Extracellular matrix functions during neuronal migration and lamination in the mammalian central nervous system

Extracellular matrix (ECM) glycoproteins are expressed in the central nervous system (CNS) in complex and developmentally regulated patterns. The ECM provides a number of critical functions in the CNS, contributing both to the overall structural organization of the CNS and to control of individual cells. At the cellular level, the ECM affects its functions by a wide range of mechanisms, including providing structural support to cells, regulating the activity of second messenger systems, and controlling the distribution and local concentration of growth and differentiation factors. Perhaps the most well known role of the ECM is as a substrate on which motile cells can migrate. Genetic, cell biological, and biochemical studies provide strong evidence that ECM glycoproteins such as laminins, tenascins, and proteoglycans control neuronal migration and positioning in several regions of the developing and adult brain. Recent findings have also shed important new insights into the cellular and molecular mechanisms by which reelin regulates migration. Here we will summarize these findings, emphasizing the emerging concept that ECM glycoproteins promote different modes of neuronal migration such as radial, tangential, and chain migration. We also discuss several studies demonstrating that mutations in ECM glycoproteins can alter neuronal positioning by cell nonautonomous mechanisms that secondarily affect migrating neurons.     

Translation initiation factors and active sites of protein synthesis co-localize at the leading edge of migrating fibroblasts

Cell migration is a highly controlled essential cellular process, often dysregulated in tumour cells, dynamically controlled by the architecture of the cell. Studies involving cellular fractionation and microarray profiling have previously identified functionally distinct mRNA populations specific to cellular organelles and architectural compartments. However, the interaction between the translational machinery itself and cellular structures is relatively unexplored. To help understand the role for the compartmentalization and localized protein synthesis in cell migration, we have used scanning confocal microscopy, immunofluorescence and a novel ribopuromycylation method to visualize translating ribosomes. In the present study we show that eIFs (eukaryotic initiation factors) localize to the leading edge of migrating MRC5 fibroblasts in a process dependent on TGN (trans-Golgi network) to plasma membrane vesicle transport. We show that eIF4E and eIF4GI are associated with the Golgi apparatus and membrane microdomains, and that a proportion of these proteins co-localize to sites of active translation at the leading edge of migrating cells.     

Ca2+ influx through L-type Ca2+ channels controls the trailing tail contraction in growth factor-induced fibroblast cell migration

Growth factor-induced cell migration underlies various physiological and pathological processes. The mechanisms by which growth factors regulate cell migration are not completely understood. Although intracellular elevation of Ca2+ is known to be critical in cell migration, the source of this Ca2+ elevation and the mechanism by which Ca2+ modulates this process in fibroblast cells are not well defined. Here we show that increase of cellular Ca2+ through Ca2+ influx, rather than Ca2+ release from intracellular stores, is essential for growth factor-induced fibroblast cell migration. Voltage-gated L-type Ca2+ channels, previously known to exist in excitable cells such as neurons and muscle cells, are shown here to be present in fibroblasts as well. Furthermore, these channels are responsible for the Ca2+ influx. L-type Ca2+ channel inhibitors block growth factor-induced Ca2+ influx and fibroblast cell migration. One mechanism by which Ca2+ signals control cell migration is to regulate the contraction of the trailing edge of migrating fibroblasts; this process is controlled by the small GTPase Rho in fast migrating cells such as leukocytes. Downstream of Ca2+, both calmodulin and myosin light chain kinase, but not calcineurin, are involved leading to phosphorylation of the myosin light chain at the trailing end. Thus, trailing edge contraction is critically regulated by Ca2+ influx through L-type Ca2+ channels in growth factor-induced fibroblast cell migration.     

The tumor suppressor Lgl1 regulates NMII-A cellular distribution and focal adhesion morphology to optimize cell migration

The Drosophila tumor suppressor Lethal (2) giant larvae (Lgl) regulates the apical-basal polarity in epithelia and asymmetric cell division. However, little is known about the role of Lgl in cell polarity in migrating cells. In this study we show direct physiological interactions between the mammalian homologue of Lgl (Lgl1) and the nonmuscle myosin II isoform A (NMII-A). We demonstrate that Lgl1 and NMII-A form a complex in vivo and provide data that Lgl1 inhibits NMII-A filament assembly in vitro. Furthermore, depletion of Lgl1 results in the unexpected presence of NMII-A in the cell leading edge, a region that is not usually occupied by this protein, suggesting that Lgl1 regulates the cellular localization of NMII-A. Finally, we show that depletion of Lgl1 affects the size and number of focal adhesions, as well as cell polarity, membrane dynamics, and the rate of migrating cells. Collectively these findings indicate that Lgl1 regulates the polarity of migrating cells by controlling the assembly state of NMII-A, its cellular localization, and focal adhesion assembly.     

Mechanical Aspects of Microtubule Bundling in Taxane-Treated Circulating Tumor Cells

Microtubules play an important role in many cellular processes, including mitotic spindle formation and cell division. Taxane-based anticancer treatments lead to the stabilization of microtubules, thus preventing the uncontrolled proliferation of tumor cells. One of the striking physical features of taxane-treated cells is the localization of their microtubules, which can be observed via fluorescent microscopy as an intense fluorescent band and are referred to as a microtubule bundle. With the recent advances in capturing and analyzing tumor cells circulating in a patient’s blood system, there is increasing interest in using these cells to examine a patient’s response to treatment. This includes taxanes that are used routinely in clinics to treat prostate, breast, lung, and other cancers. Here, we have used a computational model of microtubule mechanics to investigate self-arrangement patterns of stabilized microtubules, which allowed for the identification of specific combinations of three physical parameters: microtubule stiffness, intracellular viscosity, and cell shape, that can prevent the formation of microtubule bundles in cells with stabilized microtubules, such as taxane-treated cells. We also developed a method to quantify bundling in the whole microtubule aster structure and a way to compare the simulated results to fluorescent images from experimental data. Moreover, we investigated microtubule rearrangement in both suspended and attached cells and showed that the observed final microtubule patterns depend on the experimental protocol. The results from our computational studies can explain the heterogeneous bundling phenomena observed via fluorescent immunostaining from a mechanical point of view without relying on heterogeneous cellular responses to the microtubule-stabilizing drug.     

Mechanical Aspects of Microtubule Bundling in Taxane-Treated Circulating Tumor Cells

Microtubules play an important role in many cellular processes, including mitotic spindle formation and cell division. Taxane-based anticancer treatments lead to the stabilization of microtubules, thus preventing the uncontrolled proliferation of tumor cells. One of the striking physical features of taxane-treated cells is the localization of their microtubules, which can be observed via fluorescent microscopy as an intense fluorescent band and are referred to as a microtubule bundle. With the recent advances in capturing and analyzing tumor cells circulating in a patient’s blood system, there is increasing interest in using these cells to examine a patient’s response to treatment. This includes taxanes that are used routinely in clinics to treat prostate, breast, lung, and other cancers. Here, we have used a computational model of microtubule mechanics to investigate self-arrangement patterns of stabilized microtubules, which allowed for the identification of specific combinations of three physical parameters: microtubule stiffness, intracellular viscosity, and cell shape, that can prevent the formation of microtubule bundles in cells with stabilized microtubules, such as taxane-treated cells. We also developed a method to quantify bundling in the whole microtubule aster structure and a way to compare the simulated results to fluorescent images from experimental data. Moreover, we investigated microtubule rearrangement in both suspended and attached cells and showed that the observed final microtubule patterns depend on the experimental protocol. The results from our computational studies can explain the heterogeneous bundling phenomena observed via fluorescent immunostaining from a mechanical point of view without relying on heterogeneous cellular responses to the microtubule-stabilizing drug.     

Simple rules for a “simple” nervous system? Molecular and biomathematical approaches to enteric nervous system formation and malformation

We review morphogenesis of the enteric nervous system from migratory neural crest cells, and defects of this process such as Hirschsprung disease, centering on cell motility and assembly, and cell adhesion and extracellular matrix molecules, along with cell proliferation and growth factors. We then review continuum and agent-based (cellular automata) models with rules of cell movement and logistical proliferation. Both movement and proliferation at the individual cell level are modeled with stochastic components from which stereotyped outcomes emerge at the population level. These models reproduced the wave-like colonization of the intestine by enteric neural crest cells, and several new properties emerged, such as colonization by frontal expansion, which were later confirmed biologically. These models predict a surprising level of clonal heterogeneity both in terms of number and distribution of daughter cells. Biologically, migrating cells form stable chains made up of unstable cells, but this is not seen in the initial model. We outline additional rules for cell differentiation into neurons, axon extension, cell-axon and cell–cell adhesions, chemotaxis and repulsion which can reproduce chain migration. After the migration stage, the cells re-arrange as a network of ganglia. Changes in cell adhesion molecules parallel this, and we describe additional rules based on Steinberg's Differential Adhesion Hypothesis, reflecting changing levels of adhesion in neural crest cells and neurons. This was able to reproduce enteric ganglionation in a model. Mouse mutants with disturbances of enteric nervous system morphogenesis are discussed, and these suggest future refinement of the models. The modeling suggests a relatively simple set of cell behavioral rules could account for complex patterns of morphogenesis. The model has allowed the proposal that Hirschsprung disease is mostly an enteric neural crest cell proliferation defect, not a defect of cell migration. In addition, the model suggests an explanations for zonal and skip segment variants of Hirschsprung disease, and also gives a novel stochastic explanation for the observed discordancy of Hirschsprung disease in identical twins.     

A Model for Short- and Long-range Interactions of Migrating Tumour Cell

We examine the consequences of long-range effects on tumour cell migration. Our starting point are previous results of ours where we have shown that the migration patterns of glioma cells are best interpreted if one assumes attractive interactions between cells. Here we complement the cellular automaton model previously introduced by the assumption of the existence of a chemorepellent produced by the main bulk of large spheroids (in the hypoxic/necrotic areas). Visible effects due to the presence of such a substance can be found in the density profiles of cells migrating out of a single spheroid as well as in the angular distribution of cells coming from two close-lying spheroids. These effects depend crucially on the diffusion speed of the chemorepellent. A comparison of the simulation results to experimental data of Werbowetski et al. allows to draw (tentative) conclusions on the existence of a chemorepellent and its properties.     

Migration of cortical interneurons relies on branched leading process dynamics

Migrating cells typically reach their targets in response to a relatively wide variety of extracellular molecules. Somehow surprisingly, most cells transduce these extracellular signals into a relatively homogeneous set of cellular changes that allow them to accurately find their target position. Here we summarize the characterization of the migratory behaviour of cortical interneurons in their journey to the cerebral cortex, which seems to represent a novel type of cellular adaptation during directional guidance. Similar to other migrating cells, cortical interneurons are highly polarized cells, with a prominent leading process and a short trailing process. However, the leading process of migrating interneurons continuously branches during the migratory cycle of these cells. Leading process branches are generated in response to the extracellular environment, and seem to serve as the main mechanism that determines the migratory direction for the cell. For each migratory cycle, the branch that is best oriented towards an attractive guidance cue will become stabilized, which in turn will allow the subcellular organelles and the nucleus to progress in the right direction. This migratory process is under the strict control, among several other molecules, of members from the small Rho GTPases family proteins. Pharmacological blocking of ROCKI/II abrogates the formation of leading process branches in migrating interneurons. The resulting cells, with a single leading process, do not efficiently modify their orientation in response to extracellular guidance cues, and so they fail to complete their migration.     

Co-Regulation of Cell Polarization and Migration by Caveolar Proteins PTRF/Cavin-1 and Caveolin-1

Caveolin-1 and caveolae are differentially polarized in migrating cells in various models, and caveolin-1 expression has been shown to quantitatively modulate cell migration. PTRF/cavin-1 is a cytoplasmic protein now established to be also necessary for caveola formation. Here we tested the effect of PTRF expression on cell migration. Using fluorescence imaging, quantitative proteomics, and cell migration assays we show that PTRF/cavin-1 modulates cellular polarization, and the subcellular localization of Rac1 and caveolin-1 in migrating cells as well as PKCα caveola recruitment. PTRF/cavin-1 quantitatively reduced cell migration, and induced mesenchymal epithelial reversion. Similar to caveolin-1, the polarization of PTRF/cavin-1 was dependent on the migration mode. By selectively manipulating PTRF/cavin-1 and caveolin-1 expression (and therefore caveola formation) in multiple cell systems, we unveil caveola-independent functions for both proteins in cell migration.     

Subcellular distribution of calcium-sensitive potassium channels (IK1) in migrating cells

Cell migration is crucial for wound healing, immune defense, or formation of tumor metastases. In addition to the cytoskeleton, Ca2+ sensitive K+ channels (IK1) are also part of the cellular "migration machinery." We showed that Ca2+ sensitive K+ channels support the retraction of the rear part of migrating MDCK-F cells by inducing a localized shrinkage at this cell pole. So far the molecular nature and in particular the subcellular distribution of these channels in MDCK-F cells is unknown. We compared the effect of IK1 channel blockers and activators on the current of a cloned IK1 channel from MDCK-F cells (cIK1) and the migratory behavior of these cells. Using IK1 channels labeled with a HA-tag or the enhanced green fluorescent protein we studied the subcellular distribution of the canine (cIK1) and the human (hIK1) channel protein in different migrating cells. The functional impact of cIK1 channel activity at the front or rear part of MDCK-F cells was assessed with a local superfusion technique and a detailed morphometric analysis. We show that it is cIK1 whose activity is required for migration of MDCK-F cells. IK1 channels are found in the entire plasma membrane, but they are concentrated at the cell front. This is in part due to membrane ruffling at this cell pole. However, there appears to be only little cIK1 channel activity at the front of MDCK-F cells. In our view this apparent discrepancy can be explained by differential regulation of IK1 channels at the front and rear part of migrating cells.     

Migrating endothelial cells are distinctly hyperglycosylated and express specific migration-associated cell surface glycoproteins

Migration of endothelial cells is one of the first cellular responses in the cascade of events that leads to re-endothelialization of an injured vessel and neovascularization of growing tissues and tumors. To examine the hypothesis that endothelial cells express a specific migration-associated phenotype, we analyzed the cell surface glycoprotein expression of migrating bovine aortic endothelial cell (BAECs). Light microscopic analysis revealed an upregulation of binding sites for the lectins Concanavalin A (Con A), wheat germ agglutinin (WGA), and peanut agglutinin after neuraminidase treatment (N-PNA) on migrating endothelial cells relative to contact-inhibited cells. These findings were confirmed and quantitated with an enzyme-linked lectin assay (ELLA) of circularly scraped BAEC monolayers. The expression of migration-associated cell surface glycoproteins was also analyzed by SDS-PAGE. The overall expression of cell surface glycoproteins was upregulated on migrating BAECs. Migrating BAECs expressed Con A- and WGA-binding glycoproteins with apparent molecular masses of 25 and 48 kD that were not expressed by contact-inhibited BAEC monolayers and, accordingly, disappeared as circularly scraped monolayers reached confluence. Subconfluent BAEC monolayers expressed the same cell surface glycoconjugate pattern as migrating endothelial cells. FACS analysis of circularly scraped BAEC monolayers showed that the phenotypic changes of cell surface glycoprotein expression after release from growth arrest occurred before the recruitment of the cells into the cell cycle (3 vs. 12 h). Suramin, which inhibits endothelial cell migration, abrogated the expression of the migration-associated phenotype and induced the expression of a prominent 28-kD Con A- and WGA-binding cell surface glycoprotein. These results indicate that endothelial cells express a specific migration-associated phenotype, which is characterized by the upregulation of distinct cellular glycoconjugates and the expression of specific migration-associated cell surface glycoproteins.     

Dependence of intracellular alkali-ion concentrations of 3T3 and SV 40-3T3 cells on growth density.

Intracellular contents of potassium and of sodium are determined for 3T3 and SV 40-3T3 cells in dependence of growth density. In parallel, total cell volume and volume of intracellular water is determined for these cells suspended in physiological buffer. Intracellular potassium concentration thus evaluated for suspended 3T3 cells exhibits a sharp decrease at cellular growth densities which lead to density dependent inhibition of cell proliferation. In the case of SV 40-3T3 cells, this drop of potassium concentration with increasing cellular growth density is not observed, which correlates well with the absence of cell density dependent inhibition of cell growth in the transformed cell line. These results support the notion that processes of stimulation of quiescent 3T3 cells or of cell density dependent inhibition of their proliferation are mediated by processes including changes of potassium transport characteristics leading to increase or decrease respectively of their intracellular potassium concentration. Furthermore, these and other results suggest, that a difference between normal and transformed cells most relevant to their different proliferation behaviour might reside in different transport characteristics for potassium of the plasma membranes of these cells.     

Netrin 1 acts as an attractive or as a repulsive cue for distinct migrating neurons during the development of the cerebellar system

Netrin 1 is a long-range diffusible factor that exerts chemoattractive or chemorepulsive effects on developing axons growing to or away from the neural midline. Here we used tissue explants to study the action of netrin 1 in the migration of several cerebellar and precerebellar cell progenitors. We show that netrin 1 exerts a strong chemoattractive effect on migrating neurons from the embryonic lower rhombic lip at E12-E14, which give rise to precerebellar nuclei. Netrin 1 promotes the exit of postmitotic migrating neurons from the embryonic lower rhombic lip and upregulates the expression of TAG-1 in these neurons. In addition, in the presence of netrin 1, the migrating neurons are not isolated but are associated with thick fascicles of neurites, typical of the neurophilic way of migration. In contrast, the embryonic upper rhombic lip, which contains tangentially migrating granule cell progenitors, did not respond to netrin 1. Finally, in the postnatal cerebellum, netrin 1 repels both the parallel fibres and migrating granule cells growing out from explants taken from the external germinal layer. The developmental patterns of expression in vivo of netrin 1 and its receptors are consistent with the notion that netrin 1 secreted in the midline acts as chemoattractive cue for precerebellar neurons migrating circumferentially along the extramural stream. Similarly, the pattern of expression in the postnatal cerebellum suggests that netrin 1 could regulate the tangential migration of postmitotic premigratory granule cells. Thus, molecular mechanisms considered as primarily involved in axonal guidance appear also to steer neuronal cell migration.     

PIP2 signaling,an integrator of cell polarity and vesicle trafficking in directionally migrating cells

Cell migration is a fundamental cellular process required for embryonic development to wound healing and also plays a key role in tumor metastasis and atherosclerosis. Migration is regulated at multiple strata, from cytoskeletal reorganization to vesicle trafficking. In migrating cells, signaling pathways are integrated with vesicle trafficking machineries in a highly coordinated fashion to accomplish the recruitment and trafficking of the trans-membrane proteins toward the leading edge. Different signaling molecules regulate cell migration in different physio-pathological contexts, among them, phosphatidylinositol-4,5-biphosphate (PIP2) is an integral component of the plasma membrane and pleiotropic lipid signaling molecule modulating diverse biological processes, including actin cytoskeletal dynamics and vesicle trafficking required for cell migration. In this commentary, we provide a brief overview of our current understandings on the phosphoinositide signaling and its implication in regulation of cell polarity and vesicle trafficking in migrating cells. In addition, we highlight the coordinated role of PIPKIγi2, a focal adhesion-targeted enzyme that synthesizes PIP2, and the exocyst complex, a PIP2-effector, in the trafficking of E-cadherin in epithelial cells and integrins in migrating cancer cells.     

PIP2 signaling,an integrator of cell polarity and vesicle trafficking in directionally migrating cells

Cell migration is a fundamental cellular process required for embryonic development to wound healing and also plays a key role in tumor metastasis and atherosclerosis. Migration is regulated at multiple strata, from cytoskeletal reorganization to vesicle trafficking. In migrating cells, signaling pathways are integrated with vesicle trafficking machineries in a highly coordinated fashion to accomplish the recruitment and trafficking of the trans-membrane proteins toward the leading edge. Different signaling molecules regulate cell migration in different physio-pathological contexts, among them, phosphatidylinositol-4,5-biphosphate (PIP2) is an integral component of the plasma membrane and pleiotropic lipid signaling molecule modulating diverse biological processes, including actin cytoskeletal dynamics and vesicle trafficking required for cell migration. In this commentary, we provide a brief overview of our current understandings on the phosphoinositide signaling and its implication in regulation of cell polarity and vesicle trafficking in migrating cells. In addition, we highlight the coordinated role of PIPKIγi2, a focal adhesion-targeted enzyme that synthesizes PIP2, and the exocyst complex, a PIP2-effector, in the trafficking of E-cadherin in epithelial cells and integrins in migrating cancer cells.     

Substrate-attached materials are enriched with tetraspanins and are analogous to the structures associated with rear-end retraction in migrating cells

Substrate-attached materials (SAMs) are cellular feet that remain on substrates after the treatment of adherent cells with EGTA. SAMs are thought to contain cell adhesion machineries, but their biochemical properties have not been addressed in detail. To gain insight into the molecular mechanisms operating in cell adhesions, we comprehensively identified the protein components of SAMs by liquid chromatography coupled with tandem mass spectrometry, followed by immunoblot analysis. We found that the tetraspanins CD9, CD81, and CD151 were enriched in SAMs along with other transmembrane proteins that are known to associate with tetraspanins. Notably, integrins were detected in SAMs, but the components of focal adhesions were scarcely detected. These observations are reminiscent of the “footprints” that remain on substrates when the retraction fibers at the rear of migrating cells are released, because such footprints have been reported to contain tetraspanins and integrins but not focal adhesion proteins. In support of this hypothesis, the formation of SAMs was attenuated by inhibitors of ROCK, myosin II and dynamin, all of which are known to participate in rear-end retraction in migrating cells. Furthermore, SAMs left on collagen-coated substrates were found by electron microscopy to be fewer and thinner than those on laminin-coated substrates, reflecting the thin and fragile retraction fibers of cells migrating on collagen. Collectively, these results indicate that SAMs closely resemble the footprints and retraction fibers of migrating cells in their protein components, and that they are yielded by similar mechanisms.     

Developmentally regulated expression of mil-1 and mil-2, mouse interferon-induced transmembrane protein like genes,during formation and differentiation of primordial germ cells

In all multicellular organisms, germ cells originating from a fertilized egg have the highly specialized role of transmitting genetic information to the next generation. In many animal species, the establishment of the germ cell lineage is regulated by the maternally inherited germplasm. In mammals, however, germline determination is not based on the unequal distribution of maternal determinants. In the processes of mammalian germ cell formation and subsequent differentiation, the molecular basis of the acquisition of germ cell status is not well understood. Since migrating primordial germ cells (PGCs) are lineage-restricted to the germline, they have already acquired a germ cell specific fate distinct from that of pluri/multi-potent stem cells. However, there have been no molecules known to be expressed in migrating PGCs but not in the inner cell mass of blastocysts. Such molecules should be involved in early germ cell development, and they should make good markers for following the process of PGC formation. To identify such molecules, we performed a subtracted cDNA screening with migrating PGCs and blastocysts in mice, and isolated 11 clones preferentially expressed in PGCs. Here, we report the identification of two genes with similarity to human interferon-induced transmembrane protein (Ifitm) genes, and expression patterns of these genes in forming and in differentiating PGCs. During germ cell formation, mouse Ifitm like (mil)-1 was expressed in putative PGC ancestors in embryos at 6.5-7.5 days post coitum. In migrating PGCs, mil-1 expression was continuously observed and mil-2 expression was first detected during germ cell differentiation.     

Developmentally regulated expression of mil-1 and mil-2, mouse interferon-induced transmembrane protein like genes, during formation and differentiation of primordial germ cells

In all multicellular organisms, germ cells originating from a fertilized egg have the highly specialized role of transmitting genetic information to the next generation. In many animal species, the establishment of the germ cell lineage is regulated by the maternally inherited germplasm. In mammals, however, germline determination is not based on the unequal distribution of maternal determinants. In the processes of mammalian germ cell formation and subsequent differentiation, the molecular basis of the acquisition of germ cell status is not well understood. Since migrating primordial germ cells (PGCs) are lineage-restricted to the germline, they have already acquired a germ cell specific fate distinct from that of pluri/multi-potent stem cells. However, there have been no molecules known to be expressed in migrating PGCs but not in the inner cell mass of blastocysts. Such molecules should be involved in early germ cell development, and they should make good markers for following the process of PGC formation. To identify such molecules, we performed a subtracted cDNA screening with migrating PGCs and blastocysts in mice, and isolated 11 clones preferentially expressed in PGCs. Here, we report the identification of two genes with similarity to human interferon-induced transmembrane protein (Ifitm) genes, and expression patterns of these genes in forming and in differentiating PGCs. During germ cell formation, mouse Ifitm like (mil)-1 was expressed in putative PGC ancestors in embryos at 6.5-7.5 days post coitum. In migrating PGCs, mil-1 expression was continuously observed and mil-2 expression was first detected during germ cell differentiation.