Quantitative Kinetic Study of the Actin-Bundling Protein L-Plastin and of Its Impact on Actin Turn-Over

BACKGROUND: Initially detected in leukocytes and cancer cells derived from solid tissues, L-plastin/fimbrin belongs to a large family of actin crosslinkers and is considered as a marker for many cancers. Phosphorylation of L-plastin on residue Ser5 increases its F-actin binding activity and is required for L-plastin-mediated cell invasion. METHODOLOGY/PRINCIPAL FINDINGS: To study the kinetics of L-plastin and the impact of L-plastin Ser5 phosphorylation on L-plastin dynamics and actin turn-over in live cells, simian Vero cells were transfected with GFP-coupled WT-L-plastin, Ser5 substitution variants (S5/A, S5/E) or actin and analyzed by fluorescence recovery after photobleaching (FRAP). FRAP data were explored by mathematical modeling to estimate steady-state reaction parameters. We demonstrate that in Vero cell focal adhesions L-plastin undergoes rapid cycles of association/dissociation following a two-binding-state model. Phosphorylation of L-plastin increased its association rates by two-fold, whereas dissociation rates were unaffected. Importantly, L-plastin affected actin turn-over by decreasing the actin dissociation rate by four-fold, increasing thereby the amount of F-actin in the focal adhesions, all these effects being promoted by Ser5 phosphorylation. In MCF-7 breast carcinoma cells, phorbol 12-myristate 13-acetate (PMA) treatment induced L-plastin translocation to de novo actin polymerization sites in ruffling membranes and spike-like structures and highly increased its Ser5 phosphorylation. Both inhibition studies and siRNA knock-down of PKC isozymes pointed to the involvement of the novel PKC-delta isozyme in the PMA-elicited signaling pathway leading to L-plastin Ser5 phosphorylation. Furthermore, the L-plastin contribution to actin dynamics regulation was substantiated by its association with a protein complex comprising cortactin, which is known to be involved in this process. CONCLUSIONS/SIGNIFICANCE: Altogether these findings quantitatively demonstrate for the first time that L-plastin contributes to the fine-tuning of actin turn-over, an activity which is regulated by Ser5 phosphorylation promoting its high affinity binding to the cytoskeleton. In carcinoma cells, PKC-delta signaling pathways appear to link L-plastin phosphorylation to actin polymerization and invasion

Rôle de la L-plastine dans l'assemblage du cytosquelette cortical d'actine et la progression des cellules tumorales

L'organisation du cytosquelette d'actine en réseaux et en faisceaux est contrôlée par une multitude de protéines structurales. La modification de ces protéines contribue à des pathologies telles que le cancer où des changements structuraux et fonctionnels du cytosquelette d'actine induisant la progression et la transmission de signaux cellulaires dérégulées. Ce travail de thèse se concentre sur l étude du rôle de la L-plastine dans l'assemblage du cytosquelette cortical d actine et dans la progression des cellules tumorales.La L-plastine appartient à une grande famille de protéines de pontage de l actine qui contribue à l'invasion des cellules tumorales d'une façon phosphorylation-dépendante. La L-plastine est localisée au niveau de structures d actine membranaires impliquées dans la locomotion, l'adhésion et la défense immunitaire. Ici, nous avons étudié la dynamique de la L-plastine/actine dans les cellules vivantes en combinant la méthode de rétablissement de fluorescence après photoblanchissement (FRAP) avec la modélisation mathématique. Nous avons démontré quantitativement que la phosphorylation de la L-plastine au niveau de son résidu Ser5 augmente sa capacité d interagir avec l actine influençant de ce fait sa localisation intracellulaire. D'autre part, nos résultats ont montré que la L-plastine fixe et stabilise le cytosquelette d'actine et affecte la dynamique de l actine d'une façon phosphorylation-dépendante. Conformément au rôle de la L-plastine dans le contrôle de la dynamique de l'actine, nos résultats ont prouvé que le traitement au PMA (Phorbol 12-Myristate 13-Acetate), par l intermédiaire de la PKC, favorise la localisation de la L-plastine au niveau des protrusions membranaires et augmente sa phosphorylation au niveau du résidu Ser5 dans les cellules de cancer du sein MCF-7. Il est intéressant de noter que nos résultats indiquent qu une des isozymes de PKC d et/ou e semblent être impliquées dans la phosphorylation de la L-plastine dans les cellules cancéreuses.En utilisant des approches de biochimie, nous avons étayé la contribution de la L-plastine sur la régulation dynamique de l'actine, en illustrant son association avec un complexe protéique contenant la cortactine. Enfin, nous avons démontré la colocalisation de la L-plastine avec des marqueurs des invadosomes, et nous avons identifié un nouveau rôle phosphorylation-dépendant de la L-plastine dans la résistance à la mort cellulaire provoquée par le facteur de nécrose tumorale-alpha (TNF-a), consolidant le rôle de la L-plastine dans la progression des cellules tumorales.The overall organisation of the actin cytoskeleton into networks and bundles is controlled by a plethora of actin-binding proteins. Functional alteration of these actin-binding proteins contributes to pathologies such as cancer where structural and functional modifications of the actin cytoskeleton are linked to uncontrolled cell movement and signalling. This thesis focuses on the study of the role of L-plastin, an actin crosslinking protein, in the assembly of the cortical cytoskeleton and the progression of cancer cells. L-plastin is an actin filament bundling protein which contributes to cancer cell invasion in a phosphorylation-dependent manner. L-plastin localises to actin-rich membrane structures involved in locomotion, adhesion and immune defense. In this work, we have investigated L-plastin/actin dynamics in live cells by combining confocal microscopy-based fluorescence recovery after photobleaching (FRAP) method with mathematical modelling. We show that phosphorylation of L-plastin on residue Ser5 increases its association rate and may thereby influence its intracellular localisation and promote its capacity to dock efficiently to the actin cytoskeleton. Importantly, we provide evidence for the first time that L-plastin modulates actin dynamics and turn-over in focal adhesions and stress fibers, increasing their F-actin content by decreasing the actin dissociation rate at actin filament ends, effects which are also enhanced by Ser5 phosphorylation. Consistent with the L-plastin role in the control of actin dynamics, our data provide evidence that Phorbol 12-Myristate 13-Acetate (PMA) treatment, through activation of PKC, induces the translocation of L-plastin to de novo actin polymerisation sites in ruffling membranes and protruding spikes and significantly enhances L-plastin phosphorylation on residue Ser5 in the breast cancer cell line MCF-7. Interestingly, our results point to an important role for novel PKC isozymes (d and/or e) in regulating L-plastin phosphorylation in cancer cells. Using biochemical experiments, we further substantiate the contribution of L-plastin to actin dynamics regulation by illustrating its association with a protein complex containing the Arp2/3-complex activating protein cortactin. Finally, we provide evidence for the colocalisation of L-plastin with well characterised markers of podosomes and invadopodia, and we identified a novel phosphorylation-dependent function for L-plastin in conferring resistance to the cytotoxic effect of tumour necrosis factor-alpha (TNF-a), further supporting the role of L-plastin in cancer progression.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Vitamin A and retinoid signaling: genomic and nongenomic effects

Vitamin A or retinol is arguably the most multifunctional vitamin in the human body, as it is essential from embryogenesis to adulthood. The pleiotropic effects of vitamin A are exerted mainly by one active metabolite, all-trans retinoic acid (atRA), which regulates the expression of a battery of target genes through several families of nuclear receptors (RARs, RXRs, and PPARbeta/delta), polymorphic retinoic acid (RA) response elements, and multiple coregulators. It also involves extranuclear and nontranscriptional effects, such as the activation of kinase cascades, which are integrated in the nucleus via the phosphorylation of several actors of RA signaling. However, vitamin A itself proved recently to be active and RARs to be present in the cytosol to regulate translation and cell plasticity. These new concepts expand the scope of the biologic functions of vitamin A and RA

Genes involved in cell adhesion and signaling: a new repertoire of retinoic acid receptor target genes in mouse embryonic fibroblasts

Nuclear retinoic acid (RA) receptors (RARalpha, beta and gamma) are ligand-dependent transcription factors that regulate the expression of a battery of genes involved in cell differentiation and proliferation. They are also phosphoproteins and we previously showed the importance of their phosphorylation in their transcriptional activity. In the study reported here, we conducted a genome-wide analysis of the genes that are regulated by RARs in mouse embryonic fibroblasts (MEFs) by comparing wild-type MEFs to MEFs lacking the three RARs. We found that in the absence of RA, RARs control the expression of several gene transcripts associated with cell adhesion. Consequently the knockout MEFs are unable to adhere and to spread on substrates and they display a disrupted network of actin filaments, compared with the WT cells. In contrast, in the presence of the ligand, RARs control the expression of other genes involved in signaling and in RA metabolism. Taking advantage of rescue cell lines expressing the RARalpha or RARgamma subtypes (either wild-type or mutated at the N-terminal phosphorylation sites) in the null background, we found that the expression of RA-target genes can be controlled either by a specific single RAR or by a combination of RAR isotypes, depending on the gene. We also selected genes that require the phosphorylation of the receptors for their regulation by RA. Our results increase the repertoire of genes that are regulated by RARs and highlight the complexity and diversity of the transcriptional programs regulated by RARs, depending on the gene

Generation of human muscle fibers and satellite-like cells from human pluripotent stem cells in vitro

International audienceProgress toward finding a cure for muscle diseases has been slow because of the absence of relevant cellular models and the lack of a reliable source of muscle progenitors for biomedical investigation. Here we report an optimized serum-free differentiation protocol to efficiently produce striated, millimeter-long muscle fibers together with satellite-like cells from human pluripotent stem cells (hPSCs) in vitro. By mimicking key signaling events leading to muscle formation in the embryo, in particular the dual modulation of Wnt and bone morphogenetic protein (BMP) pathway signaling, this directed differentiation protocol avoids the requirement for genetic modifications or cell sorting. Robust myogenesis can be achieved in vitro within 1 month by personnel experienced in hPSC culture. The differentiating culture can be subcultured to produce large amounts of myogenic progenitors amenable to numerous downstream applications. Beyond the study of myogenesis, this differentiation method offers an attractive platform for the development of relevant in vitro models of muscle dystrophies and drug screening strategies, as well as providing a source of cells for tissue engineering and cell therapy approaches

Differentiation of the human PAX7-positive myogenic precursors/satellite cell lineage in vitro

Satellite cells (SC) are muscle stem cells that can regenerate adult muscles upon injury. Most SC originate from PAX7(+) myogenic precursors set aside during development. Although myogenesis has been studied in mouse and chicken embryos, little is known about human muscle development. Here, we report the generation of human induced pluripotent stem cell (iPSC) reporter lines in which fluorescent proteins have been introduced into the PAX7 and MYOG loci. We use single cell RNA sequencing to analyze the developmental trajectory of the iPSC-derived PAX7(+) myogenic precursors. We show that the PAX7(+) cells generated in culture can produce myofibers and self-renew in vitro and in vivo Together, we demonstrate that cells exhibiting characteristics of human fetal satellite cells can be produced in vitro from iPSC, opening interesting avenues for muscular dystrophy cell therapy. This work provides significant insights into the development of the human myogenic lineage

The novel PKC-δ isozyme is necessary for PMA-induced cytoskeleton reorganization and L-plastin Ser5 phosphorylation.

<p>(A). Novel PKC isozymes regulate PMA-induced L-plastin phosphorylation in MCF-7 cells. MCF-7 cells were pretreated for 3 hours with 5 µM of GF109203X (specific for α, β1, δ, ε, and ζ) or 0.5 µM of Gö6976 (specific for α and β1) and then treated for 1 hour with or without 1 µM PMA at 37°C. Total cell extracts (50 µg) were analyzed by immunoblotting using anti-Ser5-P L-plastin (upper panel) or anti-GAPDH (lower panel) antibodies. (B). PMA-induced actin cytoskeleton remodeling and L-plastin translocation involves novel PKC isozymes. MCF-7 cells were pretreated for 3 hours with 5 µM GF109203X or 0.5 µM Gö6976, then treated for 1 hour with 1 µM PMA at 37°C. Cells were then fixed and processed for immunofluorescence. Labeled cells were analyzed with an epifluorescence microscope after staining with an anti-L-plastin antibody and Alexa 488-conjugated phalloidin. Scale bar, 10 µm. (C). SiRNA knock-down of PKC-δ decreased PMA-induced L-plastin phosphorylation in MCF-7. MCF-7 cells were transfected with either PKC-δ or PKC-ε siRNAs as well as with negative control siRNA (Ctrl) for 48 hours and then treated with PMA as indicated. Total cell extracts (50 µg) were analyzed by immunoblotting using antibodies specific for Ser5 phosphorylated L-plastin, PKC-ε, PKC-δ and total L-plastin. GAPDH was used to monitor equal protein loading.</p