David G. Drubin

Origins of Cell Polarity

The authors thank Kathryn Ayscough, Ken Beck, Fred Chang, Yih-Tai Chen, Kent Grindstaff, and Jeremy Thorner for comments on the manuscript as well as colleagues who kindly provided preprints.

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.

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.

A Pathway for Association of Receptors, Adaptors, and Actin during Endocytic Internalization

In budding yeast, many proteins involved in endocytic internalization, including adaptors and actin cytoskeletal proteins, are localized to cortical patches of differing protein composition. Using multicolor real-time fluorescence microscopy and particle tracking algorithms, we define an early endocytic pathway wherein an invariant sequence of changes in cortical patch protein composition correlates with changes in patch motility. Three Arp2/3 activators each showed a distinct behavior, suggesting distinct patch-related endocytic functions. Actin polymerization occurs late in the endocytic pathway and is required both for endocytic internalization and for patch disassembly. In cells lacking the highly conserved endocytic protein Sla2p, patch motility was arrested and actin comet tails associated with endocytic patch complexes. Fluorescence recovery after photobleaching of the actin comet tails revealed that endocytic complexes are nucleation sites for rapid actin polymerization. Attention is now focused on the mechanisms by which the order and timing of events in this endocytic pathway are achieved.

Phospho-Regulation of Kinetochore-Microtubule Attachments by the Aurora Kinase Ipl1p

The Aurora kinase Ipl1p plays a crucial role in regulating kinetochore-microtubule attachments in budding yeast, but the underlying basis for this regulation is not known. To identify Ipl1p targets, we first purified 28 kinetochore proteins from yeast protein extracts. These studies identified five previously uncharacterized kinetochore proteins and defined two additional kinetochore subcomplexes. We then used mass spectrometry to identify 18 phosphorylation sites in 7 of these 28 proteins. Ten of these phosphorylation sites are targeted directly by Ipl1p, allowing us to identify a consensus phosphorylation site for an Aurora kinase. Our systematic mutational analysis of the Ipl1p phosphorylation sites demonstrated that the essential microtubule binding protein Dam1p is a key Ipl1p target for regulating kinetochore-microtubule attachments in vivo.

Cofilin promotes rapid actin filament turnover in vivo

The ability of actin filaments to function in cell morphogenesis and motility is coupled to their capacity for rapid assembly and disassembly. Because disassembly in vitro is much slower than in vivo, cellular factors that stimulate disassembly have long been assumed to exist. Although numerous proteins can affect actin dynamics in vitro, demonstration of in vivo relevance of these effects has not been achieved. We have used genetics and an actin-inhibitor in yeast to demonstrate that rapid cycles of actin assembly and disassembly depend on the small actin-binding protein cofilin, and that cofilin stimulates filament disassembly. These results may explain why cofilin is ubiquitous in eukaryotes and is essential for viability in every organism in which its function has been tested genetically. Magnitudes of disassembly defects in cofilin mutants in vivo were found to be correlated closely with the magnitudes of disassembly defects observed in vitro, supporting our conclusions. Furthermore, these cofilin mutants provided an opportunity to distinguish in living cells those actin functions that depend specifically on filament turnover (endocytosis) from those that do not (cortical actin patch motility).

A protein interaction map for cell polarity development

Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein–protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express ∼90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein–protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed.

Essential functions and actin‐binding surfaces of yeast cofilin revealed by systematic mutagenesis

Cofilin stimulates actin filament turnover in vivo. The phenotypes of twenty yeast cofilin mutants generated by systematic mutagenesis were determined. Ten grew as well as the wild type and showed no cytoskeleton defects, seven were recessive‐lethal and three were conditional‐lethal and caused severe actin organization defects. Biochemical characterization of interactions between nine mutant yeast cofilins and yeast actin provided evidence that F‐actin binding and depolymerization are essential cofilin functions. Locating the mutated residues on the yeast cofilin molecular structure allowed several important conclusions to be drawn. First, residues required for actin monomer binding are proximal to each other. Secondly, additional residues are required for interactions with actin filaments; these residues might bind an adjacent subunit in the actin filament. Thirdly, despite striking structural similarity, cofilin interacts with actin in a different manner from gelsolin segment‐1. Fourthly, a previously unrecognized cofilin function or interaction is suggested by identification of spatially proximal residues important for cofilin function in vivo, but not for actin interactions in vitro. Finally, mutation of the cofilin N‐terminus suggests that its sequence is conserved because of its critical role in actin interactions, not because it is sometimes a target for protein kinases.

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.

Development of cell polarity in budding yeast

The development of cell polarity involves virtually every aspect of cell biology. Yeast are less complex than cells traditionally used for studies on cell polarity and are amendable to sophisticated genetic analysis. This has resulted in a growing number of molecular markers for yeast cell polarity and an increasingly well-defined progression of molecular events required for bud formation. Together, these factors provide a favorable context in which to understand how the interplay between a large number of processes can polarize a cell. Many genes required for morphogenesis have been identified, and genetic interactions provide evidence that the products of these genes function together. Studies on cell polarity development in S. cerevisiae have demonstrated a requirement for small GTP-binding proteins and have established functional relationships between temporally coincident events. With the continued identification and analysis of genes required for morphogenesis, and the pursuit of these studies on a cytological and biochemical level, studies on yeast will continue to contribute to our understanding of cell polarity development.