STRUCTURES, STABILITÉ ET FONCTIONS DU CYTOSQUELETTE D'ACTINE DANS LES OSTÉOCLASTES MÂTURES

Osteoclasts are hematopoietic cells specialized in bone resorption. Actin cytoskeleton organisation depends on substrates: podosomes are observed on glass or plastic slides, whereas an homogenous actin belt, the sealing zone (SZ) is organized on resorption substrates. Study of WIP deficient cells allows us to demontrate existence of 2 actin domains: podosomes cores surrounded by actin cloud. These 2 domains are induced by different receptors, CD44 and Beta3, polymerized by 2 distinct pathways, and are implicated in adhesion and contraction os osteoclast. Podosomes cores and actin cloud reorganized into an unique structure, the SZ, when osteoclast is seeded on apatite or dentin slides. We have also demonstrated that stability of podosomes belt depends on microtubules.L'ostéoclaste est une cellule spécialisée dans la résorption de la matrice osseuse. Elle présente une organisation différente de l'actine selon les substrats sur lesquels elle est ensemencée. Sur verre ou plastique, l'actine est sous forme de "podosomes", alors que sur un substrat résorbable, elle se ré-organise en une ceinture conttinue, la zone de scellement (SZ). L'étude de cellules déficientes en WIP nous a permis de démontrer qu'un ostéoclaste sur verre forme 2 domaines d'actine distincts: les coeurs de podosomes et le nuage. Ces 2 domaines sont induits par différents récepteurs, CD44 et Beta3, polymérisés par des voies distinctes, et ont pour fonction l'adhérence et la contraction. Ils se ré-organisent en une unique structure, la SZ, quand l'ostéoclaste est transféré sur un substrat résorbable. Nous avons également démontré que le maintien de la ceinture de podosomes dépend d'un réseau de microtubules intact

Structures, stabilité et fonctions du cytosquelette d actine dans les ostéoclastes mâtures

L'ostéoclaste est une cellule spécialisée dans la résorption de la matrice osseuse. Elle présente une organisation différente de l'actine selon les substrats sur lesquels elle est ensemencée. Sur verre ou plastique, l'actine est sous forme de "podosomes", alors que sur un substrat résorbable, elle se ré-organise en une ceinture conttinue, la zone de scellement (SZ). L'étude de cellules déficientes en WIP nous a permis de démontrer qu'un ostéoclaste sur verre forme 2 domaines d'actine distincts: les coeurs de podosomes et le nuage. Ces 2 domaines sont induits par différents récepteurs, CD44 et Beta3, polymérisés par des voies distinctes, et ont pour fonction l'adhérence et la contraction. Ils se ré-organisent en une unique structure, la SZ, quand l'ostéoclaste est transféré sur un substrat résorbable. Nous avons également démontré que le maintien de la ceinture de podosomes dépend d'un réseau de microtubules intact.Osteoclasts are hematopoietic cells specialized in bone resorption. Actin cytoskeleton organisation depends on substrates: podosomes are observed on glass or plastic slides, whereas an homogenous actin belt, the sealing zone (SZ) is organized on resorption substrates. Study of WIP deficient cells allows us to demontrate existence of 2 actin domains: podosomes cores surrounded by actin cloud. These 2 domains are induced by different receptors, CD44 and Beta3, polymerized by 2 distinct pathways, and are implicated in adhesion and contraction os osteoclast. Podosomes cores and actin cloud reorganized into an unique structure, the SZ, when osteoclast is seeded on apatite or dentin slides. We have also demonstrated that stability of podosomes belt depends on microtubules.LYON-ENS Sciences (693872304) / SudocSudocFranceF

Paxillin Phosphorylation Controls Invadopodia/Podosomes Spatiotemporal Organization

In Rous sarcoma virus (RSV)-transformed baby hamster kidney (BHK) cells, invadopodia can self-organize into rings and belts, similarly to podosome distribution during osteoclast differentiation. The composition of individual invadopodia is spatiotemporally regulated and depends on invadopodia localization along the ring section: the actin core assembly precedes the recruitment of surrounding integrins and integrin-linked proteins, whereas the loss of the actin core was a prerequisite to invadopodia disassembly. We have shown that invadopodia ring expansion is controlled by paxillin phosphorylations on tyrosine 31 and 118, which allows invadopodia disassembly. In BHK-RSV cells, ectopic expression of the paxillin mutant Y31F-Y118F induces a delay in invadopodia disassembly and impairs their self-organization. A similar mechanism is unraveled in osteoclasts by using paxillin knockdown. Lack of paxillin phosphorylation, calpain or extracellular signal-regulated kinase inhibition, resulted in similar phenotype, suggesting that these proteins belong to the same regulatory pathways. Indeed, we have shown that paxillin phosphorylation promotes Erk activation that in turn activates calpain. Finally, we observed that invadopodia/podosomes ring expansion is required for efficient extracellular matrix degradation both in BHK-RSV cells and primary osteoclasts, and for transmigration through a cell monolayer

CD44 and β3 Integrin Organize Two Functionally Distinct Actin-based Domains in Osteoclasts

The actin cytoskeleton of mature osteoclasts (OCs) adhering to nonmineralized substrates is organized in a belt of podosomes reminiscent of the sealing zone (SZ) found in bone resorbing OCs. In this study, we demonstrate that the belt is composed of two functionally different actin-based domains: podosome cores linked with CD44, which are involved in cell adhesion, and a diffuse cloud associated with β3 integrin, which is involved in cell adhesion and contraction. Wiskott Aldrich Syndrome Protein (WASp) Interacting Protein (WIP)−/− OCs were devoid of podosomes, but they still exhibited actin clouds. Indeed, WIP−/− OCs show diminished expression of WASp, which is required for podosome formation. CD44 is a novel marker of OC podosome cores and the first nonintegrin receptor detected in these structures. The importance of CD44 is revealed by showing that its clustering restores podosome cores and WASp expression in WIP−/− OCs. However, although CD44 signals are sufficient to form a SZ, the presence of WIP is indispensable for the formation of a fully functional SZ