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Biomedical subjects

David G Drubin

Publications and source records attributed to David G Drubin.

At least 19 recordsLinked to original sources
PubMed2006

Negative regulation of yeast Eps15-like Arp2/3 complex activator, Pan1p, by the Hip1R-related protein, Sla2p, during endocytosis.

Control of actin assembly nucleated by the Arp2/3 complex plays a crucial role during budding yeast endocytosis. The yeast Eps15-related Arp2/3 complex activator, Pan1p, is essential for endocytic internalization and proper actin organization. Pan1p activity is negatively regulated by Prk1 kinase phosphorylation after endocytic internalization. Phosphorylated Pan1p is probably then dephosphorylated in the cytosol. Pan1p is recruited to endocytic sites approximately 25 s before initiation of actin polymerization, suggesting that its Arp2/3 complex activation activity is kept inactive during early stages of endocytosis by a yet-to-be-identified mechanism. However, how Pan1p is maintained in an inactive state is not clear. Using tandem affinity purification-tagged Pan1p, we identified End3p as a stoichiometric component of the Pan1p complex, and Sla2p, a yeast Hip1R-related protein, as a novel binding partner of Pan1p. Interestingly, Sla2p specifically inhibited Pan1p Arp2/3 complex activation activity in vitro. The coiled-coil region of Sla2p was important for Pan1p inhibition, and a pan1 partial loss-of-function mutant suppressed the temperature sensitivity, endocytic phenotypes, and actin phenotypes observed in sla2DeltaCC mutant cells that lack the coiled-coil region. Overall, our results establish that Sla2p's regulation of Pan1p plays an important role in controlling Pan1p-stimulated actin polymerization during endocytosis.

Actin-Related Protein 2-3 Complex↗
PubMed2006

The budding yeast endocytic pathway.

Recent live-cell imaging studies, coupled with powerful genetic, biochemical and pharmacological tests of function, have expanded our understanding of the molecular events that underlie clathrin/actin mediated-endocytosis in budding yeast. Many features of this pathway are evolutionarily conserved (Engqvist-Goldstein and Drubin, 2003; Kaksonen et al, 2006). Therefore, insights into the intricate molecular choreography of endocytic events in budding yeast will provide a basis for elucidating such mechanisms in more complex organisms. This poster depicts our current understanding of the dynamics of endocytosis in budding yeast.

Actins↗
PubMed2006

Arp2/3 ATP hydrolysis-catalysed branch dissociation is critical for endocytic force generation.

The Arp2/3 complex, which is crucial for actin-based motility, nucleates actin filaments and organizes them into y-branched networks. The Arp2 subunit has been shown to hydrolyse ATP, but the functional importance of Arp2/3 ATP hydrolysis is not known. Here, we analysed an Arp2 mutant in Saccharomyces cerevisiae that is defective in ATP hydrolysis. Arp2 ATP hydrolysis and Arp2/3-dependent actin nucleation occur almost simultaneously. However, ATP hydrolysis is not required for nucleation. In addition, Arp2 ATP hydrolysis is not required for the release of a WASP-like activator from y-branches. ATP hydrolysis by Arp2, and possibly Arp3, is essential for efficient y-branch dissociation in vitro. In living cells, both Arp2 and Arp3 ATP-hydrolysis mutants exhibit defects in endocytic internalization and actin-network disassembly. Our results suggest a critical feature of dendritic nucleation in which debranching and subsequent actin-filament remodelling and/or depolymerization are important for endocytic vesicle morphogenesis.

Actin-Related Protein 2↗
PubMed2006

Endocytic vesicle scission by lipid phase boundary forces.

Endocytosis in budding yeast is thought to occur in several phases. First, the membrane invaginates and then elongates into a tube. A vesicle forms at the end of the tube, eventually pinching off to form a "free" vesicle. Experiments show that actin polymerization is an active participant in the endocytic process, along with a number of membrane-associated proteins. Here we investigate the possible roles of these components in driving vesiculation by constructing a quantitative model of the process beginning at the stage where the membrane invagination has elongated into a tube encased in a sheath of membrane-associated protein. This protein sheath brings about the scission step where the vesicle separates from the tube. When the protein sheath is dynamin, it is commonly assumed that scission is brought about by the constriction of the sheath. Here, we show that an alternative scenario can work as well: The protein sheath acts as a "filter" to effect a phase separation of lipid species. The resulting line tension tends to minimize the interface between the tube region and the vesicle region. Interestingly, large vesicle size can further facilitate the reduction of the interfacial diameter down to a few nanometers, small enough so that thermal fluctuations can fuse the membrane and pinch off the vesicle. To deform the membrane into the tubular vesicle shape, the membrane elastic resistance forces must be balanced by some additional forces that we show can be generated by actin polymerization and/or myosin I. These active forces are shown to be important in successful scission processes as well.

Endocytosis↗
PubMed2006

Spatial dynamics of receptor-mediated endocytic trafficking in budding yeast revealed by using fluorescent alpha-factor derivatives.

Much progress defining the order and timing of endocytic internalization events has come as a result of real-time, live-cell fluorescence microscopy. Although the availability of numerous endocytic mutants makes yeast an especially valuable organism for functional analysis of endocytic dynamics, a serious limitation has been the lack of a fluorescent cargo for receptor-mediated endocytosis. We have now synthesized biologically active fluorescent mating-pheromone derivatives and demonstrated that receptor-mediated endocytosis in budding yeast occurs via the clathrin- and actin-mediated endocytosis pathway. We found that endocytic proteins first assemble into patches on the plasma membrane, and then alpha-factor associates with the patches. Internalization occurs next, concomitant with actin assembly at patches. Additionally, endocytic vesicles move toward early endosomes on actin cables. Early endosomes also associate with actin cables, and they actively move toward endocytic sites to capture vesicles being released from the plasma membrane. Thus, endocytic vesicle formation and capture of the newly released vesicles by early endosomes occur in a highly concerted manner, mediated by the actin cytoskeleton.

Actins↗
PubMed2006

The Dam1 kinetochore ring complex moves processively on depolymerizing microtubule ends.

Chromosomes interact through their kinetochores with microtubule plus ends and they are segregated to the spindle poles as the kinetochore microtubules shorten during anaphase A of mitosis. The molecular natures and identities of coupling proteins that allow microtubule depolymerization to pull chromosomes to poles during anaphase have long remained elusive. In budding yeast, the ten-protein Dam1 complex is a critical microtubule-binding component of the kinetochore that oligomerizes into a 50-nm ring around a microtubule in vitro. Here we show, with the use of a real-time, two-colour fluorescence microscopy assay, that the ring complex moves processively for several micrometres at the ends of depolymerizing microtubules without detaching from the lattice. Electron microscopic analysis of 'end-on views' revealed a 16-fold symmetry of the kinetochore rings. This out-of-register arrangement with respect to the 13-fold microtubule symmetry is consistent with a sliding mechanism based on an electrostatically coupled ring-microtubule interface. The Dam1 ring complex is a molecular device that can translate the force generated by microtubule depolymerization into movement along the lattice to facilitate chromosome segregation.

Cell Cycle Proteins↗
PubMed2006

A role for cortactin in Listeria monocytogenes invasion of NIH 3T3 cells, but not in its intracellular motility.

Cortactin is an F-actin binding protein that binds to the Arp2/3 complex, stimulates its actin nucleation activity, and inhibits actin filament debranching. Using RNA interference directed against cortactin, we explored the importance of cortactin for several processes involving dynamic actin assembly. Silencing cortactin expression was efficiently achieved in HeLa and NIH 3T3 cells, with less than 5% of cortactin expression in siRNA-treated cells. Surprisingly, endocytosis in HeLa and NIH 3T3 cells, and cell migration rates, were not altered by RNAi-mediated cortactin silencing. Listeria utilizes actin-based motility to move within and spread among mammalian host cells; its actin-clouds and tails recruit cortactin. We explored the role of cortactin during the Listeria life cycle in cortactin "knockdown" NIH 3T3 cells. Interestingly, cortactin siRNA-treated cells showed a significant reduction in the efficiency of the bacteria invasion in NIH 3T3 cells. However, cortactin depletion did not interfere with assembly of Listeria actin clouds or actin tails, or Listeria intracellular motility or speed. Therefore, our findings suggest that cortactin plays a role in Listeria internalization, but not in the formation of actin clouds and tails, or in bacteria intracellular motility.

Actins↗
PubMed2006

Endocytic internalization in budding yeast requires coordinated actin nucleation and myosin motor activity.

Actin polymerization essential for endocytic internalization in budding yeast is controlled by four nucleation promoting factors (NPFs) that each exhibits a unique dynamic behavior at endocytic sites. How each NPF functions and is regulated to restrict actin assembly to late stages of endocytic internalization is not known. Quantitative analysis of NPF biochemical activities, and genetic analysis of recruitment and regulatory mechanisms, defined a linear pathway in which protein composition changes at endocytic sites control actin assembly and function. We show that yeast WASP initiates actin assembly at endocytic sites and that this assembly and the recruitment of a yeast WIP-like protein by WASP recruit a type I myosin with both NPF and motor activities. Importantly, type I myosin motor and NPF activities are separable, and both contribute to endocytic coat inward movement, which likely represents membrane invagination. These results reveal a mechanism in which actin nucleation and myosin motor activity cooperate to promote endocytic internalization.

Actins↗
PubMed2006

Harnessing actin dynamics for clathrin-mediated endocytosis.

Actin polymerization often occurs at the plasma membrane to drive the protrusion of lamellipodia and filopodia at the leading edge of migrating cells. A role for actin polymerization in another cellular process that involves the reshaping of the plasma membrane--namely endocytosis--has recently been established. Live-cell imaging studies are shedding light on the order and timing of the molecular events and mechanisms of actin function during endocytosis.

Actins↗
PubMed2005

A modular design for the clathrin- and actin-mediated endocytosis machinery.

Endocytosis depends on an extensive network of interacting proteins that execute a series of distinct subprocesses. Previously, we used live-cell imaging of six budding-yeast proteins to define a pathway for association of receptors, adaptors, and actin during endocytic internalization. Here, we analyzed the effects of 61 deletion mutants on the dynamics of this pathway, revealing functions for 15 proteins, and we analyzed the dynamics of 8 of these proteins. Our studies provide evidence for four protein modules that cooperate to drive coat formation, membrane invagination, actin-meshwork assembly, and vesicle scission during clathrin/actin-mediated endocytosis. We found that clathrin facilitates the initiation of endocytic-site assembly but is not needed for membrane invagination or vesicle formation. Finally, we present evidence that the actin-meshwork assembly that drives membrane invagination is nucleated proximally to the plasma membrane, opposite to the orientation observed for previously studied actin-assembly-driven motility processes.

Actin Capping Proteins↗
PubMed2005

Phased translation function revisited: structure solution of the cofilin-homology domain from yeast actin-binding protein 1 using six-dimensional searches.

A modified molecular-replacement method is described that makes use of six-dimensional searches and the phased translation function, providing a systematic examination of all possible search-model orientations in an experimental electron-density map. As an example, the structure solution of the cofilin-homology domain of the Saccharomyces cerevisiae actin-binding protein 1 (ABP1) is presented in detail. Additional examples are presented in which these tools have significantly aided structure solutions in a variety of contexts. These results suggest that this approach might be of widespread utility for challenging structures involving weak phase information, complex asymmetric units and search models with weak structural homology. Furthermore, this approach supports an exhaustive molecular-replacement strategy in cases where an appropriate search model cannot readily be identified on the basis of sequence homology. The fully automated web-based implementation of this phased translation function is described.

Actin Depolymerizing Factors↗
PubMed2005

Formation of a dynamic kinetochore- microtubule interface through assembly of the Dam1 ring complex.

How kinetochore proteins form a dynamic interface with microtubules is largely unknown. In budding yeast, the 10-protein Dam1 complex is an Aurora kinase target that plays essential roles maintaining the integrity of the mitotic spindle and regulating interactions with the kinetochore. Here, we investigated the biochemical properties of purified Dam1 complex. The complex oligomerized into rings around microtubules. Ring formation was facilitated by microtubules but could occur in their absence. Mutant alleles led to partially assembled complexes or reduced microtubule binding. The interaction between rings and microtubules is mediated by the C termini of both Dam1 and alphabeta-tubulin. Ring formation promotes microtubule assembly, stabilizes against disassembly, and promotes bundling. A GTP-tubulin lattice is the preferred binding partner for the complex, and Dam1 rings can exhibit lateral mobility on microtubules. These observations suggest a mechanism by which the kinetochore can recognize and stay attached to the plus ends of microtubules.

Animals↗
PubMed2005

Effects of Arp2 and Arp3 nucleotide-binding pocket mutations on Arp2/3 complex function.

Contributions of actin-related proteins (Arp) 2 and 3 nucleotide state to Arp2/3 complex function were tested using nucleotide-binding pocket (NBP) mutants in Saccharomyces cerevisiae. ATP binding by Arp2 and Arp3 was required for full Arp2/3 complex nucleation activity in vitro. Analysis of actin dynamics and endocytosis in mutants demonstrated that nucleotide-bound Arp3 is particularly important for Arp2/3 complex function in vivo. Severity of endocytic defects did not correlate with effects on in vitro nucleation activity, suggesting that a critical Arp2/3 complex function during endocytosis may be structural rather than catalytic. A separate class of Arp2 and Arp3 NBP mutants suppressed phenotypes of mutants defective for actin nucleation. An Arp2 suppressor mutant increased Arp2/3 nucleation activity. Electron microscopy of Arp2/3 complex containing this Arp2 suppressor identified a structural change that also occurs upon Arp2/3 activation by nucleation promoting factors. These data demonstrate the importance of Arp2 and Arp3 nucleotide binding for nucleating activity, and Arp3 nucleotide binding for maintenance of cortical actin cytoskeleton cytoarchitecture.

Actin-Related Protein 2↗
PubMed2005

Phosphoregulation of Arp2/3-dependent actin assembly during receptor-mediated endocytosis.

In both yeast and mammals, endocytic internalization is accompanied by a transient burst of actin polymerization. The yeast protein kinases Prk1p and Ark1p, which are related to the mammalian proteins GAK and AAK1, are key regulators of this process. However, the molecular mechanism(s) by which they regulate actin assembly at endocytic sites have not yet been determined. The Eps15-like yeast protein Pan1p is a Prk1p substrate that is essential for endocytic internalization and for proper actin organization. Pan1p is an Arp2/3 activator and here we show that this activity is dependent on F-actin binding. Mutation of all 15 Prk1p-targeted threonines in Pan1p to alanines mimicked the ark1Delta prk1Delta phenotype, demonstrating that Pan1p is a key Prk1p target in vivo. Moreover, phosphorylation by Prk1p inhibited the ability of Pan1p to bind to F-actin and to activate the Arp2/3 complex, thereby identifying the endocytic phosphoregulation mechanism of Prk1p. We conclude that Prk1p phosphorylation of Pan1p shuts off Arp2/3-mediated actin polymerization on endocytic vesicles, allowing them to fuse with endosomes.

Actin-Related Protein 2↗
PubMed2005

Multiple roles for cyclin G-associated kinase in clathrin-mediated sorting events.

Cyclin G-associated kinase (GAK), also known as auxilin 2, is a potential regulator of clathrin-mediated membrane trafficking. It possesses a kinase domain at its N-terminus that can phosphorylate the clathrin adaptors AP-1 and AP-2 in vitro. The GAK C-terminus can act as a cochaperaone in vitro for Hsc70, a heat-shock protein required for clathrin uncoating. Here we show that the specificity of GAK is very similar to that of adaptor-associated kinase 1, another mammalian adaptor kinase. We used siRNA to investigate GAK's in vivo function. We discovered that early stages of clathrin-mediated endocytosis (CME) were partially inhibited when GAK expression was knocked down. This defect was specifically caused by GAK knockdown because it could be rescued by expressing a rat GAK gene that could not be silenced by one of the siRNAs. To identify the GAK activity required during CME, we mutated the kinase domain and the J domain of the rat gene. Only GAK with a functional J domain could rescue the defect, suggesting that GAK is important for clathrin uncoating. Furthermore, we demonstrated that GAK plays a role in the clathrin-dependent trafficking from the trans Golgi network.

Animals↗
PubMed2005

Cytoskeletal networks and pathways involved in endocytosis.

Until recently, the actin cytoskeleton and the endocytic machinery were thought to operate independently. However, the actin cytoskeleton is an integral part of the cell cortex and there is growing evidence in diverse eukaryotes that F-actin plays a direct role during endocytic internalization. Genetic studies in Saccharomyces cerevisiae have demonstrated that Arp2/3-mediated F-actin assembly is required specifically for the internalization step of endocytosis. Using real-time image analysis, we recently defined a pathway for receptor-mediated endocytosis in budding yeast. Many features of this pathway appear to be conserved widely, indicating that principles derived from our studies in yeast will be directly applicable in more complex eukaryotes. We are pursuing our yeast studies using a combined approach involving image analysis, functional genomics, proteomics and biochemistry. These ongoing studies are providing a broader and deeper understanding of the molecular events of endocytosis, of how forces for actin polymerization are harnessed, and of how steps in the pathway are regulated. Our studies in mammalian cells provide evidence that this pathway is conserved in more complex organisms for endocytic and Golgi trafficking events.

Actins↗
PubMed2004

Interaction of Sla2p's ANTH domain with PtdIns(4,5)P2 is important for actin-dependent endocytic internalization.

A variety of studies have implicated the lipid PtdIns(4,5)P2 in endocytic internalization, but how this lipid mediates its effects is not known. The AP180 N-terminal homology (ANTH) domain is a PtdIns(4,5)P2-binding module found in several proteins that participate in receptor-mediated endocytosis. One such protein is yeast Sla2p, a highly conserved actin-binding protein essential for actin organization and endocytic internalization. To better understand how PtdIns(4,5)P2 binding regulates actin-dependent endocytosis, we investigated the functions of Sla2p's ANTH domain. A liposome-binding assay revealed that Sla2p binds to PtdIns(4,5)P2 specifically through its ANTH domain and identified specific lysine residues required for this interaction. Mutants of Sla2p deficient in PtdIns(4,5)P2 binding showed significant defects in cell growth, actin organization, and endocytic internalization. These defects could be rescued by increasing PtdIns(4,5)P2 levels in vivo. Strikingly, mutant Sla2p defective in PtdIns(4,5)P2 binding localized with the endocytic machinery at the cell cortex, establishing that the ANTH-PtdIns(4,5)P2 interaction is not necessary for this association. In contrast, multicolor real-time fluorescence microscopy and particle-tracking analysis demonstrated that PtdIns(4,5)P2 binding is required during endocytic internalization. These results demonstrate that the interaction of Sla2p's ANTH domain with PtdIns(4,5)P2 plays a key role in regulation of the dynamics of actin-dependent endocytic internalization.

Actins↗