Gonococcal invasion into epithelial cells depends on both cell polarity and ezrin

Neisseria gonorrhoeae (GC) establishes infection in women from the cervix, lined with heterogeneous epithelial cells from non-polarized stratified at the ectocervix to polarized columnar at the endocervix. We have previously shown that GC differentially colonize and transmigrate across the ecto and endocervical epithelia. However, whether and how GC invade into heterogeneous cervical epithelial cells is unknown. This study examined GC entry of epithelial cells with various properties, using human cervical tissue explant and non-polarized/polarized epithelial cell line models. While adhering to non-polarized and polarized epithelial cells at similar levels, GC invaded into non-polarized more efficiently than polarized epithelial cells. The enhanced GC invasion in non-polarized epithelial cells was associated with increased ezrin phosphorylation, F-actin and ezrin recruitment to GC adherent sites, and the elongation of GC-associated microvilli. Inhibition of ezrin phosphorylation inhibited F-actin and ezrin recruitment and microvilli elongation, leading to a reduction in GC invasion. The reduced GC invasion in polarized epithelial cells was associated with non-muscle myosin II-mediated F-actin disassembly and microvilli denudation at GC adherence sites. Surprisingly, intraepithelial GC were only detected inside epithelial cells shedding from the cervix by immunofluorescence microscopy, but not significantly in the ectocervical and the endocervical regions. We observed similar ezrin and F-actin recruitment in exfoliated cervical epithelial cells but not in those that remained in the ectocervical epithelium, as the luminal layer of ectocervical epithelial cells expressed ten-fold lower levels of ezrin than those beneath. However, GC inoculation induced F-actin reduction and myosin recruitment in the endocervix, similar to what was seen in polarized epithelial cells. Collectively, our results suggest that while GC invade non-polarized epithelial cells through ezrin-driven microvilli elongation, the apical polarization of ezrin and F-actin inhibits GC entry into polarized epithelial cells.     

Myosin VI, a new force in clathrin mediated endocytosis.

The integrity of the actin cytoskeleton and associated motor proteins are essential for the efficient functioning of clathrin mediated endocytosis at least in polarised cells. Myosin VI, the only motor protein so far identified that moves towards the minus end of actin filaments, is the first motor protein to be shown to associate with clathrin coated pits/vesicles at the plasma membrane and to modulate clathrin mediated endocytosis. Recent kinetic studies suggest that myosin VI may move processively along actin filaments providing clues about its functions in the cell. The possible role(s) of myosin VI in the sequential steps involved in receptor mediated endocytosis are discussed.     

Endocytic traffic in polarized epithelial cells: role of the actin and microtubule cytoskeleton

The cytoskeleton is required for multiple cellular events including endocytosis and the transfer of cargo within the endocytic system. Polarized epithelial cells are capable of endocytosis at either of their distinct apical or basolateral plasma membrane domains. Actin plays a role in internalization at both cell surfaces. Microtubules and actin are required for efficient transcytosis and delivery of proteins to late endosomes and lysosomes. Microtubules are also important in apical recycling pathways and, in some polarized cell types, basolateral recycling requires actin. The microtubule motor proteins dynein and kinesin and the class I unconventional myosin motors play a role in many of these trafficking steps. This review examines the endocytic pathways of polarized epithelial cells and focuses on the emerging roles of the actin cytoskeleton in these processes.     

Herpes simplex virus-1 utilizes the host actin cytoskeleton for its release from axonal growth cones

Herpes simplex virus type 1 (HSV-1) has evolved mechanisms to exploit the host cytoskeleton during entry, replication and exit from cells. In this study, we determined the role of actin and the molecular motor proteins, myosin II and myosin V, in the transport and release of HSV-1 from axon termini, or growth cones. Using compartmentalized neuronal devices, we showed that inhibition of actin polymerization, but not actin branching, significantly reduced the release of HSV-1 from axons. Furthermore, we showed that inhibition of myosin V, but not myosin II, also significantly reduced the release of HSV-1 from axons. Using confocal and electron microscopy, we determined that viral components are transported along axons to growth cones, despite actin or myosin inhibition. Overall, our study supports the role of actin in virus release from axonal growth cones and suggests myosin V as a likely candidate involved in this process.     

Myosin VI isoform localized to clathrin-coated vesicles with a role in clathrin-mediated endocytosis.

Myosin VI is involved in membrane traffic and dynamics and is the only myosin known to move towards the minus end of actin filaments. Splice variants of myosin VI with a large insert in the tail domain were specifically expressed in polarized cells containing microvilli. In these polarized cells, endogenous myosin VI containing the large insert was concentrated at the apical domain co-localizing with clathrin- coated pits/vesicles. Using full-length myosin VI and deletion mutants tagged with green fluorescent protein (GFP) we have shown that myosin VI associates and co-localizes with clathrin-coated pits/vesicles by its C-terminal tail. Myosin VI, precipitated from whole cytosol, was present in a protein complex containing adaptor protein (AP)-2 and clathrin, and enriched in purified clathrin-coated vesicles. Over-expression of the tail domain of myosin VI containing the large insert in fibroblasts reduced transferrin uptake in transiently and stably transfected cells by >50%. Myosin VI is the first motor protein to be identified associated with clathrin-coated pits/vesicles and shown to modulate clathrin-mediated endocytosis.     

Influenza entry pathways in polarized MDCK cells

In non-polarized cell culture models, influenza virus has been shown to enter host cells via multiple endocytic pathways, including classical clathrin-mediated endocytic routes (CME), clathrin- and caveolae-independent routes and macropinocytosis. However, little is known about the entry route of influenza virus in differentiated epithelia, in vivo site of infection for influenza virus. Here, we show that in polarized Madin–Darby canine kidney type II (MDCK II) cells, influenza virus has a specific utilization of the clathrin-mediated endocytic pathway and requires Eps15 for host cell entry.     

Myosin VI binds to and localises with Dab2, potentially linking receptor-mediated endocytosis and the actin cytoskeleton

Myosin VI, an actin-based motor protein, and Disabled 2 (Dab2), a molecule involved in endocytosis and cell signalling, have been found to bind together using yeast and mammalian two-hybrid screens. In polarised epithelial cells, myosin VI is known to be associated with apical clathrin-coated vesicles and is believed to move them towards the minus end of actin filaments, away from the plasma membrane and into the cell. Dab2 belongs to a group of signal transduction proteins that bind in vitro to the FXNPXY sequence found in the cytosolic tails of members of the low-density lipoprotein receptor family. The central region of Dab2, containing two DPF motifs, binds to the clathrin adaptor protein AP-2, whereas a C-terminal region contains the binding site for myosin VI. This site is conserved in Dab1, the neuronal counterpart of Dab2. The interaction between Dab2 and myosin VI was confirmed by in vitro binding assays and coimmunoprecipitation and by their colocalisation in clathrin-coated pits/vesicles concentrated at the apical domain of polarised cells. These results suggest that the myosin VI–Dab2 interaction may be one link between the actin cytoskeleton and receptors undergoing endocytosis.     

Characterization of native myosin VI isolated from sea urchin eggs

Myosin VI is a molecular motor that is ubiquitously expressed among eukaryotic cells, and thought to be involved in membrane trafficking and anchoring the organelle to actin cytoskeleton. Studies on myosin VI have been carried out using recombinant proteins, but native myosin VI has not been purified yet. Here we purified native myosin VI from sea urchin eggs and characterized its properties. We found that the native myosin VI was a monomeric and non-processive motor protein, and also showed that it moved toward the pointed end of F-actin. Ca2+ stimulated actin-activated MgATPase activity of the native myosin VI, while it lowered its motility on F-actin. Immunofluorescence microscopy showed that the myosin VI was translocated from the inner cytoplasm to the cortex after fertilization. Myosin VI may be involved in endocytic activities in fertilized eggs.     

Myosin VI is required for sorting of AP-1B-dependent cargo to the basolateral domain in polarized MDCK cells

In polarized epithelial cells, newly synthesized membrane proteins are delivered on specific pathways to either the apical or basolateral domains, depending on the sorting motifs present in these proteins. Because myosin VI has been shown to facilitate secretory traffic in nonpolarized cells, we investigated its role in biosynthetic trafficking pathways in polarized MDCK cells. We observed that a specific splice isoform of myosin VI with no insert in the tail domain is required for the polarized transport of tyrosine motif containing basolateral membrane proteins. Sorting of other basolateral or apical cargo, however, does not involve myosin VI. Site-directed mutagenesis indicates that a functional complex consisting of myosin VI, optineurin, and probably the GTPase Rab8 plays a role in the basolateral delivery of membrane proteins, whose sorting is mediated by the clathrin adaptor protein complex (AP) AP-1B. Our results suggest that myosin VI is a crucial component in the AP-1B-dependent biosynthetic sorting pathway to the basolateral surface in polarized epithelial cells.     

Convergence and extension at gastrulation require a myosin IIB-dependent cortical actin network

Force-producing convergence (narrowing) and extension (lengthening) of tissues by active intercalation of cells along the axis of convergence play a major role in axial morphogenesis during embryo development in both vertebrates and invertebrates, and failure of these processes in human embryos leads to defects including spina bifida and anencephaly. Here we use Xenopus laevis, a system in which the polarized cell motility that drives this active cell intercalation has been related to the development of forces that close the blastopore and elongate the body axis, to examine the role of myosin IIB in convergence and extension. We find that myosin IIB is localized in the cortex of intercalating cells, and show by morpholino knockdown that this myosin isoform is essential for the maintenance of a stereotypical, cortical actin cytoskeleton as visualized with time-lapse fluorescent confocal microscopy. We show that this actin network consists of foci or nodes connected by cables and is polarized relative to the embryonic axis, preferentially cyclically shortening and lengthening parallel to the axis of cell polarization, elongation and intercalation, and also parallel to the axis of convergence forces during gastrulation. Depletion of MHC-B results in disruption of this polarized cytoskeleton, loss of the polarized protrusive activity characteristic of intercalating cells, eventual loss of cell-cell and cell-matrix adhesion, and dose-dependent failure of blastopore closure, arguably because of failure to develop convergence forces parallel to the myosin IIB-dependent dynamics of the actin cytoskeleton. These findings bridge the gap between a molecular-scale motor protein and tissue-scale embryonic morphogenesis.     

Loss of myosin VI reduces secretion and the size of the Golgi in fibroblasts from Snell's waltzer mice

Golgi morphology and function are dependent on an intact microtubule and actin cytoskeleton. Myosin VI, an unusual actin-based motor protein moving towards the minus ends of actin filaments, has been localized to the Golgi complex at the light and electron microscopic level. Myosin VI is present in purified Golgi membranes as a peripheral membrane protein, targeted by its globular tail domain. To investigate the function of myosin VI at the Golgi complex, immortal fibroblastic cell lines of Snell's waltzer mice lacking myosin VI were established. In these cell lines, where myosin VI is absent, the Golgi complex is reduced in size by approximately 40% compared with wild-type cells. Furthermore, protein secretion of a reporter protein from Snell's waltzer cells is also reduced by 40% compared with wild-type cells. Rescue experiments showed that fully functional myosin VI was able to restore Golgi complex morphology and protein secretion in Snell's waltzer cells to the same level as that observed in wild-type cells.     

Myosin VI and Optineurin Are Required for Polarized EGFR Delivery and Directed Migration

The polarized trafficking of membrane proteins into the leading edge of the cell is an integral requirement for cell migration. Myosin VI and its interacting protein optineurin have previously been shown to operate in anterograde trafficking pathways, especially for the polarized delivery of cargo to the basolateral domain in epithelial cells. Here we show that in migratory cells ablation of myosin VI or optineurin inhibits the polarized delivery of the epidermal growth factor receptor (EGFR) into the leading edge and leads to profound defects in lamellipodia formation. Depletion of either myosin VI or optineurin, however, does not impair the overall ability of cells to migrate in a random migration assay, but it dramatically reduces directed migration towards a growth factor stimulus. In summary, we identified a specific role for myosin VI and optineurin in directionally persistent cell migration, which involves the polarized delivery of vesicles containing EGFR into the leading edge of the cell.     

Myosin VI regulates endocytosis of the cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-regulated Cl(-) channel expressed in the apical plasma membrane in fluid-transporting epithelia. Although CFTR is rapidly endocytosed from the apical membrane of polarized epithelial cells and efficiently recycled back to the plasma membrane, little is known about the molecular mechanisms regulating CFTR endocytosis and endocytic recycling. Myosin VI, an actin-dependent, minus-end directed mechanoenzyme, has been implicated in clathrin-mediated endocytosis in epithelial cells. The goal of this study was to determine whether myosin VI regulates CFTR endocytosis. Endogenous, apical membrane CFTR in polarized human airway epithelial cells (Calu-3) formed a complex with myosin VI, the myosin VI adaptor protein Disabled 2 (Dab2), and clathrin. The tail domain of myosin VI, a dominant-negative recombinant fragment, displaced endogenous myosin VI from interacting with Dab2 and CFTR and increased the expression of CFTR in the plasma membrane by reducing CFTR endocytosis. However, the myosin VI tail fragment had no effect on the recycling of endocytosed CFTR or on fluid-phase endocytosis. CFTR endocytosis was decreased by cytochalasin D, an actin-filament depolymerizing agent. Taken together, these data indicate that myosin VI and Dab2 facilitate CFTR endocytosis by a mechanism that requires actin filaments.     

Hepatocyte growth factor acutely perturbs actin filament anchorage at the epithelial zonula adherens

Cadherin adhesion molecules function in close cooperation with the actin cytoskeleton. At the zonula adherens (ZA) of polarized epithelial cells, E-cadherin adhesion induces the cortical recruitment of many key cytoskeletal regulators, which act in a dynamic integrated system to regulate junctional integrity and cell-cell interactions. This capacity for the cytoskeleton to support the ZA carries the implication that regulators of the junctional cytoskeleton might also be targeted to perturb junctional integrity. In this report, we now provide evidence for this hypothesis. We show that hepatocyte growth factor (HGF), which is well-known to disrupt cell-cell interactions, acutely perturbs ZA integrity much more rapidly than generally appreciated. This is accompanied by significant loss of junctional F-actin, a process that reflects loss of filament anchorage at the junctions. We demonstrate that this involves uncoupling of the unconventional motor myosin VI from junctional E-cadherin, a novel effect of HGF that is mediated by intracellular calcium. We conclude that regulators of the junctional cytoskeleton are likely to be major targets for cadherin junctions to be acutely modulated in development and perturbed in disease.     

Myosin VI is required for asymmetric segregation of cellular components during C. elegans spermatogenesis

BACKGROUND: The asymmetric division of cells and unequal allocation of cell contents is essential for correct development. This process of active segregation is poorly understood but in many instances has been shown to depend on the cytoskeleton. Motor proteins moving along actin filaments and microtubules are logical candidates to provide the motive force for asymmetric sorting of cell contents. The role of myosins in such processes has been suggested, but few examples of their involvement are known. RESULTS: Analysis of a Caenorhabditis elegans class VI myosin deletion mutant reveals a role for this motor protein in the segregation of cell components during spermatogenesis. Mutant spermatocytes cannot efficiently deliver mitochondria and endoplasmic reticulum/Golgi-derived fibrous-body membranous organelle complexes to budding spermatids, and fail to remove actin filaments and microtubules from the spermatids. The segregation defects are not due to a global sorting failure as nuclear inheritance is unaffected. CONCLUSIONS: C. elegans myosin VI has an important role in the unequal partitioning of both organelles and cytoskeletal components, a novel role for this class of motor protein.     

Hepatoma polarization limits CD81 and hepatitis C virus dynamics

Many viruses target the polarized epithelial apex during host invasion. In contrast, hepatitis C virus (HCV) engages receptors at the basal surface of hepatocytes in the polarized liver parenchyma. Hepatocyte polarization limits HCV entry by undefined mechanism(s). Given the recent reports highlighting a role for receptor mobility in pathogen entry, we studied the effect(s) of hepatocyte polarization on viral receptor and HCV pseudoparticle (HCVpp) dynamics using real‐time fluorescence recovery after photobleaching and single particle tracking. Hepatoma polarization reduced CD81 and HCVpp dynamics at the basal membrane. Since cell polarization is accompanied by changes in the actin cytoskeleton and CD81 links to actin via its C‐terminus, we studied the dynamics of a mutant CD81 lacking a C‐terminal tail (CD81_ΔC) and its effect(s) on HCVpp mobility and infection. CD81_ΔC showed an increased frequency of confined trajectories and a reduction of Brownian diffusing molecules compared to wild‐type protein in non‐polarized cells. However, these changes were notobserved in polarized cells. HCVpp showed a significant reduction in Brownian diffusion and infection of CD81_ΔC expressing non‐polarized cells. In summary, these data highlight the dynamic nature of CD81 and demonstrate a role for CD81 lateral diffusion to regulate HCV infection in a polarization‐dependent manner.     

Myosin VI altered at threonine 406 stabilizes actin filaments in vivo

Myosin VI is a minus-end directed actin-based molecular motor implicated in uncoated endocytic vesicle transport. Recent kinetic studies have shown that myosin VI displays altered ADP release kinetics under different load conditions allowing myosin VI to serve alternately as a transporter or as an actin tether. We theorized that one potential regulatory event to modulate between these kinetic choices is phosphorylation at a conserved site, threonine 406 (T406) in the myosin VI motor domain. Alterations mimicking the phosphorylated (T406E) and dephosphorylated state (T406A) were introduced into a GFP-myosin VI fusion (GFP-M6). Live cell imaging revealed that GFP-M6(T406E) expression changed the path myosin VI took in its transport of uncoated endocytic vesicles. Rather than routing vesicles inwards as seen in GFP-M6 and GFP-M6(T406A) expressing cells, GFP-M6(T406E) moved vesicles into clusters at distinct peripheral sites. GFP-M6(T406E) expression also increased the density of the actin cytoskeleton. Filaments were enriched at the vesicle cluster sites. This was not due to a gross redistribution of the actin polymerization machinery. Instead the filament density correlated to the fixed positioning of GFP-M6(T406E)-associated vesicles on F-actin, leading to inhibition of actin depolymerization. Our study suggests that phosphorylation at T406 changes the nature of myosin VI's interaction with actin in vivo.     

Myosin VI,an actin motor for membrane traffic and cell migration

The actin cytoskeleton and associated myosin motor proteins are essential for the transport and steady-state localization of vesicles and organelles and for the dynamic remodeling of the plasma membrane as well as for the maintenance of differentiated cell-surface structures. Myosin VI may be expected to have unique cellular functions, because it moves, unlike almost all other myosins, towards the minus end of actin filaments. Localization and functional studies indicate that myosin VI plays a role in a variety of different intracellular processes, such as endocytosis and secretion as well as cell migration. These diverse functions of myosin VI are mediated by interaction with a range of different binding partners .     

ArfGAP1 restricts Mycobacterium tuberculosis entry by controlling the actin cytoskeleton

The interaction of Mycobacterium tuberculosis (Mtb) with pulmonary epithelial cells is critical for early stages of bacillus colonization and during the progression of tuberculosis. Entry of Mtb into epithelial cells has been shown to depend on F‐actin polymerization, though the molecular mechanisms are still unclear. Here, we demonstrate that mycobacterial uptake into epithelial cells requires rearrangements of the actin cytoskeleton, which are regulated by ADP‐ribosylation factor 1 (Arf1) and phospholipase D1 (PLD1), and is dependent on the M3 muscarinic receptor (M₃R). We show that this pathway is controlled by Arf GTPase‐activating protein 1 (ArfGAP1), as its silencing has an impact on actin cytoskeleton reorganization leading to uncontrolled uptake and replication of Mtb. Furthermore, we provide evidence that this pathway is critical for mycobacterial entry, while the cellular infection with other pathogens, such as Shigella flexneri and Yersinia pseudotuberculosis, is not affected. Altogether, these results reveal how cortical actin plays the role of a barrier to prevent mycobacterial entry into epithelial cells and indicate a novel role for ArfGAP1 as a restriction factor of host–pathogen interactions.     

Colocalization of APC and DLG at the tips of cellular protrusions in cultured epithelial cells and its dependency on cytoskeletons

Adenomatous polyposis coli gene product (APC) is a tumor suppressor linked to familial adenomatous polyposis and is thought to be involved in cellular polarization and migration in moving epithelial cells. APC interacts with the mammalian homolog of Discs large (DLG). DLG is a member of the membrane-associated guanylate kinase superfamily and is thought to function as a scaffolding protein that coordinates the assembly of a lateral plasma membrane-localized protein complex in epithelial cells. We confirmed the suitability of several anti-APC antibodies for immunocytochemical analysis. Using these antibodies, we showed that APC clusters were colocalized with DLG protein at cellular protrusions of subconfluent MDCK cells. A portion of the clusters was found at the tips of microtubules extending into the cellular protrusions. In addition, actin stress fibers converged near the clusters. When microtubules were disrupted by nocodazole, the colocalization of APC and DLG was lost due to the disappearance of APC clusters. However, the coclusters remained after depolymerization of actin filaments with latrunculin A. This is the first report showing colocalization of APC and DLG in non-polarized epithelial cells. This colocalization suggests that DLG functions not only at the lateral cell–cell contact sites of polarized epithelial cells but also at the protrusions of non-polarized epithelial cells through the interaction with APC protein.