The induction of protrusion by neomycin in human glioma cells is correlated with a decrease followed by an increase in filamentous actin.

In this paper we describe an experimental investigation of the mechanism of motility of vertebrate cells. Human glioma cells were treated with neomycin, an inhibitor of the phosphatidylinositol cycle; and changes in cell motility and the cytoskeleton were examined by video, fluorescence, and scanning electron microscopy and by cytofluorometry. Neomycin stimulates a single protrusion of lamellipodia from the cell margin, which is correlated with an initial rapid decrease in the amount of F-actin throughout the cell, especially at the cell edge; the fragmentation of actin filaments within the lamellipodia; and the subsequent de novo polymerization of F-actin in a marginal band at the leading edge of lamellipodia. Changes in F-actin are paralleled by changes in the distribution and amount of gelsolin. These results support the hypothesis that protrusion is initiated by the gelsolin-mediated severing and subsequent depolymerization of cortical actin filaments, which weakens the cell cortex, allowing hydrostatic or gel osmotic pressure to force the cell margin to protrude. The accompanying polymerization of filaments actin at the leading edge of the protrusion may stabilize the protrusion and support its expansion.     

Actin filament polymerization regulates gliding motility by apicomplexan parasites

Host cell entry by Toxoplasma gondii depends critically on actin filaments in the parasite, yet paradoxically, its actin is almost exclusively monomeric. In contrast to the absence of stable filaments in conventional samples, rapid-freeze electron microscopy revealed that actin filaments were formed beneath the plasma membrane of gliding parasites. To investigate the role of actin filaments in motility, we treated parasites with the filament-stabilizing drug jasplakinolide (JAS) and monitored the distribution of actin in live and fixed cells using yellow fluorescent protein (YFP)-actin. JAS treatment caused YFP-actin to redistribute to the apical and posterior ends, where filaments formed a spiral pattern subtending the plasma membrane. Although previous studies have suggested that JAS induces rigor, videomicroscopy demonstrated that JAS treatment increased the rate of parasite gliding by approximately threefold, indicating that filaments are rate limiting for motility. However, JAS also frequently reversed the normal direction of motility, disrupting forward migration and cell entry. Consistent with this alteration, subcortical filaments in JAS-treated parasites occurred in tangled plaques as opposed to the straight, roughly parallel orientation observed in control cells. These studies reveal that precisely controlled polymerization of actin filaments imparts the correct timing, duration, and directionality of gliding motility in the Apicomplexa.     

The coordination between actin filaments and adhesion in mesenchymal migration

Mesenchymal cell motility is characterized by a polarized distribution of actin filaments, with a network of short branched actin filaments at the leading edge, and polymers of actin filaments arranged into distinct classes of actin stress fibres behind the leading edge. Importantly, the distinct actin filaments are characteristically associated with discrete adhesion structures and both the adhesions and the actin filaments are co-ordinately regulated during cell migration. While it has long been known that these macromolecular structures are intimately linked in cells, precisely how they are co-ordinately regulated is presently unknown. Live imaging data now suggests that the focal adhesions may act as sites of actin polymerization resulting in the generation of tension-bearing actin bundles of actin filaments (stress fibres). Moreover, a picture is emerging to suggest that the tropomyosin family of proteins that can determine actin filament dynamics may also play a key role in determining the transition between adhesion states. Molecules such as the tropomyosins are therefore tantalizing candidates to orchestrate the coordination of actin and adhesion dynamics during mesenchymal cell migration.     

C-terminus of Hsc70-interacting protein regulates profilin1 and breast cancer cell migration

Profilin1 (Pfn1) is a key mediator of actin polymerization and regulates cell migration. Low expression of Pfn1 is implicated in tumorigenesis of various cancers, including breast cancer. The regulatory mechanism behind Pfn1 levels has not yet been elucidated. In the present study, we find that Pfn1 is poly-ubiquitinated in human cell lines, and a portion of poly-ubiquitinated Pfn1 is regulated in a proteasome-dependent manner. C-terminus of Hsc70-interacting protein (CHIP), a co-chaperone E3 ligase, interacts with and ubiquitinates Pfn1, targeting it for proteasome-dependent degradation. Depletion of CHIP stabilizes Pfn1, suggesting that CHIP functions as a major E3 ligase for Pfn1. Stable expression of wild-type CHIP in the breast cancer cell line MDA-MB231 yielded downregulation of Pfn1 and enhanced cell migration. Pfn1 overexpression in MDA-MB231 cells expressing wild-type CHIP suppressed the enhanced cell migration. Taken together, our results demonstrate that CHIP regulates Pfn1 levels as an E3 ligase, and possibly plays a role in cell migration and metastasis of breast cancer.     

BCL2 interaction with actin in vitro may inhibit cell motility by enhancing actin polymerization

In addition to its well-defined role as an antagonist in apoptosis, we propose that BCL2 may act as an intracellular suppressor of cell motility and adhesion under certain conditions. Our evidence shows that, when overexpressed in both cancer and non-cancer cells, BCL2 can form a complex with actin and gelsolin that functions to decrease gelsolin-severing activity to increase actin polymerization and, thus, suppress cell adhesive processes. The linkage between increased BCL2 and increased actin polymerization on the one hand and suppression of cell adhesion, spreading and motility on the other hand, is a novel observation that may provide a plausible explanation for why BCL2 overexpression in some tumors is correlated with improved patient survival. In addition, we have identified conditions in vitro in which F-actin polymerization can be increased while cell motility is reduced. These findings underscore the possibility that BCL2 may be involved in modulating cytoskeleton reorganization and may provide an opportunity to explore signal transduction pathways important for cell adhesion and migration and to develop small molecule therapies for suppression of cancer metastasis.Key words: BCL2, actin polymerization, cell motility, adhesion     

BCL2 interaction with actin in vitro may inhibit cell motility by enhancing actin polymerization

In addition to its well-defined role as an antagonist in apoptosis, we propose that BCL2 may act as an intracellular suppressor of cell motility and adhesion under certain conditions. Our evidence shows that, when over-expressed in both cancer and non-cancer cells, BCL2 can form a complex with actin and gelsolin that functions to decrease gelsolin-severing activity to increase actin polymerization, and, thus, suppress cell adhesive processes. The linkage between increased BCL2 and increased actin polymerization on the one hand, and suppression of cell adhesion, spreading, and motility on the other hand, is a novel observation that may provide a plausible explanation for why BCL2 over-expression in some tumors is correlated with improved patient survival. In addition, we have identified conditions in vitro in which F-actin polymerization can be increased while cell motility is reduced. These findings underscore the possibility that BCL2 may be involved in modulating cytoskeleton reorganization, and may provide an opportunity to explore signal transduction pathways important for cell adhesion and migration and to develop small molecule therapies for suppression of cancer metastasis.     

How depolymerization can promote polymerization: the case of actin and profilin

Rapid polymerization and depolymerization of actin filaments in response to extracellular stimuli is required for normal cell motility and development. Profilin is one of the most important actin‐binding proteins; it regulates actin polymerization and interacts with many cytoskeletal proteins that link actin to extracellular membrane. The molecular mechanism of profilin has been extensively considered and debated in the literature for over two decades. Here we discuss several accepted hypotheses regarding the mechanism of profilin function as well as new recently emerged possibilities. Thermal noise is routine in molecular world and unsurprisingly, nature has found a way to utilize it. An increasing amount of theoretical and experimental research suggests that fluctuation‐based processes play important roles in many cell events. Here we show how a fluctuation‐based process of exchange diffusion is involved in the regulation of actin polymerization.     

Reorganization of the actin cytoskeleton in the protruding lamellae of human fibroblasts.

To investigate the mechanisms of protrusion in vertebrate cells, the primary event in cell motility, human fibroblasts were treated with neomycin, an inhibitor of the phosphatidylinositol cycle, to induce protrusion. Changes in cell motility and the cytoskeleton were examined by video, fluorescence, scanning electron, and confocal microscopy and by cytofluorometry. Protrusion in neomycin-treated human fibroblasts is correlated with a transient overall decrease in F-actin followed by an increase in F-actin at the leading edge of the protruding lamella. In growing lamellae, F-actin is organized in a marginal band at the leading edge. Although actin is present in the lamella behind the leading edge, very little of it is F-actin. Scanning electron microscopy of detergent-extracted cells reveals a band of dense filaments at the leading edge, corresponding to the marginal band of F-actin seen in fluorescently labeled cells, and a sparse population of short, fragmented filaments, in the rest of the lamella. Gelsolin is colocalized with F-actin in the marginal band and is also present in the lamella where F-actin is largely absent. The data support the hypothesis that the protrusion is initiated by the breakdown of cortical actin filaments, possibly mediated by gelsolin, whereas expansion of the protrusion requires de novo polymerization of actin filaments at the leading edge.     

Role of cofilin in epidermal growth factor-stimulated actin polymerization and lamellipod protrusion

Stimulation of metastatic MTLn3 cells with epidermal growth factor (EGF) causes a rapid and transient increase in actin nucleation activity resulting from the appearance of free barbed ends at the extreme leading edge of extending lamellipods. To investigate the role of cofilin in EGF-stimulated actin polymerization and lamellipod extension in MTLn3 cells, we examined in detail the temporal and spatial distribution of cofilin relative to free barbed ends and characterized the actin dynamics by measuring the changes in the number of actin filaments. EGF stimulation triggers a transient increase in cofilin in the leading edge near the membrane, which is precisely cotemporal with the appearance of free barbed ends there. A deoxyribonuclease I binding assay shows that the number of filaments per cell increases by 1.5-fold after EGF stimulation. Detection of pointed ends in situ using deoxyribonuclease I binding demonstrates that this increase in the number of pointed ends is confined to the leading edge compartment, and does not occur within stress fibers or in the general cytoplasm. Using a light microscope severing assay, cofilin's severing activity was observed directly in cell extracts and shown to be activated after stimulation of the cells with EGF. Microinjection of function-blocking antibodies against cofilin inhibits the appearance of free barbed ends at the leading edge and lamellipod protrusion after EGF stimulation. These results support a model in which EGF stimulation recruits cofilin to the leading edge where its severing activity is activated, leading to the generation of short actin filaments with free barbed ends that participate in the nucleation of actin polymerization.     

VASP governs actin dynamics by modulating filament anchoring

Actin filament dynamics at the cell membrane are important for cell-matrix and cell-cell adhesions and the protrusion of the leading edge. Since actin filaments must be connected to the cell membrane to exert forces but must also detach from the membrane to allow it to move and evolve, the balance between actin filament tethering and detachment at adhesion sites and the leading edge is key for cell shape changes and motility. How this fine tuning is performed in cells remains an open question, but possible candidates are the Drosophila enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family of proteins, which localize to dynamic actin structures in the cell. Here we study VASP-mediated actin-related proteins 2/3 (Arp2/3) complex-dependent actin dynamics using a substrate that mimics the fluid properties of the cell membrane: an oil-water interface. We show evidence that polymerization activators undergo diffusion and convection on the fluid surface, due to continual attachment and detachment to the actin network. These dynamics are enhanced in the presence of VASP, and we observe cycles of catastrophic detachment of the actin network from the surface, resulting in stop-and-go motion. These results point to a role for VASP in the modulation of filament anchoring, with implications for actin dynamics at cell adhesions and at the leading edge of the cell.     

Lysyl oxidase regulates actin filament formation through the p130(Cas)/Crk/DOCK180 signaling complex

We have previously demonstrated that lysyl oxidase (LOX) is expressed in invasive breast cancer cells compared to poorly invasive cells. Additionally, we have recently shown that LOX regulates cell migration, a key step in the invasion process, through a hydrogen peroxide-dependent mechanism involving the focal adhesion kinase (FAK)/Src signaling complex. Here we further elucidate the role of LOX in cell motility/migration by examining the role of LOX in actin filament polymerization. We demonstrate that inhibition of LOX leads to an increase in phalloidin staining, directly associated with an increase in actin stress fiber formation. This increase in staining was confirmed by activity assays showing an increase in Rho activity with decreased LOX activity. Additionally, Rac and Cdc42 activity decreased with the reduction in LOX activity. Taken together, these data demonstrate a loss of a motogenic phenotype with decreased LOX activity. Finally, in order to elucidate the mechanism by which LOX regulates actin polymerization, we have demonstrated that LOX facilitates p130(Cas) phosphorylation, which allows for the binding to CAS related kinase (Crk) and formation of the p130(Cas)/Crk/DOCK180 signaling complex. Formation of this complex leads to an increase in Rac-GTP, which decreases actin stress fiber formation and increases formation of lamellipodium. These data demonstrate that LOX regulates cell motility/migration through changes in actin filament polymerization, which involve the regulation of the p130(Cas)/Crk/DOCK180 signaling pathway. Elucidating the role of LOX in the regulation of cell motility will allow the development of more effective therapeutic strategies to treat invasive/metastatic breast cancer.     

The comings and goings of actin: coupling protrusion and retraction in cell motility

Cells utilize actin filaments to produce protrusive and contractile arrays that cooperate to drive cell motility. The generation of the two arrays and the coupling between them result from the unique properties of the lamellipodium, a protrusive leaflet of cytoplasm at the cell edge. From the lamellipodium into the lamella behind, there is a transition from a fast retrograde flow of actin polymer driven by polymerization to a slow flow driven by the interaction of anti-parallel arrays of actin with myosin. In addition to driving protrusion, the lamellipodium appears to play a role in supplying filaments to the lamella for the assembly of the contractile network required for traction.     

Pointed-end capping by tropomodulin3 negatively regulates endothelial cell motility

Actin filament pointed-end dynamics are thought to play a critical role in cell motility, yet regulation of this process remains poorly understood. We describe here a previously uncharacterized tropomodulin (Tmod) isoform, Tmod3, which is widely expressed in human tissues and is present in human microvascular endothelial cells (HMEC-1). Tmod3 is present in sufficient quantity to cap pointed ends of actin filaments, localizes to actin filament structures in HMEC-1 cells, and appears enriched in leading edge ruffles and lamellipodia. Transient overexpression of GFP-Tmod3 leads to a depolarized cell morphology and decreased cell motility. A fivefold increase in Tmod3 results in an equivalent decrease in free pointed ends in the cells. Unexpectedly, a decrease in the relative amounts of F-actin, free barbed ends, and actin-related protein 2/3 (Arp2/3) complex in lamellipodia are also observed. Conversely, decreased expression of Tmod3 by RNA interference leads to faster average cell migration, along with increases in free pointed and barbed ends in lamellipodial actin filaments. These data collectively demonstrate that capping of actin filament pointed ends by Tmod3 inhibits cell migration and reveal a novel control mechanism for regulation of actin filaments in lamellipodia.     

Adenosine arrests breast cancer cell motility by A3 receptor stimulation

In neutrophils, adenosine triphosphate (ATP) release and autocrine purinergic signaling regulate coordinated cell motility during chemotaxis. Here, we studied whether similar mechanisms regulate the motility of breast cancer cells. While neutrophils and benign human mammary epithelial cells (HMEC) form a single leading edge, MDA-MB-231 breast cancer cells possess multiple leading edges enriched with A3 adenosine receptors. Compared to HMEC, MDA-MB-231 cells overexpress the ectonucleotidases ENPP1 and CD73, which convert extracellular ATP released by the cells to adenosine that stimulates A3 receptors and promotes cell migration with frequent directional changes. However, exogenous adenosine added to breast cancer cells or the A3 receptor agonist IB-MECA dose-dependently arrested cell motility by simultaneous stimulation of multiple leading edges, doubling cell surface areas and significantly reducing migration velocity by up to 75 %. We conclude that MDA-MB-231 cells, HMEC, and neutrophils differ in the purinergic signaling mechanisms that regulate their motility patterns and that the subcellular distribution of A3 adenosine receptors in MDA-MB-231 breast cancer cells contributes to dysfunctional cell motility. These findings imply that purinergic signaling mechanisms may be potential therapeutic targets to interfere with the motility of breast cancer cells in order to reduce the spread of cancer cells and the risk of metastasis.     

The role of nitric oxide in reducing deformability of Lewis lung tumor cell stimulated by inflammatory cytokines

Highly metastatic cells, especially in the lungs, are known to be resistant to nitric oxide (NO)-mediated cytotoxicity, compared with poorly or non-metastatic cells. However, the precise mechanisms connecting NO and metastasis remain to be determined. To clarify the role of NO in the characteristic changes in NO-resistant cells in response to inflammatory cytokines, we used Lewis lung tumor (LLT) cells, which are known to be highly metastatic NO-resistant cells, and determined the changes in cell deformability and the gene expression profile after the cells were stimulated using cytokine mixture or an NO donor. Both exogenous NO and endogenous NO via inducible NO synthase produced by cytokines decreased cell deformability by enhancing actin polymerization. The expression of several genes associated with actin polymerization was changed so as to increase actin filaments in the cells by enhancing actin polymerization and by suppressing actin depolymerization, actin filament severing, and barbed-end actin filament capping. In conclusion, inflammatory cytokine stimulation reduces deformability of LLT cells and enhances actin polymerization which is mainly controlled by the same genes induced by NO.     

Quantification of Cell Edge Velocities and Traction Forces Reveals Distinct Motility Modules during Cell Spreading

Actin-based cell motility and force generation are central to immune response, tissue development, and cancer metastasis, and understanding actin cytoskeleton regulation is a major goal of cell biologists. Cell spreading is a commonly used model system for motility experiments – spreading fibroblasts exhibit stereotypic, spatially-isotropic edge dynamics during a reproducible sequence of functional phases: 1) During early spreading, cells form initial contacts with the surface. 2) The middle spreading phase exhibits rapidly increasing attachment area. 3) Late spreading is characterized by periodic contractions and stable adhesions formation. While differences in cytoskeletal regulation between phases are known, a global analysis of the spatial and temporal coordination of motility and force generation is missing. Implementing improved algorithms for analyzing edge dynamics over the entire cell periphery, we observed that a single domain of homogeneous cytoskeletal dynamics dominated each of the three phases of spreading. These domains exhibited a unique combination of biophysical and biochemical parameters – a motility module. Biophysical characterization of the motility modules revealed that the early phase was dominated by periodic, rapid membrane blebbing; the middle phase exhibited continuous protrusion with very low traction force generation; and the late phase was characterized by global periodic contractions and high force generation. Biochemically, each motility module exhibited a different distribution of the actin-related protein VASP, while inhibition of actin polymerization revealed different dependencies on barbed-end polymerization. In addition, our whole-cell analysis revealed that many cells exhibited heterogeneous combinations of motility modules in neighboring regions of the cell edge. Together, these observations support a model of motility in which regions of the cell edge exhibit one of a limited number of motility modules that, together, determine the overall motility function. Our data and algorithms are publicly available to encourage further exploration.     

WIP: a multifunctional protein involved in actin cytoskeleton regulation

Knowledge of the dynamics of actin-based structures is a major key to understanding how cells move and respond to their environment. The ability to reorganize actin filaments in a spatial and temporal manner to integrate extracellular signals is at the core of cell adhesion and cell migration. Several proteins have been described as regulators of actin polymerization: this review will focus on the role of WASP-interacting protein (WIP), an actin-binding protein that participates in actin polymerization regulation and signal transduction. WIP is widely expressed and interacts with Wiskott-Aldrich syndrome protein (WASP) (a hematopoietic-specific protein) and its more widely expressed homologue neural WASP (N-WASP), to regulate WASP/N-WASP function in Arp2/3-mediated actin polymerization. WIP also interacts with profilin, globular and filamentous actin (G- and F-actin, respectively) and stabilizes actin filaments. In vivo WIP participates in filopodia and lamellipodia formation, in T and B lymphocyte activation, in mast cell degranulation and signaling through the Fcepsilon receptor (FcepsilonR), in microbial motility and in Syk protein stability.     

The essential role of profilin in the assembly of actin for microspike formation.

Profilin was first identified as an actin monomer binding protein; however, recent reports indicate its involvement in actin polymerization. To date, there is no direct evidence of a functional role in vivo for profilin in actin cytoskeletal reorganization. Here, we prepared a profilin mutant (H119E) defective in actin binding, but retaining the ability to bind to other proteins. This mutant profilin I suppresses actin polymerization in microspike formation induced by N-WASP, the essential factor in microspike formation. Profilin associates both in vivo and in vitro with N-WASP at proline-rich sites different from those to which Ash/Grb2 binds. This association between profilin and N-WASP is required for N-WASP-induced efficient microspike elongation. Moreover, we succeeded in reconstituting microspike formation in permeabilized cells using profilin I combined with N-WASP and its regulator, Cdc42. These findings provide the first evidence that profilin is a key molecule linking a signaling network to rapid actin polymerization in microspike formation.     

Individual actin filaments in a microfluidic flow reveal the mechanism of ATP hydrolysis and give insight into the properties of profilin

The hydrolysis of ATP associated with actin and profilin-actin polymerization is pivotal in cell motility. It is at the origin of treadmilling of actin filaments and controls their dynamics and mechanical properties, as well as their interactions with regulatory proteins. The slow release of inorganic phosphate (Pi) that follows rapid cleavage of ATP gamma phosphate is linked to an increase in the rate of filament disassembly. The mechanism of Pi release in actin filaments has remained elusive for over 20 years. Here, we developed a microfluidic setup to accurately monitor the depolymerization of individual filaments and determine their local ADP-Pi content. We demonstrate that Pi release in the filament is not a vectorial but a random process with a half-time of 102 seconds, irrespective of whether the filament is assembled from actin or profilin-actin. Pi release from the depolymerizing barbed end is faster (half-time of 0.39 seconds) and further accelerated by profilin. Profilin accelerates the depolymerization of both ADP- and ADP-Pi-F-actin. Altogether, our data show that during elongation from profilin-actin, the dissociation of profilin from the growing barbed end is not coupled to Pi release or to ATP cleavage on the terminal subunit. These results emphasize the potential of microfluidics in elucidating actin regulation at the scale of individual filaments.