Actin remodeling to facilitate membrane fusion

AbstractActin and its associated proteins participate in several intracellular trafficking mechanisms. This review assesses recent work that shows how actin participates in the terminal trafficking event of membrane bilayer fusion. A recent flurry of reports defines a role for Rho proteins in membrane fusion and also demonstrates that this role is distinct from any vesicle transport mechanism. Rho proteins are well known to govern actin remodeling, which implicates this process as a condition of membrane fusion. A small but significant body of work examines actin-regulated events of intracellular membrane fusion, exocytosis and endocytosis. In general, actin has been shown to act as a negative regulator of exocytosis. Cortical actin filaments act as a barrier that requires transient removal to allow vesicles to undergo docking at the plasma membrane. However, once docked, F-actin synthesis may act as a positive regulator to give the final stimulus to drive membrane fusion. F-actin synthesis is clearly needed for endocytosis and intracellular membrane fusion events. What may seem like dissimilar results are perhaps snapshots of a single mechanism of membranous actin remodeling (i.e. dynamic disassembly and reassembly) that is universally needed for all membrane fusion events

Remodeling of organelle-bound actin is required for yeast vacuole fusion

Actin participates in several intracellular trafficking pathways. We now find that actin, bound to the surface of purified yeast vacuoles in the absence of cytosol or cytoskeleton, regulates the last compartment mixing stage of homotypic vacuole fusion. The Cdc42p GTPase is known to be required for vacuole fusion. We now show that proteins of the Cdc42p-regulated actin remodeling cascade (Cdc42p → Cla4p → Las17p/Vrp1p → Arp2/3 complex → actin) are enriched on isolated vacuoles. Vacuole fusion is dramatically altered by perturbation of the vacuole-bound actin, either by mutation of the ACT1 gene, addition of specific actin ligands such as latrunculin B or jasplakinolide, antibody to the actin regulatory proteins Las17p (yeast Wiskott-Aldrich syndrome protein) or Arp2/3, or deletion of actin regulatory genes. On docked vacuoles, actin is enriched at the “vertex ring” membrane microdomain where fusion occurs and is required for the terminal steps leading to membrane fusion. This role for actin may extend to other trafficking systems

Genomic Analysis of Homotypic Vacuole Fusion

Yeast vacuoles undergo fission and homotypic fusion, yielding one to three vacuoles per cell at steady state. Defects in vacuole fusion result in vacuole fragmentation. We have screened 4828 yeast strains, each with a deletion of a nonessential gene, for vacuole morphology defects. Fragmented vacuoles were found in strains deleted for genes encoding known fusion catalysts as well as 19 enzymes of lipid metabolism, 4 SNAREs, 12 GTPases and GTPase effectors, 9 additional known vacuole protein-sorting genes, 16 protein kinases, 2 phosphatases, 11 cytoskeletal proteins, and 28 genes of unknown function. Vacuole fusion and vacuole protein sorting are catalyzed by distinct, but overlapping, sets of proteins. Novel pathways of vacuole priming and docking emerged from this deletion screen. These include ergosterol biosynthesis, phosphatidylinositol (4,5)-bisphosphate turnover, and signaling from Rho GTPases to actin remodeling. These pathways are supported by the sensitivity of the late stages of vacuole fusion to inhibitors of phospholipase C, calcium channels, and actin remodeling. Using databases of yeast protein interactions, we found that many nonessential genes identified in our deletion screen interact with essential genes that are directly involved in vacuole fusion. Our screen reveals regulatory pathways of vacuole docking and provides a genomic basis for studies of this reaction

A Ypt/Rab Effector Complex Containing the Sec1 Homolog Vps33p is Required for Homotypic Vacuole Fusion

Yeast vacuoles undergo priming, docking, and homotypic fusion, although little has been known of the connections between these reactions. Vacuole-associated Vam2p and Vam6p (Vam2/6p) are components of a 65S complex containing SNARE proteins. Upon priming by Sec18p/NSF and ATP, Vam2/6p is released as a 38S subcomplex that binds Ypt7p to initiate docking. We now report that the 38S complex consists of both Vam2/6p and the class C Vps proteins [Reider, S. E. and Emr, S. D. (1997) Mol. Biol. Cell 8, 2307-2327]. This complex includes Vps33p, a member of the Sec1 family of proteins that bind t-SNAREs. We term this 38S complex HOPS, for homotypic fusion and vacuole protein sorting. This unexpected finding explains how Vam2/6p associates with SNAREs and provides a mechanistic link of the class C Vps proteins to Ypt/Rab action. HOPS initially associates with vacuole SNAREs in cis and, after release by priming, hops to Ypt7p, activating this Ypt/Rab switch to initiate docking

Proteomic analysis of secretagogue-stimulated neutrophils implicates a role for actin and actin-interacting proteins in Rac2-mediated granule exocytosis

<p>Abstract</p> <p>Background</p> <p>Neutrophils are abundant leukocytes that play a primary role in defence against pathogens. Neutrophils enter sites of infection where they eliminate pathogens via phagocytosis and the release of antimicrobial mediators via degranulation. Rho GTPases, particularly Rac2, play a key role in neutrophil degranulation. The purpose of this study was to identify Rac2-dependent changes in protein abundance in stimulated neutrophils.</p> <p>Methods</p> <p>We performed a proteomic analysis on secretagogue-stimulated bone marrow neutrophils that were isolated from wild-type and Rac2^-/-mice. Protein abundance was analyzed by 2-dimensional SDS-PAGE of fluorescently labelled samples which allowed the detection ~3500 proteins.</p> <p>Results</p> <p>We identified 22 proteins that showed significant changes in abundance after secretagogue-stimulation of wild-type neutrophils, which did not occur in neutrophils isolated from Rac2^-/-mice. As expected, the abundance of several granule proteins was reduced in wild-type cells; this did not occur in Rac2^-/-neutrophils which confirms the requirement for Rac2 in degranulation. We also found changes in abundance of many actin remodelling proteins including coronin-1A, β-actin and the F-actin capping protein, (CapZ-β). Coronin-1A showed elevated levels of several isoforms after stimulation of neutrophils from wild-type, but not from Rac2^-/-mice. These isoforms were immunoreactive with anti-phospho-threonine antibodies, suggesting that neutrophil stimulation triggers a Rac2-dependent kinase cascade that results in the phosphorylation of coronin-1A.</p> <p>Conclusion</p> <p>The control of Rac2-mediated degranulation in neutrophils likely functions through actin remodelling via activation of several actin-binding proteins. We found coronin-1A to be a novel downstream effector protein of this pathway that is threonine phosphorylated in response to secretagogue stimulation.</p

Pex3 peroxisome biogenesis proteins function in peroxisome inheritance as class V myosin receptors

Pex3 links peroxisome formation and inheritance. By binding to class V myosin, biogenesis protein Pex3 also directs the organelles into daughter cells

Myosin-driven peroxisome partitioning in S. cerevisiae

In Saccharomyces cerevisiae, the class V myosin motor Myo2p propels the movement of most organelles. We recently identified Inp2p as the peroxisome-specific receptor for Myo2p. In this study, we delineate the region of Myo2p devoted to binding peroxisomes. Using mutants of Myo2p specifically impaired in peroxisome binding, we dissect cell cycle–dependent and peroxisome partitioning–dependent mechanisms of Inp2p regulation. We find that although total Inp2p levels oscillate with the cell cycle, Inp2p levels on individual peroxisomes are controlled by peroxisome inheritance, as Inp2p aberrantly accumulates and decorates all peroxisomes in mother cells when peroxisome partitioning is abolished. We also find that Inp2p is a phosphoprotein whose level of phosphorylation is coupled to the cell cycle irrespective of peroxisome positioning in the cell. Our findings demonstrate that both organelle positioning and cell cycle progression control the levels of organelle-specific receptors for molecular motors to ultimately achieve an equidistribution of compartments between mother and daughter cells

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery