Veronica Astro

Liprin-α1 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-α1 is stably associated with the cell membrane. Lipin-α1 shows a localization that is distinct from that of activated β1 integrins at the edge of spreading cells. Depletion of liprin-α1 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-α1 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.

Liprin-α1 regulates breast cancer cell invasion by affecting cell motility, invadopodia and extracellular matrix degradation

Migration of cells and degradation of the extracellular matrix (ECM) are required for efficient tumor cell invasion, but the underlying molecular mechanisms are only partially known. The PPFIA1 gene for liprin-α1 is frequently amplified in human breast cancers. We recently demonstrated that liprin-α1 is an important regulator of cell edge dynamics during motility. We show, herein, that the liprin-α1 protein is highly expressed in human breast tumors. Functional analysis shows that liprin-α1 is specifically required for the migration and invasion of highly invasive human breast cancer MDA-MB-231 cells. We used time-lapse analysis to demonstrate defects in the motility of liprin-α1-depleted cells that include a striking instability of the lamellipodia. Liprin-α1 levels altered by either RNA interference or overexpression affected also cell spreading and the number of invadopodia per cell, but not the density of invadopodia per unit of surface area. On the other hand, silencing of liprin-α1 inhibited the degradation of the ECM, whereas its overexpression enhanced degradation, resulting in significant negative or positive effects, respectively, on the area of degradation per invadopodium. Transfection of fluorescent-labeled cortactin revealed that depletion of liprin-α1 also affected the assembly and disassembly of invadopodia, with decrease of their lifetime. Our results strongly support a novel important role of liprin-α1 in the regulation of human tumor cell invasion.

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

ABSTRACT 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 LL5&agr; and LL5&bgr; (encoded by PHLDB1 and PHLDB2, respectively) are required, together with liprin-&agr;1, 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 &bgr;1 receptors needed for adhesion turnover at the front of the cell. Liprin-&agr;1, 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.

Plasma membrane-associated platforms: Dynamic scaffolds that organize membrane-associated events

A core of four proteins organizes sites of exocytosis, extracellular matrix remodeling, and the leading edge of moving cells. Gloss Different cell types have specialized regions of the plasma membrane that are required to perform specific functions. These specialized regions constitute large plasma membrane–associated platforms that regulate signaling events associated with the trafficking of molecules to and from these membrane regions. The platforms form by the interaction of cytosolic proteins with integral components of the plasma membrane that link the extracellular environment to the intracellular cytoskeleton. The analysis of the molecular components of different platforms reveals that a few cytosolic proteins form a core complex that is shared among platforms with different cellular functions, and this core complex is important for their assembly and function. This Review, which has 6 figures and 121 references, discusses the role of individual components of the core complex in different platforms and proposes a model for their formation and disassembly. Specialized regions of the plasma membrane dedicated to diverse cellular processes, such as vesicle exocytosis, extracellular matrix remodeling, and cell migration, share a few cytosolic scaffold proteins that associate to form large plasma membrane–associated platforms (PMAPs). PMAPs organize signaling events and trafficking of membranes and molecules at specific membrane domains. On the basis of the intrinsic disorder of the proteins constituting the core of these PMAPs and of the dynamics of these structures at the periphery of motile cells, we propose a working model for the assembly and turnover of these platforms.

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 β1 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.

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.

Effects of the scaffold proteins liprin-α1, β1 and β2 on invasion by breast cancer cells

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.

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 βPIX. 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.

Biochemical and functional characterization of the interaction between liprin-α1 and GIT1: Implications for the regulation of cell motility

We have previously identified the scaffold protein liprin-α1 as an important regulator of integrin-mediated cell motility and tumor cell invasion. Liprin-α1 may interact with different proteins, and the functional significance of these interactions in the regulation of cell motility is poorly known. Here we have addressed the involvement of the liprin-α1 partner GIT1 in liprin-α1-mediated effects on cell spreading and migration. GIT1 depletion inhibited spreading by affecting the lamellipodia, and prevented liprin-α1-enhanced spreading. Conversely inhibition of the formation of the liprin-α1-GIT complex by expression of liprin-ΔCC3 could still enhance spreading, although to a lesser extent compared to full length liprin-α1. No cumulative effects were observed after depletion of both liprin-α1 and GIT1, suggesting that the two proteins belong to the same signaling network in the regulation of cell spreading. Our data suggest that liprin-α1 may compete with paxillin for binding to GIT1, while binding of βPIX to GIT1 was unaffected by the presence of liprin-α1. Interestingly, GIT and liprin-α1 reciprocally regulated their subcellular localization, since liprin-α1 overexpression, but not the GIT binding-defective liprin-ΔCC3 mutant, affected the localization of endogenous GIT at peripheral and mature central focal adhesions, while the expression of a truncated, active form of GIT1 enhanced the localization of endogenous liprin-α1 at the edge of spreading cells. Moreover, GIT1 was required for liprin-α1-enhanced haptotatic migration, although the direct interaction between liprin-α1 and GIT1 was not needed. Our findings show that the functional interaction between liprin-α1 and GIT1 cooperate in the regulation of integrin-dependent cell spreading and motility on extracellular matrix. These findings and the possible competition of liprin-α1 with paxillin for binding to GIT1 suggest that alternative binding of GIT1 to either liprin-α1 or paxillin plays distinct roles in different phases of the protrusive activity in the cell.

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.