Leukocyte polarization in cell migration and immune interactions.

Cell migration plays a key role in a wide variety of biological phenomena. This process is particularly important for leukocyte function and the inflammatory response. Prior to migration leukocytes undergo polarization, with the formation of a lamellipodium at the leading edge and a uropod at the trailing edge. This cell shape allows them to convert cytoskeletal forces into net cell-body displacement. Leukocyte chemoattractants, including chemokines, provide directional cues for leukocyte motility, and concomitantly induce polarization. Chemoattractant receptors, integrins and other adhesion molecules, cytoskeletal proteins and intracellular regulatory molecules change their cellular localization during cell polarization. A complex system of signal transduction molecules, including tyrosine kinases, lipid kinases, second messengers and members of the Rho family of small GTPases is thought to regulate the cytoskeletal rearrangements underlying leukocyte polarization and migration. The elucidation of the mechanisms and signals that control this complex reorganization will lead to a better understanding of critical questions in cell biology of leukocyte migration and polarity.     

Role of the PI3K regulatory subunit in the control of actin organization and cell migration

Cell migration represents an important cellular response that utilizes cytoskeletal reorganization as its driving force. Here, we describe a new signaling cascade linking PDGF receptor stimulation to actin rearrangements and cell migration. We demonstrate that PDGF activates Cdc42 and its downstream effector N-WASP to mediate filopodia formation, actin stress fiber disassembly, and a reduction in focal adhesion complexes. Induction of the Cdc42 pathway is independent of phosphoinositide 3-kinase (PI3K) enzymatic activity, but it is dependent on the p85alpha regulatory subunit of PI3K. Finally, data are provided showing that activation of this pathway is required for PDGF-induced cell migration on collagen. These observations show the essential role of the PI3K regulatory subunit p85alpha in controlling PDGF receptor-induced cytoskeletal changes and cell migration, illustrating a novel signaling pathway that links receptor stimulation at the cell membrane with actin dynamics.     

Cytoskeletal rearrangement during migration and activation of T lymphocytes.

T lymphocytes have an inherent ability to migrate along a chemotactic gradient, which enables them to exit the bloodstream and reach different tissues. Motile T cells display a polarized morphology with two distinct cell compartments: the leading edge and the uropod. During cell polarization, chemoattractant receptors, cell-adhesion molecules and cytoskeletal proteins are redistributed within these cellular compartments. The polarity of T lymphocytes changes during the establishment of antigen-specific cell-cell interactions, and this involves rearrangement of cytoskeletal proteins. This article discusses the regulation of these cytoskeletal rearrangements, and their role in the activation, migration and effector function of T cells.     

The Arabidopsis SPIKE1 gene is required for normal cell shape control and tissue development

Regulated growth and cell shape control are fundamentally important to the function of plant cells, tissues, and organs. The signal transduction cascades that control localized growth and cell shape, however, are not known. To better understand the relationship between cytoskeletal organization, organelle positioning, and regulated vesicle transport, we conducted a forward genetic screen to identify genes that regulate cytoskeletal organization in plants. Because of the distinct requirements for microtubules and actin filaments during leaf trichome development, a trichome-based morphology screen is an efficient approach to identify genes that affect cytoplasmic organization. The seedling lethal spike1 mutant was identified based on trichome, cotyledon, and leaf-shape defects. The predicted SPIKE1 protein shares amino acid identity with a large family of adapter proteins present in humans, flies, and worms that integrate extracellular signals with cytoskeletal reorganization. Both the trichome phenotype and immunolocalization data suggest that SPIKE1 also is involved in cytoskeletal reorganization. The assembly of laterally clustered foci of microtubules and polarized growth are early events in cotyledon development, and both processes are misregulated in spike1 epidermal cells.     

Clinical neurosciences in the decade of the brain: hypotheses in neuro-oncology. VEG/PF acts upon the actin cytoskeleton and is inhibited by dexamethasone: relevance to tumor angiogenesis and vasogenic edema.

HYPOTHESIS: We have proposed that VEG/PF acts by transforming the cytoskeletal architecture of microvascular endothelial cells. BACKGROUND: Evidence supporting a pivotal role for vascular endothelial growth/permeability factor (VEG/PF) in tumor angiogenesis and edemagenesis is compelling. VEG/PF exhibits specific endothelial cell mitogenicity and is expressed by brain tumors exhibiting increased vascularity and microvascular extravasation. The mechanistic cascade that follows VEG/PF-tyrosine kinase receptor binding remains uncertain, however. Actin is a cytoskeletal protein that regulates cellular motility, shape and vesicular transport. Regulation of actin stress fibers, cell-surface focal adhesions and plasmalemmal "ruffles" is mediated by tyrosine kinase activation of GTP-binding proteins that are in turn linked to intracellular calcium flux. As VEG/PF is known to induce cytosolic calcium ion transients in endothelial cells, actin microfilaments would appear to be logical candidates for study of a cytocontractile response mediated by calcium signal transduction. METHODS: VEG/PF-induced endothelial actin cytoskeletal changes were studied using rhodamine phalloidin staining and fluorescence photomicrography. RESULTS: When exposed to VEG/PF, cultured endothelial cells from human umbilical veins and rat brain microvessels exhibited a reversible, dose-related reorganization of actin stress fibers, cell contraction and rounding, and widening of the intercellular spaces. VEG/PF perturbation also induced plasmalemmal "ruffling". All VEG/PF-induced cytoskeletal changes were inhibited by preincubating endothelial cells with dexamethasone or anti-VEG/PF IgG antibody. CONCLUSION: The findings support a role for VEG/PF-induced cytoskeletal alterations in the pathophysiology of brain tumor angiogenesis and edemagenesis. These observations are likely to be directly linked to VEG/PF-induced endothelial cytosolic calcium flux. Insight into the mechanism of dexamethasone''s clinical efficacy is also provided.     

5-Fluorouracil interferes with actin organization, stress fiber formation and cell migration in corneal endothelial cells during wound repair along the natural basement membrane

Corneal endothelial cells respond to a circular freeze wound by undergoing actin cytoskeletal reorganization that is mainly characterized by the disappearance of circumferential microfilament bundles (CMBs) and the subsequent appearance of distinct stress fibers. This cytoskeletal rearrangement is associated with changes in cell shape as migrating cells lose their polyhedral appearance, spread out, and assume a stellate morphology with cell processes extending outward into the injured area. We report here that in the presence of low concentrations (0.01-0.l mM) of the anti-metabolite 5-fluorouracil (5-FU), characteristic actin organization becomes disrupted and migrating cells do not display elongated processes typical of control tissues and translocation into the injury zone is retarded, but not inhibited. Rhodamine phalloidin staining revealed no evidence of stress fiber formation. A higher concentration of 5-FU (1.0 mM) not only prevented formation of discernible stress fibers but also resulted in a more restricted cell movement during wound repair. That this was not a cytotoxic effect was demonstrated by transferring tissues back into standard medium allowing endothelia to reinitiate migration and undergo complete wound healing by 72 h post-transfer. Overnight incubation of endothelia in 4 muM phallacidin resulted in limited CMB disruption the extent of which was dependent on the 5-FU concentration. The effects of 5-FU on the actin cytoskeleton are reversible and by 24 h after placing treated endothelia into medium without 5-FU, actin begins to become re-established and by 48 h microfilament patterns in the tissue resemble those of non-treated endothelia. Similarly, when non-injured tissues are cultured in the presence of 5-FU for 24 h, subsequently injured and returned to standard medium, they exhibit no stress fibers when observed at 24 h post-wounding. However, by 48 h post-injury these cells now display stress fibers and extend processes into the wound area. Biochemical studies on isolated muscle actin demonstrated that actin polymerization is unaffected in the presence of either 0.01 or 1 mM 5-FU as determined by the F-actin sedimentation and falling ball viscosity techniques. Thus, the mechanism(s) by which 5-FU exerts its actions on the actin cytoskeleton appears to be one of an indirect nature.     

Interaction of PC-3 cells with fibronectin adsorbed on sulfonated polystyrene surfaces

The ability of cancer cells to invade neighboring tissues is crucial for cell dissemination and tumor metastasis. It is generally assumed that cell adhesion to extracellular matrix proteins is an important stage of cancer progression. Hence, adhesion of cancer cells under in vitro conditions to proteins adsorbed on a substratum surface has been studied to provide a better understanding of cell-protein interaction mechanisms. A protein, adsorbed in an appropriate conformation on a substratum surface, creates a biologically active layer that regulates such cell functions as adhesion, spreading, proliferation and migration. In our study, we examined the interaction of PC-3 cells under in vitro conditions with fibronectin adsorbed on sulfonated polystyrene surfaces of a defined chemical composition and topography. We investigated cell adhesion to fibronectin and cell spreading. Using automatic, sequential microscopic image registration, we are the first to present observations of the dynamics of PC-3 cell spreading and the cell shape during this process. Our results show that cell adhesion and the shape of spreading cells strongly depend on the time interaction with fibronectin. The analysis of images of cytoskeletal protein distribution in the cell region near the cell-substratum interface revealed that induction of a signal cascade took place, which led to the reorganization of the cytoskeletal proteins and the activation of focal adhesion kinase (FAK).     

Phospholipase D2 induces stress fiber formation through mediating nucleotide exchange for RhoA

Phospholipase D (PLD) is involved in diverse cellular processes including cell movement, adhesion, and vesicle trafficking through cytoskeletal rearrangements. However, the mechanism by which PLD induces cytoskeletal reorganization is still not fully understood. Here, we describe a new link to cytoskeletal changes that is mediated by PLD2 through direct nucleotide exchange on RhoA. We found that PLD2 induces RhoA activation independent of its lipase activity. PLD2 directly interacted with RhoA, and the PX domain of PLD2 specifically recognized nucleotide-free RhoA. Finally, we found that the PX domain of PLD2 has guanine nucleotide-exchange factor (GEF) activity for RhoA in vitro. In addition, we verified that overexpression of the PLD2-PX domain induces RhoA activation, thereby provoking stress fiber formation. Together, our findings suggest that PLD2 functions as an upstream regulator of RhoA, which enables us to understand how PLD2 regulates cytoskeletal reorganization in a lipase activity-independent manner.     

Shaping up for shipping out: PLCgamma signaling of morphology changes in EGF-stimulated fibroblast migration

For effective migration, cells must establish an asymmetry in cell/substratum biophysical interactions permitting cellular protrusive and contractile motive forces to produce net cell body translocation; often this is superficially manifested as a polarized cell shape. This change is most easily noted for epithelial cells, which typically undergo a mesenchymal transition prior to rapid motility, and for hematopoietic cells, which must transition from non-adherent to adherent states. These two situations entail dramatic changes that also involve cell-cell contact and differentiation-related changes, and thus introduce confounding events and signals in defining control elements. Hence, a simpler biochemical and biophysical model system may be useful for gaining fundamental insights into the underlying mechanisms. Fortunately, even relatively "uniform" fibroblasts also undergo an initial shape change to commence locomotion. Investigators have recently begun to probe underlying signals that contribute to the reorganization of the actin cytoskeleton. We describe here a model for fibroblast shape changes involved in epidermal growth factor (EGF) stimulation of motility, focusing on signals through EGF receptor (EGFR) -mediated pathways influencing cytoskeletal organization and cell/substratum adhesion. We present new data addressing specifically phospholipase C-gamma (PLCgamma) pathway activation of actin-modifying proteins, including gelsolin, that contributes to these changes and promotes cell migration by increasing the fraction of cells in a motility-permissive morphology and the time spent in such a state.     

Vitreous-induced cytoskeletal rearrangements via the Rac1 GTPase-dependent signaling pathway in human retinal pigment epithelial cells

Proliferative vitreoretinopathy (PVR) is mainly caused by retinal pigment epithelial (RPE) cell migration, invasion, proliferation and transformation into fibroblast-like cells that produce the extracellular matrix (ECM). The vitreous humor is known to play an important role in PVR. An epithelial-to-mesenchymal transdifferentiation (EMT) of human RPE cells induced by 25% vitreous treatment has been linked to stimulation of the mesenchymal phenotype, migration and invasion. Here, we characterized the effects of the vitreous on the cell morphology and cytoskeleton in human RPE cells. The signaling pathway that mediates these effects was investigated. Serum-starved RPE cells were incubated with 25% vitreous, and the morphological changes were examined by phase-contrast microscopy. Filamentous actin (F-actin) was examined by immunofluorescence and confocal microscopy. Protein phosphorylation of AKT, ERK1/2, Smad2/3, LIM kinase (LIMK) 1 and cofilin was analyzed by Western blot analysis. Vitreous treatment induced cytoskeletal rearrangements, activated Rac1 and enhanced the phosphorylation of AKT, ERK1/2 and Smad2/3. When the cells were treated with a Rac activation-specific inhibitor, the cytoskeletal rearrangements were prevented, and the phosphorylation of Smad2/3 was blocked. Vitreous treatment also enhanced the phosphorylation of LIMK1 and cofilin and the Rac inhibitor blocked this effect. We propose that vitreous-transformed human RPE cells undergo cytoskeletal rearrangements via Rac1 GTPase-dependent pathways that modulate LIMK1 and cofilin activity. The TGFβ-like activity of the vitreous may participate in this effect. Actin polymerization causes the cytoskeletal rearrangements that lead to the plasticity of vitreous-transformed RPE cells in PVR.     

PDR-1/hParkin negatively regulates the phagocytosis of apoptotic cell corpses in Caenorhabditis elegans

Apoptotic cell death is an integral part of cell turnover in many tissues, and proper corpse clearance is vital to maintaining tissue homeostasis in all multicellular organisms. Even in tissues with high cellular turnover, apoptotic cells are rarely seen because of efficient clearance mechanisms in healthy individuals. In Caenorhabditis elegans, two parallel and partly redundant conserved pathways act in cell corpse engulfment. The pathway for cytoskeletal rearrangement requires the small GTPase CED-10 Rac1 acting for an efficient surround of the dead cell. The CED-10 Rac pathway is also required for the proper migration of the distal tip cells (DTCs) during the development of the C. elegans gonad. Parkin, the mammalian homolog of the C. elegans PDR-1, interacts with Rac1 in aged human brain and it is also implicated with actin dynamics and cytoskeletal rearrangements in Parkinsons''s disease, suggesting that it might act on engulfment. Our genetic and biochemical studies indicate that PDR-1 inhibits apoptotic cell engulfment and DTC migration by ubiquitylating CED-10 for degradation.     

Endothelial cells undergo morphological, biomechanical, and dynamic changes in response to tumor necrosis factor-α

The immune response triggers a complicated sequence of events, one of which is release of the cytokine tumor necrosis factor-α (TNF-α) from stromal cells, for example monocytes and macrophages. In this work we investigated the biophysical effects of TNF-α on endothelial cells (ECs), including changes in cell morphology, biomechanics, migration, and cytoskeletal dynamics. We found that TNF-α induces a wide distribution of cell area and aspect ratio, with these properties increasing on average during treatment. Interestingly, aspect ratio peaks after approximately 10?h of exposure to TNF-α, corresponding also to a peak in exerted traction forces. Meanwhile, ECs treated with TNF-α soften, and we associate this with significant increases in estimated cellular volume. In addition, our evaluation of migratory dynamics revealed an inverse correlation between cell aspect ratio and migration speed after TNF-α treatment, suggesting that cell shape may be an important functional regulator of EC migration during an inflammatory response. Finally, we addressed the basic mechanics of how the reorganization of F-actin filaments occurs during TNF-α treatment, and observed a dynamic shift of existing actin filaments. Together, our results suggest a functional link between EC morphology, biomechanics, migration, and cytoskeletal dynamics during an inflammatory response.     

ROCK蛋白与肿瘤

ROCK蛋白作为Rho亚家族下游最重要的效应分子之一,主要通过调节肌动蛋白在调控细胞的形态、极性、细胞骨架重构和细胞迁移等多个方面发挥生理功能。研究发现ROCK蛋白在肿瘤的发生发展中起着重要作用,主要参与调控肿瘤细胞的生存与凋亡,以及恶性肿瘤的侵袭与转移。文章论述了ROCK蛋白与肿瘤关系的研究进展,为寻找新的抗癌药物治疗靶点提供依据。     

Effect of cyclic AMP on human blood platelets: induction of pseudopod formation and protein phosphorylation

A variety of techniques, including immunofluorescence, electron microscopy and biochemical analysis, were used to examine shape changes and cytoskeletal reorganization of human blood platelets during treatment with N6,O2-dibutyryl adenosine 3',5'-cyclic monophosphoric acid (dbcAMP), and agent known to elevate the intracellular level of cyclic AMP (cAMP). Cytochemical analysis shows that the unstimulated platelets have a discoid shape with no obvious membrane projections. Platelets treated with dbcAMP produce pseudopod-like structures containing cytoskeletal proteins such as actin and microtubules. Biochemical analysis reveals that a 125,000 dalton phosphoprotein (P-125) is preferentially recruited into cytoskeletal fractions of platelets treated with dbcAMP. This protein, which is one of the substrates for cAMP-dependent kinase(s) and/or is closely associated with the cytoskeleton, may play an important role in regulating the shape changes and cytoskeletal reorganization that occur during the early stages of platelet activation.     

FGF signaling regulates cytoskeletal remodeling during epithelial morphogenesis

Changes in the cytoskeletal architecture underpin the dynamic changes in tissue shape that occur during development. It is clear that such changes must be coordinated so that individual cell behaviors are synchronized; however, the mechanisms by which morphogenesis is instructed and coordinated are unknown. After its induction in non-neural ectoderm, the inner ear undergoes morphogenesis, being transformed from a flat ectodermal disk on the surface of the embryo to a hollowed sphere embedded in the head. We provide evidence that this shape change relies on extrinsic signals subsequent to genetic specification. By using specific inhibitors, we find that local fibroblast growth factor (FGF) signaling triggers a phosphorylation cascade that activates basal myosin II through the activation of phospholipase Cgamma. Myosin II exhibits a noncanonical activity that results in the local depletion of actin filaments. Significantly, the resulting apical actin enrichment drives morphogenesis of the inner ear. Thus, FGF signaling directly exerts profound cytoskeletal effects on otic cells, coordinating the morphogenesis of the inner ear. The iteration of this morphogenetic signaling system suggests that it is a more generally applicable mechanism in other epithelial tissues undergoing shape change.     

A critical role for cortactin phosphorylation by Abl-family kinases in PDGF-induced dorsal-wave formation

Proper regulation of cell morphogenesis and migration by adhesion and growth-factor receptors requires Abl-family tyrosine kinases [1-3]. Several substrates of Abl-family kinase have been identified, but they are unlikely to mediate all of the downstream actions of these kinases on cytoskeletal structure. We used a human protein microarray to identify the actin-regulatory protein cortactin as a novel substrate of the Abl and Abl-related gene (Arg) nonreceptor tyrosine kinases. Cortactin stimulates cell motility [4-6], and its upregulation in several cancers correlates with poor prognosis [7]. Even though cortactin can be tyrosine phosphorylated by Src-family kinases in vitro [8], we show that Abl and Arg are more adept at binding and phosphorylating cortactin. Importantly, we demonstrate that platelet-derived growth-factor (PDGF)-induced cortactin phosphorylation on three tyrosine residues requires Abl or Arg. Cortactin triggers F-actin-dependent dorsal waves in fibroblasts after PDGF treatment and thus results in actin reorganization and lamellipodial protrusion [9]. We provide evidence that Abl/Arg-mediated phosphorylation of cortactin is required for this PDGF-induced dorsal-wave response. Our results reveal that Abl-family kinases target cortactin as an effector of cytoskeletal rearrangements in response to PDGF.     

Cytoskeletal-associated proteins in the migration of cortical neurons

Neuronal migration is a hallmark of cerebral cortical development as neurons born deep within the brain migrate to the surface in a highly choreographed process. The cytoskeleton extends throughout the cell, mediating the dramatic morphological changes that accompany migration. On a cellular level, proper migration is accompanied by polarization of the cytoskeleton and cellular contents and by dynamic reorganization that generates the force for cell locomotion. Genetic analyses of human brain malformations, as well as genetically engineered mouse mutants, have highlighted a number of cytoskeletal-associated proteins underlying these functions, which are necessary for proper cortical development. While these proteins are involved in diverse molecular mechanisms, disruption during development results in the ectopic placement of neurons in the cortex. We review key cytoskeletal events and the critical cytoskeletal-associated proteins involved in cortical neuronal migration.     

Nuclear mechanics during cell migration

During cell migration, the movement of the nucleus must be coordinated with the cytoskeletal dynamics at the leading edge and trailing end, and, as a result, undergoes complex changes in position and shape, which in turn affects cell polarity, shape, and migration efficiency. We here describe the steps of nuclear positioning and deformation during cell polarization and migration, focusing on migration through three-dimensional matrices. We discuss molecular components that govern nuclear shape and stiffness, and review how nuclear dynamics are connected to and controlled by the actin, tubulin and intermediate cytoskeleton-based migration machinery and how this regulation is altered in pathological conditions. Understanding the regulation of nuclear biomechanics has important implications for cell migration during tissue regeneration, immune defence and cancer.     

Tec kinases: shaping T-cell activation through actin

Following stimulation, T cells undergo marked actin-dependent changes in shape that are required for productive cellular interactions and movement during immune responses. Reorganization of the actin cytoskeletal is also necessary for the formation of an immunological synapse - the convergence of several signaling molecules at the plasma membrane that occurs after effective T-cell receptor (TCR) signaling. Much emerging evidence indicates that the Tec family of tyrosine kinases has a role in actin cytoskeleton reorganization. Specifically, T cells that lack or express mutant versions of the Tec kinase Itk show impaired TCR-induced actin polymerization, cell polarization and regulation of the signaling events involved in cytoskeletal reorganization. These data, as well as other findings, support roles for Tec kinases in actin cytoskeleton regulation.