Calcium-induced changes in cytoskeleton and motility of cultured human keratinocytes

In normal epidermis keratinocytes migrate upward from the basal layer as they undergo terminal differentiation, yet they also have the capacity for lateral movement during wound healing. The purpose of our experiments was to investigate these two types of movement by manipulating the calcium ion concentration of the medium so that keratinocytes formed monolayers (0.1 mM calcium) or stratified sheets (2.0 mM calcium). Time-lapse video recording indicated that keratinocytes in low-calcium medium were laterally more motile than keratinocytes in normal medium. This was consistent with the ultrastructural appearance of the cells and the lack of desmosomal junctions, determined by scanning and transmission electron microscopy. During calcium-induced stratification keratinocytes moved upward from the basal layer by gliding over their neighbors and forming contacts with other suprabasal cells. Keratinocytes in low-calcium medium migrated into wounds made in the cultures, a process which was inhibited by monensin; however, stratified keratinocytes in normal medium did not enter wounds. Cytochalasin D caused rapid cell rounding and disruption of actin filaments in keratinocytes grown in low-calcium but not in normal medium, indicating more rapid treadmilling of actin and consistent with the greater motility of keratinocytes in low-calcium medium. Our results suggest that desmosome formation may place constraints on the movement of individual keratinocytes and that the actomyosin cytoskeleton is involved in lateral migration.     

The actin cytoskeleton in normal and pathological cell motility

Cell motility is crucial for tissue formation and for development of organisms. Later on cell migration remains essential throughout the lifetime of the organism for wound healing and immune responses. The actin cytoskeleton is the cellular engine that drives cell motility downstream of a complex signal transduction cascade. The basic molecular machinery underlying the assembly and disassembly of actin filaments consists of a variety of actin binding proteins that regulate the dynamic behavior of the cytoskeleton in response to different signals. The multitude of proteins and regulatory mechanisms partaking in this system makes it vulnerable to mutations and alterations in expression levels that ultimately may cause diseases. The most familiar one is cancer that in later stages is characterized by active aberrant cell migration. Indeed tumor invasion and metastasis are increasingly being associated with deregulation of the actin system.     

Trophoblast cell line resistance to NK lysis mainly involves an HLA class I-independent mechanism

The lack of classical HLA molecules on trophoblast prevents allorecognition by maternal T lymphocytes, but poses the problem of susceptibility to NK lysis. Expression of the nonclassical class I molecule, HLA-G, on cytotrophoblast may provide the protective effect. However, the class I-negative syncytiotrophoblast escapes NK lysis by maternal PBL. In addition, while HLA-G-expressing transfectants of LCL.721.221 cells are protected from lymphokine-activated killer lysis, extravillous cytotrophoblast cells and HLA-G-expressing choriocarcinoma cells (CC) are not. The aim of this work was therefore to clarify the role of HLA class I expression on trophoblast cell resistance to NK lysis and on their susceptibility to lymphokine-activated killer lysis. Our results showed that both JAR (HLA class I-negative) and JEG-3 (HLA-G- and HLA-Cw4-positive) cells were resistant to NK lysis by PBL and were equally lysed by IL-2-stimulated PBL isolated from a given donor. In agreement, down-regulating HLA class I expression on JEG-3 cells by acid treatment, masking these molecules or the putative HLA-G (or HLA-E) receptor CD94/NKG2 and the CD158a/p58.1 NKR with mAbs, and inducing self class I molecule expression on JAR cells did not affect NK or LAK lysis of CC. These results demonstrate that the resistance of CC to NK lysis mainly involves an HLA class I-independent mechanism(s). In addition, we show that the expression of a classical class I target molecule (HLA-B7) on JAR cells is insufficient to induce lysis by allospecific polyclonal CTL.     

Interactions between laminin receptor and the cytoskeleton during translation and cell motility

Human laminin receptor acts as both a component of the 40S ribosomal subunit to mediate cellular translation and as a cell surface receptor that interacts with components of the extracellular matrix. Due to its role as the cell surface receptor for several viruses and its overexpression in several types of cancer, laminin receptor is a pathologically significant protein. Previous studies have determined that ribosomes are associated with components of the cytoskeleton, however the specific ribosomal component(s) responsible has not been determined. Our studies show that laminin receptor binds directly to tubulin. Through the use of siRNA and cytoskeletal inhibitors we demonstrate that laminin receptor acts as a tethering protein, holding the ribosome to tubulin, which is integral to cellular translation. Our studies also show that laminin receptor is capable of binding directly to actin. Through the use of siRNA and cytoskeletal inhibitors we have shown that this laminin receptor-actin interaction is critical for cell migration. These data indicate that interactions between laminin receptor and the cytoskeleton are vital in mediating two processes that are intimately linked to cancer, cellular translation and migration.     

Rapamycin inhibits cytoskeleton reorganization and cell motility by suppressing RhoA expression and activity

The mammalian target of rapamycin (mTOR) functions in cells at least as two complexes, mTORC1 and mTORC2. Intensive studies have focused on the roles of mTOR in the regulation of cell proliferation, growth, and survival. Recently we found that rapamycin inhibits type I insulin-like growth factor (IGF-1)-stimulated lamellipodia formation and cell motility, indicating involvement of mTOR in regulating cell motility. This study was set to further elucidate the underlying mechanism. Here we show that rapamycin inhibited protein synthesis and activities of small GTPases (RhoA, Cdc42, and Rac1), crucial regulatory proteins for cell migration. Disruption of mTORC1 or mTORC2 by down-regulation of raptor or rictor, respectively, inhibited the activities of these proteins. However, only disruption of mTORC1 mimicked the effect of rapamycin, inhibiting their protein expression. Ectopic expression of rapamycin-resistant and constitutively active S6K1 partially prevented rapamycin inhibition of RhoA, Rac1, and Cdc42 expression, whereas expression of constitutively hypophosphorylated 4E-BP1 (4EBP1-5A) or down-regulation of S6K1 by RNA interference suppressed expression of the GTPases, suggesting that both mTORC1-mediated S6K1 and 4E-BP1 pathways are involved in protein synthesis of the GTPases. Expression of constitutively active RhoA, but not Cdc42 and Rac1, conferred resistance to rapamycin inhibition of IGF-1-stimulated lamellipodia formation and cell migration. The results suggest that rapamycin inhibits cell motility at least in part by down-regulation of RhoA protein expression and activity through mTORC1-mediated S6K1 and 4E-BP1-signaling pathways.     

4-Hydroxynonenal-induced MEL cell differentiation involves PKC activity translocation

4-Hydroxynonenal (HNE) is a highly reactive aldehyde, produced by cellular lipid peroxidation, able to inhibit proliferation and to induce differentiation in MEL cells at concentrations similar to those detected in several normal tissues. Inducer-mediated differentiation of murine erythroleukemia (MEL) cells is a multiple step process characterized by modulation of several genes as well as by a transient increase in the amount of membrane-associated protein kinase C (PKC) activity. Here we demonstrate that a rapid translocation of PKC activity from cytosol to the membranes occurs during the differentiation induced by HNE. When PKC is completely translocated by phorbol-12-myristate-13-acetate (TPA), the degree of HNE-induced MEL cells differentiation is highly decreased. However, if TPA is washed out from the culture medium before the exposition to the aldehyde, HNE gradually resumes its differentiative ability. The incubation of cells with a selective inhibitor of PKC activity, bisindolylmaleimide GF 109203X, partially prevents the HNE-induced differentiation in MEL cells. In conclusion, our results demonstrate that HNE-induced MEL cell differentiation is preceded by a rapid translocation of PKC activity, and that the inhibition of this phenomenon prevents the onset of terminal differentiation.     

Golgi body motility in the plant cell cortex correlates with actin cytoskeleton organization

The actin cytoskeleton is involved in the transport and positioning of Golgi bodies, but the actin-based processes that determine the positioning and motility behavior of Golgi bodies are not well understood. In this work, we have studied the relationship between Golgi body motility behavior and actin organization in intercalary growing root epidermal cells during different developmental stages. We show that in these cells two distinct actin configurations are present, depending on the developmental stage. In small cells of the early root elongation zone, fine filamentous actin (F-actin) occupies the whole cell, including the cortex. In larger cells in the late elongation zone that have almost completed cell elongation, actin filament bundles are interspersed with areas containing this fine F-actin and areas without F-actin. Golgi bodies in areas with the fine F-actin exhibit a non-directional, wiggling type of motility. Golgi bodies in areas containing actin filament bundles move up to 7 μm s?1. Since the motility of Golgi bodies changes when they enter an area with a different actin configuration, we conclude that the type of movement depends on the actin organization and not on the individual organelle. Our results show that the positioning of Golgi bodies depends on the local actin organization.     

Epithelial cell cytoskeleton and intracellular trafficking V. Confluence of membrane trafficking and motility in epithelial cell models

Migration of epithelial cells occurs in a variety of important biological processes including tissue morphogenesis, wound healing, and the metastasis of epithelial tumors. In some instances, the cells remain attached to each other and migrate together as a sheet, maintaining epithelial integrity. In others (e.g., metastasis), junctional complexes are disrupted and cells migrate individually. In both cases, motility involves the extension of membranous protrusions (filopodia and lamellipodia) in the direction of movement and the transient assembly and disassembly of integrin-mediated adhesions with the extracellular matrix. The driving force for these events is provided by regulated changes in the organization of the actin cytoskeleton, which are thought to be coordinated with alterations in intracellular membrane traffic. In this themes article, I review current hypotheses about how these processes are integrated and attempt to identify fruitful areas for future research.     

Osteogenic differentiation of hypertrophic chondrocytes involves asymmetric cell divisions and apoptosis

We have investigated the early cellular events that take place during the change in lineage commitment from hypertrophic chondrocytes to osteoblast-like cells. We have induced this osteogenic differentiation by cutting through the hypertrophic cartilage of embryonic chick femurs and culturing the explants. Immunocytochemical characterization, [3H]thymidine pulse-chase labeling, in situ nick translation or end labeling of DNA breaks were combined with ultrastructural studies to characterize the changing pattern of differentiation. The first responses to the cutting, seen after 2 d, were upregulation of alkaline phosphatase activity, synthesis of type I collagen and single-stranded DNA breaks, probably indicating a metastable state. Associated with the change from chondrogenic to osteogenic commitment was an asymmetric cell division with diverging fates of the two daughter cells, where one daughter cell remained viable and the other one died. The available evidence suggests that the viable daughter cell then divided and generated osteogenic cells, while the other daughter cell died by apoptosis. The results suggest a new concept of how changes in lineage commitment of differentiated cells may occur. The concepts also reconcile previously opposing views of the fate of the hypertrophic chondrocyte.     

Dynamic changes in chromaffin cell cytoskeleton as prelude to exocytosis

Earlier work by us as well as others has demonstrated that filamentous actin is mainly localized in the cortical surface of chromaffin cell. This F-actin network acts as a barrier to the chromaffin granules, impeding their contact with the plasma membrane. Chromaffin granules contain α-actinin, an anchorage protein that mediates F-actin association with these vesicles. Consequently, chromaffin granules crosslink and stabilize F-actin networks. Stimulation of chromaffin cell produces disassembly of F-actin and removal of the barrier. This interpretation is based on: (1) Cytochemical experiments with rhodamine-labeled phalloidin indicated that in resting chromaffin cells, the F-actin network is visualized as a strong cortical fluorescent ring; (2) Nicotinic receptor stimulation produced fragmentation of this fluorescent ring, leaving chromaffin cell cortical areas devoid of fluorescence; and (3) These changes are accompanied by a decrease in F-actin, a concomitant increase in G-actin, and a decrease in the F-actin associated with the chromaffin cell cytoskeleton (DNAse I assay). We also have demonstrated the presence in chromaffin cells of gelsolin and scinderin, two Ca²⁺-dependent actin filament-severing proteins, and suggested that chromaffin cell stimulation activates scinderin with the consequent disruption of F-actin networks. Scinderin, a protein recently isolated in our laboratory, is restricted to secretory cells and is present mainly in the cortical chromaffin cell cytoplasm. Scinderin, which is structurally different from gelsolin (different pI_s, amino acid composition, peptide maps, and so on), decreases the viscosity of actin gels as a result of its F-actin-severing properties, as demonstrated by electron microscopy. Stimulation of chromaffin cells either by nicotine (10 μM) or high K+ (56 mM) produces a redistribution of subplasmalemmal scinderin and actin disassembly, which preceded exocytosis. The redistribution of scinderin and exocytosis is Ca²⁺-dependent and is not mediated by muscarinic receptors. Furthermore, our cytochemical experiments demonstrate that chromaffin cell stimulation produces a concomitant and similar redistribution of scinderin (fluorescein-labeled antibody) and F-actin (rhodamine phalloidin fluorescence), suggesting a functional interaction between these two proteins. Stimulation-induced redistribution of scinderin and F-actin disassembly would produce subplasmalemmal areas of decreased cytoplasmic viscosity and increased mobility for chromaffin granules. Exocytosis sites, evaluated by antidopamine-β-hydroxylase (anti-DβH) surface staining, are preferentially localized in plasma membrane areas devoid of F-actin.     

The urokinase plasminogen activator receptor in the regulation of the actin cytoskeleton and cell motility

Cell migration is a complex process requiring tight control of several mechanisms including dynamic reorganization of the actin cytoskeleton and adhesion to the extracellular matrix. The GPI-anchored urokinase plasminogen activator receptor (uPAR) has an important role in the regulation of cell motility in many cell types. This is partly due to the localization of proteolytic activity on the cell surface by binding of the serine protease uPA. Results accumulated over the last decade suggest that uPAR is also involved in motility control through other mechanisms. These include induction of signal transduction events after ligation with uPA, binding to the extracellular matrix molecule vitronectin (VN), and association with integrins and other transmembrane partners. In this review these mechanisms will be discussed with a special emphasis on how the GPI-linked receptor transmits signals to the intracellular milieu and how uPAR participates in the regulation of actin cytoskeleton reorganization and cell adhesion during cell migration.     

Apicomplexan cytoskeleton and motors: Key regulators in morphogenesis, cell division, transport and motility

Protozoan parasites of the phylum Apicomplexa undergo a lytic cycle whereby a single zoite produced by the previous cycle has to encounter a host cell, invade it, multiply to differentiate into a new zoite generation and escape to resume a new cycle. At every step of this lytic cycle, the cytoskeleton and/or the gliding motility apparatus play a crucial role and recent results have elucidated aspects of these processes, especially in terms of the molecular characterization and interaction of the increasing number of partners involved, and the signalling mechanisms implicated. The present review aims to summarize the most recent findings in the field.     

Aluminum-induced rapid changes in the microtubular cytoskeleton of tobacco cell lines

Aluminum (Al) is a major factor that limits plant growth in acid soils. It causes a cessation of root growth and changes in root morphology suggesting a role of the root cytoskeleton as a target of Al-toxicity. Here we report a rapid effect of Al on the microtubular cytoskeleton of the suspension tobacco cell lines BY-2 and VBI-0. Viability studies showed that the cells were more sensitive to Al during exponential phase as compared to stationary cells. During the first hours of exposure, Al induced the formation of additional bundles of cortical microtubules (cMTs), whereas the thickness of the individual bundles decreased. Prolonged exposure resulted in disorientation of cMTs. These changes of cMTs preceded the decrease of cell viability by several hours and were accompanied by an increase in the levels of alpha-tubulin (in its tyrosinated form) and elements of the tubulin-folding chaperone CCT. These findings suggest that the microtubular cytoskeleton is one of the early targets of Al toxicity.     

Calmodulin bound to stress fibers but not microtubules involves regulation of cell morphology and motility

Calmodulin (CaM) is a major cytoplasmic calcium receptor that performs multiple functions including cell motility. To investigate the mechanism of the regulation of CaM on cell morphology and motility, first we checked the distribution of CaM in the living cells using GFP-CaM as an indicator. We found that GFP-CaM showed a fiber-like distribution pattern in the cytosol of living Potorous tridactylis kidney (PtK2) cells but not in living HeLa cells. The endogenous CaM in heavily permeabilized HeLa was also found to display a fiber-like distribution pattern. Further examination showed that the distribution pattern of GFP-CaM was same as that of stress fibers, but not microtubules. Co-immunoprecipitation also showed that CaM can interact with actin directly or indirectly. The microinjection of trp peptide, a specific inhibitor of CaM, attenuated the polymerization of stress fibers and induced the alteration of cell morphology. A wound-healing assay and a single cell tracking experiment showed that CaM in PtK2 cells could increase cell motility. The data we have got from living cells suggested that CaM affect cell morphology and motility through binding to stress fibers and regulate f-actin polymerization.