PKA negatively modulates the migration enhancing effect of Connexin 43

Connexin 43 (Cx43) expression is associated with an increased cell migration and related changes of the actin cytoskeleton (enhanced filopodia formation). These effects are mediated by the C-terminal cytoplasmic part of Cx43 in a channel-independent manner. Since this part has been shown to interact with a variety of proteins and has multiple phosphorylation sites we analyzed here a potential role of the protein kinase A (PKA) for the Cx43 mediated increase in cell migration. Mutation of the PKA-phosphorylation site (substitution of three serines by alanine or glycine) resulted in a further increase in cell motility compared to wild-type Cx43, but with a loss of directionality. Likewise, cell motility was enhanced by PKA inhibition only in Cx43 expressing cells, while reduced in the presence of the PKA activator forskolin. In contrast, cell motility remained unaffected by stimulation with forskolin in cells expressing Cx43 with the mutated PKA phosphorylation site (Cx43-PKA) as well as in Cx-deficient cells. Moreover, PKA activation resulted in increased binding of PKA and VASP to Cx43 associated with an enhanced phosphorylation of VASP, an important regulatory protein of cell polarity and directed migration. Functionally, we could confirm these results in endothelial cells endogenously expressing Cx43. A Tat-Cx43 peptide containing the PKA phosphorylation site abolished the PKA dependent reduction in endothelial cell migration. Our results indicate that PKA dependent phosphorylation of Cx43 modulates cell motility and plays a pivotal role in regulating directed cell migration.     

Connexin 43-mediated modulation of polarized cell movement and the directional migration of cardiac neural crest cells

Connexin 43 knockout (Cx43alpha1KO) mice have conotruncal heart defects that are associated with a reduction in the abundance of cardiac neural crest cells (CNCs) targeted to the heart. In this study, we show CNCs can respond to changing fibronectin matrix density by adjusting their migratory behavior, with directionality increasing and speed decreasing with increasing fibronectin density. However, compared with wild-type CNCs, Cx43alpha1KO CNCs show reduced directionality and speed, while CNCs overexpressing Cx43alpha1 from the CMV43 transgenic mice show increased directionality and speed. Altered integrin signaling was indicated by changes in the distribution of vinculin containing focal contacts, and altered temporal response of Cx43alpha1KO and CMV43 CNCs to beta1 integrin function blocking antibody treatment. High resolution motion analysis showed Cx43alpha1KO CNCs have increased cell protrusive activity accompanied by the loss of polarized cell movement. They exhibited an unusual polygonal arrangement of actin stress fibers that indicated a profound change in cytoskeletal organization. Semaphorin 3A, a chemorepellent known to inhibit integrin activation, was found to inhibit CNC motility, but in the Cx43alpha1KO and CMV43 CNCs, cell processes failed to retract with semaphorin 3A treatment. Immunohistochemical and biochemical analyses suggested close interactions between Cx43alpha1, vinculin and other actin-binding proteins. However, dye coupling analysis showed no correlation between gap junction communication level and fibronectin plating density. Overall, these findings indicate Cx43alpha1 may have a novel function in mediating crosstalk with cell signaling pathways that regulate polarized cell movement essential for the directional migration of CNCs.     

MEGAP impedes cell migration via regulating actin and microtubule dynamics and focal complex formation

Over the past several years, it has become clear that the Rho family of GTPases plays an important role in various aspects of neuronal development including cytoskeleton dynamics and cell adhesion processes. We have analysed the role of MEGAP, a GTPase-activating protein that acts towards Rac1 and Cdc42 in vitro and in vivo, with respect to its putative regulation of cytoskeleton dynamics and cell migration. To investigate the effects of MEGAP on these cellular processes, we have established an inducible cell culture model consisting of a stably transfected neuroblastoma SHSY-5Y cell line that endogenously expresses MEGAP albeit at low levels. We can show that the induced expression of MEGAP leads to the loss of filopodia and lamellipodia protrusions, whereas constitutively activated Rac1 and Cdc42 can rescue the formation of these structures. We have also established quantitative assays for evaluating actin dynamics and cellular migration. By time-lapse microscopy, we show that induced MEGAP expression reduces cell migration by 3.8-fold and protrusion formation by 9-fold. MEGAP expressing cells also showed impeded microtubule dynamics as demonstrated in the TC-7 3x-GFP epithelial kidney cells. In contrast to the wild type, overexpression of MEGAP harbouring an artificially introduced missense mutation R542I within the functionally important GAP domain did not exert a visible effect on actin and microtubule cytoskeleton remodelling. These data suggest that MEGAP negatively regulates cell migration by perturbing the actin and microtubule cytoskeleton and by hindering the formation of focal complexes.     

Betacellulin enhances ovarian cancer cell migration by up-regulating Connexin43 via MEK-ERK signaling

Epithelial ovarian cancer is the fifth common cause of cancer death in women and the most lethal gynecological malignancies. Our previous studies have shown that up-regulation of Connexin43, a gap-junction subunit crucial for cell-cell communication, enhances ovarian cancer cell migration. Betacellulin is a member of the epidermal growth factor (EGF) family which can bind to multiple EGF family receptors. Overexpression of betacellulin is found in a variety of cancers and is associated with reduced survival. However, the specific roles and molecular mechanisms of betacellulin in ovarian cancer progression are poorly understood. In the current study, we tested the hypothesis that betacellulin induces ovarian cancer cell migration by up-regulating Connexin43. Our results showed that treatment with betacellulin significantly increased Connexin43 expression and cell migration in both OVCAR4 and SKOV3 ovarian cancer cell lines. Moreover, betacellulin induced the activation of MEK-ERK signaling, and its effects on Connexin43 were inhibited by pre-treatment with U0126. Pre-treatment with AG1478 totally blocked the activation of MEK-ERK signaling but only partially inhibited betacellulin-induced Connexin43 expression and cell migration. Most importantly, betacellulin-induced cell migration was attenuated by knockdown of Connexin43, and co-treatment with gap junction inhibitor carbenoxolone did not alter this effect. Our results suggest a bilateral role of Connexin43 in ovarian cancer migration, and also demonstrate a gap junction-independent mechanism of betacellulin.     

PTTG1/securin modulates microtubule nucleation and cell migration

Pituitary tumor transforming gene 1 (PTTG1), also known as securin, has been implicated in many biological functions, including inhibition of sister chromatid separation, DNA repair, organ development, and regulation of the expression and secretion of angiogenic and metastatic factors. Although most of these functions of securin seem to depend on the localization of PTTG1 in the nucleus of the cell, a fraction of the protein has been also detected in the cytoplasm. Here we demonstrate that, in different cell types, a portion of cytoplasmic PTTG1 is associated with the cis face of the Golgi apparatus and that this localization depends on PTTG1 phosphorylation status. In this organelle, PTTG1 forms a complex with proteins involved in microtubule nucleation, including GM130, AKAP450, and γ-tubulin. RNA interference-mediated depletion of PTTG1 produces a delay in centrosomal and noncentrosomal microtubule nucleation. Cells lacking PTTG1 show severe defects in both cell polarization and migration in wound-healing assays. To our knowledge, this is the first study reporting the role of PTTG1 in microtubule nucleation and cell polarization, two processes directly involved in cell migration. We believe that these findings will contribute to understanding the mechanisms underlying PTTG1-mediated biological functions.     

Survivin modulates microtubule dynamics and nucleation throughout the cell cycle

Survivin is a member of the chromosomal passenger complex implicated in kinetochore attachment, bipolar spindle formation, and cytokinesis. However, the mechanism by which survivin modulates these processes is unknown. Here, we show by time-lapse imaging of cells expressing either green fluorescent protein (GFP)-alpha-tubulin or the microtubule plus-end binding protein GFP-EB1 that depletion of survivin by small interfering RNAs (siRNAs) increased both the number of microtubules nucleated by centrosomes and the incidence of microtubule catastrophe, the transition from microtubule growth to shrinking. In contrast, survivin overexpression reduced centrosomal microtubule nucleation and suppressed both microtubule dynamics in mitotic spindles and bidirectional growth of microtubules in midbodies during cytokinesis. siRNA depletion or pharmacologic inhibition of another chromosomal passenger protein Aurora B, had no effect on microtubule dynamics or nucleation in interphase or mitotic cells even though mitosis was impaired. We propose a model in which survivin modulates several mitotic events, including spindle and interphase microtubule organization, the spindle assembly checkpoint and cytokinesis through its ability to modulate microtubule nucleation and dynamics. This pathway may affect the microtubule-dependent generation of aneuploidy and defects in cell polarity in cancer cells, where survivin is commonly up-regulated.     

AKAP350 modulates microtubule dynamics

AKAP350 is a multiply spliced type II protein kinase A-anchoring protein that localizes to the centrosomes in most cells and the Golgi apparatus in epithelial cells. Multiple studies suggest that AKAP350 is involved in microtubule nucleation at the centrosome. Our previous studies demonstrated that AKAP350 was necessary for the maintenance of Golgi apparatus integrity. These data suggested that AKAP350 might be necessary for normal cytoskeletal interactions with the Golgi. To examine the relationship of AKAP350 with the microtubule cytoskeleton, we analyzed the effect of the depletion of AKAP350 on microtubule regrowth after nocodazole treatment in HeLa cells. The decrease in AKAP350 expression with short interfering RNA induced a delay in microtubule elongation with no effect on microtubule aster formation. In contrast, overexpression of the centrosomal targeting domain of AKAP350 elicited alterations in aster formation, but did not affect microtubule elongation. RNA interference for AKAP350 also induced an increase in cdc42 activity during microtubule regrowth. Our data suggest that AKAP350 has a role in the remodeling of the microtubule cytoskeleton.     

Integrin switching modulates adhesion dynamics and cell migration

When cells are stimulated to move, for instance during development, wound healing or angiogenesis, they undergo changes in the turnover of their cell-matrix adhesions. This is often accompanied by alterations in the expression profile of integrins—the extracellular matrix receptors that mediate anchorage within these adhesions. Here, we discuss how a shift in expression between two different types of integrins that bind fibronectin can have dramatic consequences for cell-matrix adhesion dynamics and cell motility.Key words: integrin, fibronectin, migration, cytoskeleton, dynamicsCells attach to the extracellular matrix (ECM) that surrounds them in specialized structures termed “cell-matrix adhesions.” These come in different flavors including “focal complexes” (small adhesions found in membrane protrusions of spreading and migrating cells), “focal adhesions” (larger adhesions connected by F-actin stress fibers that are derived from focal complexes in response to tension), “fibrillar adhesions” (elongated adhesions associated with fibronectin matrix assembly), and proteolytically active adhesions termed “podosomes” or “invadopodia” found in osteoclasts, macrophages and certain cancer cells. Common to all these structures is the local connection between ECM proteins outside- and the actin cytoskeleton within the cell through integrin transmembrane receptors. The intracellular linkage to filamentous actin is indirect through proteins that concentrate in cell-matrix adhesions such as talin, vinculin, tensin, parvins and others.¹Cell migration is essential for embryonic development and a number of processes in the adult, including immune cell homing, wound healing, angiogenesis and cancer metastasis. In moving cells, cell-matrix adhesion turnover is spatiotemporally controlled.² New adhesions are made in the front and disassembled in the rear of cells that move along a gradient of motogenic factors or ECM proteins. This balance between formation and breakdown of cell-matrix adhesions is important for optimal cell migration. Several mechanisms regulate the turnover of cell-matrix adhesions. Proteolytic cleavage of talin has been identified as an important step in cell-matrix adhesion disassembly³ and FAK and Src family kinases are required for cell-matrix adhesion turnover and efficient cell migration.^4,5 Besides regulating phospho-tyrosine-mediated protein-protein interactions within cell-matrix adhesions, the FAK/Src complex mediates signaling downstream of integrins to Rho GTPases, thus controlling cytoskeletal organization.^6,7 The transition from a stationary to a motile state could involve (local) activation of such mechanisms.Interestingly, conditions of increased cell migration (development, wound healing, angiogenesis, cancer metastasis) are accompanied by shifts in integrin expression with certain integrins being lost and others gained. Most ECM proteins can be recognized by various different integrins. For instance, the ECM protein, fibronectin (Fn) can be recognized by nine different types of integrins and most of these bind to the Arg-Gly-Asp (RGD) motif in the central cell-binding domain. Thus, cell-matrix adhesions formed on Fn contain a mixture of different integrins and shifts in expression from one class of Fn-binding integrins to another will alter the receptor composition of such adhesions. This may provide an alternative means to shift from stationary to motile.Indeed, we have found that the type of integrins used for binding to Fn strongly affects cell migration. We made use of cells deficient in certain Fn-binding integrins and either restored their expression or compensated for their absence by overexpression of alternative Fn-binding integrins. This allowed us to compare in a single cellular background cell-matrix adhesions containing α5β1 to those containing αvβ3. Despite the fact that these integrins support similar levels of adhesion to Fn, only α5β1 was found to promote a contractile, fibroblastic morphology with centripetal orientation of cell-matrix adhesions⁸ (Fig. 1). Moreover, RhoA activity is high in the presence of α5β1 and these cells move in a random fashion with a speed of around 25 mm/h. By contrast, in cells using αvβ3 instead, adhesions distribute across the ventral surface, RhoA activity is low, and these cells move with similar speed but in a highly persistent fashion.^8,9 Finally, photobleaching experiments using GFP-vinculin and GFP-paxillin demonstrated that cell-matrix adhesions containing α5β1 are highly dynamic whereas adhesions containing αvβ3 are more static.⁹Open in a separate windowFigure 1Immunofluorescence images. GE11 cells, epithelial β1 knockout cells derived from mouse embryos chimeric for the integrin β1 subunit endogenously express various av integrins, including low levels of αvβ3 and αvβ5. Ectopic expression of β1 leads to expression of α5β1 and induced α5β1-mediated adhesion to Fn (left image) whereas ectopic expression of β3 (in the β1 null background) leads to strong expression of αvβ3 and induced αvβ3-mediated adhesion to Fn (right image). Adhesions containing either α5β1 or αvβ3 show distinct distribution and dynamics (paxillin; green) and cause different F-actin organization (phalloidin; red). Cartoons: Differences in cell-matrix adhesion dynamics may be explained by differential binding of soluble Fn molecules (blue) or different molecular determinants of the interaction with immobilized Fn (red). See text for details.It has been observed that α5β1 and αvβ3 use different recycling routes. Interfering with Rab4-mediated recycling of αvβ3 causes increased Rab11-mediated recycling of α5β1 to the cell surface. In agreement with our findings, the shift to α5β1 leads to increased Rho-ROCK activity and reduced persistence of migration.¹⁰ One possible explanation for the different types of migration promoted by these two Fn-binding integrins might involve different signaling and/or adaptor proteins interacting with specific amino acids in their cytoplasmic tails. However, this appears not to be the case: α5β1 in which the cytoplasmic tails of α5 or β1 are replaced by those of αv or β3, respectively, behaves identical to wild type α5β1: it promotes a fibroblast-like morphology with centripetal orientation of cell-matrix adhesions and it drives a non-persistent mode of migration.^8,11 Together, these findings point to differences between α5β1 and αvβ3 integrins in the mechanics of their interaction with Fn, which apparently modulates intracellular signaling pathways in control of cell-matrix adhesion dynamics and cell migration.How might this work? It turns out that although α5β1 and αvβ3 similarly support cell adhesion to immobilized (stretched) Fn, only α5β1 efficiently binds soluble, folded (“inactive”) Fn.¹¹ We have proposed that such interactions with soluble Fn molecules (possibly secreted by the cell itself) may weaken the interaction with the immobilized ligand thereby causing enhanced cell-matrix adhesion dynamics in the presence of α5β1,¹¹ (Fig. 1). Preferential binding of soluble Fn by α5β1 could be explained by differences in accessibility of the RGD binding pocket between α5β1 (more exposed) and αvβ3 (more hidden) as suggested by others.¹² If this is the case, immobilization (“stretching”) of Fn apparently leads to reorientation of the RGD motif in such a way that it is easily accessed by both integrins.The issue is considerably complicated by the fact that other recognition motifs are present in the Fn central cell-binding domain. In addition to the RGD sequence in the tenth Fn type 3 repeat (IIIFn10), binding of α5β1, but not αvβ3, also depends on the PHSRN “synergy” sequence in IIIFn9.^13–15 The relative contribution of these motifs is controversial and there is structural data pointing either towards a model in which IIIFn9 interacts with α5β1 or towards a model in which IIIFn9 exerts long-range electrostatic steering resulting in a higher affinity interaction without contacting the integrin.^16,17 Cell adhesion studies have suggested that an interaction of α5β1 with the synergy region stabilizes the binding to RGD.^14,18 Such a two-step interaction may facilitate binding to full length, folded Fn for instance by altering the tilt angle between IIIFn9 and IIIFn10 leading to optimal exposure of the RGD loop, perhaps explaining why αvβ3 (which may not interact with the synergy site) poorly binds soluble Fn.Others have shown that the RGD motif alone is sufficient for mechanical coupling of αvβ3 to Fn whereas the synergy region is required to provide mechanical strength to the α5β1-Fn bond.¹⁹ It appears that the interaction of α5β1 with Fn is particularly dynamic with various conformations of α5β1 interacting with different Fn binding surfaces, including the RGD and synergy sequences as well as other regions in IIIFn9. Thus, besides the above model based on differential binding to soluble Fn molecules, differences in the complexity and dynamics of interactions with immobilized Fn that determine functional binding strength could also underlie the different dynamics of cell-matrix adhesions containing either α5β1 or αvβ3 (Fig. 1).Precisely how mechanical differences in receptor-ligand interactions result in such remarkably distinct cellular responses is poorly understood. In addition to effects on cell-matrix adhesion dynamics and cytoskeletal organization it is also associated with different activities of Rho GTPases, indicating that mechanical differences between these two integrins must translate into differential activation of intracellular signaling pathways.^8,9,11 Possibly, different adhesion dynamics due to distinct mechanisms of receptor-ligand interaction result in different patterns of F-actin organization, which, in turn, affects the formation of signaling platforms. It is also possible that differences in the extent of integrin clustering have an impact on the conformation of one or more cytoplasmic components of the cell-matrix adhesions containing either α5β1 or αvβ3. This could lead to hiding or exposing binding sites for signaling molecules (e.g., upstream regulators of Rho GTPases) or substrates. Whatever the mechanism involved, altering the integrin composition of cell-matrix adhesions through shifts in integrin expression as observed during development, angiogenesis, wound healing and cancer progression may be a driving force in the enhanced cell migration that characterizes those processes.     

Integrin switching modulates adhesion dynamics and cell migration

When cells are stimulated to move, for instance during development, wound healing, or angiogenesis, they undergo changes in the turnover of their cell-matrix adhesions. This is often accompanied by alterations in the expression profile of integrins -- the extracellular matrix receptors that mediate anchorage within these adhesions. Here, we discuss how a shift in expression between two different types of integrins that bind fibronectin can have dramatic consequences for cell-matrix adhesion dynamics and cell motility.     

Hippo kinases maintain polarity during directional cell migration in Caenorhabditis elegans

Precise positioning of cells is crucial for metazoan development. Despite immense progress in the elucidation of the attractive cues of cell migration, the repulsive mechanisms that prevent the formation of secondary leading edges remain less investigated. Here, we demonstrate that Caenorhabditis elegans Hippo kinases promote cell migration along the anterior–posterior body axis via the inhibition of dorsal–ventral (DV) migration. Ectopic DV polarization was also demonstrated in gain‐of‐function mutant animals for C. elegans RhoG MIG‐2. We identified serine 139 of MIG‐2 as a novel conserved Hippo kinase phosphorylation site and demonstrated that purified Hippo kinases directly phosphorylate MIG‐2^S139. Live imaging analysis of genome‐edited animals indicates that MIG‐2^S139 phosphorylation impedes actin assembly in migrating cells. Intriguingly, Hippo kinases are excluded from the leading edge in wild‐type cells, while MIG‐2 loss induces uniform distribution of Hippo kinases. We provide evidence that Hippo kinases inhibit RhoG activity locally and are in turn restricted to the cell body by RhoG‐mediated polarization. Therefore, we propose that the Hippo–RhoG feedback regulation maintains cell polarity during directional cell motility.     

Angiogenin enhances cell migration by regulating stress fiber assembly and focal adhesion dynamics

Angiogenin (ANG) acts on both vascular endothelial cells and cancer cells, but the underlying mechanism remains elusive. In this study, we carried out a co-immunoprecipitation assay in HeLa cells and identified 14 potential ANG-interacting proteins. Among these proteins, β-actin, α-actinin 4, and non-muscle myosin heavy chain 9 are stress fiber components and involved in cytoskeleton organization and movement, which prompted us to investigate the mechanism of action of ANG in cell migration. Upon confirmation of the interactions between ANG and the three proteins, further studies revealed that ANG co-localized with β-actin and α-actinin 4 at the leading edge of migrating cells. Down-regulation of ANG resulted in fewer but thicker stress fibers with less dynamics, which was associated with the enlargements of focal adhesions. The focal adhesion kinase activity and cell migration capacity were significantly decreased in ANG-deficient cells. Taken together, our data demonstrated that the existence of ANG in the cytoplasm optimizes stress fiber assembly and focal adhesion formation to accommodate cell migration. The finding that ANG promoted cancer cell migration might provide new clues for tumor metastasis research.     

CYLD coordinates with EB1 to regulate microtubule dynamics and cell migration

Cylindromatosis (CYLD), a deubiquitinase involved in inflammation and tumorigenesis via the modulation of cell signaling, has recently been identified as a critical regulator of microtubule dynamics. CYLD has also been shown to stimulate cell migration and thereby contribute to normal physiological processes. However, it remains elusive how the regulation of microtubule dynamic properties by CYLD is connected to its role in mediating cell migration. In this study, we performed yeast 2-hybrid screening with CYLD as bait and identified 7 CYLD-interacting proteins, including end-binding protein 1 (EB1). The CYLD–EB1 interaction was confirmed both in cells and in vitro, and these 2 proteins colocalized at the plus ends of microtubules. Interestingly, the association of CYLD with EB1 was significantly increased upon the stimulation of cell migration. CYLD coordinated with EB1 to orchestrate tail retraction, centrosome reorientation, and leading-edge microtubule stabilization in migratory cells. In addition, CYLD acted in concert with EB1 to regulate microtubule assembly in vitro, microtubule nucleation at the centrosome, and microtubule growth at the cell periphery. These data provide mechanistic insights into the actions of CYLD in the regulation of microtubule dynamics and cell migration. These findings also support the notion that coordinated actions of microtubule-binding proteins are critical for microtubule-mediated cellular events.     

Connexin 43在细胞死亡中的作用

缝隙连接蛋白在细胞膜表面聚合形成半通道,部分两两结合构成缝隙连接通道,两者与胚胎发育、肿瘤发生及某些心脑血管疾病有关。Connexin 43(Cx43)在心肌细胞和神经细胞高表达,在多种缺血性心脑疾病及缺血再灌注损伤的病理过程中具有重要作用。近来有研究发现,Cx43也存在于线粒体和细胞核,分别参与心肌保护和细胞分化。该文以心肌细胞和神经细胞为主讨论近年来Cx43在细胞死亡中的作用的研究进展。     

LARG and mDia1 link Galpha12/13 to cell polarity and microtubule dynamics

Regulation of cell polarity is a process observed in all cells. During directed migration, cells orientate their microtubule cytoskeleton and the microtubule-organizing-center (MTOC), which involves integrins and downstream Cdc42 and glycogen synthase kinase-3beta activity. However, the contribution of G protein-coupled receptor signal transduction for MTOC polarity is less well understood. Here, we report that the heterotrimeric Galpha(12) and Galpha(13) proteins are necessary for MTOC polarity and microtubule dynamics based on studies using Galpha(12/13)-deficient mouse embryonic fibroblasts. Cell polarization involves the Galpha(12/13)-interacting leukemia-associated RhoGEF (LARG) and the actin-nucleating diaphanous formin mDia1. Interestingly, LARG associates with pericentrin and localizes to the MTOC and along microtubule tracks. We propose that Galpha(12/13) proteins exert essential functions linking extracellular signals to microtubule dynamics and cell polarity via RhoGEF and formin activity.     

An ELMO2-RhoG-ILK network modulates microtubule dynamics

ELMO2 belongs to a family of scaffold proteins involved in phagocytosis and cell motility. ELMO2 can simultaneously bind integrin-linked kinase (ILK) and RhoG, forming tripartite ERI complexes. These complexes are involved in promoting β1 integrin–dependent directional migration in undifferentiated epidermal keratinocytes. ELMO2 and ILK have also separately been implicated in microtubule regulation at integrin-containing focal adhesions. During differentiation, epidermal keratinocytes cease to express integrins, but ERI complexes persist. Here we show an integrin-independent role of ERI complexes in modulation of microtubule dynamics in differentiated keratinocytes. Depletion of ERI complexes by inactivating the Ilk gene in these cells reduces microtubule growth and increases the frequency of catastrophe. Reciprocally, exogenous expression of ELMO2 or RhoG stabilizes microtubules, but only if ILK is also present. Mechanistically, activation of Rac1 downstream from ERI complexes mediates their effects on microtubule stability. In this pathway, Rac1 serves as a hub to modulate microtubule dynamics through two different routes: 1) phosphorylation and inactivation of the microtubule-destabilizing protein stathmin and 2) phosphorylation and inactivation of GSK-3β, which leads to the activation of CRMP2, promoting microtubule growth. At the cellular level, the absence of ERI species impairs Ca²⁺-mediated formation of adherens junctions, critical to maintaining mechanical integrity in the epidermis. Our findings support a key role for ERI species in integrin-independent stabilization of the microtubule network in differentiated keratinocytes.     

Ror2 enhances polarity and directional migration of primordial germ cells

The trafficking of primordial germ cells (PGCs) across multiple embryonic structures to the nascent gonads ensures the transmission of genetic information to the next generation through the gametes, yet our understanding of the mechanisms underlying PGC migration remains incomplete. Here we identify a role for the receptor tyrosine kinase-like protein Ror2 in PGC development. In a Ror2 mouse mutant we isolated in a genetic screen, PGC migration and survival are dysregulated, resulting in a diminished number of PGCs in the embryonic gonad. A similar phenotype in Wnt5a mutants suggests that Wnt5a acts as a ligand to Ror2 in PGCs, although we do not find evidence that WNT5A functions as a PGC chemoattractant. We show that cultured PGCs undergo polarization, elongation, and reorientation in response to the chemotactic factor SCF (secreted KitL), whereas Ror2 PGCs are deficient in these SCF-induced responses. In the embryo, migratory PGCs exhibit a similar elongated geometry, whereas their counterparts in Ror2 mutants are round. The protein distribution of ROR2 within PGCs is asymmetric, both in vitro and in vivo; however, this asymmetry is lost in Ror2 mutants. Together these results indicate that Ror2 acts autonomously to permit the polarized response of PGCs to KitL. We propose a model by which Wnt5a potentiates PGC chemotaxis toward secreted KitL by redistribution of Ror2 within the cell.     

RACK1 regulates Src activity and modulates paxillin dynamics during cell migration

Receptor for Activated C Kinase, RACK1, is an adaptor protein that regulates signaling via Src and PKC-dependent pathways, and has been implicated in cell migration. In this study we demonstrate novel functions for RACK1 in regulating adhesion dynamics during cell migration. We report that cells lacking RACK1 are less motile and show reduced dynamics of paxillin and talin at focal complexes. To investigate the role of the RACK1/Src interactions in adhesion dynamics, we used RACK1 in which the putative Src binding site has been mutated (RACK Y246F). RACK1-deficient cells showed enhanced c-Src activity that was rescued by expression of wild type RACK1, but not by RACK Y246F. Expression of wild type RACK1, but not RACK Y246F, was also able to rescue the adhesion and migration defects observed in the RACK1-deficient cells. Furthermore, our findings indicate that RACK1 functions to regulate paxillin phosphorylation and that its effects on paxillin dynamics require the Src-mediated phosphorylation of tyrosine 31/118 on paxillin. Taken together, these findings support a novel role for RACK1 as a key regulator of cell migration and adhesion dynamics through the regulation of Src activity, and the modulation of paxillin phosphorylation at early adhesions.     

The tumor suppressor CYLD regulates microtubule dynamics and plays a role in cell migration

The familial cylindromatosis tumor suppressor CYLD is known to contain three cytoskeleton-associated protein glycine-rich (CAP-Gly) domains, which exist in a number of microtubule-binding proteins and are responsible for their association with microtubules. However, it remains elusive whether CYLD interacts with microtubules and, if so, whether the interaction is mediated by the CAP-Gly domains. In this study, our data demonstrate that CYLD associates with microtubules both in cells and in vitro, and the first CAP-Gly domain of CYLD is mainly responsible for the interaction. Knockdown of cellular CYLD expression dramatically delays microtubule regrowth after nocodazole washout, indicating an activity for CYLD in promoting microtubule assembly. Our data further demonstrate that CYLD enhances tubulin polymerization into microtubules by lowering the critical concentration for microtubule assembly. In addition, we have identified by wound healing assay a critical role for CYLD in mediating cell migration and found that its first CAP-Gly domain is required for this activity. Thus CYLD joins a growing list of CAP-Gly domain-containing proteins that regulate microtubule dynamics and function.     

Osteoblast connexin43 modulates skeletal architecture by regulating both arms of bone remodeling

Connexin43 (Cx43) has an important role in skeletal homeostasis, and Cx43 gene (Gja1) mutations have been linked to oculodentodigital dysplasia (ODDD), a human disorder characterized by prominent skeletal abnormalities. To determine the function of Cx43 at early steps of osteogenesis and its role in the ODDD skeletal phenotype, we have used the Dermo1 promoter to drive Gja1 ablation or induce an ODDD mutation in the chondro-osteogenic linage. Both Gja1 null and ODDD mutant mice develop age-related osteopenia, primarily due to a progressive enlargement of the medullary cavity and cortical thinning. This phenotype is the consequence of a high bone turnover state, with increased endocortical osteoclast-mediated bone resorption and increased periosteal bone apposition. Increased bone resorption is a noncell autonomous defect, caused by exuberant stimulation of osteoclastogenesis by Cx43-deficient bone marrow stromal cells, via decreased Opg production. The latter is part of a broad defect in osteoblast differentiation and function, which also results in abnormal structural and material properties of bone leading to decreased resistance to mechanical load. Thus Cx43 in osteogenic cells is a critical regulator of both arms of the bone remodeling cycle, its absence causing structural changes remindful of aged or disused bone.