank1 ankyrin repeats bind the Myo1 lever arm and the Ank1 acidic tail binds the Myo1 TH1 domain to prevent TH1-dependent Myo1 membrane binding. Indeed, Ank1 over-expression precluded Myo1 membrane localization and recombinant Ank1 blocked purified Myo1 liposome binding in vitro. Based on biochemical and cell biology analyses, we propose budding yeast Ank1 and human OSTF1 are functional Ank1 orthologs and that cytoplasmic sequestration by small ankyrin repeat proteins is a conserved mechanism regulating myosin-1s in endocytosis. 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/2023.04.18.537332v1?rss=1 Authors: Massey-Harroche, D., Conte, V., Gouirand, N., Sebbagh, M., Le Bivic, A., Bazellieres, E. Abstract: Several cellular processes during morphogenesis, tissue healing or cancer progression involve epithelial to mesenchymal plasticity that leads to collective motion (plasticity?). Even though a rich variety of EMP programs exist, a major hallmark unifying them is the initial breaking of symmetry that modifies the epithelial phenotype and axis of polarity. During this process, the actin cytoskeleton and cellular junctions are extensively remodelled correlating with the build-up of mechanical forces. As the collective migration proceeds, mechanical forces generated by the actin cytoskeleton align with the direction of migration ensuring an organized and efficient collective cell behaviour, but how forces are regulated during the breaking of symmetry at the onset of EMP remains an unaddressed question. It is known that the polarity complex CRB3/PALS1/PATJ, and in particular, CRB3 regulates the organization of the actin cytoskeleton associated to the apical domain thus pointing at a potential role of CRB3 in controlling mechanical forces. Whether and how CRB3 influences epithelial biomechanics during the epithelial-mesenchymal plasticity remains, however, largely unexplored. Here, we systematically combine mechanical and molecular analyses to show that CRB3 regulates the biomechanical properties of collective epithelial cells during the initial breaking of symmetry of the EMP. CRB3 interacts with ARP2/3 and controls the remodelling of actin throughout the monolayer via the modulation of the Rho-/Rac-GTPase balance. Taken together, our results identified CRB3, a polarity protein, as a regulator of epithelial monolayer mechanics during EMP Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

A new editorial paper was published in Oncotarget's Volume 14 on March 21, 2023, entitled, “Impact of cortactin in cancer progression on Wnt5a/ROR1 signaling pathway.” In this editorial, researchers Kamrul Hasan and Thomas J. Kipps from the University of California discuss cortactin—an intracellular cytoskeletal protein that can undergo tyrosine phosphorylation upon external stimulation and promote polymerization and the assembly of the actin filament that is required for cell migration. Upon stimulation, cortactin binds and activates actin related protein Arp2/3 complex, a de novo actin nucleator that can induce F (filamentous)-actin polymerization [1, 2]. Cortactin (also known as EMS1 or CTTN) is expressed broadly in a variety of cancers, for which it plays an apparent role in cellular protrusions, which include lamellipodia and filopodia formation to promote migration and metastasis. Moreover, cortactin is expressed in (i) primary chronic lymphocytic leukemia (CLL) and primary breast-cancer cells, (ii) at least 15% of metastatic breast carcinomas, and (iii) CLL or breast-cancer cell-lines [3, 4]. In structure, cortactin contains a SH3 domain that allows it to bind characteristic motifs (-P-X-X-P-), which can be found in the proline-rich-domains (PRD) of other proteins, including receptor-tyrosine-kinase-like orphan receptor 1 (ROR1) [1, 3, 4]. “We have found (as have other investigators) that ROR1 is expressed by a variety of human cancers, which include CLL and breast cancer, suggesting that ROR1 may play a role in cancer pathogenesis [3–5].” Editorial paper: DOI: https://doi.org/10.18632/oncotarget.28386 Correspondence to: Thomas J. Kipps - tkipps@ucsd.edu Keywords: cortactin, Wnt5a, ROR1, migration, metastasis Subscribe for free publication alerts from Oncotarget - https://www.oncotarget.com/subscribe/ About Oncotarget Oncotarget is a primarily oncology-focused, peer-reviewed, open access journal. Papers are published continuously within yearly volumes in their final and complete form, and then quickly released to Pubmed. On September 15, 2022, Oncotarget was accepted again for indexing by MEDLINE. Oncotarget is now indexed by Medline/PubMed and PMC/PubMed. To learn more about Oncotarget, please visit https://www.oncotarget.com and connect with us: SoundCloud - https://soundcloud.com/oncotarget Facebook - https://www.facebook.com/Oncotarget/ Twitter - https://twitter.com/oncotarget Instagram - https://www.instagram.com/oncotargetjrnl/ YouTube - https://www.youtube.com/@OncotargetJournal LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Media Contact MEDIA@IMPACTJOURNALS.COM 18009220957

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.14.532631v1?rss=1 Authors: Bhanja, A., Lazzaro, M., Upadhyaya, A., Song, W. Abstract: Antigen-induced B-cell receptor (BCR) signaling is critical for initiating and regulating B-cell activation. The actin cytoskeleton plays essential roles in BCR signaling. Upon encountering cell-surface antigens, actin-driven B-cell spreading amplifies signaling, while B-cell contraction following spreading leads to signal attenuation. However, the mechanism by which actin dynamics switch BCR signaling from amplification to attenuation is unknown. Here, we show that Arp2/3-mediated branched actin polymerization is required for B-cell contraction. Contracting B-cells generate centripetally moving actin foci from lamellipodial F-actin networks in the B-cell plasma membrane region contacting antigen-presenting surfaces. Actin polymerization driven by N-WASP, but not WASP, generates these actin foci. N-WASP-dependent actin foci facilitate non-muscle myosin II recruitment to the contact zone to create actomyosin ring-like structures. Furthermore, B-cell contraction increases BCR molecular density in individual clusters, leading to decreased BCR phosphorylation. Increased BCR molecular density reduced levels of the stimulatory kinase Syk, the inhibitory phosphatase SHIP-1, and their phosphorylated forms in individual BCR clusters. These results suggest that N-WASP-activated Arp2/3 generates centripetally moving foci and contractile actomyosin ring-like structures from lamellipodial networks, enabling contraction. B-cell contraction attenuates BCR signaling by pushing out both stimulatory kinases and inhibitory phosphatases from BCR clusters, providing novel insights into actin-facilitated signal attenuation. 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/2023.02.24.529907v1?rss=1 Authors: Basant, A., Way, M. Abstract: Vaccinia virus exiting from host cells activates Src/Abl kinases to phosphorylate A36, an integral membrane viral protein. Phosphorylated A36 binds the adaptors Nck and Grb2 which recruit N-WASP to activate Arp2/3-driven actin polymerisation to promote viral spread. A36 also recruits intersectin, which enhances actin polymerization via AP-2/clathrin and Cdc42. To obtain a better quantitative understanding of this signalling network we still need to know the absolute numbers of the key molecules. To achieve this goal, we have now used fluorescent molecule counting approaches in live cells. There are 1156{+/-}120 A36 molecules on virus particles inducing actin polymerization in HeLa cells. This number, however, is over 2000 in mouse embryonic fibroblasts (MEFs), suggesting that A36 levels on the virion are not fixed. In MEFs, viruses recruit 1032{+/-}200 Nck and 434{+/-}10 N-WASP molecules, suggesting a ratio of 4:2:1 for the A36:Nck:N-WASP signalling network. Loss of A36 binding to either Grb2 or intersectin results in a 1.3- and 2.5-fold reduction in Nck respectively. Despite recruiting comparable numbers of the Arp2/3 activator, N-WASP (245{+/-}26 and 276{+/-}66), these mutant viruses move at different speeds that inversely correlate with the number of Nck molecules. Our analysis has uncovered two unexpected new aspects of Vaccinia egress, A36 levels can vary in the viral membrane and the velocity of virus movement depends on Nck. 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/2023.02.02.526815v1?rss=1 Authors: Hosseini, K., Frenzel, A., Fischer-Friedrich, E. Abstract: Epithelial-mesenchymal transition (EMT) is a key cellular transformation for many physiological and pathological processes ranging from cancer over wound healing to embryogenesis. Changes in cell migration, cell morphology and cellular contractility were identified as hallmarks of EMT. These cellular properties are known to be tightly regulated by the actin cytoskeleton. EMT-induced changes of actin-cytoskeletal regulation were demonstrated by previous reports of cell-cycle-dependent changes of actin cortex mechanics in conjunction with characteristic modifications of cortex-associated f-actin and myosin. However, at the current state, the changes of upstream actomyosin signalling that lead to corresponding mechanical and structural changes of the cortex are not well understood. In this work, we show in breast epithelial cancer cells MCF-7 that EMT results in characteristic changes of the cortical signalling of Rho-GTPases Rac1, RhoA and RhoC and downstream actin regulators cofilin, mDia1 and Arp2/3. In the light of our findings, we propose that cell-cycle-dependent EMT-induced changes in cortical mechanics rely on two hitherto unknown signalling paths - i) a cell-cycle-dependent feedback between Rac1 and RhoC and ii) a negative feedback between Arp2/3 activity and cortical association of myosin II. 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/2023.01.05.522888v1?rss=1 Authors: Cazzagon, G., Roubinet, C., Baum, B. Abstract: While the Formin-nucleated actomyosin cortex has been shown to drive the changes in cell shape that accompany cell division in both symmetric and asymmetric cell divisions, it is not clear whether or not Arp2/3-nucleated branched actin filament networks also play a role. In order to look for mitotic roles of the Arp2/3 complex, here we use Drosophila neural stem cells as a model system. These cells are unusual in that they divide asymmetrically to produce a large and small daughter cell with different fates. Our analysis identifies a pool of Arp2/3-dependent actin-based membrane protrusions that form at the apical cortex of these cells as they enter mitosis. Strikingly, at metaphase, these protrusions co-localise with components of the SCAR complex. By perturbing Arp2/3 complex activity we show that this apical pool of actin likely functions to limit the accumulation of apical Myosin in metaphase. Following the onset of anaphase, the loss of these SCAR and Arp2/3 dependent structures then leads to a delay in the clearance of apical Myosin and to cortical instability at cytokinesis. These data point to a role for a polarised branched actin filament network in fine tuning the apical actomyosin cortex to enable the precise control of cell shape during asymmetric cell division. 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/2022.12.27.522022v1?rss=1 Authors: Zhang, Y., Ripley, B. M., Ouyang, W., Sturtz, M., Upton, E., Luhmann, E., Vessely, M., Coloma, R., Schwery, N., Anthony, S. M., Goeken, A., Moninger, T. O., Harty, J. T., Klingelhutz, A. J., Lundberg, E., Meyerholz, D. K., Manicassamy, B., Stipp, C. S., Guerra, S., Radoshevich, L. Abstract: The ubiquitin-like protein, ISG15, can act as a cytokine or can covalently modify host and pathogen-derived proteins. The consequences of ISG15 modification on substrate fate remain unknown. Here we reveal that ISGylation of the Arp2/3 complex slows actin filament formation and stabilizes Arp2/3 dependent structures including cortical actin and lamella. When properly controlled, this serves as an antibacterial and antiviral host defense strategy to directly restrict actin-mediated pathogen spread. However, Listeria monocytogenes takes advantage in models of dysregulated ISGylation, leading to increased mortality due to augmented spread. The underlying molecular mechanism responsible for the ISG15-dependent impact on actin-based motility is due to failed bacterial separation after division. This promotes spread by enabling the formation of multi-headed bacterial bazookas with stabilized comet tails that can disseminate deeper into tissues. A bacterial mutant that cannot recruit Arp2/3 or a non-ISGylatable mutant of Arp3 is sufficient to rescue slowed comet tail speed and restrict spread. Importantly, ISG15-deficient neonatal mice have aberrant epidermal epithelia characterized by keratinocytes with diffuse cortical actin, which could underlie observed defects in wound healing in human patients who lack ISG15. Ultimately, our discovery links host innate immune responses to cytoskeletal dynamics with therapeutic implications for viral infection and metastasis. 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/2022.11.29.518248v1?rss=1 Authors: Cuentas-Condori, A., Chen, S., Krout, M., Gallick, K., Tipps, J., Flautt, L., Richmond, J. E. E., Miller, D. M. Abstract: Presynaptic terminals are actively relocated during development to refine circuit function, but the underlying cell biological mechanisms are largely unknown. In C. elegans, the presynaptic boutons of GABAergic DD neurons are moved to new locations during early larval development. We show that developmentally regulated expression of a presynaptic Epithelial Na+ Channel (ENaC), UNC-8, promotes a Ca2+-dependent mechanism, resembling Activity-Dependent Bulk Endocytosis (ADBE), that dismantles presynaptic material for reassembly at nascent DD synapses. ADBE normally functions in highly active neurons to accelerate local recycling of synaptic vesicles. We show that DD presynaptic remodeling depends on canonical features of ADBE including elevated intracellular Ca2+, the phosphatase Calcineurin and its targets, dynamin and the F-BAR protein syndapin, and Arp2/3-driven actin polymerization. Thus, our findings suggest that a native mechanism (ADBE) for maintaining neurotransmitter release at local synapses has been repurposed, in this case, to dismantle presynaptic terminals for reassembly at new locations. 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/2022.11.21.517374v1?rss=1 Authors: Spencer, W. J., Schneider, N. F., Skiba, N. P., Arshavsky, V. Y. Abstract: The photoreceptor outer segment is a modified cilium filled with hundreds of flattened disc membranes responsible for efficient light capture. To maintain photoreceptor health and functionality, outer segments are continuously renewed through the addition of new discs at their base. This process is driven by branched actin polymerization nucleated by the Arp2/3 complex. To induce actin polymerization, Arp2/3 requires a nucleation promoting factor. Here, we show that the nucleation promoting factor driving disc morphogenesis is the pentameric WAVE complex and identify all protein subunits of this complex. We further demonstrate that the knockout of one of them, WASF3, abolishes actin polymerization at the site of disc morphogenesis leading to formation of disorganized membrane lamellae emanating from the photoreceptor cilium instead of an outer segment. These data establish that, despite the intrinsic ability of photoreceptor ciliary membranes to form lamellar structures, WAVE-dependent actin polymerization is essential for organizing these membranes into a proper outer segment. 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/2022.11.16.516846v1?rss=1 Authors: Davidson, K. A., Nakamura, M., Verboon, J. M., Parkhurst, S. M. Abstract: Nuclear envelope (NE) budding is a nuclear pore independent nuclear export pathway, analogous to the egress of herpesviruses, and required for protein quality control, synapse development and mitochondrial integrity. The physical formation of NE buds is dependent on the Wiskott-Aldrich Syndrome protein Wash, its regulatory complex (SHRC), and Arp2/3, and requires Wash actin nucleation activity. However, the machinery governing cargo recruitment and organization within the NE bud remains unknown. Here, we identify Pavarotti (Pav) and Tumbleweed (Tum) as new molecular components of NE budding. Pav and Tum interact directly with Wash and define a second nuclear Wash-containing complex required for NE budding. Interestingly, we find that the actin bundling activities of Wash and Pav are required, suggesting a structural role in the physical and/or organizational aspects of NE buds. Thus, Pav and Tum are providing exciting new entry points into the physical machineries of this alternative nuclear export pathway for large cargos during cell differentiation and development. 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/2022.11.14.515780v1?rss=1 Authors: Li, D., Yang, Y., Wang, Y., Chao, X., Huang, J., Singh, S. P., Zhang, C., Lou, J., Gao, P., Huang, S., Cai, H. Abstract: The actin-rich cortex plays a fundamental role in many cellular processes. Its architecture and molecular composition vary across cell types and physiological states. The full complement of actin assembly factors driving cortex formation and how their activities are spatiotemporally regulated remain to be fully elucidated. Using Dictyostelium as a model for polarized and rapidly migrating cells, we show that GxcM, a RhoGEF localized specifically in the rear of migrating cells, functions together with F-BAR protein Fbp17, a small GTPase RacC, and the actin nucleation-promoting factor WASP to coordinately promote Arp2/3 complex-mediated cortical actin assembly. Over-activation of this signaling cascade leads to excessive actin polymerization in the rear cortex, whereas its disruption causes defects in cortical integrity and function. Therefore, different from its well-defined role in the formation of the front protrusions, the Arp2/3 complex-based actin carries out a previously unappreciated function in building the rear cortical subcompartment in rapidly migrating cells. 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/2022.10.19.512957v1?rss=1 Authors: Jiu, Y., Zhang, Y., Zhang, X., Li, Z., Yang, H., Tang, D., Zhao, S., Zhang, Q., Li, B., Lappalainen, P., Cui, Z., Liu, H., Li, H., Zhao, W. Abstract: Emerging COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a great threat to human health and economics. Although SARS-CoV-2 entry mechanism has been explored, little is known about how SARS-CoV-2 regulates the host cell remodeling to facilitate virus invasion process. Here we unveil that SARS-CoV-2 boosts and repurposes filopodia for entry to the target cells. Using SARS-CoV-2 virus-like particle (VLP), real-time live-cell imaging and simulation of active gel model, we reveal that VLP-induced Cdc42 activation leads to the formation of filopodia, which reinforce the viral entry to host cells. By single-particle tracking and sparse deconvolution algorithm, we uncover that VLP particles utilize filopodia to reach the entry site in two patterns, surfing and grabbing, which are more efficient and faster than entry via flat plasma membrane regions. Furthermore, the entry process via filopodia is dependent on the actin cytoskeleton and actin-associated proteins fascin, formin, and Arp2/3. Importantly, either inhibition the actin cross-linking protein fascin or the active level of Cdc42 could significantly hinders both the VLP and the authentic SARS-CoV-2 entry. Together, our results highlight that the spatial-temporal regulation of the actin cytoskeleton by SARS-CoV-2 infection makes filopodia as a highway for virus entry, which emerges as an antiviral target. 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/2022.10.12.511969v1?rss=1 Authors: Bogucka-Janczi, K., Harms, G., May-Coissieux, M., Bentires-Alj, M., Thiede, B., Rajalingam, K. Abstract: The actin cytoskeleton is tightly controlled by RhoGTPases, actin binding proteins and nucleation-promoting factors to perform fundamental cellular functions. Here, we show that ERK3, an atypical MAPK, directly acts as a guanine nucleotide exchange factor for Cdc42 and phosphorylates the ARP3 subunit of the ARP2/3 complex at S418 to promote filopodia formation and actin polymerization, respectively. Consistently, depletion of ERK3 prevented both basal and EGF-dependent Rac1 and Cdc42 activation, maintenance of F-actin content, filopodia formation and epithelial cell migration. Further, ERK3 protein binds directly to the purified ARP2/3 complex and augments polymerization of actin in vitro. ERK3 kinase activity is required for the formation of actin-rich protrusions in mammalian cells. These findings unveil a fundamentally unique pathway employed by cells to control actin-dependent cellular functions. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

As discussed in previous episodes, the actin cytoskeleton is vital to allow cells to move. But what about the specifics? In this episode, we're going to be dissecting the lamellipodium- a meshwork actin structure that some cells use to move. Sources for this episode: 1) Alberts, Johnson, Lewis, Raff, Roberts, and Walter (2008), Molecular Biology of the Cell, Fifth Edition. Abingdon: Garland Science, Taylor and Francis Group LLC. 2) Berro, Michelot, Blanchoin, Kovar and Martiel (2007), Attachment Conditions Control Actin Filament Buckling and the Production of Forces. Biophysical Journal 92(7): 2546- 2558. 3) Kiuchi, Ohashi, Kurita and Mizuno (2007), Cofilin promotes stimulus-induced lamellipodium formation by generating an abundant supply of actin monomers. The Journal of Cell Biology 177(3): 465- 476. 4) ‘Mechanobiology Institute, Singapore', YouTube (2013), Arp2/3 complex mediated actin nucleation (online) [Accessed 23/11/2020]

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.16.384099v1?rss=1 Authors: Kundu, T., Das, S. S., Kumar, D. S., Sewatkar, L. K., Ghose, A. Abstract: Interstitial collateral branching of axons is a critical component in the development of functional neural circuits. Axon collateral branches are established through a series of cellular processes initiated by the development of a specialized, focal F-actin network in axons. The formation, maintenance and remodelling of this F-actin patch is critical for the initiation of axonal protrusions that are subsequently consolidated to form a collateral branch. However, the mechanisms regulating F-actin patch dynamics are poorly understood. Fmn2 is a formin family member implicated in multiple neurodevelopmental disorders. We find that Fmn2 regulates the initiation of axon collateral protrusions. Fmn2 localises to the protrusion-initiating axonal F-actin patches and regulates the lifetime and size of these F-actin networks. The F-actin nucleation activity of Fmn2 is necessary for F-actin patch stability but not for initiating patch formation. We show that Fmn2 insulates the F-actin patches from disassembly by the actin-depolymerizing factor, ADF, and promotes long-lived, larger patches that are competent to initiate axonal protrusions. The regulation of axonal branching can contribute to the neurodevelopmental pathologies associated with Fmn2 and the dynamic antagonism between Fmn2 and ADF may represent a general mechanism of formin-dependent protection of Arp2/3-initiated F-actin networks from disassembly. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.07.330126v1?rss=1 Authors: Dube, S., Racz, B., Brown, W. E., Gao, Y., Soderblom, E. J., Yasuda, R., Soderling, S. H. Abstract: In contrast to their postsynaptic counterparts, the contributions of activity-dependent cytoskeletal signaling to presynaptic plasticity remain controversial and poorly understood. To identify and evaluate these signaling pathways, we conducted a proteomic analysis of the presynaptic cytomatrix using in vivo biotin identification (iBioID). The resultant proteome was heavily enriched for actin cytoskeleton regulators, including Rac1, a Rho GTPase that activates the Arp2/3 complex to nucleate branched actin filaments. Strikingly, we find Rac1 and Arp2/3 are closely associated with presynaptic vesicle membranes and negatively regulate synaptic vesicle replenishment at both excitatory and inhibitory synapses. Using optogenetics and fluorescence lifetime imaging, we show this pathway bidirectionally sculpts short-term synaptic depression and that its presynaptic activation is coupled to action potentials by voltage-gated calcium influx. Thus, this study provides a new proteomic framework for understanding presynaptic physiology and uncovers a previously unrecognized mechanism of actin-regulated short-term presynaptic plasticity that is conserved across cell types. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.02.868505v1?rss=1 Authors: Francois, J., Kandasamy, A., Yeh, Y.-T., Ayala, C., Meili, R., Chien, S., Lasheras, J. C., del Alamo, J. C. Abstract: Three-dimensional (3-D) neutrophil migration is essential for immune surveillance and inflammatory responses. During 3-D migration, especially through extravascular spaces, neutrophils rely on frontal protrusions and rear contractions to squeeze and maneuver through extracellular matrices containing narrow pores. However, the role of matrix density and the cells' ability to probe and remodel matrix pores during 3-D chemotaxis are far from being understood. We investigated these processes by tracking the trajectories of over 20,000 neutrophils in a 3-D migration device containing collagen matrices of varying concentrations and analyzing the shape of these trajectories at multiple scales. Additionally, we quantified the transient 3-D matrix deformations caused by the migrating cells. The mean pore size of our reconstituted collagen matrices decreased when the collagen concentration ([col]) was increased. In low-[col] matrices, neutrophils exerted large transient deformations and migrated in relatively straight trajectories. In contrast, they were not able to appreciably deform high-[col] matrices and adapted to this inability by turning more often to circumvent these narrow matrix pores. While this adaptation resulted in slower migration, the cells were able to balance the more frequent turning with the long-range directional bias necessary for chemotaxis. Based on our statistical analysis of cell trajectories, we postulate that neutrophils achieve this balance by using matrix obstacles as pivoting points to steer their motion towards the chemoattractant. Inhibiting myosin-II contractility or Arp2/3-mediated pseudopod protrusions not only compromised the cells' ability to deform the matrix, but also made them switch to increased turning in more restrictive matrices when compared to untreated control cells. Both myosin-II contractility and Arp2/3-mediated branched polymerization of actin played a role in fast migration, but Arp2/3 was also crucial for neutrophils when coordinating the orientations of successive turns to prevent veering away from the chemotactic path. These results may contribute to an improved understanding of the mechanisms employed by migrating neutrophils in confined 3-D environments, as well as the molecular and environmental regulators for maintaining persistent motion. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.29.177758v1?rss=1 Authors: Mandal, P., Belapurkar, V., Nair, D., Ramanan, N. Abstract: Axon growth requires coordination of the actin cytoskeleton by actin-binding proteins in the growth cones of the extending neurites. The actin-binding protein vinculin (Vcl) is a major constituent of focal adhesion but its role in neuronal migration and axon growth is poorly understood. We found that vinculin deletion in mouse neocortical neurons attenuated axon growth both in vitro and in vivo. Using functional mutants of vinculin, we found that various domains of vinculin affect cell migration and axon growth differently. While expression of a constitutively active vinculin significantly enhanced axon growth, the head-neck domain had a moderate inhibitory effect. Contrary to previous findings, vinculin-talin interaction was dispensable for axon growth and neuronal migration. Strikingly, expression of the tail domain delayed neuronal migration and caused increased branching, enlarged soma and highly stunted axon both in vitro and in vivo. Inhibition of the Arp2/3 complex completely reversed the branching phenotype caused by tail domain expression without affecting axon length. Similarly, abolishing the tail domain interaction with actin reversed the enlarged cell soma and branching phenotypes but not axon length. Super-resolution microscopy showed increased mobile fraction of actin in tail domain expressing neurons. Our results provide novel insights into the role of vinculin and its functional domains in regulating neuronal migration and axon growth. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.04.134924v1?rss=1 Authors: Schiweck, J., Murk, K., Ledderose, J., Muenster-Wandowski, A., Vida, I., Eickholt, B. J. Abstract: The brain of mammals lacks a significant ability to regenerate neurons and is thus particularly vulnerable. To protect the brain from injury and disease, damage control by astrocytes through astrogliosis and scar formation is vital. Here, we show that brain injury triggers an ad hoc upregulation of the actin-binding protein Drebrin (DBN) in astrocytes, which is essential for the formation and maintenance of glial scars in vivo. In turn, DBN loss leads to defective glial scar formation and excessive neurodegeneration following mild brain injuries. At the cellular level, DBN switches actin homeostasis from ARP2/3-dependent arrays to microtubule-compatible scaffolds and facilitates the formation of RAB8-positive membrane tubules. This injury-specific RAB8 membrane compartment serves as hub for the trafficking of surface proteins involved in astrogliosis and adhesive responses, such as {beta}1-integrin. Our work identifies DBN as pathology-specific actin regulator, and establishes DBN-dependent membrane trafficking as crucial mechanism in protecting the brain from escalating damage following traumatic injuries. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.05.31.124941v1?rss=1 Authors: Drew, J., Arancibia-Carcamo, L., Jolivet, R. B., Lopez-Domenech, G., Attwell, D., Kittler, J. T. Abstract: Microglia use a highly complex and dynamic network of processes to sense and respond to their surroundings. Microglial dynamics differ throughout development and in neurological and neuropsychiatric disease, though mechanistic insight into these changes is lacking. Here we identify novel roles for regulators of the actin cytoskeleton in controlling microglial behaviour. We show that the actin branching complex Arp2/3 is critical for maintaining microglial morphology and required for surveillance but not chemotactic motility. Neuropsychiatric disease-associated Cyfip1, a core component of the WAVE regulatory complex that links Rac1 signalling to Arp2/3 activation, is highly expressed in microglia but has unknown function. We report that conditional deletion of Cyfip1 in mouse microglia impairs morphological complexity and surveillance of brain parenchyma, and increases activation state as defined by CD68 expression. Thus, altered actin-dependent microglial dynamics mediated by Cyfip1 and Arp2/3 may contribute to neuropsychiatric disease. Copy rights belong to original authors. Visit the link for more info

Drubin explains how endocytosis can be used as a system to dissect the mechanisms of actin dynamics in yeast.  He focuses on his lab's research on three key processes: actin disassembly, filament capping and Arp2/3 nucleation.  By giving an overview of the importance of actin dynamics in regulating a single cell process, endocytosis, Drubin highlights the crucial role of actin assembly and disassembly in both yeast and mammalian cells.

Actin forms many cellular structures and regulates a variety of critical biological processes. Dr. David Drubin's lab focuses on studying actin in the context of membrane trafficking. In his first iBiology seminar, Drubin recounts seminal research done using the intracellular pathogen Listeria that uncovered how the bacteria harnesses phagocytosis and actin polymerization to facilitate motility. These initial studies led to the discovery of key regulators of actin filament formation including Arp2/3 and N-WASP. Advances in yeast genetics, biochemistry and imaging then allowed Drubin and others to expand their studies to actin dynamics and endocytosis in yeast.

Confocal microscopy is known for its capability to produce exceptional 3D images, even in living tissue. At the same time, it is a powerful spectroscopic tool, facilitating fluores- cence methods such as Fluorescence Correlation Spectroscopy (FCS) or single-molecule Förster Resonance Energy Transfer (FRET). It is heavily used to investigate a wide range of biological problems. This holds true especially for protein properties such as ligand binding, complex formation, conformational changes, or the intracellular distribution of the species in question. In this work, I will describe the assembly of two instruments: The first is a multi- parameter fluorescence detection (MFD) setup. It is a purely spectroscopic tool that offers the capability to characterize a fluorescent molecule, delivering information like fluorescence lifetime, anisotropy or the speed of its diffusion in free solution. When the molecule of interest is labelled with two fluorophores, additional information, like the energy transfer in-between them, becomes accessible and the correct distance between these two fluorophores can be calculated. If the two fluorophores are attached to different molecules, the MFD setup can detect interactions of these molecules in the range from pM up to μM with the help of Fluorescence Cross-Correlation Spectroscopy (FCCS). The second instrument, a stimulated emission depletion setup, combines some of the mentioned techniques, like FCS, with the superior image capability of a confocal micro- scope. One particular problem of fluorescent microscopes, though, is that image resolution is always restricted to the diffraction limit of the wavelength of the laser light. The STED setup utilizes the effect of stimulated emission in order to circumvent the diffraction bar- rier and allows images with a three-fold resolution increase, down to 75nm. These two setups will be used for several applications: The first will be centered around the molecular conformation of proteins, which are sensitive to the nature of the aqueous environment. In particular, the presence of ions can stabilize or destabilize (denature) protein secondary structure. The underlying mechanisms of these actions are still not fully understood. I will apply single-pair FRET to a small 29 amino acid long model peptide to investigate unfolding mechanisms of different unfolding reagents from the Hofmeister series, like sodium perchlorate or guanidinium chloride. The results show that certain salts, which are commonly summarized as denaturing agents, achieve the unfolding by either collapsing the molecule to a compressed state or swelling it to a denatured state. 7 The second application of the MFD setup is the investigation of the enhanced green fluorescent protein (EGFP). Although highly used in biochemistry and biophysics, for example to read out the expression level of genes, it is still not fully known what percentage of EGFP is fluorescent. This lack of knowledge makes it nearly impossible to make quantitative statements. With the help of FCCS, it is shown that the folding efficiencies range from 40 − 90%, depending on the environment of the fluorescent protein and which particular mutant is used. In the third application, the focus will be shifted to nucleation- and polymerization- behavior of actin. The actin cytoskeleton is a central mediator of cellular morphogenesis, and rapid actin reorganization drives essential processes such as cell migration and cell di- vision. In order to compare results of confocal spectroscopy methods with well-established bulk essays, we successfully ported the standard bulk essay to the confocal microscope, allowing for the first time to follow the decrease of monomer concentration and appear- ance of small filaments. Also, the formation of dimers or other small oligomers below the critical concentration is proven for the first time, using FCCS. The last application will utilize the STED setup in order to carry out the first steps towards the investigation of the nucleation and branching behavior of actin in cooperation with the actin related protein 2/3 (ARP2/3). This protein complex preferentially attaches to actin filaments that are located at the leading edge of a cell and forms branched filamentous structures. The exact conditions under which this process occurs are not well characterized. This part of the work will deal with the steps that are necessary to follow the polymerization process on the STED setup.

Tumor cells WASH away the extracellular matrix Tumor cells invade through extracellular matrices by forming actin-rich structures called invadopodia, which contain the transmembrane matrix metalloproteinase MT1-MMP. Monteiro et al. reveal that the Arp2/3 activating protein WASH works with the exocyst complex to deliver MT1-MMP from late endosomes to the invadopodial plasma membrane. This biosights episode presents the paper by Monteiro et al. from the December 23, 2013, issue of The Journal of Cell Biology and includes an interview with senior author Philippe Chavrier (Institut Curie, Paris, 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

Breaking egg symmetry During the first meiotic division of mammalian oocytes, the meiotic spindle and chromosomes move from the interior to the periphery of the cell. Yi et al. reveal that chromosome migration occurs in two sequential phases driven by the actin nucleators Fmn2 and the Arp2/3 complex. This biosights episode presents the paper by Yi et al. from the March 4, 2013, issue of The Journal of Cell Biology and includes an interview with senior author Rong Li (Stowers Institute for Medical Research, Kansas City, MO). 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

Der Hauptregulator der vaskulären Homöostase ist das Endothel, welches eine Vielzahl an vasoprotektiven Effekten ausübt. Die Integrität und Regulation des endothelialen Zellayers der Blutgefäße ist von großer Bedeutung bei physiologischen und pathologischen Prozessen. Die Basis dieser Phänomene ist in der dynamischen und exakt kontrollierten Regulation des Zytoskeletts begründet. Die wichtigsten Regulatoren des Zytoskeletts stellen die GTPasen der Rho-Familie und deren Effektoren dar. Im Rahmen dieser Doktorarbeit untersuchten wir in primären humanen Nabelschnurendothelzellen, eine neu in Erscheinung tretende Zytoskelettstruktur, der wir in Anlehnung an ähnliche Proteingruppierungen in monozytären Zellen den Namen HUVEC-Podosomen gaben. HUVEC-Podosomen sind aufgrund ihrer Komponenten mit klassischen Podosomen vergleichbar. Allerdings gibt es zwischen beiden Strukturen auch Unterschiede, denn während klassische Podosomen aus Ring und Kern bestehen, zeigen HUVEC-Podosomen eine zweischichtige Architektur. Wie wir weiterhin nachweisen konnten liegen Podosomen der Endothelzellen an der ventralen Plasmamembran und haben engen Kontakt mit der extrazellulären Matrix.. Somit fungieren sie, wie auch die klassischen Podosomen, als Adhäsionsstrukturen. Sie dienen aber nicht nur der Adhäsion, denn wie bei FITC-markierten Monolayer-Versuchen gezeigt werden konnte, haben sie auch eine proteolytische Aktivität, die insbesondere beim Matrixverdau und der daran anschließenden Migration von Bedeutung ist. Ferner können wir zeigen, daß HUVEC-Podosomen in ruhenden, konfluenten Zellayern nicht beobachtet werden können. Sie lassen sich aber in migratorischen (subkonfluent oder nach Verwundung) HUVEC, in hoher Anzahl und diversen ringförmigen Formationen, vorwiegend in Bereichen nahe dem Leitsaum nachweisen. Wie wir mit Hilfe von live cell imaging-Experimenten zeigen konnten, sind diese Strukturen hochdynamisch und breiten sich wellenartig mit einem weiten Radius innerhalb einer Zelle aus. Scheinbar dispergieren diese Formationen oder fusionieren mit der Zellplasma, wodurch sie die enthaltenen Proteine für viele andere zytoplasmatische oder membranöse Prozesse freigeben könnten. Durch Experimente, in denen Zytokine wie VEGF, bzw. Zytokin-produzierende Zellen wie Monozyten den HUVEC-Kulturen zugegeben wurden, konnten wir zeigen, daß diese die Bildung von Podosomen induzieren und sogar erheblich steigern. Unsere Arbeiten mit konstitutiv aktiven und dominant negativen GTPase-Mutanten zeigten weiterhin, daß diese bei der Organisation und Entstehung der HUVEC-Podosomen von entscheidender Bedeutung sind. Ferner konnte mit Hilfe von Mikroinjektionsversuchen von einer Teildomäne (A) des N-WASP-Proteins verifiziert werden, daß der Mechanismus zur Bildung der HUVEC-Podosomen eine Arp2/3-abhängige Aktinnukleation beinhaltet. Weiterhin ist die Bildung dieser Adhäsionsstrukturen auch von Src Tyrosinkinasen und PI3-Kinase abhängig. Eine der Komponenten von HUVEC-Podosomen ist das Markerprotein Drebrin. Drebrin kann nur in diesen Strukturen und an Zell-Zell-Kontakten in HUVEC detektiert werden. Mikroinjektionsversuche von diversen Konstrukten der unterschiedlichen Regionen von Drebrin zeigen, daß dieses Protein von großer Bedeutung für die Bildung und Struktur der HUVEC-Podosomen ist. Die einzelnen Protein-Protein-Interaktionen von podosomalen Komponenten untereinander und mit Drebrin wurden mit Hilfe von Immunpräzipitation getestet. Es ist uns jedoch nicht gelungen einen Drebrin-Interaktionspartner zu finden. Eine Interaktion von Drebrin konnten wir nur mit Drebrin selbst in Form einer Dimerisierung bzw. mit F-Aktin nachgeweisen. Es ist sehr wahrscheinlich, daß es sich bei HUVEC-Podosomen um ein multifunktionelles Organell handelt. Wie wir in dieser Arbeit darstellen, sind HUVEC-Podosomen Adhäsionsstrukturen. Sie können am häufigsten am Leitsaum detektiert werden, wobei ihre Generierung nur in Zellen mit migratorischen Phänotyp (in Zellen am Wundrand oder in subkonfluenten layern) detektiert werden kann. Beide Tatsachen sprechen dafür, daß HUVEC-Podosomen den Prozeß der Migration unterstützen. Zudem können diese Adhäsionsstrukturen die Matrix degradieren, wodurch sie so wiederum zur Migration aber auch invasiven Prozessen beitragen könnten. HUVEC-Podosomen könnten auch eine Funktion als Speicherform ihrer Komponenten ausüben. Sie fusionieren mit der Zellmembran und liefern so möglicherweise notwendige Proteine und Signale, die die Induktion von Protrusionen ermöglichen und so migratorische Prozesse unterstützen könnten. Durch die Involvierung u. a. von Drebrin, das an Zell-Grenzen detektiert werden kann, können HUVEC-Podosomen möglicherweise einen Einfluß auf Zell-Zell-Kontakte und Vorgänge wie Angiogenese ausüben. Dies bestätigt auch die Tatsache, daß Zytokine die Anzahl an Zellen erhöhen, die HUVEC-Podosomen generieren können und Vorgänge wie Wundheilung beschleunigt ablaufen lassen und so u. U. eine klinische Relevanz haben könnten.

Die Polymerisation von monomerem G-Aktin zu filamentösem F-Aktin, die Organisation von Aktin-Filamenten zu spezifischen Zytoskelettstrukturen, sowie die F-Aktin Depolymerisation stellen in ihrem dynamischen Zusammenspiel die Triebkraft vieler zellulärer Bewegungsvorgänge dar. Dies sind u.a. Zell-Polarisation, Zell-Migration, Zell-Teilung, Zell-Zell-Interaktionen, Phagozytose und intrazellulärer Vesikeltransport. Das Verständnis der molekularen Grundlagen der Aktin Organisation hat 1999 einen entscheidenden Fortschritt gemacht: damals wurde entdeckt, dass der sieben Untereinheiten umfassende Arp2/3 Komplex nach Aktivierung durch Proteine der Wiskott-Aldrich Syndrom Protein- (WASp-) Familie Aktin-Filamente de novo polymerisieren kann. Die Experimente für diese Dissertationsarbeit hatten zum Ziel, den Mechanismus der WASp-abhängigen Aktin Nukleation in vitro und in humanen Makrophagen genauer zu untersuchen. Einerseits sollten die Regionen von WASp genauer charakterisiert werden, die für die Aktivierung des Arp2/3 Komplexes verantwortlich sind. Andererseits sollte die Bedeutung von WASp und Arp2/3 Komplex bei der Aktin Nukleation in spezialisierten Adhäsionsstrukturen von Makrophagen, sogenannten Podosomen, geklärt werden. Podosomen sind essentiell für Adhäsion, Migration und vermutlich auch Gewebeinvasion von Makrophagen und ihnen verwandten Zellarten, aber sie finden sich auch in einer Vielzahl weiterer Zelltypen einschließlich metastasierender Tumorzellen. Die Bedeutung von Podosomen wird dadurch verdeutlicht, dass ihr Fehlen bei unter Wiskott-Aldrich Syndrom leidenden Patienten mit klinisch relevanten Immundefekten assoziiert ist. Zur Identifizierung der für die Aktin Nukleation minimal notwendigen WASp Regionen wurden verschiedene GST-Fusionskonstrukte der konstitutiv aktiven VCA (“Verprolin-like“, “Central“, “Acidic“) Domäne hergestellt. Durch Anisotropie Messungen und in GST-“Pulldown“ Versuchen wurde die Bindung von Arp2/3 Komplex und G-Aktin an die verschiedenen Konstrukte in vitro bestimmt. In einem zweiten Schritt wurde getestet, welche Regionen für die Arp2/3 Komplex Aktivierung notwendig sind. Dabei wurde GST-VC als die minimal notwendige Region für die Aktivierung der Aktin Nukleation identifiziert. Durch mikroskopische Analyse von in vitro nukleierten Aktin-Filamenten stellten wir fest, dass der durch GST-VC aktivierte Arp2/3 Komplex auch zur Ausbildung von Aktin-Filament Verzweigungen fähig ist. Die zellulären Effekte der VCA Konstrukte wurden mittels Mikroinjektion in primäre humane Makrophagen untersucht. Aktive Konstrukte führten zum Auftreten von prominenten Aktin-Aggregaten im Zytoplasma und zur Zerstörung von Podosomen. Auch hierbei war GST-VC das kürzeste Konstrukt, das vermutlich durch Aktivierung von zellulärem Arp2/3 Komplex, zur Zunahme des Gehaltes an intrazellulärem polymerisiertem Aktin führte. Obwohl die WASp-A Region als hochaffine Arp2/3 Komplex Bindungsstelle beschrieben worden war (Machesky und Insall, 1998), ist sie für die Arp2/3 Komplex Aktivierung nicht essentiell. Durch Koinjektion von WASp-A oder N-WASP-A mit aktiven GTPasen konnte ein erster experimenteller Hinweise für eine “Priming“- (Sensibilisierungs-) Funktion der A Region am Arp2/3 Komplex gefunden werden. Bei der Untersuchung der Aktin Nukleation und Organisation in Podosomen zeigte sich ein enger funktioneller Zusammenhang zwischen Aktin- und Tubulin-Zytoskelett. So konnte durch Depolymerisierung der Mikrotubuli in adhärierenden Monozyten die Ausbildung von Podosomen verhindert werden. Da bekannt war, dass WASp über seine Polyprolin Domäne an CIP4 (“CDC42 Interacting Protein“) bindet und dieses wiederum mit Mikrotubuli assoziiert, wurde der Einfluss der isolierten WASp-Polyprolin Domäne, aber auch von CIP4 Deletions-Konstrukten, denen entweder die Mikrotubuli oder WASp Bindungsstelle fehlten, auf die Podosomen-Ausbildung untersucht. Einem auf den so erhaltenen Ergebnissen basierendem Modell zufolge könnte WASp durch CIP4 an Mikrotubuli rekrutiert und anschließend via Mikrotubuli an Orte der Podosomen-Bildung transportiert werden. Dort könnte WASp dann zur Aktivierung von Arp2/3 Komplex und zur Nukleation von neuen Aktin-Filamenten führen.

Myosine sind molekulare Motoren, die an einer Vielzahl von zellulären Prozessen wie Bewegung, Zellteilung oder Polarität beteiligt sind. Ihr Grundaufbau gliedert sich in Motordomäne, Hals- und Schwanzdomäne. Der Motor interagiert ATP-abhängig mit dem Aktinzytoskelett und ist die krafterzeugende Komponente. Vergleicht man die verschiedenen Myosine miteinander, zeigt der Kopfbereich die höchste Konservierung. An den Motor schliesst sich der Halsbereich an, der die Bindestellen für regulatorische Untereinheiten wie z.B. Calmodulin beinhaltet. Der Schwanzbereich dient zum einem der Interaktion mit der transportierten Fracht und zum anderen der Dimerisierung oder Organisation in Filamente. In der Hefe Saccharomyces cerevisiae findet man fünf Myosine aus drei verschiedenen Klassen. Myo1p ist das einzige Klasse II Myosinund gehört zu den muskelähnlichen Myosinen, die sich in Filamenten organisieren. Myo2p und Myo4p gehören zu den Klasse V Myosinen und vermitteln Prozesse wie Vesikel-Transport, mRNALokalisation und Vererbung von Organellen und Endoplasmatischen Retikulum. Es wird vermutet, dass sie Dimere bilden, die als prozessive Motoren, also eigenständig, durch die Zelle wandern und so ihre Fracht an den Ort ihrer Bestimmung bringen. Myo3p und Myo5p sind in ihrer Funktion redundant und vermitteln als Klasse I Myosine die Endozytose, sowie die Integrität und Polarität des kortikalen Aktinzytoskeletts. Sie liegen als Monomere vor und interagieren über spezifische Domänen in ihren Schwanzbereich mit einer Vielzahl von Proteinen wie z.B. Verprolin oder Komponenten des Arp2/3-Komplexes. Die rekombinante Expression von Myosinen stellt sich als sehr problematisch dar, da sich die Motordomäne nicht spontan in eine funktionelle Konformation falten kann. Verschiedene Publikationen deuten daraufhin, dass für die Faltung dieser Multidomänenstruktur die UCS-Proteine notwendig sind. UCS leitet sich von den Namen der zuerst identifizierten Mitglieder ab (UNC-45 aus C. elegans, Cro1p aus P. anserina und She4p aus S. cerevisiae), welche lediglich die C-terminale UCS-Domäne gemeinsam haben. Für UNC-45 konnte bereits gezeigt werden, das es über die UCS-Domäne mit der Motordomäne von Muskelmyosin interagiert und als Chaperon dessen thermale Aggregation verhindert. Ausserdem interagiert UNC-45 über eine N-terminale TPR-Domäne mit Hsp90 und über den zentralen Bereich mit Hsp70. Im Rahmen meiner Arbeit wurde der Einfluss von She4p auf die Funktion der Myosine untersucht. Es wurde gezeigt, dass She4p über die UCS-Domäne mit der Motordomäne von Klasse I und Klasse V Myosinen interagiertund somit die Lokalisation von Myo3p, Myo4p und Myo5p ermöglicht. Mit Hilfe eines Aktin Pelleting Assays konnte gezeigt werden, dass die Misslokalisation der Klasse I Myosine im she4! Hintergrund durch einen Defekt in der Aktinbindedomäne im Motorbereich verursacht wird. Die Spezifität von She4p für verschiedene Myosinklassen spiegelt sich in der zellulären Verteilung des Proteins wieder. Das UCS-Protein wird Myo2p-abhängig in die Knospenspitze transportiert, um dort die Interaktion zwischen Klasse I Myosinen und dem Aktinzytoskelett zu vermitteln. Im Gegensatz dazu benötigt Myo4p lediglich funktionelles She4p innerhalb der Zelle, da dieses Myosin durch Mutter- und Tochterzelle wandert und somit seinen Regulator überall benötigt. Die Tatsache, ob She4p wie UNC-45 als Chaperon an der Faltung der Motordomäne beteiligt ist, ist weiterhin unklar. Es konnte jedoch in einem Pulldown Experiment und einer Immunpräzipitation eine Interaktion zwischen She4p und Hsp90 festgestellt werden. Es ist daher durchaus möglich, dass She4p als Kochaperon das Hsp90 System zum Myosin rekrutiert, damit die Motordomäne in eine funktionelle Konformation gefaltet wird. Neben der zytoplasmatischen Funktion von She4p scheint es noch eine nukleäre zu geben, da im Pulldown Experiment zahlreiche Proteine gefunden wurden, die Teil des Processosomes der kleinen ribosomalen Untereinheit sind und im Nucleolus lokalisieren. Die Funktion von She4p in diesem Prozess ist noch unbekannt.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.04.15.042143v1?rss=1 Authors: Desale, S. E., Chinnathambi, S. Abstract: Background: Seeding effect of extracellular Tau species is an emerging aspect to study the Tauopathies in Alzheimers disease. Tau seeds enhance the propagation of disease along with its contribution to microglia-mediated inflammation. Omega-3 fatty acids are known to exert the anti-inflammatory property to microglia by modulating cell membrane compositions. The immunomodulatory function of omega-3 fatty acids exerts anti-inflammatory property to microglia. Owing to the imparted anti-inflammatory nature enhance phagocytosis and increased migration property has been observed in microglia. The dietary omega-3 fatty acids are found to change the lipid composition of the cell membrane that predominated many signaling cascade and by modulating specific receptor response. Thus the omega-3 fatty acids influence microglial response in Tauopathy. Methods: N9 microglia cells were exposed to extracellular full-length Tau monomer and aggregates along with ALA (alpha Linolenic acid) to study the internalization of exposed Tau. The degradation of internalized Tau studied with the endosomal markers Rab5 and Rab7. The final degradation step in phagocytosis has been studied with LAMP-2A as lysosomal markers. The changes in the rate of migration of microglia were assessed by wound-scratch assay along with Microtubule organizing center (MTOC) reorientation were studied after exposure of Tau and ALA as the property of highly migratory microglia. The role of actin in phagocytosis and migration was observed with the study of actin structures lamellipodia, filopodia, and membrane ruffling. The formation of extensive actin branching in lamellipodia and membrane ruffling was studied with the help of ARP2/3 complex for nucleating actin network. Results: The increased phagocytosis of extracellular Tau monomer and aggregates has been observed upon ALA exposure to microglia cells. The intracellular degradation of internalized Tau species was targeted by early and late endosomal markers Rab5 and Rab7. The increase levels of LAMP-2A and colocalization with internalized Tau indicated the degradation via lysosome. These results indicate the degradation of internalized Tau species in the presence of ALA instead of getting accumulated in the cell. The enhanced migratory ability of microglia in the presence of ALA induces the MTOC repolarization and reduces the nuclear-centrosomal axis polarity and favorable anterior positioning of MTOC. The increased migration also complemented with the enhance actin remodeling through lamellipodia, filopodia and membrane ruffles formation along with Iba-1 protein. The high density of ARP2/3 complex at the leading ends of migratory microglia confirmed the extensive branching of actin filaments on ALA exposure. Conclusions: Tau seeds greatly contributes to the spread of disease, one way to reduce the spreading is to reduce the presence of extracellular Tau seed. Microglia could be influenced to reduce extracellular Tau seed with dietary fatty acids. Our results suggest that dietary fatty acids ALA significantly enhance phagocytosis and intracellular degradation of internalized Tau. The actin dynamics and enhanced migration supports the phagocytosis process. Our approach provides the insights of beneficial role of ALA as anti-inflammatory dietary supplement to treat AD. Copy rights belong to original authors. Visit the link for more info