N-cadherin as a key regulator of collective cell migration in a 3D environment

Cell migration is a critical step of normal developmental processes and disease progression. Often, migrating cells interact and maintain contact with neighboring cells. However, the precise roles of cell-cell adhesion in cell migration have thus far been poorly defined. Often in aggressive cancers, N-cadherin is prominently upregulated, yet, these highly motile cells have limited cell-cell adhesion when plated on a stiff 2D substrate. But, the same cells in a 3D matrix migrate as a multicellular cluster. This new observation suggests that N-cadherin-mediated cell-cell adhesion supports cell interactions between migrating cells in a more physiologically relevant 3D matrix, but not on a 2D substrate. While N-cadherin is an integral part of neural synapses, the ectopic expression of N-cadherin in transformed epithelial cells plays an equally important part in initiating pro-migratory signaling, and providing strong yet flexible cell cohesion essential for persistent cell migration in a 3D matrix. The 3D cell migration analysis for studying cell-to-cell interactions exposes the roles of N-cadherin in multicellular migration, and reveals novel insights into cell migration-dependent normal and pathological processes.     

N-cadherin as a key regulator of collective cell migration in a 3D environment

Cell migration is a critical step of normal developmental processes and disease progression. Often, migrating cells interact and maintain contact with neighboring cells. However, the precise roles of cell-cell adhesion in cell migration have thus far been poorly defined. Often in aggressive cancers, N-cadherin is prominently upregulated, yet, these highly motile cells have limited cell-cell adhesion when plated on a stiff 2D substrate. But, the same cells in a 3D matrix migrate as a multicellular cluster. This new observation suggests that N-cadherin-mediated cell-cell adhesion supports cell interactions between migrating cells in a more physiologically relevant 3D matrix, but not on a 2D substrate. While N-cadherin is an integral part of neural synapses, the ectopic expression of N-cadherin in transformed epithelial cells plays an equally important part in initiating pro-migratory signaling, and providing strong yet flexible cell cohesion essential for persistent cell migration in a 3D matrix. The 3D cell migration analysis for studying cell-to-cell interactions exposes the roles of N-cadherin in multicellular migration, and reveals novel insights into cell migration-dependent normal and pathological processes.     

Endocytosis regulates cell soma translocation and the distribution of adhesion proteins in migrating neurons

Newborn neurons migrate from their birthplace to their final location to form a properly functioning nervous system. During these movements, young neurons must attach and subsequently detach from their substrate to facilitate migration, but little is known about the mechanisms cells use to release their attachments. We show that the machinery for clathrin-mediated endocytosis is positioned to regulate the distribution of adhesion proteins in a subcellular region just proximal to the neuronal cell body. Inhibiting clathrin or dynamin function impedes the movement of migrating neurons both in vitro and in vivo. Inhibiting dynamin function in vitro shifts the distribution of adhesion proteins to the rear of the cell. These results suggest that endocytosis may play a critical role in regulating substrate detachment to enable cell body translocation in migrating neurons.     

Myosin-X is critical for migratory ability of Xenopus cranial neural crest cells

The neural crest is a highly migratory cell population, unique to vertebrates, that forms much of the craniofacial skeleton and peripheral nervous system. In exploring the cell biological basis underlying this behavior, we have identified an unconventional myosin, myosin-X (Myo10) that is required for neural crest migration. Myo10 is highly expressed in both premigratory and migrating cranial neural crest (CNC) cells in Xenopus embryos. Disrupting Myo10 expression using antisense morpholino oligonucleotides leads to impaired neural crest migration and subsequent cartilage formation, but only a slight delay in induction. In vivo grafting experiments reveal that Myo10-depleted CNC cells migrate a shorter distance and fail to segregate into distinct migratory streams. Finally, in vitro cultures and cell dissociation-reaggregation assays suggest that Myo10 may be critical for cell protrusion and cell-cell adhesion. These results demonstrate an essential role for Myo10 in normal cranial neural crest migration and suggest a link to cell-cell interactions and formation of processes.     

Cadherin-2 Controls Directional Chain Migration of Cerebellar Granule Neurons

Long distance migration of differentiating granule cells from the cerebellar upper rhombic lip has been reported in many vertebrates. However, the knowledge about the subcellular dynamics and molecular mechanisms regulating directional neuronal migration in vivo is just beginning to emerge. Here we show by time-lapse imaging in live zebrafish (Danio rerio) embryos that cerebellar granule cells migrate in chain-like structures in a homotypic glia-independent manner. Temporal rescue of zebrafish Cadherin-2 mutants reveals a direct role for this adhesion molecule in mediating chain formation and coherent migratory behavior of granule cells. In addition, Cadherin-2 maintains the orientation of cell polarization in direction of migration, whereas in Cadherin-2 mutant granule cells the site of leading edge formation and centrosome positioning is randomized. Thus, the lack of adhesion leads to impaired directional migration with a mispositioning of Cadherin-2 deficient granule cells as a consequence. Furthermore, these cells fail to differentiate properly into mature granule neurons. In vivo imaging of Cadherin-2 localization revealed the dynamics of this adhesion molecule during cell locomotion. Cadherin-2 concentrates transiently at the front of granule cells during the initiation of individual migratory steps by intramembraneous transport. The presence of Cadherin-2 in the leading edge corresponds to the observed centrosome orientation in direction of migration. Our results indicate that Cadherin-2 plays a key role during zebrafish granule cell migration by continuously coordinating cell-cell contacts and cell polarity through the remodeling of adherens junctions. As Cadherin-containing adherens junctions have been shown to be connected via microtubule fibers with the centrosome, our results offer an explanation for the mechanism of leading edge and centrosome positioning during nucleokinetic migration of many vertebrate neuronal populations.     

The Rho-mDia1 pathway regulates cell polarity and focal adhesion turnover in migrating cells through mobilizing Apc and c-Src

Directed cell migration requires cell polarization and adhesion turnover, in which the actin cytoskeleton and microtubules work critically. The Rho GTPases induce specific types of actin cytoskeleton and regulate microtubule dynamics. In migrating cells, Cdc42 regulates cell polarity and Rac works in membrane protrusion. However, the role of Rho in migration is little known. Rho acts on two major effectors, ROCK and mDia1, among which mDia1 produces straight actin filaments and aligns microtubules. Here we depleted mDia1 by RNA interference and found that mDia1 depletion impaired directed migration of rat C6 glioma cells by inhibiting both cell polarization and adhesion turnover. Apc and active Cdc42, which work together for cell polarization, localized in the front of migrating cells, while active c-Src, which regulates adhesion turnover, localized in focal adhesions. mDia1 depletion impaired localization of these molecules at their respective sites. Conversely, expression of active mDia1 facilitated microtubule-dependent accumulation of Apc and active Cdc42 in the polar ends of the cells and actin-dependent recruitment of c-Src in adhesions. Thus, the Rho-mDia1 pathway regulates polarization and adhesion turnover by aligning microtubules and actin filaments and delivering Apc/Cdc42 and c-Src to their respective sites of action.     

Collective cell migration in morphogenesis and cancer

The movement of cells that maintain cell-cell junctions yet protrude along or within tissues is an important mechanism for cell positioning in morphogenesis, tissue repair and cancer. Collective cell migration shares similarities but also important differences to individually migrating cells. Coherent groups of cells are arranged and held together by cell-cell adhesion molecules, including cadherins, integrins, ALCAM and NCAM. Integrins of the beta 1 and beta 3 families further provide polarized interactions with the extracellular tissue environment, while matrix-degrading proteases become focalized to substrate contacts to widen tissue space for the advancing cell mass. By generating one functional unit, in contrast to individual cell migration, collective migration provides the active and passive translocation of mobile and non-mobile cells, respectively. This review highlights cellular and molecular principles of collective migration in the context of morphogenic tissue patterning and tumor cell invasion.     

NudE and NudEL are required for mitotic progression and are involved in dynein recruitment to kinetochores

NudE and NudEL are related proteins that interact with cytoplasmic dynein and LIS1. Their functional relationship and involvement in LIS1 and dynein regulation are not completely understood. We find that NudE and NudEL each localize to mitotic kinetochores before dynein, dynactin, ZW10, and LIS1 and exhibit additional temporal and spatial differences in distribution from the motor protein. Inhibition of NudE and NudEL caused metaphase arrest with misoriented chromosomes and defective microtubule attachment. Dynein and dynactin were both displaced from kinetochores by the injection of an anti-NudE/NudEL antibody. Dynein but not dynactin interacted with NudE surprisingly through the dynein intermediate and light chains but not the motor domain. Together, these results identify a common function for NudE and NudEL in mitotic progression and identify an alternative mechanism for dynein recruitment to and regulation at kinetochores.     

APC nuclear membrane association and microtubule polarity.

BACKGROUND INFORMATION: Directional cell migration is a fundamental feature of embryonic development, the inflammatory response and the metastatic spread of cancer. Migrating cells have a polarized morphology with an asymmetric distribution of signalling molecules and of the actin and microtubule cytoskeletons. The dynamic reorganization of the actin cytoskeleton provides the major driving force for migration in all mammalian cell types, but microtubules also play an important role in many cells, most notably neuronal precursors. RESULTS: We previously showed, using primary fibroblasts and astrocytes in in vitro scratch-induced migration assays, that the accumulation of APC (adenomatous polyposis coli; the APC tumour suppressor protein) at microtubule plus-ends promotes their association with the plasma membrane at the leading edge. This is required for polarization of the microtubule cytoskeleton during directional migration. Here, we have examined the organization of microtubules in the soma of migrating neurons and fibroblasts. CONCLUSIONS: We find that APC, through a direct interaction with the NPC (nuclear pore complex) protein Nup153 (nucleoporin 153), promotes the association of microtubules with the nuclear membrane.     

Cohort migration of carcinoma cells: differentiated colorectal carcinoma cells move as coherent cell clusters or sheets.

Active migration of tumor cells is usually assessed as single cell locomotion in vitro using Boyden chamber-type assays. In vivo, however, carcinoma cells, malignant cells of epithelial origin, frequently invade the surrounding tissue as coherent clusters or nests of cells. We have called this type of movement "cohort migration". In our work, the invasion front of colon carcinomas consisted of compact tumor glands, partially resolved glands or markedly resolved glands with scattered tumor cell clusters or single cells lying ahead. In the former two types, which constituted about a half of all cases, cohort migration seems to be the predominant mechanism, whereas both cohort migration and single cell locomotion may be involved in the last one. In this light, it is very advantageous to investigate the mechanisms involved in the cohort migration. In this review, we present a two-dimensional motility assay as a cohort migration model, in which human colorectal carcinoma cells move outwards from the cell islands mainly as localized coherent sheets of cells when stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor/scatter factor (HGF/SF). Within the migrating cell sheets, wide intercellular gaps occur at the lower portion of the cells to allow the cells to extend leading lamellae forward while close cell-cell contacts remain at the upper portion of the cells. This localized modulation of cell-cell adhesion at the lower portion of the cells is associated with increased tyrosine phosphorylation of the E-cadherin-catenin complex in TPA-induced cohort migration and with reduced alpha-catenin complexed with E-cadherin in HGF/SF-induced cohort migration. Furthermore, fibronectin deposited by migrating cells is essential for their movement, and on the gelatin-coated substrate even degradation and remodeling of the substrate by matrix metalloproteinases are also needed. Thus, in cohort migration it is likely that cells are released from cell-cell adhesion only at the lower portion of the cells via modulation of E-cadherin-catenin-based mechanism, and this change allows the cells to extend leading lamellae onto the extracellular matrix substrate remodeled by deposition of fibronectin and organized digestion.     

Activated armadillo/beta-catenin does not play a general role in cell migration and process extension in Drosophila.

Human beta-catenin and its fly homolog Armadillo are best known for their roles in cadherin-based cell-cell adhesion and in transduction of Wingless/Wnt signals. It has been hypothesized that beta-catenin may also regulate cell migration and cell shape changes, possibly by regulating the microtubule cytoskeleton via interactions with APC. This hypothesis was based on experiments in which a hyperstable mutant form of beta-catenin was expressed in MDCK cells, where it altered their migratory properties and their ability to send out long cellular processes. We tested the generality of this hypothesis in vivo in Drosophila. We utilized three model systems in which cell migration and/or process extension are known to play key roles during development: the migration of the border cells during oogenesis, the extension of axons in the nervous system, and the migration and cell process extension of tracheal cells. In all cases, cells expressing activated Armadillo were able to migrate and extend cell processes essentially normally. The one alteration from normal involved an apparent cell fate change in certain tracheal cells. These results suggest that only certain cells are affected by activation of Armadillo/beta-catenin, and that Armadillo/beta-catenin does not play a general role in inhibiting cell migration or process extension.     

The Role of Microtubules and Their Dynamics in Cell Migration

Although microtubules have long been implicated in cell locomotion, the mechanism of their involvement remains controversial. Most studies have concluded that microtubules play a positive role by regulating actin polymerization, transporting membrane vesicles to the leading edge, and/or facilitating the turnover of adhesion plaques. Here we used wild-type and mutant CHO cell lines with alterations in tubulin to demonstrate that microtubules can also act to restrain cell motility. Tubulin mutations or low concentrations of drugs that suppress microtubule dynamics without affecting the amount of microtubule polymer inhibited the rate of migration by preventing microtubule reorganization in the trailing portion of the cells where the more dynamic microtubules are normally found. Under these conditions, cells along the edge of a wound still extended lamellipodia and elongated toward the wound but were inhibited in their ability to retract their tails, thus retarding forward progress. The idea that microtubules normally act to restrain cell locomotion was confirmed by treating cells with high concentrations of nocodazole to depolymerize the microtubule network. In the absence of microtubules, wild-type CHO and HeLa cells could still move at near normal speeds, but the movement became more random. We conclude that microtubules act both to restrain cell movement and to establish directionality.     

Regulation of dynein localization and centrosome positioning by Lis-1 and asunder during Drosophila spermatogenesis

Dynein, a microtubule motor complex, plays crucial roles in cell-cycle progression in many systems. The LIS1 accessory protein directly binds dynein, although its precise role in regulating dynein remains unclear. Mutation of human LIS1 causes lissencephaly, a developmental brain disorder. To gain insight into the in vivo functions of LIS1, we characterized a male-sterile allele of the Drosophila homolog of human LIS1. We found that centrosomes do not properly detach from the cell cortex at the onset of meiosis in most Lis-1 spermatocytes; centrosomes that do break cortical associations fail to attach to the nucleus. In Lis-1 spermatids, we observed loss of attachments between the nucleus, basal body and mitochondria. The localization pattern of LIS-1 protein throughout Drosophila spermatogenesis mirrors that of dynein. We show that dynein recruitment to the nuclear surface and spindle poles is severely reduced in Lis-1 male germ cells. We propose that Lis-1 spermatogenesis phenotypes are due to loss of dynein regulation, as we observed similar phenotypes in flies null for Tctex-1, a dynein light chain. We have previously identified asunder (asun) as another regulator of dynein localization and centrosome positioning during Drosophila spermatogenesis. We now report that Lis-1 is a strong dominant enhancer of asun and that localization of LIS-1 in male germ cells is ASUN dependent. We found that Drosophila LIS-1 and ASUN colocalize and coimmunoprecipitate from transfected cells, suggesting that they function within a common complex. We present a model in which Lis-1 and asun cooperate to regulate dynein localization and centrosome positioning during Drosophila spermatogenesis.     

Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons.

Doublecortin (DCX) is required for normal migration of neurons into the cerebral cortex, since mutations in the human gene cause a disruption of cortical neuronal migration. To date, little is known about the distribution of DCX protein or its function. Here, we demonstrate that DCX is expressed in migrating neurons throughout the central and peripheral nervous system during embryonic and postnatal development. DCX protein localization overlaps with microtubules in cultured primary cortical neurons, and this overlapping expression is disrupted by microtubule depolymerization. DCX coassembles with brain microtubules, and recombinant DCX stimulates the polymerization of purified tubulin. Finally, overexpression of DCX in heterologous cells leads to a dramatic microtubule phenotype that is resistant to depolymerization. Therefore, DCX likely directs neuronal migration by regulating the organization and stability of microtubules.     

The position of the microtubule-organizing center relative to the nucleus is independent of the direction of cell migration in Dictyostelium discoideum

Dictyostelium amoebae can migrate in several different modes. We tested for correlations of the direction of cell locomotion with the relative positions of the nucleus and microtubule-organizing center (MTOC). Five cases were analyzed on electron micrographs with a microcomputer. Each mode of movement showed characteristic locations of the MTOC relative to the nucleus; however, they differed in the various cases. In randomly migrating interphase amoebae, the number of cells with the MTOC located behind the nucleus was twice as great as those with the MTOC located ahead of the nucleus. During chemotactic migration toward folic acid, cells with the MTOC behind the nucleus were more numerous, with a concomitant reduction of anterior MTOCs. When amoebae aggregated on agar plates, a posterior location of the MTOC was most strikingly favored, whereas in cells aggregating under submerged conditions, the MTOC was indifferently anterior or posterior to the nucleus. (It may be significant that EDTA-resistant cell-cell adhesion was fully expressed in the former cells, but weaker in the latter.) Finally, in the case of chemotactically migrating cells from dissociated pseudoplasmodia, which adhere by means of other molecules, the MTOC was consistently ahead of the nucleus. Thus the MTOC shows no necessary preferential position anterior or posterior to the nucleus; its position, rather, correlates with the type of migration and perhaps with the nature of cell-cell adhesion.     

APC nuclear membrane association and microtubule polarity

Background information. Directional cell migration is a fundamental feature of embryonic development, the inflammatory response and the metastatic spread of cancer. Migrating cells have a polarized morphology with an asymmetric distribution of signalling molecules and of the actin and microtubule cytoskeletons. The dynamic reorganization of the actin cytoskeleton provides the major driving force for migration in all mammalian cell types, but microtubules also play an important role in many cells, most notably neuronal precursors. Results. We previously showed, using primary fibroblasts and astrocytes in in vitro scratch‐induced migration assays, that the accumulation of APC (adenomatous polyposis coli; the APC tumour suppressor protein) at microtubule plus‐ends promotes their association with the plasma membrane at the leading edge. This is required for polarization of the microtubule cytoskeleton during directional migration. Here, we have examined the organization of microtubules in the soma of migrating neurons and fibroblasts. Conclusions. We find that APC, through a direct interaction with the NPC (nuclear pore complex) protein Nup153 (nucleoporin 153), promotes the association of microtubules with the nuclear membrane.     

aPKC controls microtubule organization to balance adherens junction symmetry and planar polarity during development

Tissue morphogenesis requires assembling and disassembling individual cell-cell contacts without losing epithelial integrity. This requires dynamic control of adherens junction (AJ) positioning around the apical domain, but the mechanisms involved are unclear. We show that atypical Protein Kinase C (aPKC) is required for symmetric AJ positioning during Drosophila embryogenesis. aPKC is dispensable for initial apical AJ recruitment, but without aPKC, AJs form atypical planar-polarized puncta at gastrulation. Preceding this, microtubules fail to dissociate from centrosomes, and at gastrulation abnormally persistent centrosomal microtubule asters cluster AJs into the puncta. Dynein enrichment at the puncta suggests it may draw AJs and microtubules together and microtubule disruption disperses the puncta. Through cytoskeletal disruption in wild-type embryos, we find a balance of microtubule and actin interactions controls AJ symmetry versus planar polarity during normal gastrulation. aPKC apparently regulates this balance. Without aPKC, abnormally strong microtubule interactions break AJ symmetry and epithelial structure is lost.     

Polarized interfacial tension induces collective migration of cells,as a cluster,in a 3D tissue

In embryogenesis and cancer invasion, cells collectively migrate as a cluster in 3D tissues. Many studies have elucidated mechanisms of either individual or collective cell migration on 2D substrates; however, it remains unclear how cells collectively migrate as a cluster through 3D tissues. To address this issue, we considered the interfacial tension at cell-cell boundaries expressing cortical actomyosin contractions and cell-cell adhesive interactions. The strength of this tension is polarized; i.e., spatially biased within each cell according to a chemoattractant gradient. Using a 3D vertex model, we performed numerical simulations of multicellular dynamics in 3D space. The simulations revealed that the polarized interfacial tension enables cells to migrate collectively as a cluster through a 3D tissue. In this mechanism, interfacial tension induces unidirectional flow of each cell surface from the front to the rear along the cluster surface. Importantly, this mechanism does not necessarily require convection of cells, i.e., cell rearrangement, within the cluster. Moreover, several migratory modes were induced, depending on the strengths of polarity, adhesion, and noise; i.e., cells migrate either as single cells, as a cluster, or aligned like beads on a string, as occurs in embryogenesis and cancer invasion. These results indicate that the simple expansion and contraction of cell-cell boundaries enables cells to move directionally forward and to produce the variety of collective migratory movements observed in living systems.     

Functional role for stable microtubules in lens fiber cell elongation

The process of tissue morphogenesis, especially for tissues reliant on the establishment of a specific cytoarchitecture for their functionality, depends a balanced interplay between cytoskeletal elements and their interactions with cell adhesion molecules. The microtubule cytoskeleton, which has many roles in the cell, is a determinant of directional cell migration, a process that underlies many aspects of development. We investigated the role of microtubules in development of the lens, a tissue where cell elongation underlies morphogenesis. Our studies with the microtubule depolymerizing agent nocodazole revealed an essential function for the acetylated population of stable microtubules in the elongation of lens fiber cells, which was linked to their regulation of the activation state of myosin. Suppressing myosin activation with the inhibitor blebbistatin could attenuate the loss of acetylated microtubules by nocodazole and rescue the effect of this microtubule depolymerization agent on both fiber cell elongation and lens integrity. Our results also suggest that acetylated microtubules impact lens morphogenesis through their interaction with N-cadherin junctions, with which they specifically associate in the region where lens fiber cell elongate. Disruption of the stable microtubule network increased N-cadherin junctional organization along lateral borders of differentiating lens fiber cells, which was prevented by suppression of myosin activity. These results reveal a role for the stable microtubule population in lens fiber cell elongation, acting in tandem with N-cadherin cell-cell junctions and the actomyosin network, giving insight into the cooperative role these systems play in tissue morphogenesis.     

Formation and preservation of cortical layers in slice cultures.

During cortical development, neurons generated at the same time in the ventricular zone migrate out into the cortical plate and form a cortical layer (Berry and Eayrs, 1963, Nature 197:984-985; Berry and Rogers, 1965, J. Anat. 99:691-709). We have been studying both the formation and maintenance of cortical layers in slice cultures from rat cortex. The bromodeoxyuridine (BrdU) method was used to label cortical neurons on their birthday in vivo. When slice cultures were prepared from animals at different embryonic and postnatal ages, all cortical layers that have already been established in vivo remained preserved for several weeks in vitro. In slice cultures prepared during migration in the cortex, cells continued to migrate towards the pial side of the cortical slice, however, migration ceased after about 1 week in culture. Thus, cortical cells reached their final laminar position only in slice cultures from postnatal animals, whereas in embryonic slice, migrating cells became scattered throughout the cortex. Previous studies demonstrated that radial glia fibers are the major substrate for migrating neurons (Rakic, 1972, J. Comp. Neurol. 145:61-84; Hatten and Mason, 1990, Experientia 46:907-916). Using antibodies directed against the intermediate filament Vimentin, radial glial cells were detected in all slice cultures where cell migration did occur. Comparable to the glia development in vivo, radial glial fibers disappeared and astrocytes containing the glia fibrillary-associated protein (GFAP) differentiated in slice cultures from postnatal cortex, after the neurons have completed their migration. In contrast, radial glial cells were detected over the whole culture period, and very few astrocytes differentiated in embryonic slices, where cortical neurons failed to finish their migration. The results of this study indicate that the local environment is sufficient to sustain the layered organization of the cortex and support the migration of cortical neurons. In addition, our results reveal a close relationship between cell migration and the developmental status of glial cells.