Cortical branched actin determines cell cycle progression

International audienc

Rôle d’une protéine trimérique à superhélice dans l’assemblage du complexe WASH

The Arp2/3 complex generates branched actin networks, which produces a pushing force that helps the cell to remodel its membranes. The WASH complex activates the Arp2/3 complex at the surface of endosomes and thereby, facilitates the membrane scission of the transport intermediates containing internalized receptors such as α5β1 integrins. Hence, by promoting integrin recycling, the WASH complex plays a crucial role in tumor cell invasion during cancer progression. However, how cells assemble the WASH complex at first is unknown. Here we report the identification of the first assembly factor of the WASH complex. We identified HSBP1 in a proteomics screen for proteins binding to precursor forms of subunits, but not to the fully assembled WASH complex. Through biochemical reconstitution and molecular modeling, we found that HSBP1 associates with the precursor CCDC53 trimer, dissociates it and forms a heterotrimer that will eventually contribute a single CCDC53 molecule to the assembling WASH complex. The role of HSBP1 in WASH complex assembly is well conserved since WASH is similarly destabilized upon HSBP1 knock-down in mammalian cells or upon HSBP1 knock-out in Dictyostelium amoeba. In line with the defective assembly of the WASH complex, the HSBP1 knock-out closely phenocopies WASH knock-out in amoeba. In human mammary carcinoma cells, HSBP1 depletion results in impaired integrin recycling to the plasma membrane leading to the defective development of focal adhesions and reduced invasion abilities. Moreover, HSBP1 was found to localize at the centrosome and was required for the polarization associated with the migration. On the other end, in mammary breast tumors, we found that HSBP1 was often overexpressed and that its overexpression was associated with increased levels of the WASH complex and with poor prognosis for breast cancer patients. Hence, HSBP1 is a conserved assembly factor that controls the levels of the WASH complex.Le complexe Arp2/3 génère des réseaux d’actine branchés, qui produisent une forcée de poussée permettant à la cellule de remodeler ses membranes. Le complexe WASH active le complexe Arp2/3 à la surface des endosomes et facilite ainsi la scission membranaire des intermédiaires de transports contenants des récepteurs internalisés tels que les intégrines α5β1. De ce fait, le complexe WASH en favorisant le recyclage des intégrines, joue un rôle crucial dans l’invasion des cellules tumorales durant la progression tumorale. Cependant, le mécanisme d’assemblage du complexe WASH est inconnu. Dans cette étude, nous rapportons l’identification du premier facteur d’assemblage du complexe WASH. Nous avons identifié la protéine HSBP1 grâce à un crible des protéines qui se lient aux formes précurseurs des sous-unités mais plus au complexe une fois assemblé. La reconstitution biochimique et la modélisation moléculaire nous a permis de montrer que HSBP1 est associé avec le précurseur trimérique CCDC53, le dissocie et forme un hétérotrimère qui va éventuellement libérer une forme monomérique de CCDC53 pour l’assemblage du complexe WASH. Le rôle de HSBP1 dans l’assemblage du complexe WASH est conservé. En effet, WASH est déstabilisé dans des cellules mammaires par le knock-down de HSBP1 et dans l’amibe Dictyostelium par le knock-out de HSBP1. La déstabilisation du complexe WASH par le knock-out de HSBP1 phénocopie la déplétion de WASH dans l’amibe Dictyostelium. Dans des cellules humaines de carcinomes mammaires l’inhibition de l’expression de HSBP1 altère le recyclage des intégrines à la membrane plasmidique. Il en résulte des adhésions focales défectueuses et des capacités invasives réduites. De plus, HSBP1 est localisé aux centrosomes et est requis pour la polarité des cellules lors de la migration. Enfin, nous avons trouvé que la surexpression de HSBP1 dans des tumeurs mammaires est associée à une augmentation des niveaux du complexe WASH et à un mauvais pronostic pour les patientes atteintes de cancer du sein. En conclusion, HSBP1 est un facteur d’assemblage conservé qui contrôle les niveaux du complexe WASH

Analysis of Random Migration of Cancer Cells in 3D

International audienceThe ability of cancer cells to migrate through a complex three-dimensional (3D) environment is a hallmark event of cancer metastasis. Therefore, an in vitro migration assay to evaluate cancer cell migration in a 3D setting is valuable to examine cancer progression. Here, we describe such a simple migration assay in a 3D collagen-fibronectin gel for observing cell morphology and comparing the migration abilities of cancer cells. We describe below how to prepare the collagen-fibronectin gel castings, how to set up time-lapse recording, how to draw single-cell trajectories from movies and extract key parameters that characterize cell motility, such as cell speed, directionality, mean square displacement, and directional persistence. In our setup , cells are sandwiched in a single plane between two collagen-fibronectin gels. This trick facilitates the analysis of cell tracks, which are for the most part 2D, at least in the beginning, but in a 3D environment. This protocol has been previously published in Visweshwaran et al. (2018) and is described here in more detail

DataSheet1_Ena/VASP proteins at the crossroads of actin nucleation pathways in dendritic cell migration.PDF

As sentinels of our immune system dendritic cells (DCs) rely on efficient cell migration for patrolling peripheral tissues and delivering sampled antigens to secondary lymphoid organs for the activation of T-cells. Dynamic actin polymerization is key to their macropinocytic and migratory properties. Both major actin nucleation machineries, formins and the Arp2/3 complex, are critical for different aspects of DC functionality, by driving the generation of linear and branched actin filaments, respectively. However, the importance of a third group of actin nucleators, the Ena/VASP family, has not been addressed yet. Here, we show that the two family members Evl and VASP are expressed in murine DCs and that their loss negatively affects DC macropinocytosis, spreading, and migration. Our interactome analysis reveals Ena/VASP proteins to be ideally positioned for orchestrating the different actin nucleation pathways by binding to the formin mDia1 as well as to the WAVE regulatory complex, a stimulator of Arp2/3. In fact, Evl/VASP deficient murine DCs are more vulnerable to inhibition of Arp2/3 demonstrating that Ena/VASP proteins contribute to the robustness and efficiency of DC migration.</p

Table1_Ena/VASP proteins at the crossroads of actin nucleation pathways in dendritic cell migration.XLSX

As sentinels of our immune system dendritic cells (DCs) rely on efficient cell migration for patrolling peripheral tissues and delivering sampled antigens to secondary lymphoid organs for the activation of T-cells. Dynamic actin polymerization is key to their macropinocytic and migratory properties. Both major actin nucleation machineries, formins and the Arp2/3 complex, are critical for different aspects of DC functionality, by driving the generation of linear and branched actin filaments, respectively. However, the importance of a third group of actin nucleators, the Ena/VASP family, has not been addressed yet. Here, we show that the two family members Evl and VASP are expressed in murine DCs and that their loss negatively affects DC macropinocytosis, spreading, and migration. Our interactome analysis reveals Ena/VASP proteins to be ideally positioned for orchestrating the different actin nucleation pathways by binding to the formin mDia1 as well as to the WAVE regulatory complex, a stimulator of Arp2/3. In fact, Evl/VASP deficient murine DCs are more vulnerable to inhibition of Arp2/3 demonstrating that Ena/VASP proteins contribute to the robustness and efficiency of DC migration.</p

The trimeric coiled‐coil HSBP 1 protein promotes WASH complex assembly at centrosomes

International audienceThe Arp2/3 complex generates branched actin networks that exert pushing forces onto different cellular membranes. WASH complexes activate Arp2/3 complexes at the surface of endosomes and thereby fission transport intermediates containing endocy-tosed receptors, such as a5b1 integrins. How WASH complexes are assembled in the cell is unknown. Here, we identify the small coiled-coil protein HSBP1 as a factor that specifically promotes the assembly of a ternary complex composed of CCDC53, WASH, and FAM21 by dissociating the CCDC53 homotrimeric precursor. HSBP1 operates at the centrosome, which concentrates the building blocks. HSBP1 depletion in human cancer cell lines and in Dictyos-telium amoebae phenocopies WASH depletion, suggesting a critical role of the ternary WASH complex for WASH functions. HSBP1 is required for the development of focal adhesions and of cell polarity. These defects impair the migration and invasion of tumor cells. Overexpression of HSBP1 in breast tumors is associated with increased levels of WASH complexes and with poor prognosis for patients

The trimeric coiled-coil HSBP1 protein promotes WASH complex assembly at centrosomes

The Arp2/3 complex generates branched actin networks that exert pushing forces onto different cellular membranes. WASH complexes activate Arp2/3 complexes at the surface of endosomes and thereby fission transport intermediates containing endocytosed receptors, such as α5β1 integrins. How WASH complexes are assembled in the cell is unknown. Here, we identify the small coiled‐coil protein HSBP1 as a factor that specifically promotes the assembly of a ternary complex composed of CCDC53, WASH, and FAM21 by dissociating the CCDC53 homotrimeric precursor. HSBP1 operates at the centrosome, which concentrates the building blocks. HSBP1 depletion in human cancer cell lines and in Dictyostelium amoebae phenocopies WASH depletion, suggesting a critical role of the ternary WASH complex for WASH functions. HSBP1 is required for the development of focal adhesions and of cell polarity. These defects impair the migration and invasion of tumor cells. Overexpression of HSBP1 in breast tumors is associated with increased levels of WASH complexes and with poor prognosis for patients