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 (Rac1N) in a Rac3-deficient (Rac3KO) background causes motor behavioural defects, epilepsy, and premature mouse death around postnatal day 13. We report here that Rac1N/Rac3KO mice display a progressive loss of immune-competence. Comparative longitudinal analysis of lymphoid organs from control, single Rac1N or Rac3KO, and double Rac1N/Rac3KO mutant animals showed that thymus development is preserved up to postnatal day 9 in all animals, but is impaired in Rac1N/Rac3KO 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

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

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

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

Abstract 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

Hyperphosphorylation of GIT1-N by Src and pervanadate does not affect its binding <i>in vitro</i> to full length GIT1 proteins.

<p>(<b>A–C</b>) COS7 cells were transfected with full length GFP-GIT1 or GIT1-N constructs (WT, FF, or EE), or with wildtype or mutant FLAG-GIT1-N fragments, alone or together with c-Src. The cells cotransfected with c-Src were incubated 20 min at 37°C with 1 mM pervanadate before lysis (pV). Aliquots of the lysates (200 μg of protein) were immunoprecipitated (IP) with anti-FLAG (<b>A,C</b>) or anti-GFP (<b>B</b>) antibodies. For pulldowns shown in (<b>A</b>) and (<b>C</b>): FLAG-immunoprecipitates were washed and incubated for 2 h at 4°C with lysates (250 μg of protein) from cells transfected with the indicated full length GFP-GIT1 constructs. Equal amounts of lysates (25 μg of protein), and the pulldowns performed with GIT1-N without (<b>A</b>) or with c-Src and pervanadate treatment (<b>C</b>) were blotted for the detection of the indicated antigens. In (<b>B</b>) the immunoprecipitations with anti-GFP antibody (right) were immunoblotted to detect the levels of GFP-GIT1-N protein (upper filter) and of its tyrosine phosphorylation (lower filter). (<b>D</b>) COS7 cells were transfected to express the indicated GFP-GIT1 mutants, or transfected with the HA-GIT1-N fragment alone or together with c-Src. The cells co-transfected with c-Src were treated as in (<b>A,C</b>). Aliquots of the lysates from cells expressing GFP-GIT1 mutants (250 μg of protein) were immunoprecipitated with anti-GFP. Pulldowns: GFP-immunoprecipitates were washed and incubated for 2 h at 4°C with lysates (400 μg of protein) from cells transfected with HA-GIT1-N alone, or together with c-Src. Equal amounts of lysates (25 μg of protein), and the pulldowns were blotted for the detection of the indicated antigens.</p

Mutations Y246E and Y293E of GIT1 enhance binding to paxillin.

<p>(<b>A</b>) Schematic representation of human GIT1 (NP 001078923.1). GFP-tagged wild type GIT1 was used to introduce phosphomimetic mutations. Tyrosine (Y) or serine (S) and threonine (T) residues were mutated into glutamic acid or aspartic acid, respectively. White and black stars indicate the locations of the mutated tyrosine and serine/threonine residues, respectively. ArfGAP, Arf GTPase-activating protein; ANK, ankyrin repeats; SHD, Spa2-homology domain; LZ, leucine zipper; PBS, paxillin binding site; SLD, synaptic localization domain. (<b>B</b>) Aliquots of lysates (400 μg) from cells transfected with the indicated constructs were used for immunoprecipitation of endogenous paxillin. Filters with immunoprecipitates (IP), and equal amounts (80 μg) of the respective lysates (Ly) or unbound fractions after immunoprecipitation (Ub) were blotted with anti-GFP (for GIT1) or anti-paxillin antibodies. Molecular weight markers are indicated to the right of each blot. (<b>C</b>) The double substitution of residues Y246 and Y293 with either two glutamic acid (EE) or two alanine residues (AA) enhanced GIT1 binding to paxillin. Aliquots of lysates (200 μg protein) from cells transfected with the indicated constructs were immunoprecipitated with anti-paxillin antibody. Filters with immunoprecipitates (IP), and lysates (40 μg) were blotted as indicated, using anti-GFP (for GIT1) or anti-paxillin antibodies. (<b>D</b>) Model for GIT1 activation: see details in the text.</p

Expression of the “active” GIT1-Y246E/Y293E mutant increases the efficiency of migration.

<p>(<b>A</b>) COS7 cells transfected with GFP or with GFP-tagged GIT1 constructs were used for haptotactic cell migration towards fibronectin. Images show fields with cells migrated to the lower side of the filters coated with fibronectin. (<b>B,C</b>) Quantification of the number of cells migrated to the lower side of the wells. Bars are normalized mean values ±SEM from 12–19 fields from 2–3 independent experiments. **P<0.02. (<b>D–F</b>) Wound-healing assay. COS7 cells were transfected as in (<b>A</b>). Confluent monolayers were wounded and followed by time lapse imaging. (<b>D</b>) Fluorescent images (left column) showing GFP-positive transfected COS7 cells at time 0. The other columns show the phase contrast images of the same fields, at the beginning (t = 0) and the end (t = 6 h) of the assay. Bar, 100 μm. (<b>E</b>) Tracks of GFP-positive cells transfected with either GFP-GIT1 or GFP-GIT1-Y246E/Y293E. Tracks were taken during the wound healing assays and refer to an interval of 8 h. (<b>F</b>) Quantification of the euclidean distance covered by cells during the wound healing assay. Bars are mean values ±SEM from 90 cells from 3 different experiments. *P<0.05; ***P<0,002.</p

Mutation of tyrosines 246 and 293 does not affect the binding of GIT1 to βPIX.

<p>(<b>A</b>) Lysates from mock-transfected cells or from cells transfected with the indicated GIT1 constructs were immunoprecipitated for GIT1 with anti-GFP antibody. Filters with immunoprecipitates (IP, from 200 μg of protein lysate), and lysates (25 μg) were blotted with anti-GFP (for GIT1), anti-paxillin, and anti-βPIX antibodies. (<b>B</b>) Binding of GIT1-N to GIT1-Y246E/Y293E does not affect the interaction of GIT1-Y246E/Y293E to βPIX. Lysates from COS7 cells co-transfected to express the HA-GIT1-N fragment and the full length GFP-GIT1-WT or GIT1-Y246E/Y293E proteins were immunoprecipitated (IP, from 175 μg of protein lysate) with anti-GFP. Lysates (25 μg, oƒn the lef) and IP (right) were blotted to reveal the full length proteins (anti-GFP), the HA-GIT1-N fragment (anti-HA mAb 12CA5), and endogenous βPIX. (<b>C</b>) Our data support the hypothesis that the tyrosine 293 of the SHD domain of GIT1 is required for the intramolecular interaction, but not for the interaction of the SHD domain of GIT1 with βPIX.</p

Efficient binding of the amino-terminal portion of GIT1 to the carboxy-terminal portion requires the inclusion of the first Spa2 region of the SHD.

<p>(<b>A</b>) Scheme of the constructs of avian GIT1 used in the following experiment. To be noted that tyrosine 284 of avian GIT1 corresponds to tyrosine 293 of human GIT1. (<b>B</b>) COS7 cells were cotransfected with the indicated combination of HA-tagged amino-terminal and FLAG-tagged carboxy-terminal fragments of GIT1. Lysates were immunoprecipitated with the anti-FLAG M2-conjugated beads. Filters with immunoprecipitates (IP, from 250 μg of protein lysate) and lysates (50 μg) were incubated with anti-HA or anti-FLAG antibodies. Amino-terminal fragments in the lysates are indicated by asterisks.</p