RÉSUMÉ
Humans express 15 formins that play crucial roles in actin-based processes, including cytokinesis, cell motility and mechanotransduction1,2. However, the lack of structures bound to the actin filament (F-actin) has been a major impediment to understanding formin function. Whereas formins are known for their ability to nucleate and elongate F-actin3-7, some formins can additionally depolymerize, sever or bundle F-actin. Two mammalian formins, inverted formin 2 (INF2) and diaphanous 1 (DIA1, encoded by DIAPH1), exemplify this diversity. INF2 shows potent severing activity but elongates weakly8-11 whereas DIA1 has potent elongation activity but does not sever4,8. Using cryo-electron microscopy (cryo-EM) we show five structural states of INF2 and two of DIA1 bound to the middle and barbed end of F-actin. INF2 and DIA1 bind differently to these sites, consistent with their distinct activities. The formin-homology 2 and Wiskott-Aldrich syndrome protein-homology 2 (FH2 and WH2, respectively) domains of INF2 are positioned to sever F-actin, whereas DIA1 appears unsuited for severing. These structures also show how profilin-actin is delivered to the fast-growing barbed end, and how this is followed by a transition of the incoming monomer into the F-actin conformation and the release of profilin. Combined, the seven structures presented here provide step-by-step visualization of the mechanisms of F-actin severing and elongation by formins.
Sujet(s)
Cytosquelette d'actine , Actines , Formines , Animaux , Humains , Souris , Cytosquelette d'actine/métabolisme , Cytosquelette d'actine/composition chimique , Cytosquelette d'actine/ultrastructure , Actines/composition chimique , Actines/métabolisme , Actines/ultrastructure , Protéines adaptatrices de la transduction du signal/métabolisme , Protéines adaptatrices de la transduction du signal/composition chimique , Sites de fixation , Cryomicroscopie électronique , Formines/composition chimique , Formines/métabolisme , Formines/ultrastructure , Protéines des microfilaments/métabolisme , Protéines des microfilaments/composition chimique , Protéines des microfilaments/ultrastructure , Modèles moléculaires , Profilines/composition chimique , Profilines/métabolisme , Profilines/ultrastructure , Liaison aux protéinesRÉSUMÉ
Formins control the assembly of actin filaments (F-actin) that drive cell morphogenesis and motility in eukaryotes. However, their molecular interaction with F-actin and their mechanism of action remain unclear. In this work, we present high-resolution cryo-electron microscopy structures of F-actin barbed ends bound by three distinct formins, revealing a common asymmetric formin conformation imposed by the filament. Formation of new intersubunit contacts during actin polymerization sterically displaces formin and triggers its translocation. This "undock-and-lock" mechanism explains how actin-filament growth is coordinated with formin movement. Filament elongation speeds are controlled by the positioning and stability of actin-formin interfaces, which distinguish fast and slow formins. Furthermore, we provide a structure of the actin-formin-profilin ring complex, which resolves how profilin is rapidly released from the barbed end during filament elongation.
Sujet(s)
Cytosquelette d'actine , Actines , Formines , Cytosquelette d'actine/composition chimique , Actines/composition chimique , Cryomicroscopie électronique , Formines/composition chimique , Formines/génétique , Profilines/composition chimique , Mutation , SchizosaccharomycesRÉSUMÉ
Highly specialized cells, such as neurons and podocytes, have arborized morphologies that serve their specific functions. Actin cytoskeleton and its associated proteins are responsible for the distinctive shapes of cells. The mechanism of their cytoskeleton regulation - contributing to cell shape maintenance - is yet to be fully clarified. Inverted formin 2 (INF2), one of the modulators of the cytoskeleton, is an atypical formin that can both polymerize and depolymerize actin filaments depending on its molar ratio to actin. Prior work has established that INF2 binds to the sides of actin filaments and severs them. Drebrin is another actin-binding protein that also binds filaments laterally and stabilizes them, but the interplay between drebrin and INF2 on actin filament stabilization is not well understood. Here, we have used biochemical assays, electron microscopy, and total internal reflection fluorescence microscopy imaging to show that drebrin protects actin filaments from severing by INF2 without inhibiting its polymerization activity. Notably, truncated drebrin - DrbA1-300 - is sufficient for this protection, though not as effective as the full-length protein. INF2 and drebrin are abundantly expressed in highly specialized cells and are crucial for the temporal regulation of their actin cytoskeleton, consistent with their involvement in peripheral neuropathy.
Sujet(s)
Actines , Formines , Neuropeptides , Cytosquelette d'actine/composition chimique , Actines/composition chimique , Formines/composition chimique , Neuropeptides/composition chimique , Prolongements cytoplasmiques/composition chimique , Neurones/métabolisme , Microscopie électroniqueRÉSUMÉ
Formin proteins catalyze actin nucleation and microfilament polymerization. Inverted formin 2 (INF2) is an atypical diaphanous-related formin characterized by polymerization and depolymerization of actin. Accumulating evidence showed that INF2 is associated with kidney disease focal segmental glomerulosclerosis and cancers, such as colorectal and thyroid cancer where it functions as a tumor suppressor, glioblastoma, breast, prostate, and gastric cancer, via its oncogenic function. However, studies on the underlying molecular mechanisms of the different roles of INF2 in diverse cancers are limited. This review comprehensively describes the structure, biochemical features, and primary pathogenic mutations of INF2.
Sujet(s)
Formines/génétique , Formines/métabolisme , Gènes suppresseurs de tumeur , Glomérulonéphrite segmentaire et focale/génétique , Glomérulonéphrite segmentaire et focale/métabolisme , Tumeurs/génétique , Tumeurs/métabolisme , Oncogènes , Actines/métabolisme , Formines/composition chimique , Humains , Protéines des microfilaments/métabolisme , Mitochondries/métabolisme , Mutation , Domaines protéiques , Maturation post-traductionnelle des protéines , Transduction du signalRÉSUMÉ
The actin cytoskeleton is crucial for plant morphogenesis, and organization of actin filaments (AF) is dynamically regulated by actin-binding proteins. However, the roles of actin-binding proteins, particularly type II formins, in this process remain poorly understood in plants. Here, we report that a type II formin in rice, Oryza sativa formin homolog 3 (OsFH3), acts as a major player to modulate AF dynamics and contributes to rice morphogenesis. osfh3 mutants were semi-dwarf with reduced size of seeds and unchanged responses to light or gravity compared with mutants of osfh5, another type II formin in rice. osfh3 osfh5 mutants were dwarf with more severe developmental defectiveness. Recombinant OsFH3 could nucleate actin, promote AF bundling, and cap the barbed end of AF to prevent elongation and depolymerization, but in the absence of profilin, OsFH3 could inhibit AF elongation. Different from other reported type II formins, OsFH3 could bind, but not bundle, microtubules directly. Furthermore, its N-terminal phosphatase and tensin homolog domain played a key role in modulating OsFH3 localization at intersections of AF and punctate structures of microtubules, which differed from other reported plant formins. Our results, thus, provide insights into the biological function of type II formins in modulating plant morphology by acting on AF dynamics.
Sujet(s)
Formines/génétique , Formines/métabolisme , Oryza/croissance et développement , Cytosquelette d'actine/métabolisme , Formines/composition chimique , Morphogenèse , Mutation , Taille d'organe , Oryza/génétique , Oryza/métabolisme , Protéines végétales/composition chimique , Protéines végétales/génétique , Protéines végétales/métabolisme , Domaines protéiques , Graines/génétique , Graines/croissance et développement , Graines/métabolismeRÉSUMÉ
One of the earliest mapped human deafness genes, DIAPH1, encodes the formin DIAPH1. To date, at least three distinct mutations in the C-terminal domains and two additional mutations in the N-terminal region are associated with autosomal dominant hearing loss. The underlying molecular mechanisms are not known, and the role of formins in the inner ear is not well understood. In this study, we use biochemical assays to test the hypotheses that autoinhibition and/or actin assembly activities are disrupted by DFNA1 mutations. Our results indicate that C-terminal mutant forms of DIAPH1 are functional in vitro and promote actin filament assembly. Similarly, N-terminal mutants are well-folded and have quaternary structures and thermal stabilities similar to those of the wild-type (WT) protein. The strength of the autoinhibitory interactions varies widely among mutants, with the ttaa, A265S, and I530S mutations having an affinity similar to that of WT and the 1213x and Δag mutations completely abolishing autoinhibition. These data indicate that, in some cases, hearing loss may be linked to weakened inhibition of actin assembly.
Sujet(s)
Formines/génétique , Mutation , Actines/métabolisme , Lignée cellulaire , Formines/composition chimique , Formines/métabolisme , Perte d'audition/génétique , Perte d'audition/métabolisme , Humains , Modèles moléculaires , Pliage des protéines , Stabilité protéiqueRÉSUMÉ
Diaphanous related formins are highly conserved proteins regulated by Rho-GTPases that act as actin nucleation and assembly factors. Here we report the functional characterization of a non-inherited heterozygous FMNL2 p.L136P mutation carried by a patient who presented with severe very early onset inflammatory bowel disease (IBD). We found that the FMNL2 L136P protein displayed subcellular mislocalization and deregulated protein autoinhibition indicating gain-of-function mechanism. Expression of FMNL2 L136P impaired cell spreading as well as filopodia formation. THP-1 macrophages expressing FMNL2 L136P revealed dysregulated podosome formation and a defect in matrix degradation. Our data indicate that the L136P mutation affects cellular actin dynamics in fibroblasts and immune cells such as macrophages.
Sujet(s)
Formines/génétique , Maladies inflammatoires intestinales/génétique , Différenciation cellulaire , Lignée cellulaire , Maladie chronique , Formines/composition chimique , Formines/métabolisme , Humains , Maladies inflammatoires intestinales/anatomopathologie , Macrophages/cytologie , Macrophages/métabolisme , Podosomes/métabolisme , Polymorphisme de nucléotide simple , Pseudopodes/métabolisme , Pseudopodes/anatomopathologieRÉSUMÉ
The nuclear position in eukaryotes is controlled by a nucleo-cytoskeletal network, critical in cell differentiation, division, and movement. Forces are transmitted through conserved Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes that traverse the nuclear envelope and engage on either side of the membrane with diverse binding partners. Nesprin-2-giant (Nes2G), a LINC element in the outer nuclear membrane, connects to the actin directly as well as through FHOD1, a formin primarily involved in actin bundling. Here, we report the crystal structure of Nes2G bound to FHOD1 and show that the presumed G-binding domain of FHOD1 is rather a spectrin repeat (SR) binding enhancer for the neighboring FH3 domain. The structure reveals that SR binding by FHOD1 is likely not regulated by the diaphanous-autoregulatory domain helix of FHOD1. Finally, we establish that Nes1G also has one FHOD1 binding SR, indicating that these abundant, giant Nesprins have overlapping functions in actin-bundle recruitment for nuclear movement.
Sujet(s)
Protéines du cytosquelette/métabolisme , Protéines foetales/composition chimique , Protéines foetales/métabolisme , Formines/composition chimique , Formines/métabolisme , Protéines des microfilaments/métabolisme , Protéines de tissu nerveux/métabolisme , Motifs d'acides aminés , Animaux , Cristallographie aux rayons X , Protéines du cytosquelette/composition chimique , Protéines du cytosquelette/génétique , Cellules HEK293 , Humains , Souris , Protéines des microfilaments/composition chimique , Protéines des microfilaments/génétique , Modèles moléculaires , Cellules NIH 3T3 , Protéines de tissu nerveux/composition chimique , Protéines de tissu nerveux/génétique , Liaison aux protéines , Conformation des protéines , Domaines protéiquesRÉSUMÉ
Nucleation-promoting proteins tightly regulate actin polymerization in cells. Whereas many of these proteins bind actin monomers directly, formins use the actin-binding protein profilin to dynamically load actin monomers onto their flexible Formin Homology 1 (FH1) domains. Following binding, FH1 domains deliver profilin-actin complexes to filament ends. To investigate profilin's role as an adaptor protein in formin-mediated elongation, we engineered a chimeric formin that binds actin monomers directly via covalent attachment of profilin to its binding site in the formin. This formin mediates slow filament elongation owing to a high probability of profilin binding at filament ends. Varying the position at which profilin is tethered to the formin alters the elongation rate by modulating profilin occupancy at the filament end. By regulating the availability of the barbed end, we propose that profilin binding establishes a secondary point of control over the rate of filament elongation mediated by formins. Profilin's differential affinities for actin monomers, barbed ends and polyproline are thus tuned to adaptively bridge actin and formins and optimize the rate of actin polymerization.
Sujet(s)
Cytosquelette d'actine/génétique , Actines/composition chimique , Formines/génétique , Profilines/génétique , Cytosquelette d'actine/composition chimique , Actines/génétique , Sites de fixation/génétique , Cytosquelette/génétique , Cytosquelette/ultrastructure , Formines/composition chimique , Protéines des microfilaments/composition chimique , Protéines des microfilaments/génétique , Microscopie de fluorescence , Peptides/composition chimique , Peptides/génétique , Profilines/composition chimique , Liaison aux protéines , Domaines protéiques/génétique , Structure tertiaire des protéines , Saccharomyces cerevisiae/composition chimique , Saccharomyces cerevisiae/génétique , Protéines de Saccharomyces cerevisiae/composition chimique , Protéines de Saccharomyces cerevisiae/génétiqueRÉSUMÉ
Filopodia are actin-built finger-like dynamic structures that protrude from the cell cortex. These structures can sense the environment and play key roles in migration and cell-cell interactions. The growth-retraction cycle of filopodia is a complex process exquisitely regulated by intra- and extra-cellular cues, whose nature remains elusive. Filopodia present wide variation in length, lifetime and growth rate. Here, we investigate the features of filopodia patterns in fixed prostate tumor cells by confocal microscopy. Analysis of almost a thousand filopodia suggests the presence of two different populations: one characterized by a narrow distribution of lengths and the other with a much more variable pattern with very long filopodia. We explore a stochastic model of filopodial growth which takes into account diffusion and reactions involving actin and the regulatory proteins formin and capping, and retrograde flow. Interestingly, we found an inverse dependence between the filopodial length and the retrograde velocity. This result led us to propose that variations in the retrograde velocity could explain the experimental lengths observed for these tumor cells. In this sense, one population involves a wider range of retrograde velocities than the other population, and also includes low values of this velocity. It has been hypothesized that cells would be able to regulate retrograde flow as a mechanism to control filopodial length. Thus, we propound that the experimental filopodia pattern is the result of differential retrograde velocities originated from heterogeneous signaling due to cell-substrate interactions or prior cell-cell contacts.
Sujet(s)
Communication cellulaire , Formines/composition chimique , Myosines/composition chimique , Pseudopodes/physiologie , Actines , Algorithmes , Mouvement cellulaire , Simulation numérique , Cytoplasme/métabolisme , Diffusion , Humains , Microscopie confocale , Cellules PC-3 , Probabilité , Transduction du signal , Processus stochastiquesRÉSUMÉ
Chloride intracellular channel 4 (CLIC4) functions in diverse actin-dependent processes. Upon Rho activation, CLIC4 reversibly translocates from the cytosol to the plasma membrane to regulate cell adhesion and migration. At the plasma membrane, CLIC4 counters the formation of filopodia, which requires actin assembly by the formin mammalian Diaphanous (mDia)2. To this end, mDia2 must be activated through conversion from the closed to the open conformation. Thus, CLIC4 could harness the activation or the open conformation of mDia2 to inhibit filopodium formation. Here, we find that CLIC4 silencing enhances the filopodia induced by two constitutively active mDia2 mutants. Furthermore, we report that CLIC4 binds the actin-regulatory region of mDia2 in vitro. These results suggest that CLIC4 modulates the activity of the open conformation of mDia2, shedding new light into how cells may control filopodia.
Sujet(s)
Canaux chlorure/métabolisme , Formines/génétique , Formines/métabolisme , Mutation , Pseudopodes/métabolisme , Actines/métabolisme , Canaux chlorure/déficit , Canaux chlorure/génétique , Formines/composition chimique , Cellules HeLa , HumainsRÉSUMÉ
Migration of meiosis-I (MI) spindle from the cell center to a sub-cortical location is a critical step for mouse oocytes to undergo asymmetric meiotic cell division. In this study, we investigate the mechanism by which formin-2 (FMN2) orchestrates the initial movement of MI spindle. By defining protein domains responsible for targeting FMN2, we show that spindle-periphery localized FMN2 is required for spindle migration. The spindle-peripheral FMN2 nucleates short actin bundles from vesicles derived likely from the endoplasmic reticulum (ER) and concentrated in a layer outside the spindle. This layer is in turn surrounded by mitochondria. A model based on polymerizing actin filaments pushing against mitochondria, thus generating a counter force on the spindle, demonstrated an inherent ability of this system to break symmetry and evolve directional spindle motion. The model is further supported through experiments involving spatially biasing actin nucleation via optogenetics and disruption of mitochondrial distribution and dynamics.
Sujet(s)
Actines/métabolisme , Méiose , Ovocytes/cytologie , Organites/métabolisme , Appareil du fuseau/métabolisme , Animaux , Division cellulaire asymétrique , Vésicules cytoplasmiques/métabolisme , Réticulum endoplasmique/métabolisme , Femelle , Formines/composition chimique , Formines/génétique , Formines/métabolisme , Souris , Mitochondries/métabolisme , Modèles biologiques , Protéines de tissu nerveux/composition chimique , Protéines de tissu nerveux/génétique , Protéines de tissu nerveux/métabolisme , Ovocytes/métabolisme , Domaines protéiques , Délétion de séquenceRÉSUMÉ
Formins are one of the central players in the assembly of most actin networks in cells. The sensitivity of these processive molecular machines to mechanical tension is now well established. However, how the activity of formins is affected by geometrical constraints related to network architecture, such as filament cross-linking and formin spatial confinement, remains largely unknown. Combining microfluidics and micropatterning, we reconstituted in vitro mDia1 formin-elongated filament bundles induced by fascin, with different geometrical constraints on the formins, and measured the impact of these constraints on formin elongation rate and processivity. When filaments are not bundled, the anchoring details of formins have only a mild impact on their processivity and do not affect their elongation rate. When formins are unanchored, we show that filament bundling by fascin reduces both their elongation rate and their processivity. Strikingly, when filaments elongated by surface-anchored formins are cross-linked together, formin elongation rate immediately decreases and processivity is reduced up to 24-fold depending on the cumulative impact of formin rotational and translational freedom. Our results reveal an unexpected crosstalk between the constraints at the filament and the formin levels. We anticipate that in cells the molecular details of formin anchoring to the plasma membrane strongly modulate formin activity at actin filament barbed ends.
Sujet(s)
Cytosquelette d'actine/métabolisme , Membrane cellulaire/métabolisme , Cytosquelette/métabolisme , Formines/métabolisme , Cytosquelette d'actine/composition chimique , Animaux , Membrane cellulaire/composition chimique , Cytosquelette/composition chimique , Formines/composition chimique , HumainsRÉSUMÉ
To establish a strategy for identifying protein-N-myristoylation-dependent phosphorylation of cellular proteins, Phos-tag SDS-PAGE was performed on wild-type (WT) and nonmyristoylated mutant (G2A-mutant) FMNL2 and FMNL3, phosphorylated N-myristoylated model proteins expressed in HEK293 cells. The difference in the banding pattern in Phos-tag SDS-PAGE between the WT and G2A-mutant FMNL2 indicated the presence of N-myristoylation-dependent phosphorylation sites in FMNL2. Phos-tag SDS-PAGE of FMNL2 mutants in which the putative phosphorylation sites listed in PhosphoSitePlus (an online database of phosphorylation sites) were changed to Ala revealed that Ser-171 and Ser-1072 are N-myristoylation-dependent phosphorylation sites in FMNL2. Similar experiments with FMNL3 demonstrated that N-myristoylation-dependent phosphorylation occurs at a single Ser residue at position 174, which is a Ser residue conserved between FMNL2 and FMNL3, corresponding to Ser-171 in FMNL2. The facts that phosphorylation of Ser-1072 in FMNL2 has been shown to play a critical role in integrin ß1 internalization mediated by FMNL2 and that Ser-171 in FMNL2 and Ser-174 in FMNL3 are novel putative phosphorylation sites conserved between FMNL2 and FMNL3 indicate that the strategy used in this study is a useful tool for identifying and characterizing physiologically important phosphorylation reactions occurring on N-myristoylated proteins.
Sujet(s)
Formines/métabolisme , Pyridines/composition chimique , Sérine/composition chimique , Animaux , Cellules COS , Chlorocebus aethiops , Électrophorèse sur gel de polyacrylamide , Formines/composition chimique , Formines/génétique , Cellules HEK293 , Humains , Mutation , PhosphorylationRÉSUMÉ
In addition to its role in erythrocyte invasion, Plasmodium falciparum actin is implicated in endocytosis, cytokinesis and inheritance of the chloroplast-like organelle called the apicoplast. Previously, the inability to visualise filamentous actin (F-actin) dynamics had restricted the characterisation of both F-actin and actin regulatory proteins, a limitation we recently overcame for Toxoplasma (Periz et al, 2017). Here, we have expressed and validated actin-binding chromobodies as F-actin-sensors in Plasmodium falciparum and characterised in-vivo actin dynamics. F-actin could be chemically modulated, and genetically disrupted upon conditionally deleting actin-1. In a comparative approach, we demonstrate that Formin-2, a predicted nucleator of F-actin, is responsible for apicoplast inheritance in both Plasmodium and Toxoplasma, and additionally mediates efficient cytokinesis in Plasmodium. Finally, time-averaged local intensity measurements of F-actin in Toxoplasma conditional mutants revealed molecular determinants of spatiotemporally regulated F-actin flow. Together, our data indicate that Formin-2 is the primary F-actin nucleator during apicomplexan intracellular growth, mediating multiple essential functions.
Sujet(s)
Cytosquelette d'actine/métabolisme , Cytocinèse/génétique , Formines/composition chimique , Paludisme à Plasmodium falciparum/génétique , Cytosquelette d'actine/composition chimique , Actines/génétique , Actines/métabolisme , Apicoplastes/composition chimique , Apicoplastes/métabolisme , Endocytose/génétique , Érythrocytes/composition chimique , Érythrocytes/parasitologie , Formines/génétique , Régulation de l'expression des gènes/génétique , Humains , Paludisme à Plasmodium falciparum/métabolisme , Paludisme à Plasmodium falciparum/parasitologie , Plasmodium falciparum/composition chimique , Plasmodium falciparum/métabolisme , Liaison aux protéines , Toxoplasma/métabolisme , Toxoplasma/pathogénicitéRÉSUMÉ
OBJECTIVE: Long non-coding RNAs (lncRNAs) have been verified to participate in the regulation of colorectal cancer (CRC). However, the role of LINC00707 in CRC still remains unknown. Here, we aim to study the role of LINC00707 in CRC. PATIENTS AND METHODS: LINC00707 expression in 97 pairs of CRC tissues and adjacent normal tissues was determined by the quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). LINC00707 overexpression or knockdown in SW620 or HCT116 cells was achieved by lentivirus transfection. The proliferation and cell circle progression of established cells were detected by cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. Cell invasion and migration abilities were studied by transwell assay. Dual-luciferase assay and Western blot was used to verify the underlying mechanism of LINC00707 in CRC. Nude mice were obtained to identify the in vivo function of LINC00707 in CRC. RESULTS: LINC00707 was significantly over-expressed in CRC tissues and cell lines. Up-regulation of LINC00707 promoted cell proliferation, cell cycle progression, invasion, and migration of SW620 cells. Conversely, down-regulation of LINC00707 reduced cell growth and metastasis of HCT116 cells. MiR-206 was verified as a direct target of LINC00707, and its function was inhibited by LINC00707. FMNL2 was a target for miR-206 in CRC cells. Meanwhile, LINC00707 promoted tumor growth of CRC in vivo. CONCLUSIONS: LINC00707 was up-regulated in CRC tissues and cells, which promoted cell proliferation and metastasis via sponging miR-206 to increase FMNL2 expression. This might provide a novel target for the biological treatment of CRC.
Sujet(s)
Tumeurs colorectales/anatomopathologie , Formines/métabolisme , microARN/métabolisme , ARN long non codant/métabolisme , Régions 3' non traduites , Animaux , Sites de fixation , Lignée cellulaire tumorale , Mouvement cellulaire , Prolifération cellulaire , Tumeurs colorectales/traitement médicamenteux , Tumeurs colorectales/génétique , Formines/composition chimique , Formines/génétique , Humains , Souris , Souris nude , microARN/composition chimique , microARN/génétique , Interférence par ARN , ARN long non codant/antagonistes et inhibiteurs , ARN long non codant/génétique , Petit ARN interférent/métabolisme , Petit ARN interférent/usage thérapeutique , Transplantation hétérologue , Régulation positiveRÉSUMÉ
The formin family of proteins contributes to spatiotemporal control of actin cytoskeletal rearrangements during motile cell activities. The FMNL subfamily exhibits multiple mechanisms of linear actin filament formation and organization. Here we report novel actin-modifying functions of FMNL1 in breast adenocarcinoma migration models. FMNL1 is required for efficient cell migration and its three isoforms exhibit distinct localization. Suppression of FMNL1 protein expression results in a significant impairment of cell adhesion, migration, and invasion. Overexpression of FMNL1É£, but not FMNL1ß or FMNL1α, enhances cell adhesion independent of the FH2 domain and FMNL1É£ rescues migration in cells depleted of all three endogenous isoforms. While FMNL1É£ inhibits actin assembly in vitro, it facilitates bundling of filamentous actin independent of the FH2 domain. The unique interactions of FMNL1É£ with filamentous actin provide a new understanding of formin domain functions and its effect on motility of diverse cell types suggest a broader role than previously realized.
Sujet(s)
Cytosquelette d'actine/métabolisme , Actines/métabolisme , Mouvement cellulaire , Cytosquelette/métabolisme , Formines/métabolisme , Adénocarcinome/génétique , Adénocarcinome/métabolisme , Adénocarcinome/anatomopathologie , Séquence d'acides aminés , Tumeurs du sein/génétique , Tumeurs du sein/métabolisme , Tumeurs du sein/anatomopathologie , Adhérence cellulaire , Lignée cellulaire tumorale , Formines/composition chimique , Formines/génétique , Humains , Microscopie de fluorescence , Isoformes de protéines/composition chimique , Isoformes de protéines/génétique , Isoformes de protéines/métabolisme , Interférence par ARNRÉSUMÉ
In this study, we analyzed the functional role of the formin Drosophila Homologue of Diaphanous2 (Diaph2) in colorectal cancer cells. We show that stable down-regulation of Diaph2 expression in HT29 cells decreased chromosome alignment and the velocity of chromosome movement during M-phase, thus reducing the proliferation rate and colony formation. In interphase cells, Diaph2 was diffusely distributed in the cytosol, while in metaphase cells the protein was located to spindle microtubules (MTs). Diaph2-depletion increased the concentration of stable spindle MTs, showing that the formin is required to control spindle MT-dynamics. Our cellular data indicate that Diaph2-controls spindle MT-dynamics independent of Cdc42 activity and our in vitro results reveal that bacterially produced full-length (FL) Diaph2 strongly altered MT-dynamics in absence of Cdc42, where its actin-nucleating activity is auto-inhibited. FL-Diaph2 mediates a 10-fold increase in MT-polymerization compared to the Diaph2-FH2-domain. Interestingly, a Diaph2-mutant lacking the FH2-domain (ΔFH2) increased MT-polymerization to a similar extent as the FH2-domain, indicating the existence of a second MT-binding domain. However, in contrast to FL-Diaph2 and the FH2-domain, ΔFH2 did not alter the density of taxol-stabilized MTs. Thus, the FH2-domain and the second Diaph2-binding domain appear to control MT-dynamics by different mechanisms. In summary, our data indicate that Diaph2 controls M-phase progression under basal conditions by regulating spindle MT-dynamics. In addition, a region outside of the canonical MT-regulating FH2-domain is involved in Diaph2-mediated control of MT-dynamics.