Regulation of cell migration during tracheal development in Drosophila melanogaster

Most of the knowledge concerning the intracellular mechanisms involved in cell locomotion have been obtained from in vitro studies of cells in culture. Many of the concepts derived from these studies have been partially confirmed in in vivo systems but numerous questions regarding the developmental control of cell migration remain to be addressed. Tracheal morphogenesis in Drosophila melanogaster embryos represents an in vivo model system to study the genetic control of cell migration. We review what is known about tracheal development and regulation of tracheal cell migration. We try to link these in vivo studies and the movement of cells over two dimensional substrates and elaborate on important questions which remain to be addressed in the future.     

Computational modelling and analysis of mechanical conditions on cell locomotion and cell–cell interaction

Between other parameters, cell migration is partially guided by the mechanical properties of its substrate. Although many experimental works have been developed to understand the effect of substrate mechanical properties on cell migration, accurate 3D cell locomotion models have not been presented yet. In this paper, we present a novel 3D model for cells migration. In the presented model, we assume that a cell follows two main processes: in the first process, it senses its interface with the substrate to determine the migration direction and in the second process, it exerts subsequent forces to move. In the presented model, cell traction forces are considered to depend on cell internal deformation during the sensing step. A random protrusion force is also considered which may change cell migration direction and/or speed. The presented model was applied for many cases of migration of the cells. The obtained results show high agreement with the available experimental and numerical data.     

Modulators of neuronal migration and neurite growth.

A multitude of molecules have been identified over the past few years that promote neurite outgrowth in vitro. The concept that these molecules work mainly by providing an adhesive surface for neuronal growth cones has been challenged by evidence from recent experiments. Some of the substrate molecules have diverse actions on cell migration and neurite growth. In addition, there is now evidence that there are molecules that specifically inhibit growth cone locomotion. This has given rise to the hypothesis that growth cones integrate a variety of growth-promoting and inhibitory signals and translate them into directed locomotion.     

细胞运动、细胞迁移与细胞骨架研究进展

细胞定向运动与细胞骨架的动态循环密切相关。运动细胞在其伪足前沿依靠细胞骨架的不断聚合推动细胞膜的前进,在基部靠近细胞体部位通过细胞骨架的不断解聚收缩拖拉细胞体向前运动,细胞骨架的聚合与解聚通过伪足与支撑表面的吸附与解吸附而偶连。肌动蛋白组成的微丝骨架是大多数运动细胞的主要成分。外界刺激引起微丝细胞骨架动态变化的信号通路已逐步明了。线虫精子细胞的运动行为与阿米巴变形运动相似,但是在线虫精子细胞中没有肌动蛋白,而是以精子主要蛋白为基础形成细胞骨架驱动精子细胞的运动。与肌动蛋白不同,精子主要蛋白没有分子极性、ATP结合位点和马达蛋白。通过比较研究以上两种运动体系将有助于在分子水平上进一步阐明细胞运动的机理。     

Dynamic modes of the cortical actomyosin gel during cell locomotion and division

Tight regulation of the contractility of the actomyosin cortex is essential for proper cell locomotion and division. Enhanced contractility leads, for example, to aberrations in the positioning of the mitotic spindle or to anomalous migration modes that allow tumor cells to escape anti-dissemination treatments. Spherical membrane protrusions called blebs occasionally appear during cell migration, cell division or apoptosis. We have shown that the cortex ruptures at sites where actomyosin cortical contractility is increased, leading to the formation of blebs. Here, we propose that bleb formation, which releases cortical tension, can be used as a reporter of cortical contractility. We go on to analyze the implications of spontaneous cortical contractile behaviors on cell locomotion and division and we particularly emphasize that variations in actomyosin contractility can account for a variety of migration modes.     

Stimulation of Cortical Myosin Phosphorylation by p114RhoGEF Drives Cell Migration and Tumor Cell Invasion

Actinomyosin activity is an important driver of cell locomotion and has been shown to promote collective cell migration of epithelial sheets as well as single cell migration and tumor cell invasion. However, the molecular mechanisms underlying activation of cortical myosin to stimulate single cell movement, and the relationship between the mechanisms that drive single cell locomotion and those that mediate collective cell migration of epithelial sheets are incompletely understood. Here, we demonstrate that p114RhoGEF, an activator of RhoA that associates with non-muscle myosin IIA, regulates collective cell migration of epithelial sheets and tumor cell invasion. Depletion of p114RhoGEF resulted in specific spatial inhibition of myosin activation at cell-cell contacts in migrating epithelial sheets and the cortex of migrating single cells, but only affected double and not single phosphorylation of myosin light chain. In agreement, overall elasticity and contractility of the cells, processes that rely on persistent and more constant forces, were not affected, suggesting that p114RhoGEF mediates process-specific myosin activation. Locomotion was p114RhoGEF-dependent on Matrigel, which favors more roundish cells and amoeboid-like actinomyosin-driven movement, but not on fibronectin, which stimulates flatter cells and lamellipodia-driven, mesenchymal-like migration. Accordingly, depletion of p114RhoGEF led to reduced RhoA, but increased Rac activity. Invasion of 3D matrices was p114RhoGEF-dependent under conditions that do not require metalloproteinase activity, supporting a role of p114RhoGEF in myosin-dependent, amoeboid-like locomotion. Our data demonstrate that p114RhoGEF drives cortical myosin activation by stimulating myosin light chain double phosphorylation and, thereby, collective cell migration of epithelial sheets and amoeboid-like motility of tumor cells.     

Microarchitecture of three-dimensional scaffolds influences cell migration behavior via junction interactions

Cell migration plays a critical role in a wide variety of physiological and pathological phenomena as well as in scaffold-based tissue engineering. Cell migration behavior is known to be governed by biochemical stimuli and cellular interactions. Biophysical processes associated with interactions between the cell and its surrounding extracellular matrix may also play a significant role in regulating migration. Although biophysical properties of two-dimensional substrates have been shown to significantly influence cell migration, elucidating factors governing migration in a three-dimensional environment is a relatively new avenue of research. Here, we investigate the effect of the three-dimensional microstructure, specifically the pore size and Young's modulus, of collagen-glycosaminoglycan scaffolds on the migratory behavior of individual mouse fibroblasts. We observe that the fibroblast migration, characterized by motile fraction as well as locomotion speed, decreases as scaffold pore size increases across a range from 90 to 150 μm. Directly testing the effects of varying strut Young's modulus on cell motility showed a biphasic relationship between cell speed and strut modulus and also indicated that mechanical factors were not responsible for the observed effect of scaffold pore size on cell motility. Instead, in-depth analysis of cell locomotion paths revealed that the distribution of junction points between scaffold struts strongly modulates motility. Strut junction interactions affect local directional persistence as well as cell speed at and away from the junctions, providing a new biophysical mechanism for the governance of cell motility by the extracellular microstructure.     

Competition amongst Eph receptors regulates contact inhibition of locomotion and invasiveness in prostate cancer cells

Metastatic cancer cells typically fail to halt migration on contact with non-cancer cells. This invasiveness is in contrast to normal mesenchymal cells that retract on contact with another cell. Why cancer cells are defective in contact inhibition of locomotion is not understood. Here, we analyse the dynamics of prostate cancer cell lines co-cultured with fibroblasts, and demonstrate that a combinatorial code of Eph receptor activation dictates whether cell migration will be contact inhibited. The unimpeded migration of metastatic PC-3 cells towards fibroblasts is dependent on activation of EphB3 and EphB4 by ephrin-B2, which we show activates Cdc42 and cell migration. Knockdown of EphB3 and EphB4 restores contact inhibition of locomotion to PC-3 cells. Conversely, homotypic collisions between two cancer cells results in contact inhibition of locomotion, mediated by EphA-Rho-Rho kinase (ROCK) signalling. Thus, the migration of cancer cells can switch from restrained to invasive, depending on the Eph-receptor profile of the cancer cell and the reciprocal ephrin ligands expressed by neighbouring cells.     

Are hyaluronan receptors involved in three-dimensional cell migration?

Hyaluronan (HA), an unbranched polysaccharide consisting of repeated glucuronic acid/N-acetylglucosamine disaccharide units, is ubiquitously present in the extracellular matrix of many tissues (for a more comprehensive review see: Fraser et al., 1997). Increased amounts of hyaluronan are produced by solid tumors and tumor-associated fibroblasts, and tumor-induced HA is correlated with poor prognosis. HA is well known to stimulate the migration of a large variety of cell types. Stimulation of cell migration by HA has been explained by different mechanisms. HA was shown to specifically bind to cell surface receptors, and inhibition of HA-receptor function was demonstrated to decrease cell migration and tumor growth. On the other hand, HA as a large hydrophilic molecule is also known to modulate the extracellular packing of collagen and fibrin, leading to increased fiber size and porosity of extracellular substrates. Hence a modified matrix architecture might similarly account for increased locomotion of cells. In this review, we attempted to summarize the available data on HA-induced cell migration, with particular emphasis on the role of HA receptors in three-dimensional cell migration. Although the HA receptor CD44 has been shown to mediate migration of cells over two-dimensional hyaluronan-coated surfaces in vitro, there is only little evidence that HA-binding to CD44 or other HA receptors has major impact on the locomotion of cells through three-dimensional matrices in vivo. We showed recently that the promigratory effect of HA in fibrin gels is largely due to HA-mediated modulation of fibrin polymerization. By increasing the porosity of fibrin gels, HA strongly accelerates cell migration. The porosity of matrices therefore appears as an important and probably underestimated determinant of cell migration and tumor spread.     

Tumor cell locomotion: differential dynamics of spontaneous and induced migration in a 3D collagen matrix

Although great strides have recently been made in elucidating the factors initiating tumor cell migration and the relevant cellular pathways involved, the constituent components of migratory dynamics for individual tumor cell motion have still not been resolved. Utilizing a three-dimensional (3D) collagen assay and computer-assisted, continuous single cell tracking, we investigated the basic parameters for both the spontaneous and norepinephrine-induced migration of highly metastatic MBA-MB-468 breast, PC-3 prostate, and SW 480 colon carcinoma cells. We show that tumor cells do not migrate with uniform migrational structure and speed as previously thought, but rather, the induction of locomotion elicits significant increases in speed, break frequency, and total cell displacement, but decreases in break length and no change in the recruitment of nonlocomotory cells. We furthermore illustrate the corresponding morphological changes of induced tumor cell migration with emphasis on motion in a collagen matrix. These results demonstrate the complexity of tumor cell migration, and the compulsion for incorporating not only knowledge of intracellular pathways, but also fundamental parameters of migratory behavior into any expansive theory of tumor cell migration and metastasis formation. We furthermore establish the analytical methodology of investigating both the stimulation and potential pharmaceutical inhibition of tumor cell migration.     

T lymphocytes and neutrophil granulocytes differ in regulatory signaling and migratory dynamics with regard to spontaneous locomotion and chemotaxis

Chemotactic migration of T lymphocytes and neutrophil granulocytes within a three-dimensional collagen matrix is distinct from spontaneous, matrix-induced migration concerning dynamic parameters and regulatory intracellular signaling. Both spontaneous T lymphocyte locomotion and stromal-cell-derived factor-1 (SDF-1)-induced chemotaxis-involved protein tyrosine kinase (PTK) activity, whereas only SDF-1-induced migration was protein kinase C (PKC) dependent. Spontaneous locomotion of neutrophil granulocytes was independent of PKC and PTK activity, but formyl-methionyl-leucyl-phenylalanine-induced migration involved PKC activity. In addition, the microtubule cytoskeleton was not changed after induction of chemotaxis in both cell types. T lymphocytes had a well-developed microtubule cytoskeleton with the microtubule organizing center located in the uropod, whereas neutrophil granulocytes revealed a clustered tubulin distribution at the leading edge of the migrating cell. Therefore, differences of the microtubule cytoskeleton might contribute to differences in locomotion between T lymphocytes and neutrophil granulocytes but not to differences between spontaneous locomotion and chemotaxis.     

Neural crest cell migration in relation to extracellular matrix organization in the embryonic axolotl trunk

Temporal and regional aspects of early neural crest cell migration in relation to extracellular matrix (ECM) organization and distribution in the embryonic axolotl trunk were studied by light microscopy, TEM, and SEM. The dominating structure of the interstitial ECM is a complex network of fibrils, which are indicated by ruthenium red staining to consist of collagen in association with ruthenium red-positive components, probably including glycosaminoglycans. The ECM fibrils, which are largely used as substratum for locomotion by the crest cells, have a temporally and regionally specific organization and distribution. Increase in ECM fibrils on the neural tube, ahead of the crest cell front, is correlated with initiation of crest cell emigration, and it is suggested that the fibrils may stimulate this process by providing a suitable substratum for cell locomotion. An increase in ECM fibrils in extracellular spaces surrounding the crest cell population is correlated with an expansion of these spaces and with progressing crest cell migration into them. It is proposed that the spatial organization of the ECM fibrils influences crest cell shape and orientation during early migration.     

Effects of injected inhibitors of microfilament and microtubule function on the gastrulation movement in Xenopus laevis

It was suggested recently that gastrulation movements in amphibian embryos are caused by the active cell locomotion of individual cells. In order to elucidate the role of microfilaments and microtubules in the cell locomotion occurring during gastrulation, cytochalasin B, colchicine, and other microtubule-disrupting drugs were injected into the blastocoel of early gastrulae of Xenopus laevis. Hypertonic solutions of sorbitol were also injected to elucidate the influence of the internal hydrostatic pressure on the migrating cells. The effects were examined in 1-μm Epon sections of serially fixed embryos and by transmission electron microscopy. Cytochalasin B strongly inhibits cell migration even under conditions that do not cause dissociation into single cells. The cells become round, and have only a few thin cell processes. Electron microscopy shows an alteration in the cortical microfilament network. Colchicine and other microtubule-disrupting drugs have little effect on the rate of cell migration before they cause the accumulation of many mitotic cells and the dissociation of the embryo. The interphase cells are angular and have thin processes like those in the control embryos. The microtubules disappear, and bundles of 10-nm filaments are observed in the cytoplasm of colchicine-injected embryos. Hypertonic sorbitol solutions strongly inhibit cell migration.     

Computational model for cell migration in three-dimensional matrices

Although computational models for cell migration on two-dimensional (2D) substrata have described how various molecular and cellular properties and physiochemical processes are integrated to accomplish cell locomotion, the same issues, along with certain new ones, might contribute differently to a model for migration within three-dimensional (3D) matrices. To address this more complicated situation, we have developed a computational model for cell migration in 3D matrices using a force-based dynamics approach. This model determines an overall locomotion velocity vector, comprising speed and direction, for individual cells based on internally generated forces transmitted into external traction forces and considering a timescale during which multiple attachment and detachment events are integrated. Key parameters characterize cell and matrix properties, including cell/matrix adhesion and mechanical and steric properties of the matrix; critical underlying molecular properties are incorporated explicitly or implicitly. Model predictions agree well with experimental results for the limiting case of migration on 2D substrata as well as with recent experiments in 3D natural tissues and synthetic gels. Certain predicted features such as biphasic behavior of speed with density of matrix ligands for 3D migration are qualitatively similar to their 2D counterparts, but new effects generally absent in 2D systems, such as effects due to matrix sterics and mechanics, are now predicted to arise in many 3D situations. As one particular sample manifestation of these effects, the optimal levels of cell receptor expression and matrix ligand density yielding maximal migration are dependent on matrix mechanical compliance.     

Cortical Factor Feedback Model for Cellular Locomotion and Cytofission

Eukaryotic cells can move spontaneously without being guided by external cues. For such spontaneous movements, a variety of different modes have been observed, including the amoeboid-like locomotion with protrusion of multiple pseudopods, the keratocyte-like locomotion with a widely spread lamellipodium, cell division with two daughter cells crawling in opposite directions, and fragmentations of a cell to multiple pieces. Mutagenesis studies have revealed that cells exhibit these modes depending on which genes are deficient, suggesting that seemingly different modes are the manifestation of a common mechanism to regulate cell motion. In this paper, we propose a hypothesis that the positive feedback mechanism working through the inhomogeneous distribution of regulatory proteins underlies this variety of cell locomotion and cytofission. In this hypothesis, a set of regulatory proteins, which we call cortical factors, suppress actin polymerization. These suppressing factors are diluted at the extending front and accumulated at the retracting rear of cell, which establishes a cellular polarity and enhances the cell motility, leading to the further accumulation of cortical factors at the rear. Stochastic simulation of cell movement shows that the positive feedback mechanism of cortical factors stabilizes or destabilizes modes of movement and determines the cell migration pattern. The model predicts that the pattern is selected by changing the rate of formation of the actin-filament network or the threshold to initiate the network formation.     

A calcium dependent de-adhesion mechanism regulates the direction and rate of cell migration: a mathematical model

Cell migration has long been studied by a variety of techniques and many proteins have been implicated in its regulation. Integrins, key proteins that link the cell to the extracellular matrix, are central to adhesion complexes whose turnover defines the rate of cell locomotion. The formation and disassembly of these adhesions is regulated by both intracellular and extracellular factors. In this study we have focused on the Ca2+-dependent protein network (module) that disassembles the adhesion complexes. We have developed a mathematical model that includes the Ca2+-dependent enzymes micro-calpain and phospholipase C (PLC) as well as IP3 receptors and stretch activated Ca2+ channels, all of which have been reported to regulate migration. The model also considers the spatial effects of Ca2+ propagation into lamella. Our model predicts differential activation of calpain at the leading and trailing edges of the cell. Since disassembly of integrin adhesive contacts is proportional to the degree of calpain activation, this leads to cell migration in a preferred direction. We show how the dynamics of Ca2+ spiking affects calpain activation and thus changes the disassembly rate of adhesions. The spiking is controlled by PLC activity and currents through stretch-activated Ca2+ channels. Our model thus combines the effects of various molecular factors and leads to a consistent explanation of the regulation of the rate and direction of cell migration.     

Electric field-directed fibroblast locomotion involves cell surface molecular reorganization and is calcium independent

Directional cellular locomotion is thought to involve localized intracellular calcium changes and the lateral transport of cell surface molecules. We have examined the roles of both calcium and cell surface glycoprotein redistribution in the directional migration of two murine fibroblastic cell lines, NIH 3T3 and SV101. These cell types exhibit persistent, cathode directed motility when exposed to direct current electric fields. Using time lapse phase contrast microscopy and image analysis, we have determined that electric field-directed locomotion in each cell type is a calcium independent process. Both exhibit cathode directed motility in the absence of extracellular calcium, and electric fields cause no detectable elevations or gradients of cytosolic free calcium. We find evidence suggesting that galvanotaxis in these cells involves the lateral redistribution of plasma membrane glycoproteins. Electric fields cause the lateral migration of plasma membrane concanavalin A receptors toward the cathode in both NIH 3T3 and SV101 fibroblasts. Exposure of directionally migrating cells to Con A inhibits the normal change of cell direction following a reversal of electric field polarity. Additionally, when cells are plated on Con A- coated substrata so that Con A receptors mediate cell-substratum adhesion, cathode-directed locomotion and a cathodal accumulation of Con A receptors are observed. Immunofluorescent labeling of the fibronectin receptor in NIH 3T3 fibroblasts suggests the recruitment of integrins from large clusters to form a more diffuse distribution toward the cathode in field-treated cells. Our results indicate that the mechanism of electric field directed locomotion in NIH 3T3 and SV101 fibroblasts involves the lateral redistribution of plasma membrane glycoproteins involved in cell-substratum adhesion.     

Capping protein is essential for cell migration in vivo and for filopodial morphology and dynamics

Capping protein (CP) binds to barbed ends of growing actin filaments and inhibits elongation. CP is essential for actin-based motility in cell-free systems and in Dictyostelium. Even though CP is believed to be critical for creating the lamellipodial actin structure necessary for protrusion and migration, CP''s role in mammalian cell migration has not been directly tested. Moreover, recent studies have suggested that structures besides lamellipodia, including lamella and filopodia, may have unappreciated roles in cell migration. CP has been postulated to be absent from filopodia, and thus its role in filopodial activity has remained unexplored. We report that silencing CP in both cultured mammalian B16F10 cells and in neurons of developing neocortex impaired cell migration. Moreover, we unexpectedly observed that low levels of CP were detectable in the majority of filopodia. CP depletion decreased filopodial length, altered filopodial shape, and reduced filopodial dynamics. Our results support an expansion of the potential roles that CP plays in cell motility by implicating CP in filopodia as well as in lamellipodia, both of which are important for locomotion in many types of migrating cells.     

Autotaxin and lysophosphatidic acid stimulate intestinal cell motility by redistribution of the actin modifying protein villin to the developing lamellipodia

Autotaxin (ATX) is a potent tumor cell motogen that can produce lysophosphatidic acid (LPA) from lysophosphatidylcholine. LPA is a lipid mediator that has also been shown to modulate tumor cell invasion. Autotaxin mRNA is expressed at significant levels in the intestine. Likewise, LPA2 receptor levels have been shown to be elevated in colon cancers. The molecular mechanism of ATX/LPA-induced increase in intestinal cell migration however, remains poorly understood. Villin is an intestinal and renal epithelial cell specific actin regulatory protein that modifies epithelial cell migration. In this study we demonstrate that both Caco-2 (endogenous villin) and MDCK (exogenous villin) cells, which express primarily LPA2 receptors, show enhanced cell migration in response to ATX/LPA. ATX and LPA treatment results in the rapid formation of lamellipodia and redistribution of villin to these cell surface structures, suggesting a role for villin in regulating this initial event of cell locomotion. The LPA-induced increase in cell migration required activation of c-src kinase and downstream tyrosine phosphorylation of villin by c-src kinase. LPA stimulated cell motility was determined to be insensitive to pertussis toxin, but was regulated by activation of PLC-gamma 1. Together, our results show that in epithelial cells ATX and LPA act as strong stimulators of cell migration by recruiting PLC-gamma 1 and villin, both of which participate in the initiation of protrusion.     

L1cam Is Crucial for Cell Locomotion and Terminal Translocation of the Soma in Radial Migration during Murine Corticogenesis

L1cam (L1) is a cell adhesion molecule associated with a spectrum of human neurological diseases, the most well-known being X-linked hydrocephalus. Although we recently demonstrated that L1 plays an important role in neuronal migration during cortical histogenesis, the mechanisms of delayed migration have still not been clarified. In this study, we found that cell locomotion in the intermediate zone and terminal translocation in the primitive cortical zone (PCZ) were affected by L1-knockdown (L1-KD). Time-lapse analyses revealed that L1-KD neurons produced by in utero electroporation of shRNA targeting L1 (L1-shRNAs) molecules showed decreased locomotion velocity in the intermediate zone, compared with control neurons. Furthermore, L1-KD neurons showed longer and more undulated leading processes during translocation through the primitive cortical zone. The curvature index, a quantitative index for curvilinearity, as well as the length of the leading process, were increased, whereas the somal movement was decreased in L1-KD neurons during terminal translocation in the PCZ. These results suggest that L1 has a role in radial migration of cortical neurons.