ARHGAP18-ezrin functions as an autoregulatory module for RhoA in the assembly of distinct actin-based structures.

The location of different actin-based structures is largely regulated by Rho GTPases through specific effectors. We use the apical aspect of epithelial cells as a model system to investigate how RhoA is locally regulated to contribute to two distinct adjacent actin-based structures. Assembly of the non-muscle myosin-2 filaments in the terminal web is dependent on RhoA activity, and assembly of the microvilli also requires active RhoA for phosphorylation and activation of ezrin. We show that the RhoGAP, ARHGAP18, is localized by binding active microvillar ezrin, and this interaction enhances ARHGAP18's RhoGAP activity. We present a model where ezrin-ARHGAP18 acts as a negative autoregulatory module to locally reduce RhoA activity in microvilli. Consistent with this model, loss of ARHGAP18 results in disruption of the distinction between microvilli and the terminal web including aberrant assembly of myosin-2 filaments forming inside microvilli. Thus, ARHGAP18, through its recruitment and activation by ezrin, fine-tunes the local level of RhoA to allow for the appropriate distribution of actin-based structures between the microvilli and terminal web. As RhoGAPs vastly outnumber Rho GTPases, this may represent a general mechanism whereby individual Rho effectors drive specific actin-based structures.

High-resolution secretory timeline from vesicle formation at the Golgi to fusion at the plasma membrane in S. cerevisiae.

Most of the components in the yeast secretory pathway have been studied, yet a high-resolution temporal timeline of their participation is lacking. Here, we define the order of acquisition, lifetime, and release of critical components involved in late secretion from the Golgi to the plasma membrane. Of particular interest is the timing of the many reported effectors of the secretory vesicle Rab protein Sec4, including the myosin-V Myo2, the exocyst complex, the lgl homolog Sro7, and the small yeast-specific protein Mso1. At the trans-Golgi network (TGN) Sec4's GEF, Sec2, is recruited to Ypt31-positive compartments, quickly followed by Sec4 and Myo2 and vesicle formation. While transported to the bud tip, the entire exocyst complex, including Sec3, is assembled on to the vesicle. Before fusion, vesicles tether for 5 s, during which the vesicle retains the exocyst complex and stimulates lateral recruitment of Rho3 on the plasma membrane. Sec2 and Myo2 are rapidly lost, followed by recruitment of cytosolic Sro7, and finally the SM protein Sec1, which appears for just 2 s prior to fusion. Perturbation experiments reveal an ordered and robust series of events during tethering that provide insights into the function of Sec4 and effector exchange.

Generation and characterization of conditional yeast mutants affecting each of the 2 essential functions of the scaffolding proteins Boi1/2 and Bem1.

Boi1 and Boi2 are closely related yeast scaffolding proteins, either of which can perform an essential function. Previous studies have suggested a role in cell polarity, interacting with lipids, components of the late secretory pathway, and actin nucleators. We report detailed studies of their localization, dynamics, and the generation and characterization of conditional mutants. Boi1/2 are present on the plasma membrane in dynamic patches, then at the bud neck during cytokinesis. These distributions are unaffected by perturbation of the actin cytoskeleton or the secretory pathway. We identify 2 critical aromatic residues, present in both Boi1 and Boi2, in the essential C-terminal Pleckstrin-Homology domain, that cause temperature-sensitive growth resulting in defects in polarized growth leading to cell lysis. The scaffolding protein, Bem1, colocalizes with Boi1 in patches at the growing bud, and at the bud neck, the latter requiring the N-terminal SH3 domain of Boi1p. Loss of function of Boi1-SH3 domain renders Bem1 essential, which can be fully replaced by a fusion of the SH3b and PB1 domains of Bem1. Thus, the 2 essential functions of the Boi1/2/Bem1 proteins can be satisfied by Bem1-SH3b-PB1 and Boi1-Pleckstrin-Homology. Generation and characterization of conditional mutations in the essential function of Bem1 reveal a slow onset of defects in polarized growth, which is difficult to define a specific initial defect. This study provides more details into the functions of Boi1/2 and their relationship with Bem1 and presents the generation of conditional mutants that will be useful for future genetic analysis.

The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells †.

Here we report on the related TBC/RabGAPs EPI64A and EPI64B and show that they function to organize the apical aspect of epithelial cells. EPI64A binds the scaffolding protein EBP50/NHERF1, which itself binds active ezrin in epithelial cell microvilli. Epithelial cells additionally express EPI64B that also localizes to microvilli. However, EPI64B does not bind EBP50 and both proteins are shown to have a microvillar localization domain that spans the RabGAP domains. CRISPR/Cas9 was used to inactivate expression of each protein individually or both in Jeg-3 and Caco2 cells. In Jeg-3 cells, loss of EPI64B resulted in a reduction of apical microvilli, and a further reduction was seen in the double knockout, mostly likely due to misregulation of Rab8 and Rab35. In addition, apical junctions were partially disrupted in cells lacking EPI64A and accentuated in the double knockout. In Caco2 loss of EPI64B resulted in wavy junctions, whereas loss of both EPI64A and EPI64B had a severe phenotype often resulting in cells with a stellate apical morphology. In the knockout cells, the basal region of the cell remained unchanged, so EPI64A and EPI64B specifically localize to and regulate the morphology of the apical domain of polarized epithelial cells.

Effector-mediated ERM activation locally inhibits RhoA activity to shape the apical cell domain.

Activated ezrin-radixin-moesin (ERM) proteins link the plasma membrane to the actin cytoskeleton to generate apical structures, including microvilli. Among many kinases implicated in ERM activation are the homologues LOK and SLK. CRISPR/Cas9 was used to knock out all ERM proteins or LOK/SLK in human cells. LOK/SLK knockout eliminates all ERM-activating phosphorylation. The apical domains of cells lacking LOK/SLK or ERMs are strikingly similar and selectively altered, with loss of microvilli and with junctional actin replaced by ectopic myosin-II-containing apical contractile structures. Constitutively active ezrin can reverse the phenotypes of either ERM or LOK/SLK knockouts, indicating that a central function of LOK/SLK is to activate ERMs. Both knockout lines have elevated active RhoA with concomitant enhanced myosin light chain phosphorylation, revealing that active ERMs are negative regulators of RhoA. As RhoA-GTP activates LOK/SLK to activate ERM proteins, the ability of active ERMs to negatively regulate RhoA-GTP represents a novel local feedback loop necessary for the proper apical morphology of epithelial cells.

Yeast Rgd3 is a phospho-regulated F-BAR-containing RhoGAP involved in the regulation of Rho3 distribution and cell morphology.

Polarized growth requires the integration of polarity pathways with the delivery of exocytic vesicles for cell expansion and counterbalancing endocytic uptake. In budding yeast, the myosin-V Myo2 is aided by the kinesin-related protein Smy1 in carrying out the essential Sec4-dependent transport of secretory vesicles to sites of polarized growth. Overexpression suppressors of a conditional myo2 smy1 mutant identified a novel F-BAR (Fes/CIP4 homology-Bin-Amphiphysin-Rvs protein)-containing RhoGAP, Rgd3, that has activity primarily on Rho3, but also Cdc42. Internally tagged Rho3 is restricted to the plasma membrane in a gradient corresponding to cell polarity that is altered upon Rgd3 overexpression. Rgd3 itself is localized to dynamic polarized vesicles that, while distinct from constitutive secretory vesicles, are dependent on actin and Myo2 function. In vitro Rgd3 associates with liposomes in a PIP2-enhanced manner. Further, the Rgd3 C-terminal region contains several phosphorylatable residues within a reported SH3-binding motif. An unphosphorylated mimetic construct is active and highly polarized, while the phospho-mimetic form is not. Rgd3 is capable of activating Myo2, dependent on its phospho state, and Rgd3 overexpression rescues aberrant Rho3 localization and cell morphologies seen at the restrictive temperature in the myo2 smy1 mutant. We propose a model where Rgd3 functions to modulate and maintain Rho3 polarity during growth.

Regulation of actin-based apical structures on epithelial cells.

Cells of transporting epithelia are characterized by the presence of abundant F-actin-based microvilli on their apical surfaces. Likewise, auditory hair cells have highly reproducible rows of apical stereocilia (giant microvilli) that convert mechanical sound into an electrical signal. Analysis of mutations in deaf patients has highlighted the critical components of tip links between stereocilia, and related structures that contribute to the organization of microvilli on epithelial cells have been found. Ezrin/radixin/moesin (ERM) proteins, which are activated by phosphorylation, provide a critical link between the plasma membrane and underlying actin cytoskeleton in surface structures. Here, we outline recent insights into how microvilli and stereocilia are built, and the roles of tip links. Furthermore, we highlight how ezrin is locally regulated by phosphorylation, and that this is necessary to maintain polarity. Localized phosphorylation is achieved through an intricate coincidence detection mechanism that requires the membrane lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and the apically localized ezrin kinase, lymphocyte-oriented kinase (LOK, also known as STK10) or Ste20-like kinase (SLK). We also discuss how ezrin-binding scaffolding proteins regulate microvilli and how, despite these significant advances, it remains to be discovered how the cell polarity program ultimately interfaces with these processes.

Yeast Aim21/Tda2 both regulates free actin by reducing barbed end assembly and forms a complex with Cap1/Cap2 to balance actin assembly between patches and cables.

Yeast Aim21 is recruited by the SH3-containing proteins Bbc1 and Abp1 to patches and, with Tda2, reduces barbed end assembly to balance the distribution of actin between patches and cables. Aim21/Tda2 also interacts with Cap1/Cap2, revealing a complex interplay between actin assembly regulators.

Ezrin activation by LOK phosphorylation involves a PIP2-dependent wedge mechanism.

How cells specify morphologically distinct plasma membrane domains is poorly understood. Prior work has shown that restriction of microvilli to the apical aspect of epithelial cells requires the localized activation of the membrane-F-actin linking protein ezrin. Using an in vitro system, we now define a multi-step process whereby the kinase LOK specifically phosphorylates ezrin to activate it. Binding of PIP2 to ezrin induces a conformational change permitting the insertion of the LOK C-terminal domain to wedge apart the membrane and F-actin-binding domains of ezrin. The N-terminal LOK kinase domain can then access a site 40 residues distal from the consensus sequence that collectively direct phosphorylation of the appropriate threonine residue. We suggest that this elaborate mechanism ensures that ezrin is only phosphorylated at the plasma membrane, and with high specificity by the apically localized kinase LOK.

Kinesin-related Smy1 enhances the Rab-dependent association of myosin-V with secretory cargo.

The mechanisms by which molecular motors associate with specific cargo is a central problem in cell organization. The kinesin-like protein Smy1 of budding yeast was originally identified by the ability of elevated levels to suppress a conditional myosin-V mutation (myo2-66), but its function with Myo2 remained mysterious. Subsequently, Myo2 was found to provide an essential role in delivery of secretory vesicles for polarized growth and in the transport of mitochondria for segregation. By isolating and characterizing myo2 smy1 conditional mutants, we uncover the molecular function of Smy1 as a factor that enhances the association of Myo2 with its receptor, the Rab Sec4, on secretory vesicles. The tail of Smy1-which binds Myo2-its central dimerization domain, and its kinesin-like head domain are all necessary for this function. Consistent with this model, overexpression of full-length Smy1 enhances the number of Sec4 receptors and Myo2 motors per transporting secretory vesicle. Rab proteins Sec4 and Ypt11, receptors for essential transport of secretory vesicles and mitochondria, respectively, bind the same region on Myo2, yet Smy1 functions selectively in the transport of secretory vesicles. Thus a kinesin-related protein can function intimately with a myosin-V and its receptor in the transport of a specific cargo.

Structure, regulation, and functional diversity of microvilli on the apical domain of epithelial cells.

Microvilli are actin-based structures found on the apical aspect of many epithelial cells. In this review, we discuss different types of microvilli, as well as comparisons with actin-based sensory stereocilia and filopodia. Much is known about the actin-bundling proteins of these structures; we summarize recent studies that focus on the components of the microvillar membrane. We pay special attention to mechanisms of membrane microfilament attachment by the ezrin/radixin/moesin family and regulation of this protein family. We also discuss the NHERF family of scaffolding proteins that are found in microvilli and their role in microvilli regulation. Microvilli on cultured cells are not static structures, and their dynamics and those of their components are discussed. Finally, we mention diseases related to microvilli and outline questions that our current knowledge will allow the field to address in the near future.

The function and dynamics of the apical scaffolding protein E3KARP are regulated by cell-cycle phosphorylation.

We examine the dynamics and function of the apical scaffolding protein E3KARP/NHERF2, which consists of two PDZ domains and a tail containing an ezrin-binding domain. The exchange rate of E3KARP is greatly enhanced during mitosis due to phosphorylation at Ser-303 in its tail region. Whereas E3KARP can substitute for the function of the closely related scaffolding protein EBP50/NHERF1 in the formation of interphase microvilli, E3KARP S303D cannot. Moreover, the S303D mutation enhances the in vivo dynamics of the E3KARP tail alone, whereas in vitro the interaction of E3KARP with active ezrin is unaffected by S303D, implicating another factor regulating dynamics in vivo. A-Raf is found to be required for S303 phosphorylation in mitotic cells. Regulation of the dynamics of EBP50 is known to be dependent on its tail region but modulated by PDZ domain occupancy, which is not the case for E3KARP. Of interest, in both cases, the mechanisms regulating dynamics involve the tails, which are the most diverged region of the paralogues and probably evolved independently after a gene duplication event that occurred early in vertebrate evolution.

Tracking individual secretory vesicles during exocytosis reveals an ordered and regulated process.

Post-Golgi secretory vesicle trafficking is a coordinated process, with transport and regulatory mechanisms to ensure appropriate exocytosis. While the contributions of many individual regulatory proteins to this process are well studied, the timing and dependencies of events have not been defined. Here we track individual secretory vesicles and associated proteins in vivo during tethering and fusion in budding yeast. Secretory vesicles tether to the plasma membrane very reproducibly for ∼18 s, which is extended in cells defective for membrane fusion and significantly lengthened and more variable when GTP hydrolysis of the exocytic Rab is delayed. Further, the myosin-V Myo2p regulates the tethering time in a mechanism unrelated to its interaction with exocyst component Sec15p. Two-color imaging of tethered vesicles with Myo2p, the GEF Sec2p, and several exocyst components allowed us to document a timeline for yeast exocytosis in which Myo2p leaves 4 s before fusion, whereas Sec2p and all the components of the exocyst disperse coincident with fusion.

Head-to-tail regulation is critical for the in vivo function of myosin V.

Cell organization requires regulated cargo transport along cytoskeletal elements. Myosin V motors are among the most conserved organelle motors and have been well characterized in both yeast and mammalian systems. Biochemical data for mammalian myosin V suggest that a head-to-tail autoinhibitory interaction is a primary means of regulation, but the in vivo significance of this interaction has not been studied. Here we generated and characterized mutations in the yeast myosin V Myo2p to reveal that it is regulated by a head-to-tail interaction and that loss of regulation renders the myosin V constitutively active. We show that an unregulated motor is very deleterious for growth, resulting in severe defects in Myo2-mediated transport processes, including secretory vesicle transport, mitochondrial inheritance, and nuclear orientation. All of the defects associated with motor misregulation could be rescued by artificially restoring regulation. Thus, spatial and temporal regulation of myosin V in vivo by a head-to-tail interaction is critical for the normal delivery functions of the motor.

Rapid glucose depletion immobilizes active myosin V on stabilized actin cables.

Polarization of eukaryotic cells requires organelles and protein complexes to be transported to their proper destinations along the cytoskeleton. When nutrients are abundant, budding yeast grows rapidly transporting secretory vesicles for localized growth and actively segregating organelles. This is mediated by myosin Vs transporting cargos along F-actin bundles known as actin cables. Actin cables are dynamic structures regulated by assembly, stabilization, and disassembly. Polarized growth and actin filament dynamics consume energy. For most organisms, glucose is the preferred energy source and generally represses alternative carbon source usage. Thus, upon abrupt glucose depletion, yeast shuts down pathways consuming large amounts of energy, including the vacuolar-ATPase, translation, and phosphoinositide metabolism. Here we show that glucose withdrawal rapidly (<1 min) depletes ATP levels and that the yeast myosin V, Myo2, responds by relocalizing to actin cables, making it the fastest response documented. Myo2 immobilized on cables releases its secretory cargo, defining a new rigor-like state of a myosin V in vivo. Only actively transporting Myo2 can be converted to the rigor-like state. Glucose depletion has differential effects on the actin cytoskeleton, resulting in disassembly of actin patches with concomitant inhibition of endocytosis and strong stabilization of actin cables, thereby revealing a selective and previously unappreciated ATP requirement for actin cable disassembly. A similar response is seen in HeLa cells to ATP depletion. These findings reveal a new fast-acting energy conservation strategy halting growth by immobilizing myosin V in a newly described state on selectively stabilized actin cables.

The surprising dynamics of scaffolding proteins.

The function of scaffolding proteins is to bring together two or more proteins in a relatively stable configuration, hence their name. Numerous scaffolding proteins are found in nature, many having multiple protein-protein interaction modules. Over the past decade, examples of scaffolding complexes long thought to be stable have instead been found to be surprisingly dynamic. These studies are scattered among different biological systems, and so the concept that scaffolding complexes might not always represent stable entities and that their dynamics can be regulated has not garnered general attention. We became aware of this issue in our studies of a scaffolding protein in microvilli, which forced us to reevaluate its contribution to their structure. The purpose of this Perspective is to draw attention to this phenomenon and discuss why complexes might show regulated dynamics. We also wish to encourage more studies on the dynamics of "stable" complexes and to provide a word of caution about how functionally important dynamic associations may be missed in biochemical and proteomic studies.

Cordon Bleu serves as a platform at the basal region of microvilli, where it regulates microvillar length through its WH2 domains.

Cordon Bleu (Cobl) is a WH2-containing protein believed to act as an actin nucleator. We show that it has a very specific localization in epithelial cells at the basal region of microvilli, a localization unlikely to be involved in actin nucleation. The protein is localized by a central region between the N-terminal COBL domain and the three C-terminal WH2 domains. Ectopic expression of Cobl shortens apical microvilli, and this requires functional WH2 domains. Proteomic studies reveal that the COBL domain binds several BAR-containing proteins, including SNX9, PACSIN 2/syndapin 2, and ASAP1. ASAP1 is recruited to the base of microvilli by binding the COBL domain through its SH3. We propose that Cobl is localized to the basal region of microvilli both to participate in length regulation and to recruit BAR proteins that associate with the curved membrane found at the microvillar base.

Dynamics of ezrin and EBP50 in regulating microvilli on the apical aspect of epithelial cells.

Microvilli are found on the apical surface of epithelial cells. Recent studies on the microvillar proteins ezrin and EBP50 (ezrin/radixin/moesin-binding phosphoprotein of 50 kDa) have revealed both the dynamics and the regulation of microvillar components, and how a dynamic ezrin phosphocycle is necessary to confine microvilli to the apical membrane. In the present review, we first summarize the background to allow us to place these advances in context.

Interactome analysis reveals ezrin can adopt multiple conformational states.

Ezrin, a member of the ezrin-radixin-moesin family (ERM), is an essential regulator of the structure of microvilli on the apical aspect of epithelial cells. Ezrin provides a linkage between membrane-associated proteins and F-actin, oscillating between active/open and inactive/closed states, and is regulated in part by phosphorylation of a C-terminal threonine. In the open state, ezrin can bind a number of ligands, but in the closed state the ligand-binding sites are inaccessible. In vitro analysis has proposed that there may be a third hyperactivated form of ezrin. To gain a better understanding of ezrin, we conducted an unbiased proteomic analysis of ezrin-binding proteins in an epithelial cell line, Jeg-3. We refined our list of interactors by comparing the interactomes using quantitative mass spectrometry between wild-type ezrin, closed ezrin, open ezrin, and hyperactivated ezrin. The analysis reveals several novel interactors confirmed by their localization to microvilli, as well as a significant class of proteins that bind closed ezrin. Taken together, the data indicate that ezrin can exist in three different conformational states, and different ligands "perceive" ezrin conformational states differently.