Bsp1, a fungal CPI motif protein, regulates actin filament capping in endocytosis and cytokinesis.

The capping of barbed filament ends is a fundamental mechanism for actin regulation. Capping protein controls filament growth and actin turnover in cells by binding to the barbed ends of the filaments with high affinity and slow off-rate. The interaction between capping protein and actin is regulated by capping protein interaction (CPI) motif proteins. We identified a novel CPI motif protein, Bsp1, which is involved in cytokinesis and endocytosis in budding yeast. We demonstrate that Bsp1 is an actin binding protein with a high affinity for capping protein via its CPI motif. In cells, Bsp1 regulates capping protein at endocytic sites and is a major recruiter of capping protein to the cytokinetic actin ring. Lastly, we define Bsp1-related proteins as a distinct fungi-specific CPI protein group. Our results suggest that Bsp1 promotes actin filament capping by the capping protein. This study establishes Bsp1 as a new capping protein regulator and promising candidate to regulate actin networks in fungi.

The cellular slime mold Fonticula alba forms a dynamic, multicellular collective while feeding on bacteria.

Multicellularity evolved in fungi and animals, or the opisthokonts, from their common amoeboflagellate ancestor but resulted in strikingly distinct cellular organizations. The origins of this multicellularity divergence are not known. The stark mechanistic differences that underlie the two groups and the lack of information about ancestral cellular organizations limits progress in this field. We discovered a new type of invasive multicellular behavior in Fonticula alba, a unique species in the opisthokont tree, which has a simple, bacteria-feeding sorocarpic amoeba lifestyle. This invasive multicellularity follows germination dependent on the bacterial culture state, after which amoebae coalesce to form dynamic collectives that invade virgin bacterial resources. This bacteria-dependent social behavior emerges from amoeba density and allows for rapid and directed invasion. The motile collectives have animal-like properties but also hyphal-like search and invasive behavior. These surprising findings enrich the diverse multicellularities present within the opisthokont lineage and offer a new perspective on fungal origins.

Specialization of actin isoforms derived from the loss of key interactions with regulatory factors.

A paradox of eukaryotic cells is that while some species assemble a complex actin cytoskeleton from a single ortholog, other species utilize a greater diversity of actin isoforms. The physiological consequences of using different actin isoforms, and the molecular mechanisms by which highly conserved actin isoforms are segregated into distinct networks, are poorly known. Here, we sought to understand how a simple biological system, composed of a unique actin and a limited set of actin-binding proteins, reacts to a switch to heterologous actin expression. Using yeast as a model system and biomimetic assays, we show that such perturbation causes drastic reorganization of the actin cytoskeleton. Our results indicate that defective interaction of a heterologous actin for important regulators of actin assembly limits certain actin assembly pathways while reinforcing others. Expression of two heterologous actin variants, each specialized in assembling a different network, rescues cytoskeletal organization and confers resistance to external perturbation. Hence, while species using a unique actin have homeostatic actin networks, actin assembly pathways in species using several actin isoforms may act more independently.

A potential Rho GEF and Rac GAP for coupled Rac and Rho cycles during mesenchymal-to-epithelial-like transitions.

The leading edge-to-cadherin contact transitions that occur during metazoan developmental processes and disease states require fine coordination of Rac and Rho pathways. Recently the elmo-mbc complex, a Rac GEF and RhoGAP19D, a Rho GAP were identified as key, conserved regulators that link Rac and Rho during these transitions. The corresponding Rho GEF and Rac GAP remain hidden amongst the large family of GEF and GAP proteins. Identification of these regulators is essential to understand GTPase coordination during these transitions. Here we find two candidates based on the mammalian literature and use RNAi to explore the fly ortholog effects on the dorsal closure epidermis. RhoGEF64C and RhoGAP92B are strong contenders to couple Rac and Rho during mesenchymal-to-epithelial-like transitions.

The elmo-mbc complex and rhogap19d couple Rho family GTPases during mesenchymal-to-epithelial-like transitions.

Many metazoan developmental processes require cells to transition between migratory mesenchymal- and adherent epithelial-like states. These transitions require Rho GTPase-mediated actin rearrangements downstream of integrin and cadherin pathways. A regulatory toolbox of GEF and GAP proteins precisely coordinates Rho protein activities, yet defining the involvement of specific regulators within a cellular context remains a challenge due to overlapping and coupled activities. Here we demonstrate that Drosophila dorsal closure is a powerful model for Rho GTPase regulation during transitions from leading edges to cadherin contacts. During these transitions a Rac GEF elmo-mbc complex regulates both lamellipodia and Rho1-dependent, actomyosin-mediated tension at initial cadherin contacts. Moreover, the Rho GAP Rhogap19d controls Rac and Rho GTPases during the same processes and genetically regulates the elmo-mbc complex. This study presents a fresh framework to understand the inter-relationship between GEF and GAP proteins that tether Rac and Rho cycles during developmental processes.

An Elmo-Dock complex locally controls Rho GTPases and actin remodeling during cadherin-mediated adhesion.

Cell-cell contact formation is a dynamic process requiring the coordination of cadherin-based cell-cell adhesion and integrin-based cell migration. A genome-wide RNA interference screen for proteins required specifically for cadherin-dependent cell-cell adhesion identified an Elmo-Dock complex. This was unexpected as Elmo-Dock complexes act downstream of integrin signaling as Rac guanine-nucleotide exchange factors. In this paper, we show that Elmo2 recruits Dock1 to initial cell-cell contacts in Madin-Darby canine kidney cells. At cell-cell contacts, both Elmo2 and Dock1 are essential for the rapid recruitment and spreading of E-cadherin, actin reorganization, localized Rac and Rho GTPase activities, and the development of strong cell-cell adhesion. Upon completion of cell-cell adhesion, Elmo2 and Dock1 no longer localize to cell-cell contacts and are not required subsequently for the maintenance of cell-cell adhesion. These studies show that Elmo-Dock complexes are involved in both integrin- and cadherin-based adhesions, which may help to coordinate the transition of cells from migration to strong cell-cell adhesion.

A genome-wide screen identifies conserved protein hubs required for cadherin-mediated cell-cell adhesion.

Cadherins and associated catenins provide an important structural interface between neighboring cells, the actin cytoskeleton, and intracellular signaling pathways in a variety of cell types throughout the Metazoa. However, the full inventory of the proteins and pathways required for cadherin-mediated adhesion has not been established. To this end, we completed a genome-wide (~14,000 genes) ribonucleic acid interference (RNAi) screen that targeted Ca(2+)-dependent adhesion in DE-cadherin-expressing Drosophila melanogaster S2 cells in suspension culture. This novel screen eliminated Ca(2+)-independent cell-cell adhesion, integrin-based adhesion, cell spreading, and cell migration. We identified 17 interconnected regulatory hubs, based on protein functions and protein-protein interactions that regulate the levels of the core cadherin-catenin complex and coordinate cadherin-mediated cell-cell adhesion. Representative proteins from these hubs were analyzed further in Drosophila oogenesis, using targeted germline RNAi, and adhesion was analyzed in Madin-Darby canine kidney mammalian epithelial cell-cell adhesion. These experiments reveal roles for a diversity of cellular pathways that are required for cadherin function in Metazoa, including cytoskeleton organization, cell-substrate interactions, and nuclear and cytoplasmic signaling.

Analysis of yeast endocytic site formation and maturation through a regulatory transition point.

The earliest stages of endocytic site formation and the regulation of endocytic site maturation are not well understood. Here we analyzed the order in which the earliest proteins are detectable at endocytic sites in budding yeast and found that an uncharacterized protein, Pal1p/Ydr348cp, is also present at the initial stages of endocytosis. Because Ede1p (homologue of Eps15) and clathrin are the early-arriving proteins most important for cargo uptake, their roles during the early stages of endocytosis were examined more comprehensively. Ede1p is necessary for efficient recruitment of most early-arriving proteins, but not for the recruitment of the adaptor protein Yap1802p, to endocytic sites. The early-arriving proteins, as well as the later-arriving proteins Sla2p and Ent1/2p (homologues of Hip1R and epsins), were found to have longer lifetimes in CLC1-knockout yeast, which indicates that clathrin light chain facilitates the transition from the intermediate to late coat stages. Cargo also arrives during the early stages of endocytosis, and therefore its effect on endocytic machinery dynamics was investigated. Our results are consistent with a role for cargo in regulating the transition of endocytic sites from the early stages of formation to the late stages during which vesicle formation occurs.

Bayesian modeling of the yeast SH3 domain interactome predicts spatiotemporal dynamics of endocytosis proteins.

SH3 domains are peptide recognition modules that mediate the assembly of diverse biological complexes. We scanned billions of phage-displayed peptides to map the binding specificities of the SH3 domain family in the budding yeast, Saccharomyces cerevisiae. Although most of the SH3 domains fall into the canonical classes I and II, each domain utilizes distinct features of its cognate ligands to achieve binding selectivity. Furthermore, we uncovered several SH3 domains with specificity profiles that clearly deviate from the two canonical classes. In conjunction with phage display, we used yeast two-hybrid and peptide array screening to independently identify SH3 domain binding partners. The results from the three complementary techniques were integrated using a Bayesian algorithm to generate a high-confidence yeast SH3 domain interaction map. The interaction map was enriched for proteins involved in endocytosis, revealing a set of SH3-mediated interactions that underlie formation of protein complexes essential to this biological pathway. We used the SH3 domain interaction network to predict the dynamic localization of several previously uncharacterized endocytic proteins, and our analysis suggests a novel role for the SH3 domains of Lsb3p and Lsb4p as hubs that recruit and assemble several endocytic complexes.

Early-arriving Syp1p and Ede1p function in endocytic site placement and formation in budding yeast.

Recent studies have revealed the detailed timing of protein recruitment to endocytic sites in budding yeast. However, little is understood about the early stages of their formation. Here we identify the septin-associated protein Syp1p as a component of the machinery that drives clathrin-mediated endocytosis in budding yeast. Syp1p arrives at endocytic sites early in their formation and shares unique dynamics with the EH-domain protein Ede1p. We find that Syp1p is related in amino acid sequence to several mammalian proteins one of which, SGIP1-alpha, is an endocytic component that binds the Ede1p homolog Eps15. Like Syp1p, SGIP1-alpha arrives early at sites of clathrin-mediated endocytosis, suggesting that Syp1p/Ede1p and SGIP1-alpha/Eps15 may have a conserved function. In yeast, both Syp1p and Ede1p play important roles in the rate of endocytic site turnover. Additionally, Ede1p is important for endocytic site formation, whereas Syp1p acts as a polarized factor that recruits both Ede1p and endocytic sites to the necks of emerging buds. Thus Ede1p and Syp1p are conserved, early-arriving endocytic proteins with roles in the formation and placement of endocytic sites, respectively.

Multiple pathways regulate endocytic coat disassembly in Saccharomyces cerevisiae for optimal downstream trafficking.

Recently, a pathway involving the highly choreographed recruitment of endocytic proteins to sites of clathrin/actin-mediated endocytosis has been revealed in budding yeast. Here, we investigated possible roles for candidate disassembly factors in regulation of the dynamics of the endocytic coat proteins Sla2p, Ent1p, Ent2p, Sla1p, Pan1p and End3p, each of which has mammalian homologues. Live cell imaging analysis revealed that in addition to the synaptojanin, Sjl2p, the Ark1p and Prk1p protein kinases, the putative Arf GTPase-activating protein, Gts1p and the Arf GTPase-interacting protein, Lsb5p, also arrive at endocytic sites late in the internalization pathway, consistent with roles in coat disassembly. Analysis of coat dynamics in various mutant backgrounds revealed that multiple pathways, including the ones mediated by an Arf guanosine triphosphatase and a synaptojanin, facilitate efficient disassembly of different endocytic coat proteins. In total, at least four separate processes are important for disassembly of endocytic complexes and efficient downstream trafficking of endocytic cargo.

Two binding partners cooperate to activate the molecular motor Kinesin-1.

The regulation of molecular motors is an important cellular problem, as motility in the absence of cargo results in futile adenosine triphosphate hydrolysis. When not transporting cargo, the microtubule (MT)-based motor Kinesin-1 is kept inactive as a result of a folded conformation that allows autoinhibition of the N-terminal motor by the C-terminal tail. The simplest model of Kinesin-1 activation posits that cargo binding to nonmotor regions relieves autoinhibition. In this study, we show that binding of the c-Jun N-terminal kinase-interacting protein 1 (JIP1) cargo protein is not sufficient to activate Kinesin-1. Because two regions of the Kinesin-1 tail are required for autoinhibition, we searched for a second molecule that contributes to activation of the motor. We identified fasciculation and elongation protein zeta1 (FEZ1) as a binding partner of kinesin heavy chain. We show that binding of JIP1 and FEZ1 to Kinesin-1 is sufficient to activate the motor for MT binding and motility. These results provide the first demonstration of the activation of a MT-based motor by cellular binding partners.

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.

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.

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.

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.

A novel centrosome-associated protein with affinity for microtubules.

We have identified a novel mammalian protein, MIR1, with microtubule-binding activity. MIR1 is a relative of MID1/midin, the protein implicated in Opitz G/BBB syndrome. In tissue culture cells, MIR1 is enriched at the centrosome. MIR1 dissociates from centrosomes at the G2/M transition and is recruited back to spindle poles during anaphase. When overexpressed during interphase, MIR1 binds along microtubule filaments, which become stabilized, bundled and detached from the centrosome. In mitosis, overexpressed MIR1 dissociates from microtubules but still affects the normally focused localization of gamma-tubulin in spindle poles. Tight binding to microtubules in interphase appears to require an oligomeric state of MIR1, and phosphorylation in mitosis at predicted cyclin-dependent kinase (cdk) sites weakens the interaction.