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.

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.

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.

The ERC1 scaffold protein implicated in cell motility drives the assembly of a liquid phase.

Several cellular processes depend on networks of proteins assembled at specific sites near the plasma membrane. Scaffold proteins assemble these networks by recruiting relevant molecules. The scaffold protein ERC1/ELKS and its partners promote cell migration and invasion, and assemble into dynamic networks at the protruding edge of cells. Here by electron microscopy and single molecule analysis we identify ERC1 as an extended flexible dimer. We found that ERC1 scaffolds form cytoplasmic condensates with a behavior that is consistent with liquid phases that are modulated by a predicted disordered region of ERC1. These condensates specifically host partners of a network relevant to cell motility, including liprin-α1, which was unnecessary for the formation of condensates, but influenced their dynamic behavior. Phase separation at specific sites of the cell periphery may represent an elegant mechanism to control the assembly and turnover of dynamic scaffolds needed for the spatial localization and processing of molecules.

Identification of a membrane-less compartment regulating invadosome function and motility.

Depletion of liprin-α1, ERC1 or LL5 scaffolds inhibits extracellular matrix degradation by invasive cells. These proteins co-accumulate near invadosomes in NIH-Src cells, identifying a novel invadosome-associated compartment distinct from the core and adhesion ring of invadosomes. Depletion of either protein perturbs the organization of invadosomes without influencing the recruitment of MT1-MMP metalloprotease. Liprin-α1 is not required for de novo formation of invadosomes after their disassembly by microtubules and Src inhibitors, while its depletion inhibits invadosome motility, thus affecting matrix degradation. Fluorescence recovery after photobleaching shows that the invadosome-associated compartment is dynamic, while correlative light immunoelectron microscopy identifies bona fide membrane-free invadosome-associated regions enriched in liprin-α1, which is virtually excluded from the invadosome core. The results indicate that liprin-α1, LL5 and ERC1 define a novel dynamic membrane-less compartment that regulates matrix degradation by affecting invadosome motility.

A Method to Culture GABAergic Interneurons Derived from the Medial Ganglionic Eminence.

Understanding the mechanisms guiding interneuron development is a central aspect of the current research on cortical/hippocampal interneurons, which is highly relevant to brain function and pathology. In this methodological study we have addressed the setup of protocols for the reproducible culture of dissociated cells from murine medial ganglionic eminences (MGEs), to provide a culture system for the analysis of interneurons in vitro. This study includes the detailed protocols for the preparation of the dissociated cells, and for their culture on optimal substrates for cell migration or differentiation. These cultures enriched in interneurons may allow the investigation of the migratory behavior of interneuron precursors and their differentiation in vitro, up to the formation of morphologically identifiable GABAergic synapses. Live imaging of MGE-derived cells plated on proper substrates shows that they are useful to study the migratory behavior of the precursors, as well as the behavior of growth cones during the development of neurites. Most MGE-derived precursors develop into polarized GABAergic interneurons as determined by axonal, dendritic, and GABAergic markers. We present also a comparison of cells from WT and mutant mice as a proof of principle for the use of these cultures for the analysis of the migration and differentiation of GABAergic cells with different genetic backgrounds. The culture enriched in interneurons described here represents a useful experimental system to examine in a relatively easy and fast way the morpho-functional properties of these cells under physiological or pathological conditions, providing a powerful tool to complement the studies in vivo.

Liprin-α1 and ERC1 control cell edge dynamics by promoting focal adhesion turnover.

Liprin-α1 and ERC1 are interacting scaffold proteins regulating the motility of normal and tumor cells. They act as part of plasma membrane-associated platforms at the edge of motile cells to promote protrusion by largely unknown mechanisms. Here we identify an amino-terminal region of the liprin-α1 protein (liprin-N) that is sufficient and necessary for the interaction with other liprin-α1 molecules. Similar to liprin-α1 or ERC1 silencing, expression of the liprin-N negatively affects tumor cell motility and extracellular matrix invasion, acting as a dominant negative by interacting with endogenous liprin-α1 and causing the displacement of the endogenous ERC1 protein from the cell edge. Interfering with the localization of ERC1 at the cell edge inhibits the disassembly of focal adhesions, impairing protrusion. Liprin-α1 and ERC1 proteins colocalize with active integrin ß1 clusters distinct from those colocalizing with cytoplasmic focal adhesion proteins, and influence the localization of peripheral Rab7-positive endosomes. We propose that liprin-α1 and ERC1 promote protrusion by displacing cytoplasmic adhesion components to favour active integrin internalization into Rab7-positive endosomes.

Identification of a Protein Network Driving Neuritogenesis of MGE-Derived GABAergic Interneurons.

Interneurons are essential modulators of brain activity and their abnormal maturation may lead to neural and intellectual disabilities. Here we show that cultures derived from murine medial ganglionic eminences (MGEs) produce virtually pure, polarized γ-aminobutyric acid (GABA)-ergic interneurons that can form morphologically identifiable inhibitory synapses. We show that Rac GTPases and a protein complex including the GIT family scaffold proteins are expressed during maturation in vitro, and are required for the normal development of neurites. GIT1 promotes neurite extension in a conformation-dependent manner, while affecting its interaction with specific partners reduces neurite branching. Proteins of the GIT network are concentrated at growth cones, and interaction mutants may affect growth cone behavior. Our findings identify the PIX/GIT1/liprin-α1/ERC1 network as critical for the regulation of interneuron neurite differentiation in vitro, and show that these cultures represent a valuable system to identify the molecular mechanisms driving the maturation of cortical/hippocampal interneurons.

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.

Identification of two tyrosine residues required for the intramolecular mechanism implicated in GIT1 activation.

GIT1 is an ArfGAP and scaffolding protein regulating cell adhesion and migration. The multidomain structure of GIT1 allows the interaction with several partners. Binding of GIT1 to some of its partners requires activation of the GIT1 polypeptide. Our previous studies indicated that binding of paxillin to GIT1 is enhanced by release of an intramolecular interaction between the amino-terminal and carboxy-terminal portions that keeps the protein in a binding-incompetent state. Here we have addressed the mechanism mediating this intramolecular inhibitory mechanism by testing the effects of the mutation of several formerly identified GIT1 phosphorylation sites on the binding to paxillin. We have identified two tyrosines at positions 246 and 293 of the human GIT1 polypeptide that are needed to keep the protein in the inactive conformation. Interestingly, mutation of these residues to phenylalanine did not affect binding to paxillin, while mutation to either alanine or glutamic acid enhanced binding to paxillin, without affecting the constitutive binding to the Rac/Cdc42 exchange factor ßPIX. The involvement of the two tyrosine residues in the intramolecular interaction was supported by reconstitution experiments showing that these residues are important for the binding between the amino-terminal fragment and carboxy-terminal portions of GIT1. Either GIT1 or GIT1-N tyrosine phosphorylation by Src and pervanadate treatment to inhibit protein tyrosine phosphatases did not affect the intramolecular binding between the amino- and carboxy-terminal fragments, nor the binding of GIT1 to paxillin. Mutations increasing the binding of GIT1 to paxillin positively affected cell motility, measured both by transwell migration and wound healing assays. Altogether these results show that tyrosines 246 and 293 of GIT1 are required for the intramolecular inhibitory mechanism that prevents the binding of GIT1 to paxillin. The data also suggest that tyrosine phosphorylation may not be sufficient to release the intramolecular interaction that keeps GIT1 in the inactive conformation.

Rac1 and Rac3 GTPases regulate the development of hilar mossy cells by affecting the migration of their precursors to the hilus.

We have previously shown that double deletion of the genes for Rac1 and Rac3 GTPases during neuronal development affects late developmental events that perturb the circuitry of the hippocampus, with ensuing epileptic phenotype. These effects include a defect in mossy cells, the major class of excitatory neurons of the hilus. Here, we have addressed the mechanisms that affect the loss of hilar mossy cells in the dorsal hippocampus of mice depleted of the two Rac GTPases. Quantification showed that the loss of mossy cells was evident already at postnatal day 8, soon after these cells become identifiable by a specific marker in the dorsal hilus. Comparative analysis of the hilar region from control and double mutant mice revealed that synaptogenesis was affected in the double mutants, with strongly reduced presynaptic input from dentate granule cells. We found that apoptosis was equally low in the hippocampus of both control and double knockout mice. Labelling with bromodeoxyuridine at embryonic day 12.5 showed no evident difference in the proliferation of neuronal precursors in the hippocampal primordium, while differences in the number of bromodeoxyuridine-labelled cells in the developing hilus revealed a defect in the migration of immature, developing mossy cells in the brain of double knockout mice. Overall, our data show that Rac1 and Rac3 GTPases participate in the normal development of hilar mossy cells, and indicate that they are involved in the regulation of the migration of the mossy cell precursor by preventing their arrival to the dorsal hilus.

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.