Cell surface actin remodeling.
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Actin Cytoskeleton↗
Biomedical subjects
Gabriel Fenteany
Publications and source records attributed to Gabriel Fenteany.
Glycogen synthase kinase-3 acts upstream of ADP-ribosylation factor 6 and Rac1 to regulate epithelial cell migration.
Cell sheet movement during epithelial wound closure is a complex process involving collective cell migration. We have found that glycogen synthase kinase-3 (GSK-3) activity is required for membrane protrusion and crawling of cells at the wound edge and those behind it in wounded Madin-Darby canine kidney (MDCK) epithelial cell monolayers. RNA interference-based silencing of GSK-3alpha and GSK-3beta expression also results in slowed cell sheet migration, with the effect being more pronounced with knockdown of GSK-3beta. Both GSK-3alpha and GSK-3beta are in activated states during the most active phase of cell migration. In addition to having a positive control or permissive, rather than negative, function in MDCK cell migration, GSK-3 appears to act upstream of the small GTPases ADP-ribosylation factor 6 (ARF6) and Rac1. Expression of constitutively active ARF6 restores a protrusive, migratory phenotype in cells treated with GSK-3 inhibitors. It does not, however, restore to normal levels the directional polarization of cells behind the wound edge toward the wound area, implying the existence of a separate ARF6-independent branch of the GSK-3 pathway that regulates proper wound-directed polarization of these cells. Finally, inhibition of GSK-3 also strongly reduces activation of Rac1 and cell scatter in response to hepatocyte growth factor/scatter factor, which triggers dispersal and migration of cells in monolayer culture as fibroblast-like individual cells, a mode of epithelial cell motility distinct from the collective migration of wound closure.
ADP-Ribosylation Factor 6↗
Quinocarmycin analog DX-52-1 inhibits cell migration and targets radixin, disrupting interactions of radixin with actin and CD44.
In the course of screening for new small-molecule modulators of cell motility, we discovered that quinocarmycin (also known as quinocarcin) analog DX-52-1 is an inhibitor of epithelial cell migration. While it has been assumed that the main target of DX-52-1 is DNA, we identified and confirmed radixin as the relevant molecular target of DX-52-1 in the cell. Radixin is a member of the ezrin/radixin/moesin family of membrane-actin cytoskeleton linker proteins that also participate in signal transduction pathways. DX-52-1 binds specifically and covalently to the C-terminal region of radixin, which contains the domain that interacts with actin filaments. Overexpression of radixin in cells abrogates their sensitivity to DX-52-1's antimigratory activity. Small interfering RNA-mediated silencing of radixin expression reduces the rate of cell migration. Finally, we found that DX-52-1 disrupts radixin's ability to interact with both actin and the cell adhesion molecule CD44.
Actins↗
A chemical inhibitor reveals the role of Raf kinase inhibitor protein in cell migration.
Raf kinase inhibitor protein (RKIP) is a modulator of cell signaling that functions as an endogenous inhibitor of multiple kinases. We demonstrate here a positive role for RKIP in the regulation of cell locomotion. We discovered that RKIP is the relevant cellular target of locostatin, a cell migration inhibitor. Locostatin abrogates RKIP's ability to bind and inhibit Raf-1 kinase, and it acts by disrupting a protein-protein interaction, an uncommon mode of action for a small molecule. Small interfering RNA-mediated silencing of RKIP expression also reduces cell migration rate. Overexpression of RKIP converts epithelial cells to a highly migratory fibroblast-like phenotype, with dramatic reduction in the sensitivity of cells to locostatin. RKIP is therefore the compound's valid target and a key regulator of cell motility.
Androgen-Binding Protein↗
Multiple rows of cells behind an epithelial wound edge extend cryptic lamellipodia to collectively drive cell-sheet movement.
The mechanism by which epithelial, endothelial and other strongly cell-cell adhesive cells migrate collectively as continuous sheets is not clear, even though this process is crucial for embryonic development and tissue repair in virtually all multicellular animals. Wound closure in Madin-Darby canine kidney (MDCK) epithelial cell monolayers involves Rac GTPase-dependent migration of cells both at and behind the wound edge. We report here for the first time that cells behind the margin of wounded MDCK cell monolayers, even hundreds of microns from the edge, extend 'cryptic' lamellipodia against the substratum beneath cells in front of them, toward the wound, as determined by confocal, two-photon and transmission electron microscopy. These so-called submarginal cells nevertheless strictly maintain their more apical cell-cell contacts when they migrate as part of a coherent cell sheet, hiding their basal protrusions from conventional microscopy. The submarginal protrusions display the hallmarks of traditional lamellipodia based on morphology and dynamics. Cells behind the margin therefore actively crawl, instead of just moving passively when cells at the margin pull on them. The rate of migration is inversely proportional to the distance from the margin, and cells move co-ordinately, yet still in part autonomously, toward the wound area. We also clarify the ancillary role played by nonprotrusive contractile actin bundles that assemble in a Rho GTPase-dependent manner at the margin after wounding. In addition, some cell proliferation occurs at a delay after wounding but does not contribute to closure. Instead, it apparently serves to replace damaged cells so that intact spread cells can revert to their normal cuboidal morphology and the original cell density of the unbroken sheet can be restored.
Actins↗
c-Jun N-terminal kinase regulates lamellipodial protrusion and cell sheet migration during epithelial wound closure by a gene expression-independent mechanism.
c-Jun N-terminal kinase (JNK) is emerging as an important regulator of cell migration. Perturbing the JNK signaling pathway with three structurally and mechanistically distinct inhibitors that selectively target either JNKs themselves or the upstream mixed-lineage kinases, we found dramatic inhibition of membrane protrusion and cell sheet migration during wound closure in Madin-Darby canine kidney (MDCK) epithelial cell monolayers. Extension of lamellipodia is blocked from the earliest times after wounding in the presence of JNK pathway inhibitors, whereas assembly of non-protrusive actin bundles at the wound margin is unaffected. Inhibitors of the other mitogen-activated protein kinase (MAPK) pathways, the extracellular signal-regulated kinase and p38 MAPK pathways, only have comparatively weak or marginal inhibitory effects on wound closure. Multiple splice variants of both JNK1 and JNK2 are expressed in MDCK cells, and JNK1 and JNK2 are rapidly and transiently activated upon wounding. Phosphorylation of c-Jun does not appear relevant to MDCK wound closure, and membrane protrusion directly after wounding is not affected by inhibitors of RNA or protein synthesis. While most known substrates of JNK are transcription factors or proteins regulating apoptosis, our data indicate that JNK regulates protrusion and migration in a gene expression-independent manner and suggest an important cytoplasmic role for JNK in the control of cell motility.
Animals↗
Cytoskeletal remodeling in leukocyte function.
PURPOSE OF REVIEW: This review focuses on recent developments in understanding the roles and regulation of the cytoskeleton in the function of leukocytes. RECENT FINDINGS: New studies have shed light on the regulation and dynamics of actin and microtubules in leukocytes relevant both to cell motility generally and to immune function specifically. The roles of cytoskeletal dynamics in processes such as cell activation, cell migration, and phagocytosis are being elucidated. Dramatic progress has been made recently in understanding the mechanisms of leukocyte directional sensing, polarization, and chemotaxis. SUMMARY: Leukocytes need to be activated, polarize, change shape, move, or phagocytose in response to their environment. Leukocytes accomplish these processes by remodeling their cytoskeleton, the active musculoskeletal system of the cell that is not just the ultimate effector of motile responses but is also a dynamic framework for subcellular organization and regional signaling. Active areas of research include the direct and indirect reciprocal interactions between the cytoskeleton and the membrane and among cytoskeletal elements. The pervasive and multi-layered roles played by small GTPases of the Rho family and phosphoinositides in leukocyte function are also becoming clearer.
Actin Cytoskeleton↗
Small-molecule inhibitors of actin dynamics and cell motility.
Cell motility is a central feature of a range of normal and pathological processes, including embryonic development, tissue repair, immune cell function, angiogenesis, and cancer metastasis. The dynamics of the actin cytoskeleton power cell migration. A large number of proteins are known or suspected to play roles in regulating actin dynamics. While there are now many available small molecules that target the actin cytoskeleton directly, there is a paucity of specific inhibitors of actin-binding proteins and other immediate regulators of actin dynamics and cell movement. This makes the field of exceptional interest as a meeting place between the goals of chemical biology and the needs of cell biology. Furthermore, while regulators of the cell cycle have been recognized for some time as targets for anti-cancer drug development, controlling actin dynamics and cell motility as a therapeutic approach has received scant attention in comparison until recently. This review deals with small-molecule inhibitors of actin dynamics as they relate to cell shape change and motility, from compounds targeting actin directly to those targeting proteins involved in the fundamental control of the actin cytoskeleton.
Actins↗
A non-antibacterial oxazolidinone derivative that inhibits epithelial cell sheet migration.
We have developed a high-throughput assay for screening chemical libraries for compounds that affect cell sheet migration during wound closure in epithelial cell monolayers. By using this assay, we have discovered a new inhibitor of cell sheet migration. This compound (UIC-1005) is a 3,4-disubstituted oxazolidinone that bears an electrophilic alpha,beta-unsaturated N-acyl group required for activity. UIC-1005 also inhibits growth in an epithelial cell proliferation assay. The molecule does not display general toxicity at concentrations at which it potently inhibits cell sheet migration and growth. Unlike certain 3,5-disubstituted oxazolidinones, it exhibits no antibacterial activity. UIC-1005 therefore represents a new class of bioactive oxazolidinone derivative that may prove useful as a probe for signaling pathways leading to cell motility.
Animals↗