BAR domain competition during directional cellular migration

While directed cellular migration facilitates the coordinated movement of cells during development and tissue repair, the precise mechanisms regulating the interplay between the extracellular environment, the actin cytoskeleton and the overlying plasma membrane remain inadequately understood. The BAR domain family of lipid binding, actin cytoskeletal regulators are gaining greater appreciation for their role in these critical processes. BAR domain proteins are involved as both positive and negative regulators of endocytosis, membrane plasticity and directional cell migration. This review focuses on the functional relationship between different classes of BAR domain proteins and their role in guiding cell migration through regulation of the endocytic machinery. Competition for key signaling substrates by positive and negative BAR domain endocytic regulators appears to mediate control of directional cell migration, and may have wider applicability to other trafficking functions associated with development and carcinogenesis.Key words: BAR domain, MIM, directed cell migration, competition, membrane dynamics, actin cytoskeleton, endocytosis     

Superoxide plays critical roles in electrotaxis of fibrosarcoma cells via activation of ERK and reorganization of the cytoskeleton

Direct-current electrical field (DCEF) induces directional migration in many cell types by activating intracellular signaling pathways. However, the mechanisms coupling the extracellular electric stimulation to the intracellular signals remain largely unknown. In this study, we show that DCEF directs migration of HT-1080 fibrosarcoma cells to the cathode, stimulates generation of hydrogen peroxide and superoxide through the activation of NADPH oxidase, induces anode-facing cytoskeleton polarization, and activates ERK signaling. Subsequent studies demonstrate that the electrotaxis of HT-1080 fibrosarcoma cells is abolished by NADPH oxidase inhibitor and overexpression of manganese superoxide dismutase (MnSOD), an enzyme that hydrolyzes superoxide. In contrast, overexpression of catalases, which hydrolyze hydrogen peroxide, does not affect electrotaxis. MnSOD overexpression also eliminates cytoskeleton polarization as well as the activation of AKT, ERKs, and p38. In contrast, under catalase overexpression, the cytoskeleton still polarizes and p38 activation is affected. Finally, we show that inhibition of ERK activation also abolishes DCEF-induced directional migration and cytoskeleton polarization. Collectively, our results indicate that superoxide plays critical roles in DCEF-induced directional migration of fibrosarcoma cells, possibly by regulating the activation of ERKs. This study provides novel insights into the current understanding of DCEF-mediated cancer cell directional migration and metastasis.     

Caveolin-1 in cell polarization and directional migration

Migration is a complex process in which cells move in a given direction either in response to changes in the extracellular environment or as a consequence of an intrinsic propensity for directional movement. Migration plays key roles in many physiological and pathological processes, including development, angiogenesis, tissue regeneration and metastasis. An important role in migration is played by caveolin-1 and caveolae. Caveolae compartmentalize intracellular signalling pathways to orchestrate cell migration. Caveolin-1 presents a polarized distribution in migrating cells and is linked to the cytoskeleton, and changes in its expression modulate migration. Although there are some discrepancies regarding the regulatory effect of caveolin-1, most studies show that it promotes cell movement and polarity. The importance of caveolin-1 has recently been reinforced by studies with Cav1(-/-) cells, which indicate that it establishes polarity during directional migration by coordinating Src kinase and Rho GTPase signalling.     

BAR domain competition during directional cellular migration

While directed cellular migration facilitates the coordinated movement of cells during development and tissue repair, the precise mechanisms regulating the interplay between the extracellular environment, the actin cytoskeleton, and the overlying plasma membrane remain inadequately understood. The BAR domain family of lipid binding, actin cytoskeletal regulators are gaining greater appreciation for their role in these critical processes. BAR domain proteins are involved as both positive and negative regulators of endocytosis, membrane plasticity, and directional cell migration. This review focuses on the functional relationship between different classes of BAR domain proteins and their role in guiding cell migration through regulation of the endocytic machinery. Competition for key signaling substrates by positive and negative BAR domain endocytic regulators appears to mediate control of directional cell migration, and may have wider applicability to other trafficking functions associated with development and carcinogenesis.     

Symmetry-breaking in mammalian cell cohort migration during tissue pattern formation: role of random-walk persistence

Coordinated, cohort cell migration plays an important role in the morphogenesis of tissue patterns in metazoa. However, individual cells intrinsically move in a random walk-like fashion when studied in vitro. Hence, in the absence of an external orchestrating influence or template, the emergence of cohort cell migration must involve a symmetry-breaking event. To study this process, we used a novel experimental system in which multiple capillary endothelial cells exhibit spontaneous and robust cohort migration in the absence of chemical gradients when cultured on micrometer-scale extracellular matrix islands fabricated using microcontact printing. A computational model suggested that directional persistence of random-walk and dynamic mechanical coupling of adjacent cells are the critical control parameters for this symmetry-breaking behavior that is induced in spatially-constrained cell ensembles. The model predicted our finding that fibroblasts, which exhibit a much shorter motility persistence time than endothelial cells, failed to undergo symmetry breaking or produce cohort migration on the matrix islands. These findings suggest that cells have intrinsic motility characteristics that are tuned to match their role in tissue patterning. Our results underscore the importance of studying cell motility in the context of cell populations, and the need to address emergent features in multicellular organisms that arise not only from cell-cell and cell-matrix interactions, but also from properties that are intrinsic to individual cells.     

Relationships between glycosaminoglycan and receptor binding sites in chemokines-the CXCL12 example

Chemokines are small proteins, promoting directional migration and activation of different cells through binding to specific receptors. Most chemokines also bind to heparan sulfate (HS), a family of complex and highly sulfated glycosaminoglycan (GAG) found at the cell surface and in the extracellular matrix. This class of molecules has recently emerged as critical regulators of many events involving cell response to the external environment. Binding to HS is thought to be functionally important. Current models suggested that HS ensures the correct positioning of chemokines within tissues and maintains haptotactic gradients of the proteins along cell surfaces, thus providing directional cues for migrating cells. On the chemokine surface, the GAG binding epitopes can be displayed on different areas, some of which overlap the receptor binding domain, while others are clearly separated. We review here some structural aspects of the interaction between GAGs or receptors and chemokines. In particular, we will address the case of CXCL12, a chemokine whose receptor binding site is distinct from the GAG binding site and whose different isoforms display different GAG binding abilities. This chemokine system thus offers an unprecedented opportunity to ascertain the importance of chemokine/GAG interaction in the regulation of cell migration.     

Spatial distribution of filament elasticity determines the migratory behaviors of a cell

Any cellular response leading to morphological changes is highly tuned to balance the force generated from structural reorganization, provided by actin cytoskeleton. Actin filaments serve as the backbone of intracellular force, and transduce external mechanical signal via focal adhesion complex into the cell. During migration, cells not only undergo molecular changes but also rapid mechanical modulation. Here we focus on determining, the role of spatial distribution of mechanical changes of actin filaments in epithelial, mesenchymal, fibrotic and cancer cells with non-migration, directional migration, and non-directional migration behaviors using the atomic force microscopy. We found 1) non-migratory cells only generated one type of filament elasticity, 2) cells generating spatially distributed two types of filament elasticity showed directional migration, and 3) pathologic cells that autonomously generated two types of filament elasticity without spatial distribution were actively migrating non-directionally. The demonstration of spatial regulation of filament elasticity of different cell types at the nano-scale highlights the coupling of cytoskeletal function with physical characters at the sub-cellular level, and provides new research directions for migration related disease.     

Analysis of directional cell migration on defined FN gradients: role of intracellular signaling molecules

Directional cell migration on extracellular matrix (ECM) plays important roles in embryonic development and adult organisms. To study the mechanisms and signaling pathways involved in the regulation of directional cell migration, we created defined fibronectin (FN) gradients by using microfluidic systems. We found that fibroblasts exhibited haptotaxis towards higher FN concentration on the gradient. Furthermore, the net movements in the direction of FN gradients correlated with the increase in the slope of the gradient although the overall rate of migration was not correlated. Consistent with previous observations on the uniformly coated surface, local higher FN concentration led to reduced migration rate due to increased spreading. Upon transfection of N-WASP or activated Cdc42, but not FAK or Grb7, the cells showed increased directional migration. However, transfection of FAK, but not the other signaling molecules, led to an increase in the persistence of directional cell migration, which is dependent on the slope of the gradient as well as FAK interaction with PI3K. Together, these studies reveal some novel properties of directional cell migration on defined FN gradient and suggested a role for FAK signaling and N-WASP and Cdc42 in the differential regulation of the persistence and rate of directional cell migration.     

Mechanotransduction in endothelial cell migration

The migration of endothelial cells (ECs) plays an important role in vascular remodeling and regeneration. EC migration can be regulated by different mechanisms such as chemotaxis, haptotaxis, and mechanotaxis. This review will focus on fluid shear stress-induced mechanotransduction during EC migration. EC migration and mechanotransduction can be modulated by cytoskeleton, cell surface receptors such as integrins and proteoglycans, the chemical and physical properties of extracellular matrix (ECM) and cell-cell adhesions. The shear stress applied on the luminal surface of ECs can be sensed by cell membrane and associated receptor and transmitted throughout the cell to cell-ECM adhesions and cell-cell adhesions. As a result, shear stress induces directional migration of ECs by promoting lamellipodial protrusion and the formation of focal adhesions (FAs) at the front in the flow direction and the disassembly of FAs at the rear. Persistent EC migration in the flow direction can be driven by polarized activation of signaling molecules and the positive feedback loops constituted by Rho GTPases, cytoskeleton, and FAs at the leading edge. Furthermore, shear stress-induced EC migration can overcome the haptotaxis of ECs. Given the hemodynamic environment of the vascular system, mechanotransduction during EC migration has a significant impact on vascular development, angiogenesis, and vascular wound healing.     

Control of myofibroblast differentiation and function by cytoskeletal signaling

The cytoskeleton consists of three distinct types of protein polymer structures–microfilaments, intermediate filaments, and microtubules; each serves distinct roles in controlling cell shape, division, contraction, migration, and other processes. In addition to mechanical functions, the cytoskeleton accepts signals from outside the cell and triggers additional signals to extracellular matrix, thus playing a key role in signal transduction from extracellular stimuli through dynamic recruitment of diverse intermediates of the intracellular signaling machinery. This review summarizes current knowledge about the role of cytoskeleton in the signaling mechanism of fibroblast-to-myofibroblast differentiation–a process characterized by accumulation of contractile proteins and secretion of extracellular matrix proteins, and being critical for normal wound healing in response to tissue injury as well as for aberrant tissue remodeling in fibrotic disorders. Specifically, we discuss control of serum response factor and Hippo signaling pathways by actin and microtubule dynamics as well as regulation of collagen synthesis by intermediate filaments.     

A Novel Chemotaxis Assay in 3-D Collagen Gels by Time-Lapse Microscopy

The directional cell response to chemical gradients, referred to as chemotaxis, plays an important role in physiological and pathological processes including development, immune response and tumor cell invasion. Despite such implications, chemotaxis remains a challenging process to study under physiologically-relevant conditions in-vitro, mainly due to difficulties in generating a well characterized and sustained gradient in substrata mimicking the in-vivo environment while allowing dynamic cell imaging. Here, we describe a novel chemotaxis assay in 3D collagen gels, based on a reusable direct-viewing chamber in which a chemoattractant gradient is generated by diffusion through a porous membrane. The diffusion process has been analysed by monitoring the concentration of FITC-labelled dextran through epifluorescence microscopy and by comparing experimental data with theoretical and numerical predictions based on Fick''s law. Cell migration towards chemoattractant gradients has been followed by time-lapse microscopy and quantified by cell tracking based on image analysis techniques. The results are expressed in terms of chemotactic index (I) and average cell velocity. The assay has been tested by comparing the migration of human neutrophils in isotropic conditions and in the presence of an Interleukin-8 (IL-8) gradient. In the absence of IL-8 stimulation, 80% of the cells showed a velocity ranging from 0 to 1 µm/min. However, in the presence of an IL-8 gradient, 60% of the cells showed an increase in velocity reaching values between 2 and 7 µm/min. Furthermore, after IL-8 addition, I increased from 0 to 0.25 and 0.25 to 0.5, respectively, for the two donors examined. These data indicate a pronounced directional migration of neutrophils towards the IL-8 gradient in 3D collagen matrix. The chemotaxis assay described here can be adapted to other cell types and may serve as a physiologically relevant method to study the directed locomotion of cells in a 3D environment in response to different chemoattractants.     

Filamin structure,function and mechanics: are altered filamin-mediated force responses associated with human disease?

The cytoskeleton framework is essential not only for cell structure and stability but also for dynamic processes such as cell migration, division and differentiation. The F-actin cytoskeleton is mechanically stabilised and regulated by various actin-binding proteins, one family of which are the filamins that cross-link F-actin into networks that greatly alter the elastic properties of the cytoskeleton. Filamins also interact with cell membrane-associated extracellular matrix receptors and intracellular signalling proteins providing a potential mechanism for cells to sense their external environment by linking these signalling systems. The stiffness of the external matrix to which cells are attached is an important environmental variable for cellular behaviour. In order for a cell to probe matrix stiffness, a mechanosensing mechanism functioning via alteration of protein structure and/or binding events in response to external tension is required. Current structural, mechanical, biochemical and human disease-associated evidence suggests filamins are good candidates for a role in mechanosensing.     

Regulation of microtubules in cell migration

Directional cell migration is a fundamental process in all organisms that is stringently regulated during tissue development, chemotaxis and wound healing. Migrating cells have a polarized morphology with an asymmetrical distribution of signaling molecules and the cytoskeleton. Microtubules are indispensable for the directional migration of certain cells. Recent studies have shown that Rho family GTPases, which are key regulators of cell migration, affect microtubules, in addition to the actin cytoskeleton and adhesion. Rho family GTPases capture and stabilize microtubules through their effectors at the cell cortex, leading to a polarized microtubule array; in turn, microtubules modulate the activities of Rho family GTPases. In this article, we discuss how a polarized microtubule array is established and how microtubules facilitate cell migration.     

Using light to shape chemical gradients for parallel and automated analysis of chemotaxis

Numerous molecular components have been identified that regulate the directed migration of eukaryotic cells toward sources of chemoattractant. However, how the components of this system are wired together to coordinate multiple aspects of the response, such as directionality, speed, and sensitivity to stimulus, remains poorly understood. Here we developed a method to shape chemoattractant gradients optically and analyze cellular chemotaxis responses of hundreds of living cells per well in 96‐well format by measuring speed changes and directional accuracy. We then systematically characterized migration and chemotaxis phenotypes for 285 siRNA perturbations. A key finding was that the G‐protein G_iα subunit selectively controls the direction of migration while the receptor and Gβ subunit proportionally control both speed and direction. Furthermore, we demonstrate that neutrophils chemotax persistently in response to gradients of fMLF but only transiently in response to gradients of ATP. The method we introduce is applicable for diverse chemical cues and systematic perturbations, can be used to measure multiple cell migration and signaling parameters, and is compatible with low‐ and high‐resolution fluorescence microscopy.     

Shear stress stimulates integrin β1 trafficking and increases directional migration of cancer cells via promoting deacetylation of microtubules

In egress routes of malignancy, cancer cells are constantly subjected to shear stress imposed by blood/lymph flow. Increasing evidence points toward the regulatory roles of shear stress in tumor cell adhesion and motility. Although it is known that integrin endocytic trafficking governs focal adhesion (FA) turnover and cell migration, the effect and biological consequences of low shear stress (LSS) on integrin trafficking remain unclear. Here, we identified the critical role of integrin β1 trafficking and caveolin-1 (Cav-1) mediated endocytosis in LSS-induced cell directional migration. LSS altered the distribution of integrin β1 in MDA-MB-231 cells and significantly promoted its internalization and recycling, which in turn facilitated FA turnover and directional cell migration. Furthermore, LSS induced cytoskeleton remodeling, which was required for internalization of integrin β1. LSS down-regulated the acetylation level of microtubules (MTs) via activating ROCK/HDAC6 pathway, resulting in elevation of MTs dynamics, Cav-1 motility, and Cav-1-dependent integrin β1 recycling. We also showed that high HDAC6 expression was a ROCK-dependent prognostic factor, which was correlated with poor outcomes in breast cancer patients. Taken together, these results defined a novel mechanism by which LSS enhanced integrin β1 trafficking via actin cytoskeleton remodeling and ROCK/HDAC6 mediated deacetylation of MTs, thereby promoting FAs turnover and directional cell migration.     

Differential migration and proliferation of geometrical ensembles of cell clusters

Differential cell migration and growth drives the organization of specific tissue forms and plays a critical role in embryonic development, tissue morphogenesis, and tumor invasion. Localized gradients of soluble factors and extracellular matrix have been shown to modulate cell migration and proliferation. Here we show that in addition to these factors, initial tissue geometry can feedback to generate differential proliferation, cell polarity, and migration patterns. We apply layer by layer polyelectrolyte assembly to confine multicellular organization and subsequently release cells to demonstrate the spatial patterns of cell migration and growth. The cell shapes, spreading areas, and cell–cell contacts are influenced strongly by the confining geometry. Cells within geometric ensembles are morphologically polarized. Symmetry breaking was observed for cells on the circular pattern and cells migrate toward the corners and in the direction parallel to the longest dimension of the geometric shapes. This migration pattern is disrupted when actomyosin based tension was inhibited. Cells near the edge or corner of geometric shapes proliferate while cells within do not. Regions of higher rate of cell migration corresponded to regions of concentrated growth. These findings demonstrate that multicellular organization can result in spatial patterns of migration and proliferation.     

JWA as a functional molecule to regulate cancer cells migration via MAPK cascades and F-actin cytoskeleton

Mitogen activated protein kinase (MAPK) cascades are thought to mediate diverse biological functions such as cell growth, differentiation and migration. Activated MAPK may affect microtubule (MT) which is essential for cellular polarity, differentiation and motility. Data in this study show that JWA, a newly identified novel microtubule-associated protein (MAP) was essential for the rearrangement of F-actin cytoskeleton and activation of MAPK cascades induced by arsenic trioxide (As2O3) and phorbol ester (PMA). Over-expression of JWA alone in HeLa, B16 and HCCLM3 cancer cells effectively inhibited cellular migration; whereas, cellular migration was significantly accelerated when cells were deficient in JWA expression. The mechanism underlying these phenomena might be due to JWA affected F-actin rearrangement. Furthermore, JWA deficiency blocked anti-migratory effect produced by As2O3 but enhanced the migratory effect initiated by PMA in HeLa cells. JWA SDR-SLR motifs are not only critical for the MAPK cascades activation, but also for cell migration. Further studies found that JWA differentially regulated cell migration via ERK downstream effectors focal adhesion kinase (FAK) and cyclooxygenase-2 (COX-2). Therefore, JWA regulated-tumor cellular migration might involve MAPK cascades activation and F-actin cytoskeleton rearrangement mechanisms. Our data provide an unexpected role for JWA in tumor cell migration behaviors.     

Prespecification and plasticity: shifting mechanisms of cell migration

Cell migration is a universal process involving different morphologies and mechanisms in different cell types and tissue environments. Prespecified cell-type-specific patterns of cell migration can be classified into single cell migration (amoeboid, mesenchymal) and collective migration modes (cell sheets, strands, tubes, clusters). These intrinsic molecular programs are associated with a characteristic structure of the actin cytoskeleton, as well as the cell-type-specific use of integrins, matrix-degrading enzymes (matrix metalloproteinases and serine proteases), cell-cell adhesion molecules (cadherins and activated leukocyte adhesion molecule), and signaling towards the cytoskeleton (carried out by RHO GTPases). In response to the gain or loss of these key molecular determinants, significant adaptation reactions can modify the cell's shape, pattern, and migration mechanism; examples of this include the epithelial-mesenchymal transition, mesenchymal-amoeboid transition and collective-amoeboid transition.     

Biophysical interactions between components of the tumor microenvironment promote metastasis

During metastasis, tumor cells need to adapt to their dynamic microenvironment and modify their mechanical properties in response to both chemical and mechanical stimulation. Physical interactions occur between cancer cells and the surrounding matrix including cell movements and cell shape alterations through the process of mechanotransduction. The latter describes the translation of external mechanical cues into intracellular biochemical signaling. Reorganization of both the cytoskeleton and the extracellular matrix (ECM) plays a critical role in these spreading steps. Migrating tumor cells show increased motility in order to cross the tumor microenvironment, migrate through ECM and reach the bloodstream to the metastatic site. There are specific factors affecting these processes, as well as the survival of circulating tumor cells (CTC) in the blood flow until they finally invade the secondary tissue to form metastasis. This review aims to study the mechanisms of metastasis from a biomechanical perspective and investigate cell migration, with a focus on the alterations in the cytoskeleton through this journey and the effect of biologic fluids on metastasis. Understanding of the biophysical mechanisms that promote tumor metastasis may contribute successful therapeutic approaches in the fight against cancer.