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.

Organizing the cell cortex: the role of ERM proteins.

Specialized membrane domains are an important feature of almost all cells. In particular, they are essential to tissues that have a highly organized cell cortex, such as the intestinal brush border epithelium. The ERM proteins (ezrin, radixin and moesin) have a crucial role in organizing membrane domains through their ability to interact with transmembrane proteins and the cytoskeleton. In doing so, they can provide structural links to strengthen the cell cortex and regulate the activities of signal transduction pathways. Recent studies examining the structure and in vivo functions of ERMs have greatly advanced our understanding of the importance of membrane-cytoskeleton interactions.

T cell antigen receptor signaling and immunological synapse stability require myosin IIA.

Immunological synapses are initiated by signaling in discrete T cell antigen receptor microclusters and are important for the differentiation and effector functions of T cells. Synapse formation involves the orchestrated movement of microclusters toward the center of the contact area with the antigen-presenting cell. Microcluster movement is associated with centripetal actin flow, but the function of motor proteins is unknown. Here we show that myosin IIA was necessary for complete assembly and movement of T cell antigen receptor microclusters. In the absence of myosin IIA or its ATPase activity, T cell signaling was interrupted 'downstream' of the kinase Lck and the synapse was destabilized. Thus, T cell antigen receptor signaling and the subsequent formation of immunological synapses are active processes dependent on myosin IIA.

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.

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.

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.

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.

The UBX protein SAKS1 negatively regulates endoplasmic reticulum-associated degradation and p97-dependent degradation.

Endoplasmic reticulum-associated degradation (ERAD) is an essential quality control process whereby misfolded proteins are exported from the endoplasmic reticulum and degraded by the proteasome in the cytosol. The ATPase p97 acts as an essential component of this process by providing the force needed for retrotranslocation and by serving as a processing station for the substrate once in the cytosol. Proteins containing the ubiquitin regulatory X (UBX) ubiquitin-like domain function as adaptors for p97 through their direct binding with the amino terminus of the ATPase. We demonstrate that the UBX protein SAKS1 is able to act as an adaptor for p97 that negatively modulates ERAD. This requires the ability of SAKS1 to bind both polyubiquitin and p97. Moreover, the association between SAKS1 and p97 is positively regulated by polyubiquitin binding of the UBX protein. SAKS1 also negatively impacts the p97-dependent processing required for degradation of a cytosolic, non-ERAD, substrate. We find SAKS1 is able to protect polyubiquitin from the activity of deubiquitinases, such as ataxin-3, that are necessary for efficient ERAD. Thus, SAKS1 inhibits protein degradation mediated by p97 complexes in the cytosol with a component of the mechanism being the ability to shield polyubiquitin chains from ubiquitin-processing factors.

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.

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.

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.

Formins direct Arp2/3-independent actin filament assembly to polarize cell growth in yeast.

Formins have been implicated in the regulation of cytoskeletal structure in animals and fungi. Here we show that the formins Bni1 and Bnr1 of budding yeast stimulate the assembly of actin filaments that function as precursors to tropomyosin-stabilized cables that direct polarized cell growth. With loss of formin function, cables disassemble,whereas increased formin activity causes the hyperaccumulation of cable-like filaments. Unlike the assembly of cortical actin patches, cable assembly requires profilin but not the Arp2/3 complex. Thus formins control a distinct pathway for assembling actin filaments that organize the overall polarity of the cell.

EPI64 regulates microvillar subdomains and structure.

EPI64 is a TBC domain-containing protein that binds the PDZ domains of EBP50, which binds ezrin, a major actin-binding protein of microvilli. High-resolution light microscopy revealed that ezrin and EBP50 localize exclusively to the membrane-surrounded region of microvilli, whereas EPI64 localizes to variable regions in the structures. Overexpressing EPI64 results in its and EBP50's relocalization to the base of microvilli, including to the actin rootlet devoid of ezrin or plasma membrane. Uncoupling EPI64's binding to EBP50, expression of any construct mislocalizing its TBC domain, or knock down of EBP50 results in loss of microvilli. The TBC domain of EPI64 binds directly to Arf6-GTP. Overexpressing the TBC domain increases Arf6-GTP levels, and expressing dominant-active Arf6 results in microvillar loss. These data reveal that microvilli have distinct cytoskeletal subdomains and that EPI64 regulates microvillar structure.

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.

Microtubule tips redirect actin assembly.

In fission yeast, a protein complex that rides on the plus end of growing microtubules regulates establishment of new sites of actin cable assembly, necessary for cell growth, from one to both ends of the rod-shaped cells. Martin et al. describe a direct molecular link between the microtubule tip complex and the formin for3p, the nucleator for assembly of actin cables, which is necessary for this regulatory switch.

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.

The scaffold protein PDZK1 undergoes a head-to-tail intramolecular association that negatively regulates its interaction with EBP50.

PDZK1 (also known as CAP70, NHERF3, or NaPi-Cap1) is a scaffolding protein composed of four PDZ (Post-Synaptic Density-95, Discs Large, Zonula Occludens-1) domains followed by a short carboxyl-terminal tail. This scaffold acts as a mediator of localization and expression levels of multiple receptors in the kidney, liver, and endothelium. Here, we characterize the self-association properties of the protein. PDZK1 can undergo modest homodimerization in vivo and in vitro through self-association involving its third PDZ domain. In addition, the tail of PDZK1 interacts in an intramolecular fashion with the first PDZ domain, but this interaction does not contribute to dimer formation. The interaction between the tail of PDZK1 and its first PDZ domain induces the protein to adopt a more compact conformation. A head-to-tail association has also been reported for EBP50/NHERF1, a two-PDZ domain member of the same scaffolding protein family as PDZK1, and shown to regulate binding of target proteins to the EBP50 PDZ domains. As opposed to EBP50, the association of PDZK1 with specific ligands for its PDZ domains is unaffected by the intramolecular association, establishing a different mode of interaction among these two members of the same scaffolding family. However, the tail of PDZK1 interacts with the PDZ domains of EBP50, and this interaction is negatively regulated by the intramolecular association of PDZK1. Thus, we have uncovered a regulated association between the two PDZ-containing scaffolding molecules, PDZK1 and EBP50.

Polarized growth and organelle segregation in yeast: the tracks, motors, and receptors.

In yeast, growth and organelle segregation requires formin-dependent assembly of polarized actin cables. These tracks are used by myosin Vs to deliver secretory vesicles for cell growth, organelles for their segregation, and mRNA for fate determination. Several specific receptors have been identified that interact with the cargo-binding tails of the myosin Vs. A recent study implicates specific degradation in the bud of the vacuolar receptor, Vac17, as a mechanism for cell cycle-regulated segregation of this organelle.

Secretory vesicle transport velocity in living cells depends on the myosin-V lever arm length.

Myosins are molecular motors that exert force against actin filaments. One widely conserved myosin class, the myosin-Vs, recruits organelles to polarized sites in animal and fungal cells. However, it has been unclear whether myosin-Vs actively transport organelles, and whether the recently challenged lever arm model developed for muscle myosin applies to myosin-Vs. Here we demonstrate in living, intact yeast that secretory vesicles move rapidly toward their site of exocytosis. The maximal speed varies linearly over a wide range of lever arm lengths genetically engineered into the myosin-V heavy chain encoded by the MYO2 gene. Thus, secretory vesicle polarization is achieved through active transport by a myosin-V, and the motor mechanism is consistent with the lever arm model.