Action non classique des androgenes (pTHrP et cancer de la prostate)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Plexin-B2 controls the timing of differentiation and the motility of cerebellar granule neurons

International audiencePlexin-B2 deletion leads to aberrant lamination of cerebellar granule neurons (CGNs) and Purkinje cells. Although in the cerebellum Plexin-B2 is only expressed by proliferating CGN precursors in the outer external granule layer (oEGL), its function in CGN development is still elusive. Here, we used 3D imaging, in vivo electroporation and live-imaging techniques to study CGN development in novel cerebellum-specific Plexin-B2 conditional knockout mice. We show that proliferating CGNs in Plexin-B2 mutants not only escape the oEGL and mix with newborn postmitotic CGNs. Furthermore, motility of mitotic precursors and early postmitotic CGNs is altered. Together, this leads to the formation of ectopic patches of CGNs at the cerebellar surface and an intermingling of normally time-stamped parallel fibers in the molecular layer (ML), and aberrant arborization of Purkinje cell dendrites. There results suggest that Plexin-B2 restrict CGN motility and might have a function in cytokinesis

Reverse Signaling by Semaphorin-6A Regulates Cellular Aggregation and Neuronal Morphology.

The transmembrane semaphorin, Sema6A, has important roles in axon guidance, cell migration and neuronal connectivity in multiple regions of the nervous system, mediated by context-dependent interactions with plexin receptors, PlxnA2 and PlxnA4. Here, we demonstrate that Sema6A can also signal cell-autonomously, in two modes, constitutively, or in response to higher-order clustering mediated by either PlxnA2-binding or chemically induced multimerisation. Sema6A activation stimulates recruitment of Abl to the cytoplasmic domain of Sema6A and phos¡phorylation of this cytoplasmic tyrosine kinase, as well as phosphorylation of additional cytoskeletal regulators. Sema6A reverse signaling affects the surface area and cellular complexity of non-neuronal cells and aggregation and neurite formation of primary neurons in vitro. Sema6A also interacts with PlxnA2 in cis, which reduces binding by PlxnA2 of Sema6A in trans but not vice versa. These experiments reveal the complex nature of Sema6A biochemical functions and the molecular logic of the context-dependent interactions between Sema6A and PlxnA2

Pleiotropic Roles of Scavenger Receptors in Circadian Retinal Phagocytosis: A New Function for Lysosomal SR-B2/LIMP-2 at the RPE Cell Surface

International audienceThe retinal phagocytic machinery resembles the one used by macrophages to clear apoptotic cells. However, in the retina, the permanent contact between photoreceptor outer segments (POS) and retinal pigment epithelial (RPE) cells requires a tight control of this circadian machinery. In addition to the known receptors synchronizing POS internalization, several others are expressed by RPE cells. Notably, scavenger receptor CD36 has been shown to intervene in the internalization speed. We thus investigated members of the scavenger receptor family class A SR-AI and MARCO and class B CD36, SR-BI and SR-B2/LIMP-2 using immunoblotting, immunohisto- and immunocytochemistry, lipid raft flotation gradients, phagocytosis assays after siRNA/antibody inhibition, RT-qPCR and western blot analysis along the light:dark cycle. All receptors were expressed by RPE cell lines and tissues and colocalized with POS, except SR-BI. All receptors were associated with lipid rafts, and even more upon POS challenge. SR-B2/LIMP-2 inhibition suggested a role in the control of the internalization speed similar to CD36. In vivo, MARCO and CD36 displayed rhythmic gene and protein expression patterns concomitant with the phagocytic peak. Taken together, our results indicate that CD36 and SR-B2/LIMP-2 play a direct regulatory role in POS phagocytosis dynamics, while the others such as MARCO might participate in POS clearance by RPE cells either as co-receptors or via an indirect process

A plasma membrane microdomain compartmentalizes ephrin-generated cAMP signals to prune developing retinal axon arbors

International audienceThe development of neuronal circuits is controlled by guidance molecules that are hypothesized to interact with the cholesterol-enriched domains of the plasma membrane termed lipid rafts. Whether such domains enable local intracellular signalling at the submicrometre scale in developing neurons and are required for shaping the nervous system connectivity in vivo remains controversial. Here, we report a role for lipid rafts in generating domains of local cAMP signalling in axonal growth cones downstream of ephrin-A repulsive guidance cues. Ephrin-A-dependent retraction of retinal ganglion cell axons involves cAMP signalling restricted to the vicinity of lipid rafts and is independent of cAMP modulation outside of this microdomain. cAMP modulation near lipid rafts controls the pruning of ectopic axonal branches of retinal ganglion cells in vivo, a process requiring intact ephrin-A signalling. Together, our findings indicate that lipid rafts structure the subcellular organization of intracellular cAMP signalling shaping axonal arbors during the nervous system development

SpiCee: A Genetic Tool for Subcellular and Cell-Specific Calcium Manipulation

International audienc

Sema6A exerts constitutive and PlxnA2-dependent cell-autonomous functions.

<p>(<b>A-F</b>) NIH3T3 cells expressing GFP alone (<b>A and B</b>), myc-Sema6A-FL (Sema6A; <b>B and E</b>), or myc-Sema6A-K393D (Sema6A-K393D; <b>C and F</b>) were treated with purified AP-Fc (AP; <b>A-C</b>) or PlxnA2-EC-Fc (PlxnA2-EC; <b>D-F</b>). Scale bar = 20 μm. (<b>G</b>) Graph represents the cell area in cells transfected with GFP, Sema6A or Sema6A-K393D and treated with AP or PlxnA2-EC; <i>n</i> = 100–200 cells per experimental condition. Data are expressed as mean ± s.e.m; ***<i>P</i> <0.001; Student’s <i>t</i>-test.</p

Cell-contraction induced by PlxnA2-Sema6A interaction depends on Abl signaling.

<p>(<b>A-F</b>) NIH3T3 cells expressing GFP (<b>A</b> and <b>D</b>), Sema6A-FL (<b>B</b> and <b>E</b>), and Sema6A-∆Abl (<b>C</b> and <b>F</b>) were treated with purified AP-Fc (AP; <b>a-c</b>) or PlxnA2-EC-Fc (PlxnA2-EC; <b>D-F</b>). Scale bar = 20 μm. (<b>G</b>) Scheme illustrates that the cell contraction is via Abl signaling. (<b>H</b>) Graph represents the contraction of the cell area in NIH3T3 transfected with GFP, Sema6A or Sema6A-∆Abl and treated with AP or PlxnA2-EC; <i>n</i> = 100–200 cells per experimental condition. Data are expressed as mean ± s.e.m; ***<i>P</i> < 0.001; Student’s <i>t</i>-test.</p

Sema6A reverse signaling is stimulated by multimerisation.

<p>(<b>A</b>) Scheme representing how the induced multi-aggregation of the cytosolic domain of Sema6A activates signaling pathways. A chimeric construct was engineered by putting in frame the entire cytosolic domain of Sema6A (Sema6A-cyt) together with 3 self-aggregation FKBP domains, and GFP. The chimeric peptide targets the plasma membrane by the myristoylating sequence located at the N-terminal. The application of the drug FK1012 triggers the chemically induced multimerisation (CIM) of Sema6A-cyt via the self-aggregation of FKBP. (<b>B</b>) Cerebellar neurons transfected with MF3-GFP or MF3-Sema6A-cyt-GFP (MF3-Sema6A-cyt), and treated with 500nM FK1012 (“CIM”). GFP-positive aggregates are indicated (arrowheads). Scale bar = 20μm. (<b>C</b>) Axonal shaft of cerebellar neurons transfected with myc-Sema6A-FL or MF3-Sema6A-cyt-GFP (MF3-Sema6A-cyt), and treated with PlxnA2-EC or CIM respectively. Scale bar = 5 μm. (<b>D</b>) and (<b>E</b>) Graphs summarising the myc-Sema6A-FL aggregation in myc-Sema6A-FL-expressing neurons treated with PlxnA2-EC at different time points. The aggregation is represented as the average distance between Sema6A clusters, or the average size of Sema6A clusters; <i>n</i> = 50–100 cells per time point. Data are expressed as mean ± s.e.m; ***<i>P</i> < 0.001; one-way ANOVA followed by Bonferroni multiple comparison test. (<b>F</b>) MF3-Sema6A-cyt-expressing granular neurons were cultured on NIH3T3 cell layers, and treated with CIM. Scale bar = 50 μm. (<b>G</b>) Graph representing the axonal length of MF3-Sema6A-cyt-expressing cerebellar neurons treated with CIM for 24 and 48h; <i>n</i> = 50–100 cells per experimental condition. Data are expressed as mean ± s.e.m; ***<i>P</i> < 0.001; one-way ANOVA followed by Bonferroni multiple comparison test.</p

Sema6A cytoplasmic domain interacts with Abl and Mena.

<p>(<b>A</b>) Immunoprecipitations from untreated or PlxnA2-EC-treated COS-7 cells previously transfected with different combinations of myc-Sema6A, Abl-V5 and PlxnA2. Immunoprecipitations (Ip) were performed employing an anti-V5 antibody. Antibodies against V5, myc and PlxnA2 were used in the immunoblots (Ib). (<b>B</b>) Immunoprecipitations from untreated or PlxnA2-EC treated COS-7 cells previously transfected with different combinations of myc-Sema6A, Mena-V5 and PlxnA2. Immunoprecipitations (Ip) were performed employing an anti-myc antibody. Antibodies against V5, myc and PlxnA2 were used in the immunoblots (Ib). (<b>C</b>) and (<b>D</b>) Interactions were quantified by immunoblotting and densitometric analysis. Histograms represent quantification of interaction normalized to the amount of immunoprecipitated protein (Abelson-V5 for Sema6A-Abelson or Sema6A-myc for Sema6A-Mena) from three independent experiments. Students t-test *p<0,05 indicating signifiance. S6A-Abl/S6A-Abl+PlA2 <i>p</i> = 0.013, S6A-Abl-PlA2/S6A-Abl-PlA2+PlA2 <i>p</i> = 0.034. Errors bars represent standard error.</p