Myosin II isoforms promote internalization of spatially distinct clathrin-independent endocytosis cargoes through modulation of cortical tension downstream of ROCK2.

Although the actomyosin cytoskeleton has been implicated in clathrin-mediated endocytosis, a clear requirement for actomyosin in clathrin-independent endocytosis (CIE) has not been demonstrated. We discovered that the Rho-associated kinase ROCK2 is required for CIE of MHCI and CD59 through promotion of myosin II activity. Myosin IIA promoted internalization of MHCI and myosin IIB drove CD59 uptake in both HeLa and polarized Caco2 intestinal epithelial cells. In Caco2 cells, myosin IIA localized to the basal cortex and apical brush border and mediated MHCI internalization from the basolateral domain, while myosin IIB localized at the basal cortex and apical cell-cell junctions and promoted CD59 uptake from the apical membrane. Atomic force microscopy demonstrated that myosin IIB mediated apical epithelial tension in Caco2 cells. Thus, specific cargoes are internalized by ROCK2-mediated activation of myosin II isoforms to mediate spatial regulation of CIE, possibly by modulation of local cortical tension.

Arf6 and Rab22 mediate T cell conjugate formation by regulating clathrin-independent endosomal membrane trafficking.

Endosomal trafficking can influence the composition of the plasma membrane and the ability of cells to polarize their membranes. Here, we examined whether trafficking through clathrin-independent endocytosis (CIE) affects the ability of T cells to form a cell-cell conjugate with antigen-presenting cells (APCs). We show that CIE occurs in both the Jurkat T cell line and primary human T cells. In Jurkat cells, the activities of two guanine nucleotide binding proteins, Arf6 and Rab22 (also known as Rab22a), influence CIE and conjugate formation. Expression of the constitutively active form of Arf6, Arf6Q67L, inhibits CIE and conjugate formation, and results in the accumulation of vacuoles containing lymphocyte function-associated antigen 1 (LFA-1) and CD4, molecules important for T cell interaction with the APC. Moreover, expression of the GTP-binding defective mutant of Rab22, Rab22S19N, inhibits CIE and conjugate formation, suggesting that Rab22 function is required for these activities. Furthermore, Jurkat cells expressing Rab22S19N were impaired in spreading onto coverslips coated with T cell receptor-activating antibodies. These observations support a role for CIE, Arf6 and Rab22 in conjugate formation between T cells and APCs.

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.

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.

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.

Membrane dynamics correlate with formation of signaling clusters during cell spreading.

The morphology and duration of contacts between cells and adhesive surfaces play a key role in several biological processes, such as cell migration, cell differentiation, and the immune response. The interaction of receptors on the cell membrane with ligands on the adhesive surface leads to triggering of signaling pathways, which allow cytoskeletal rearrangement, and large-scale deformation of the cell membrane, which allows the cell to spread over the substrate. Despite numerous studies of cell spreading, the nanometer-scale dynamics of the membrane during formation of contacts, spreading, and initiation of signaling are not well understood. Using interference reflection microscopy, we study the kinetics of cell spreading at the micron scale, as well as the topography and fluctuations of the membrane at the nanometer scale during spreading of Jurkat T cells on antibody-coated substrates. We observed two modes of spreading, which were characterized by dramatic differences in membrane dynamics and topography. Formation of signaling clusters was closely related to the movement and morphology of the membrane in contact with the activating surface. Our results suggest that cell membrane morphology may be a critical constraint on signaling at the cell-substrate interface.