Dysregulation of myelin synthesis and actomyosin function underlies aberrant myelin in CMT4B1 neuropathy.

Charcot-Marie-Tooth type 4B1 (CMT4B1) is a severe autosomal recessive demyelinating neuropathy with childhood onset, caused by loss-of-function mutations in the myotubularin-related 2 (MTMR2) gene. MTMR2 is a ubiquitously expressed catalytically active 3-phosphatase, which in vitro dephosphorylates the 3-phosphoinositides PtdIns3P and PtdIns(3,5)P2, with a preference for PtdIns(3,5)P2 A hallmark of CMT4B1 neuropathy are redundant loops of myelin in the nerve termed myelin outfoldings, which can be considered the consequence of altered growth of myelinated fibers during postnatal development. How MTMR2 loss and the resulting imbalance of 3'-phosphoinositides cause CMT4B1 is unknown. Here we show that MTMR2 by regulating PtdIns(3,5)P2 levels coordinates mTORC1-dependent myelin synthesis and RhoA/myosin II-dependent cytoskeletal dynamics to promote myelin membrane expansion and longitudinal myelin growth. Consistent with this, pharmacological inhibition of PtdIns(3,5)P2 synthesis or mTORC1/RhoA signaling ameliorates CMT4B1 phenotypes. Our data reveal a crucial role for MTMR2-regulated lipid turnover to titrate mTORC1 and RhoA signaling thereby controlling myelin growth.

Novel role of Rac-Mid1 signaling in medial cerebellar development.

Rac signaling impacts a relatively large number of downstream targets; however, few studies have established an association between Rac pathways and pathological conditions. In the present study, we generated mice with double knockout of Rac1 and Rac3 (Atoh1-Cre;Rac1flox/flox;Rac3-/- ) in cerebellar granule neurons (CGNs). We observed impaired tangential migration at E16.5, as well as numerous apoptotic CGNs at the deepest layer of the external granule layer (EGL) in the medial cerebellum of Atoh1-Cre;Rac1flox/flox;Rac3-/- mice at P8. Atoh1-Cre;Rac1flox/flox;Rac3-/- CGNs differentiated normally until expression of p27kip1 and NeuN in the deep EGL at P5. Primary CGNs and cerebellar microexplants from Atoh1-Cre;Rac1flox/flox;Rac3-/- mice exhibited impaired neuritogenesis, which was more apparent in Map2-positive dendrites. Such findings suggest that impaired tangential migration and final differentiation of CGNs have resulted in decreased cerebellum size and agenesis of the medial internal granule layer, respectively. Furthermore, Rac depleted/deleted cells exhibited decreased levels of Mid1 and impaired mTORC1 signaling. Mid1 depletion in CGNs produced mild impairments in neuritogenesis and reductions in mTORC1 signaling. Thus, a novel Rac-signaling pathway (Rac1-Mid1-mTORC1) may be involved in medial cerebellar development.

Effects of the scaffold proteins liprin-α1, ß1 and ß2 on invasion by breast cancer cells.

BACKGROUND INFORMATION: The expression of the scaffold protein liprin-α1 is upregulated in human breast cancer. This protein is part of a molecular network that is important for tumour cell invasion in vitro. Liprin-α1 promotes invasion by supporting the protrusive activity at the leading edge of the migrating tumour cell and the degradation of the extracellular matrix by invadopodia. In this study, we have addressed the role of liprin-α1 in the invasive process in vivo and of liprin-proteins in tumor cell motility. RESULTS: The human tumour cell line MDA-MB-231 expresses liprin-α1 and is able to promote the formation of metastasis in mice. Liprin-α proteins may hetero-oligomerize with the members of the subfamily of the liprin-ß adaptor proteins. Analysis of the role of liprin-ß1 and liprin-ß2 has shown that while liprin-ß1 contributes positively to tumour cell motility in vitro; liprin-ß2 has a negative effect on both cell motility and invasion. Interestingly, we also observed differential effects on the ability of tumour cells to degrade the extracellular matrix, which is required for efficient invasion by tumour cells. In addition, analysis of the formation of lung metastases in vivo revealed that while the overexpression of liprin-α1 in MDA-MB-231 cells did not evidently affect the metastatic process, silencing of the endogenous protein strongly impaired the formation of metastases by two independent invasion assays, without inhibiting the growth of primary tumours. CONCLUSIONS: Our data support an important role of distinct liprin family members in the regulation of tumour cell invasion, highlighting pro-invasive and anti-invasive effects by liprin-α1 and liprin-ß2, respectively. SIGNIFICANCE: Our results indicate the importance of liprins in breast cancer cell invasion, and are expected to lead to future investigations on the mechanisms underlying the effects of distinct liprin proteins in different processes linked to tumor cell migration and invasion.

Loss of Either Rac1 or Rac3 GTPase Differentially Affects the Behavior of Mutant Mice and the Development of Functional GABAergic Networks.

Rac GTPases regulate the development of cortical/hippocampal GABAergic interneurons by affecting the early development and migration of GABAergic precursors. We have addressed the function of Rac1 and Rac3 proteins during the late maturation of hippocampal interneurons. We observed specific phenotypic differences between conditional Rac1 and full Rac3 knockout mice. Rac1 deletion caused greater generalized hyperactivity and cognitive impairment compared with Rac3 deletion. This phenotype matched with a more evident functional impairment of the inhibitory circuits in Rac1 mutants, showing higher excitability and reduced spontaneous inhibitory currents in the CA hippocampal pyramidal neurons. Morphological analysis confirmed a differential modification of the inhibitory circuits: deletion of either Rac caused a similar reduction of parvalbumin-positive inhibitory terminals in the pyramidal layer. Intriguingly, cannabinoid receptor-1-positive terminals were strongly increased only in the CA1 of Rac1-depleted mice. This increase may underlie the stronger electrophysiological defects in this mutant. Accordingly, incubation with an antagonist for cannabinoid receptors partially rescued the reduction of spontaneous inhibitory currents in the pyramidal cells of Rac1 mutants. Our results show that Rac1 and Rac3 have independent roles in the formation of GABAergic circuits, as highlighted by the differential effects of their deletion on the late maturation of specific populations of interneurons.

Liprin-α1, ERC1 and LL5 define polarized and dynamic structures that are implicated in cell migration.

Cell migration during development and metastatic invasion requires the coordination of actin and adhesion dynamics to promote protrusive activity at the front of the cell. The knowledge of the molecular mechanisms required to achieve such coordination is fragmentary. Here, we identify a new functional complex that drives cell motility. ERC1a (an isoform of ERC1) and the LL5 proteins LL5α and LL5ß (encoded by PHLDB1 and PHLDB2, respectively) are required, together with liprin-α1, for effective migration and tumor cell invasion, and do so by stabilizing the protrusive activity at the cell front. Depletion of either protein negatively affects invasion, migration on extracellular matrix, lamellipodial persistence and the internalization of active integrin ß1 receptors needed for adhesion turnover at the front of the cell. Liprin-α1, ERC1a and LL5 also define new highly polarized and dynamic cytoplasmic structures uniquely localized near the protruding cell edge. Our results indicate that the functional complex and the associated structures described here represent an important mechanism to drive tumor cell migration.

Rac-GTPases Regulate Microtubule Stability and Axon Growth of Cortical GABAergic Interneurons.

Cortical interneurons are characterized by extraordinary functional and morphological diversity. Although tremendous progress has been made in uncovering molecular and cellular mechanisms implicated in interneuron generation and function, several questions still remain open. Rho-GTPases have been implicated as intracellular mediators of numerous developmental processes such as cytoskeleton organization, vesicle trafficking, transcription, cell cycle progression, and apoptosis. Specifically in cortical interneurons, we have recently shown a cell-autonomous and stage-specific requirement for Rac1 activity within proliferating interneuron precursors. Conditional ablation of Rac1 in the medial ganglionic eminence leads to a 50% reduction of GABAergic interneurons in the postnatal cortex. Here we examine the additional role of Rac3 by analyzing Rac1/Rac3 double-mutant mice. We show that in the absence of both Rac proteins, the embryonic migration of medial ganglionic eminence-derived interneurons is further impaired. Postnatally, double-mutant mice display a dramatic loss of cortical interneurons. In addition, Rac1/Rac3-deficient interneurons show gross cytoskeletal defects in vitro, with the length of their leading processes significantly reduced and a clear multipolar morphology. We propose that in the absence of Rac1/Rac3, cortical interneurons fail to migrate tangentially towards the pallium due to defects in actin and microtubule cytoskeletal dynamics.

Rac1 and rac3 GTPases control synergistically the development of cortical and hippocampal GABAergic interneurons.

The intracellular mechanisms driving postmitotic development of cortical γ-aminobutyric acid (GABA)ergic interneurons are poorly understood. We have addressed the function of Rac GTPases in cortical and hippocampal interneuron development. Developing neurons express both Rac1 and Rac3. Previous work has shown that Rac1 ablation does not affect the development of migrating cortical interneurons. Analysis of mice with double deletion of Rac1 and Rac3 shows that these GTPases are required during postmitotic interneuron development. The number of parvalbumin-positive cells was affected in the hippocampus and cortex of double knockout mice. Rac depletion also influences the maturation of interneurons that reach their destination, with reduction of inhibitory synapses in both hippocampal CA1 and cortical pyramidal cells. The decreased number of cortical migrating interneurons and their altered morphology indicate a role of Rac1 and Rac3 in regulating the motility of cortical interneurons, thus interfering with their final localization. While electrophysiological passive and active properties of pyramidal neurons including membrane capacity, resting potential, and spike amplitude and duration were normal, these cells showed reduced spontaneous inhibitory currents and increased excitability. Our results show that Rac1 and Rac3 contribute synergistically to postmitotic development of specific populations of GABAergic cells, suggesting that these proteins regulate their migration and differentiation.

Cell surface dynamics - how Rho GTPases orchestrate the interplay between the plasma membrane and the cortical cytoskeleton.

Small GTPases are known to regulate hundreds of cell functions. In particular, Rho family GTPases are master regulators of the cytoskeleton. By regulating actin nucleation complexes, Rho GTPases control changes in cell shape, including the extension and/or retraction of surface protrusions and invaginations. Protrusion and invagination of the plasma membrane also involves the interaction between the plasma membrane and the cortical cytoskeleton. This interplay between membranes and the cytoskeleton can lead to an increase or decrease in the plasma membrane surface area and its tension as a result of the fusion (exocytosis) or internalization (endocytosis) of membranous compartments, respectively. For a long time, the cytoskeleton and plasma membrane dynamics were investigated separately. However, studies from many laboratories have now revealed that Rho GTPases, their modulation of the cytoskeleton, and membrane traffic are closely connected during the dynamic remodeling of the cell surface. Arf- and Rab-dependent exocytosis of specific vesicles contributes to the targeting of Rho GTPases and their regulatory factors to discrete sites of the plasma membrane. Rho GTPases regulate the tethering of exocytic vesicles and modulate their subsequent fusion. They also have crucial roles in the different forms of endocytosis, where they participate in the sorting of membrane domains as well as the sculpting and sealing of membrane flasks and cups. Here, we discuss how cell surface dynamics depend on the orchestration of the cytoskeleton and the plasma membrane by Rho GTPases.

Reduction in parvalbumin-positive interneurons and inhibitory input in the cortex of mice with experimental autoimmune encephalomyelitis.

In multiple sclerosis (MS), inflammation leads to damage of central nervous system myelin and axons. Previous studies have postulated impaired GABA transmission in MS, and recent postmortem analysis has shown that GABAergic parvalbumin (PV)-positive interneurons are decreased in the primary motor cortex (M1) of patients with MS. In this report, we present evidence for the loss of a specific population of GABAergic interneurons in the experimental autoimmune encephalomyelitis mouse model of MS. Using experimental autoimmune encephalomyelitis, we evaluated the distribution of both PV-positive interneurons and of the inhibitory presynaptic input in the M1 of experimental autoimmune encephalomyelitis and control mice. Our results demonstrate a specific decrease in the number of PV-positive interneurons in the M1 of mice with experimental autoimmune encephalomyelitis. We detected a significant reduction in the number of PV-positive interneurons in the layers II and III of the M1 of diseased mice, while there was no difference in the number of calretinin (CR)-positive cells between animals with experimental autoimmune encephalomyelitis and control animals. Moreover, we observed a significant reduction in the inhibitory presynaptic input in the M1 of treated mice. These changes were specific for the mice with elevated clinical score, while they were not detectable in the mice with low clinical score. Our results support the hypothesis that reinforcing the action of the GABAergic network may represent a therapeutic alternative to limit the progression of the neuronal damage in MS patients.

Dual specificity kinase DYRK3 regulates cell migration by influencing the stability of protrusions.

Plasma membrane-associated platforms (PMAPs) form at specific sites of plasma membrane by scaffolds including ERC1 and Liprin-α1. We identify a mechanism regulating PMAPs assembly, with consequences on motility/invasion. Silencing Ser/Thr kinase DYRK3 in invasive breast cancer cells inhibits their motility and invasive capacity. Similar effects on motility were observed by increasing DYRK3 levels, while kinase-dead DYRK3 had limited effects. DYRK3 overexpression inhibits PMAPs formation and has negative effects on stability of lamellipodia and adhesions in migrating cells. Liprin-α1 depletion results in unstable lamellipodia and impaired cell motility. DYRK3 causes increased Liprin-α1 phosphorylation. Increasing levels of Liprin-α1 rescue the inhibitory effects of DYRK3 on cell spreading, suggesting that an equilibrium between Liprin-α1 and DYRK3 levels is required for lamellipodia stability and tumor cell motility. Our results show that DYRK3 is relevant to tumor cell motility, and identify a PMAP target of the kinase, highlighting a new mechanism regulating cell edge dynamics.

Biochemical and functional characterisation of αPIX, a specific regulator of axonal and dendritic branching in hippocampal neurons.

BACKGROUND INFORMATION: PIX proteins are exchange factors for Rac and Cdc42 GTPases that are differentially expressed in the brain, where they are implicated in neuronal morphogenesis. The PIX family includes the two members αPIX and ßPIX, and the gene of αPIX is mutated in patients with intellectual disability. RESULTS: We have analysed the expression of PIX proteins in the developing brain and addressed their role during early hippocampal neuron development. Mass spectrometry identified several ßPIX isoforms and a major p75 αPIX isoform in brain and hippocampal cultures. PIX proteins expression increased with time during neuronal differentiation in vitro. The PIX partners GIT1 and GIT2 are also found in brain and their expression was increased during neuronal differentiation. We found that αPIX, but not ßPIX, was required for proper hippocampal neuron differentiation, since silencing of αPIX specifically hampered dendritogenesis and axonal branching. Interestingly, the depletion of GIT2 but not GIT1 mimicked the phenotype observed after αPIX knock-down. Over-expression of αPIX specifically enhanced dendritic branching, while both αPIX and ßPIX over-expression affected axonal morphology. Again, only over-expression of GIT2, but not GIT1, affected neuritic morphology. CONCLUSIONS: The results indicate that αPIX and GIT2 are required for neuronal differentiation, and suggest that they are part of the same pathway, while GIT1 and ßPIX are dispensable for early hippocampal neurons development.

Interfering with the ERC1-LL5ß interaction disrupts plasma membrane-Associated platforms and affects tumor cell motility.

Cell migration requires a complex array of molecular events to promote protrusion at the front of motile cells. The scaffold protein LL5ß interacts with the scaffold ERC1, and recruits it at plasma membrane-associated platforms that form at the front of migrating tumor cells. LL5 and ERC1 proteins support protrusion during migration as shown by the finding that depletion of either endogenous protein impairs tumor cell motility and invasion. In this study we have tested the hypothesis that interfering with the interaction between LL5ß and ERC1 may be used to interfere with the function of the endogenous proteins to inhibit tumor cell migration. For this, we identified ERC1(270-370) and LL5ß(381-510) as minimal fragments required for the direct interaction between the two proteins. The biochemical characterization demonstrated that the specific regions of the two proteins, including predicted intrinsically disordered regions, are implicated in a reversible, high affinity direct heterotypic interaction. NMR spectroscopy further confirmed the disordered nature of the two fragments and also support the occurrence of interaction between them. We tested if the LL5ß protein fragment interferes with the formation of the complex between the two full-length proteins. Coimmunoprecipitation experiments showed that LL5ß(381-510) hampers the formation of the complex in cells. Moreover, expression of either fragment is able to specifically delocalize endogenous ERC1 from the edge of migrating MDA-MB-231 tumor cells. Coimmunoprecipitation experiments show that the ERC1-binding fragment of LL5ß interacts with endogenous ERC1 and interferes with the binding of endogenous ERC1 to full length LL5ß. Expression of LL5ß(381-510) affects tumor cell motility with a reduction in the density of invadopodia and inhibits transwell invasion. These results provide a proof of principle that interfering with heterotypic intermolecular interactions between components of plasma membrane-associated platforms forming at the front of tumor cells may represent a new approach to inhibit cell invasion.

Absence of Rac1 and Rac3 GTPases in the nervous system hinders thymic, splenic and immune-competence development.

The nervous system influences organ development by direct innervation and the action of hormones. We recently showed that the specific absence of Rac1 in neurons (Rac1(N) ) in a Rac3-deficient (Rac3(KO) ) background causes motor behavioural defects, epilepsy, and premature mouse death around postnatal day 13. We report here that Rac1(N) /Rac3(KO) mice display a progressive loss of immune-competence. Comparative longitudinal analysis of lymphoid organs from control, single Rac1(N) or Rac3(KO) , and double Rac1(N) /Rac3(KO) mutant animals showed that thymus development is preserved up to postnatal day 9 in all animals, but is impaired in Rac1(N) /Rac3(KO) mice at later times. This is evidenced by a drastic reduction in thymic cell numbers. Cell numbers were also reduced in the spleen, leading to splenic tissue disarray. Organ involution occurs in spite of unaltered thymocyte and lymphocyte subset composition, and proper mature T-cell responses to polyclonal stimuli in vitro. Suboptimal thymus innervation by tau-positive neuronal terminals possibly explains the suboptimal thymic output and arrested thymic development, which is accompanied by higher apoptotic rates. Our results support a role for neuronal Rac1 and Rac3 in dictating proper lymphoid organ development, and suggest the existence of lymphoid-extrinsic mechanisms linking neural defects to the loss of immune-competence.

Function of liprins in cell motility.

Liprins have been known for years to play an essential role in setting up functional synapses in the nervous system. On the other hand, these proteins had been first identified in non-neuronal cells as multivalent proteins that may affect the integrin-mediated interactions of the cells with extracellular matrix ligands. Although the research on the function of liprins in non-neuronal cells has been quiescent for several years, a number of recent findings are putting them back on stage again as important players also in the regulation of non-neuronal cell motility, and possibly of tumor cell behavior. The aim of this review is to highlight the findings supporting the importance of liprins as central regulators of cell adhesion and motility, making them an interesting family of proteins to be considered for future studies on the mechanisms regulating cell migration.

Paxillin: A Hub for Mechano-Transduction from the ß3 Integrin-Talin-Kindlin Axis.

Focal adhesions are specialized integrin-dependent adhesion complexes, which ensure cell anchoring to the extracellular matrix. Focal adhesions also function as mechano-signaling platforms by perceiving and integrating diverse physical and (bio)chemical cues of their microenvironment, and by transducing them into intracellular signaling for the control of cell behavior. The fundamental biological mechanism of creating intracellular signaling in response to changes in tensional forces appears to be tightly linked to paxillin recruitment and binding to focal adhesions. Interestingly, the tension-dependent nature of the paxillin binding to adhesions, combined with its scaffolding function, suggests a major role of this protein in integrating multiple signals from the microenvironment, and accordingly activating diverse molecular responses. This minireview offers an overview of the molecular bases of the mechano-sensitivity and mechano-signaling capacity of core focal adhesion proteins, and highlights the role of paxillin as a key component of the mechano-transducing machinery based on the interaction of cells to substrates activating the ß3 integrin-talin1-kindlin.

Liquid-Liquid Phase Separation at the Plasma Membrane-Cytosol Interface: Common Players in Adhesion, Motility, and Synaptic Function.

Networks of scaffold proteins and enzymes assemble at the interface between the cytosol and specific sites of the plasma membrane, where these networks guide distinct cellular functions. Some of these plasma membrane-associated platforms (PMAPs) include shared core components that are able to establish specific protein-protein interactions, to produce distinct supramolecular assemblies regulating dynamic processes as diverse as cell adhesion and motility, or the formation and function of neuronal synapses. How cells organize such dynamic networks is still an open question. In this review we introduce molecular networks assembling at the edge of migrating cells, and at pre- and postsynaptic sites, which share molecular players that can drive the assembly of biomolecular condensates. Very recent experimental evidence has highlighted the emerging role of some of these multidomain/scaffold proteins belonging to the GIT, liprin-α and ELKS/ERC families as drivers of liquid-liquid phase separation (LLPS). The data point to an important role of LLPS: (i) in the formation of PMAPs at the edge of migrating cells, where LLPS appears to be involved in promoting protrusion and the turnover of integrin-mediated adhesions, to allow forward cell translocation; (ii) in the assembly of the presynaptic active zone and of the postsynaptic density deputed to the release and reception of neurotransmitter signals, respectively. The recent results indicate that LLPS at cytosol-membrane interfaces is suitable not only for the regulation of active cellular processes, but also for the continuous spatial rearrangements of the molecular interactions involved in these dynamic processes.

A functional interaction between liprin-α1 and B56γ regulatory subunit of protein phosphatase 2A supports tumor cell motility.

Scaffold liprin-α1 is required to assemble dynamic plasma membrane-associated platforms (PMAPs) at the front of migrating breast cancer cells, to promote protrusion and invasion. We show that the N-terminal region of liprin-α1 contains an LxxIxE motif interacting with B56 regulatory subunits of serine/threonine protein phosphatase 2A (PP2A). The specific interaction of B56γ with liprin-α1 requires an intact motif, since two point mutations strongly reduce the interaction. B56γ mediates the interaction of liprin-α1 with the heterotrimeric PP2A holoenzyme. Most B56γ protein is recovered in the cytosolic fraction of invasive MDA-MB-231 breast cancer cells, where B56γ is complexed with liprin-α1. While mutation of the short linear motif (SLiM) does not affect localization of liprin-α1 to PMAPs, localization of B56γ at these sites specifically requires liprin-α1. Silencing of B56γ or liprin-α1 inhibits to similar extent cell spreading on extracellular matrix, invasion, motility and lamellipodia dynamics in migrating MDA-MB-231 cells, suggesting that B56γ/PP2A is a novel component of the PMAPs machinery regulating tumor cell motility. In this direction, inhibition of cell spreading by silencing liprin-α1 is not rescued by expression of B56γ binding-defective liprin-α1 mutant. We propose that liprin-α1-mediated recruitment of PP2A via B56γ regulates cell motility by controlling protrusion in migrating MDA-MB-231 cells.

Liprin-alpha1 promotes cell spreading on the extracellular matrix by affecting the distribution of activated integrins.

Integrin activation is needed to link the extracellular matrix with the actin cytoskeleton during cell motility. Protrusion requires coordination of actin dynamics with focal-adhesion turnover. We report that the adaptor protein liprin-alpha1 is stably associated with the cell membrane. Lipin-alpha1 shows a localization that is distinct from that of activated beta1 integrins at the edge of spreading cells. Depletion of liprin-alpha1 inhibits the spreading of COS7 cells on fibronectin by affecting lamellipodia formation, whereas its overexpression enhances spreading, and lamellipodia and focal-adhesion formation at the cell edge. Cooperation between liprin-alpha1 and talin is needed, because either talin or liprin depletion prevents spreading in the presence of the other protein. The effects of liprin on spreading, but not its effects in the reorganization of the cell edge, are dependent on its interaction with leukocyte common antigen-related tyrosine phosphatase receptors. Therefore, liprin is an essential regulator of cell motility that contributes to the effectiveness of cell-edge protrusion.

The GIT-PIX complexes regulate the chemotactic response of rat basophilic leukaemia cells.

BACKGROUND INFORMATION: Cell motility entails the reorganization of the cytoskeleton and membrane trafficking for effective protrusion. The GIT-PIX protein complexes are involved in the regulation of cell motility and adhesion and in the endocytic traffic of members of the family of G-protein-coupled receptors. We have investigated the function of the endogenous GIT complexes in the regulation of cell motility stimulated by fMLP (formyl-Met-Leu-Phe) peptide, in a rat basophilic leukaemia RBL-2H3 cell line stably expressing an HA (haemagglutinin)-tagged receptor for the fMLP peptide. RESULTS: Our analysis shows that RBL cells stably transfected with the chemoattractant receptor expressed both GIT1-PIX and GIT2-PIX endogenous complexes. We have used silencing of the different members of the complex by small interfering RNAs to study the effects on a number of events linked to agonist-induced cell migration. We found that cell adhesion was not affected by depletion of any of the proteins of the GIT complex, whereas agonist-enhanced cell spreading was inhibited. Analysis of agonist-stimulated haptotactic cell migration indicated a specific positive effect of GIT1 depletion on trans-well migration. The internalization of the formyl-peptide receptor was also inhibited by depletion of GIT1 and GIT2. The effects of the GIT complexes on trafficking of the receptors was confirmed by an antibody-enhanced agonist-induced internalization assay, showing that depletion of PIX, GIT1 or GIT2 protein caused decreased perinuclear accumulation of internalized receptors. CONCLUSIONS: Our results show that endogenous GIT complexes are involved in the regulation of chemoattractant-induced cell motility and receptor trafficking, and support previous findings indicating an important function of the GIT complexes in the regulation of different G-protein-coupled receptors. Our results also indicate that endogenous GIT1 and GIT2 regulate distinct subsets of agonist-induced responses and suggest a possible functional link between the control of receptor trafficking and the regulation of cell motility by GIT proteins.

Liprin-alpha1 affects the distribution of low-affinity beta1 integrins and stabilizes their permanence at the cell surface.

Integrins mediate the interaction between cells and extracellular matrix by assembling adhesive structures that need to be dynamically modulated to allow cell motility. We have recently identified liprin-alpha1 as an essential regulator of integrin dynamics required for efficient cell motility. Here we investigated the effects of liprin-alpha1 expression on beta1 integrin receptors. We found that increased levels of liprin-alpha1 affected the localization of inactive, low-affinity integrins, while increasing the average size of beta1 integrin-positive focal adhesions. Although a direct interaction between beta1 integrins and liprin-alpha1 could not be revealed biochemically, a striking colocalization between redistributed inactive beta1 integrins and liprin-alpha1 was observed. The tight association of overexpressed and endogenous liprin-alpha1 to the cytoplasmic side of the ventral plasma membrane suggested a possible role of liprin in stabilizing integrin receptors at the cell surface. In support of this hypothesis, we demonstrated an inhibitory effect of liprin overexpression on antibody-induced beta1 integrin internalization. On the other hand, depletion of endogenous liprin-alpha by small interfering RNA increased the rate of integrin internalization. Overall, these results support the hypothesis that liprin-alpha1 exerts its action on focal adhesion turnover by influencing the localization and stability of integrin receptors at the cell surface.