RESUMO
The mammalian formin family comprises fifteen multi-domain proteins that regulate actin dynamics and microtubules in vitro and in cells. Evolutionarily conserved formin homology (FH) 1 and 2 domains allow formins to locally modulate the cell cytoskeleton. Formins are involved in several developmental and homeostatic processes, as well as human diseases. However, functional redundancy has long hampered studies of individual formins with genetic loss-of-function approaches and prevents the rapid inhibition of formin activities in cells. The discovery of small molecule inhibitor of formin homology 2 domains (SMIFH2) in 2009 was a disruptive change that provided a powerful chemical tool to explore formins' functions across biological scales. Here, I critically discuss the characterization of SMIFH2 as a pan-formin inhibitor, as well as growing evidence of unexpected off-target effects. By collating the literature and information hidden in public repositories, outstanding controversies and fundamental open questions about the substrates and mechanism of action of SMIFH2 emerge. Whenever possible, I propose explanations for these discrepancies and roadmaps to address the paramount open questions. Furthermore, I suggest that SMIFH2 be reclassified as a multi-target inhibitor for its appealing activities on proteins involved in pathological formin-dependent processes. Notwithstanding all drawbacks and limitations, SMIFH2 will continue to prove useful in studying formins in health and disease in the years to come.
Assuntos
Actinas , Proteínas dos Microfilamentos , Animais , Humanos , Forminas/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , Biologia , Citoesqueleto de Actina/metabolismo , Mamíferos/metabolismoRESUMO
BACKGROUND & AIMS: Oncogenic KrasG12D induces neoplastic transformation of pancreatic acinar cells through acinar-to-ductal metaplasia (ADM), an actin-based morphogenetic process, and drives pancreatic ductal adenocarcinoma (PDAC). mTOR (mechanistic target of rapamycin kinase) complex 1 (mTORC1) and 2 (mTORC2) contain Rptor and Rictor, respectively, and are activated downstream of KrasG12D, thereby contributing to PDAC. Yet, whether and how mTORC1 and mTORC2 impact on ADM and the identity of the actin nucleator(s) mediating such actin rearrangements remain unknown. METHODS: A mouse model of inflammation-accelerated KrasG12D-driven early pancreatic carcinogenesis was used. Rptor, Rictor, and Arpc4 (actin-related protein 2/3 complex subunit 4) were conditionally ablated in acinar cells to deactivate the function of mTORC1, mTORC2 and the actin-related protein (Arp) 2/3 complex, respectively. RESULTS: We found that mTORC1 and mTORC2 are markedly activated in human and mouse ADM lesions, and cooperate to promote KrasG12D-driven ADM in mice and in vitro. They use the Arp2/3 complex as a common downstream effector to induce the remodeling the actin cytoskeleton leading to ADM. In particular, mTORC1 regulates the translation of Rac1 (Rac family small GTPase 1) and the Arp2/3-complex subunit Arp3, whereas mTORC2 activates the Arp2/3 complex by promoting Akt/Rac1 signaling. Consistently, genetic ablation of the Arp2/3 complex prevents KrasG12D-driven ADM in vivo. In acinar cells, the Arp2/3 complex and its actin-nucleation activity mediated the formation of a basolateral actin cortex, which is indispensable for ADM and pre-neoplastic transformation. CONCLUSIONS: Here, we show that mTORC1 and mTORC2 attain a dual, yet nonredundant regulatory role in ADM and early pancreatic carcinogenesis by promoting Arp2/3 complex function. The role of Arp2/3 complex as a common effector of mTORC1 and mTORC2 fills the gap between oncogenic signals and actin dynamics underlying PDAC initiation.
Assuntos
Células Acinares/enzimologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Carcinoma Ductal Pancreático/enzimologia , Transformação Celular Neoplásica/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Mutação , Ductos Pancreáticos/enzimologia , Neoplasias Pancreáticas/enzimologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Células Acinares/patologia , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Metaplasia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Proteína Regulatória Associada a mTOR/genética , Proteína Regulatória Associada a mTOR/metabolismo , Transdução de SinaisRESUMO
The Arp2/3 complex assembles branched actin filaments, which are key to many cellular processes, but its organismal roles remain poorly understood. Here, we employed conditional Arpc4 knockout mice to study the function of the Arp2/3 complex in the epidermis. We found that depletion of the Arp2/3 complex by knockout of Arpc4 results in skin abnormalities at birth that evolve into a severe psoriasis-like disease hallmarked by hyperactivation of transcription factor Nrf2. Knockout of Arpc4 in cultured keratinocytes was sufficient to induce nuclear accumulation of Nrf2, upregulation of Nrf2 target genes and decreased filamentous actin levels. Furthermore, pharmacological inhibition of the Arp2/3 complex unmasked the role of branched actin filaments in Nrf2 regulation. Consistent with this, we revealed that Nrf2 associates with the actin cytoskeleton in cells and binds to filamentous actin in vitro Finally, we discovered that Arpc4 is downregulated in both human and mouse psoriatic epidermis. Thus, the Arp2/3 complex affects keratinocyte shape and transcriptome through an actin-based cell-autonomous mechanism that influences epidermal morphogenesis and homeostasis.
Assuntos
Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Actinas/metabolismo , Epiderme/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Psoríase/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/antagonistas & inibidores , Adulto , Animais , Células Cultivadas , Modelos Animais de Doenças , Ativação Enzimática/genética , Feminino , Humanos , Queratinócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Psoríase/patologiaRESUMO
Chloride intracellular channel 4 (CLIC4) is a cytosolic protein implicated in diverse actin-based processes, including integrin trafficking, cell adhesion, and tubulogenesis. CLIC4 is rapidly recruited to the plasma membrane by RhoA-activating agonists and then partly colocalizes with ß1 integrins. Agonist-induced CLIC4 translocation depends on actin polymerization and requires conserved residues that make up a putative binding groove. However, the mechanism and significance of CLIC4 trafficking have been elusive. Here, we show that RhoA activation by either lysophosphatidic acid (LPA) or epidermal growth factor is necessary and sufficient for CLIC4 translocation to the plasma membrane and involves regulation by the RhoA effector mDia2, a driver of actin polymerization and filopodium formation. We found that CLIC4 binds the G-actin-binding protein profilin-1 via the same residues that are required for CLIC4 trafficking. Consistently, shRNA-induced profilin-1 silencing impaired agonist-induced CLIC4 trafficking and the formation of mDia2-dependent filopodia. Conversely, CLIC4 knockdown increased filopodium formation in an integrin-dependent manner, a phenotype rescued by wild-type CLIC4 but not by the trafficking-incompetent mutant CLIC4(C35A). Furthermore, CLIC4 accelerated LPA-induced filopodium retraction. We conclude that through profilin-1 binding, CLIC4 functions in a RhoA-mDia2-regulated signaling network to integrate cortical actin assembly and membrane protrusion. We propose that agonist-induced CLIC4 translocation provides a feedback mechanism that counteracts formin-driven filopodium formation.
Assuntos
Proteínas de Transporte/metabolismo , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Profilinas/metabolismo , Pseudópodes/metabolismo , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/metabolismo , Membrana Celular/metabolismo , Canais de Cloreto/química , Sequência Conservada , Cristalografia por Raios X , Ativação Enzimática , Forminas , Células HeLa , Humanos , Integrinas/metabolismo , Modelos Moleculares , Profilinas/química , Ligação Proteica , Conformação Proteica , Transporte ProteicoRESUMO
Invadosomes are actin-rich membrane protrusions that degrade the extracellular matrix to drive tumor cell invasion. Key players in invadosome formation are c-Src and Rho family GTPases. Invadosomes can reassemble into circular rosette-like superstructures, but the underlying signaling mechanisms remain obscure. Here we show that Src-induced invadosomes in human melanoma cells (A375M and MDA-MB-435) undergo rapid remodeling into dynamic extracellular matrix-degrading rosettes by distinct G protein-coupled receptor agonists, notably lysophosphatidic acid (LPA; acting through the LPA1 receptor) and endothelin. Agonist-induced rosette formation is blocked by pertussis toxin, dependent on PI3K activity and accompanied by localized production of phosphatidylinositol 3,4,5-trisphosphate, whereas MAPK and Ca(2+) signaling are dispensable. Using FRET-based biosensors, we show that LPA and endothelin transiently activate Cdc42 through Gi, concurrent with a biphasic decrease in Rac activity and differential effects on RhoA. Cdc42 activity is essential for rosette formation, whereas G12/13-mediated RhoA-ROCK signaling suppresses the remodeling process. Our results reveal a Gi-mediated Cdc42 signaling axis by which G protein-coupled receptors trigger invadosome remodeling, the degree of which is dictated by the Cdc42-RhoA activity balance.
Assuntos
Endotelinas/metabolismo , Lisofosfolipídeos/metabolismo , Melanoma/metabolismo , Podossomos/metabolismo , Receptores de Ácidos Lisofosfatídicos/agonistas , Proteína cdc42 de Ligação ao GTP/agonistas , Proteínas rac1 de Ligação ao GTP/metabolismo , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Transferência Ressonante de Energia de Fluorescência , Humanos , Hidrólise , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Melanoma/enzimologia , Melanoma/patologia , Microscopia Confocal , Microscopia de Fluorescência , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Podossomos/enzimologia , Podossomos/patologia , Interferência de RNA , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Imagem com Lapso de Tempo , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/agonistas , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Protrusion of lamellipodia and ruffles requires polymerization of branched actin filaments by the Arp2/3 complex. Although regulation of Arp2/3 complex activity has been extensively investigated, the mechanism of initiation of lamellipodia and ruffles remains poorly understood. Here, we show that mDia1 acts in concert with the Arp2/3 complex to promote initiation of lamellipodia and ruffles. We find that mDia1 is an epidermal growth factor (EGF)-regulated actin nucleator involved in membrane ruffling using a combination of knockdown and rescue experiments. At the molecular level, mDia1 polymerizes linear actin filaments, activating the Arp2/3 complex, and localizes within nascent and mature membrane ruffles. We employ functional complementation experiments and optogenetics to show that mDia1 cooperates with the Arp2/3 complex in initiating lamellipodia and ruffles. Finally, we show that genetic and pharmacological interference with this cooperation hampers ruffling and cell migration. Thus, we propose that the lamellipodium- and ruffle-initiating machinery consists of two actin nucleators that act sequentially to regulate membrane protrusion and cell migration.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Estruturas da Membrana Celular/metabolismo , Pseudópodes/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células COS , Estruturas da Membrana Celular/genética , Chlorocebus aethiops , Forminas , Células HeLa , Humanos , Pseudópodes/genéticaRESUMO
mDia2 is an auto-inhibited Formin influencing actin dynamics upon conversion to the active conformation. mDia2 regulates actin-based protrusions and cell invasion, cell differentiation, vesicle trafficking, and cytokinesis. However, whether mDia2 has additional functions and how its action is functionally specified remain unknown. Here we draw the interactome of auto-inhibited and constitutively active mDia2 to address these issues. We embed mDia2 in protein networks accounting for its attributed functions and unexpectedly link it to the Ubiquitin Proteasome System. Taking FBXO3 as a test case, we show that mDia2 binds FBXO3 and p53, and regulates p53 transcriptional activity in an actin-nucleation-independent and conformation-insensitive manner. Increased mDia2 and FBXO3 levels elevate p53 activity and expression thereby sensitizing cells to p53-dependent apoptosis, whereas their decrease produces opposite effects. Thus, we discover a new role of mDia2 in p53 regulation suggesting that the closed conformation is biologically active and an FBXO3-based mechanism to functionally specify mDia2's activity.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , NADPH Desidrogenase/metabolismo , Proteômica/métodos , Homologia de Sequência de Aminoácidos , Animais , Apoptose , Cromatografia de Afinidade , Dano ao DNA , Proteínas F-Box/metabolismo , Proteínas Fetais/metabolismo , Forminas , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Espectrometria de Massas , Camundongos , Proteínas dos Microfilamentos/metabolismo , Proteínas Nucleares/metabolismo , Ligação Proteica , Conformação Proteica , Mapas de Interação de Proteínas , Reprodutibilidade dos Testes , Proteína Supressora de Tumor p53/metabolismoRESUMO
Formin mDia2 is a cytoskeleton-regulatory protein that switches reversibly between a closed, autoinhibited and an open, active conformation. Although the open conformation of mDia2 induces actin assembly thereby controlling many cellular processes, mDia2 possesses also actin-independent and conformation-insensitive scaffolding roles related to microtubules and p53, respectively. Thus, we hypothesize that mDia2 may have other unappreciated functions and regulatory modes. Here we identify and validate proteasome and Ubiquitin as mDia2-interacting partners using stable isotope labeling with amino acids in cell culture-based quantitative proteomics and biochemistry, respectively. Although mDia2 is ubiquitinated, binds ubiquitinated proteins and free Ubiquitin, it is not a proteasome substrate. Surprisingly, knockdown of mDia2 increases the activity of the proteasome in vitro, whereas mDia2 overexpression has opposite effects only when it adopts the open conformation and cannot induce actin assembly. Consistently, a combination of candidate and unbiased proteome-wide analyses indicates that mDia2 regulates the cellular levels of proteasome substrate ß-catenin and a number of ubiquitinated actin-regulatory proteins. Hence, these findings add more complexity to the mDia2 activity cycle by showing that the open conformation may control actin dynamics also through actin-independent regulation of the proteasome.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , NADPH Desidrogenase/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteômica/métodos , Actinas/metabolismo , Animais , Marcação por Isótopo , Camundongos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/fisiologia , NADPH Desidrogenase/química , NADPH Desidrogenase/fisiologia , Conformação Proteica , Mapeamento de Interação de Proteínas , Ubiquitina/metabolismoRESUMO
Formin family proteins (formins) represent an evolutionary conserved protein family encoded in the genome of a wide range of eukaryotes. Formins are hallmarked by a formin homology 1 (FH1) domain juxtaposed to an FH2 domain whereby they control actin and microtubule dynamics. Not surprisingly, formins are best known as key regulators of the cytoskeleton in a variety of morphogenetic processes. However, mounting evidence implicates several formins in the assembly and organization of actin within and around the nucleus. In addition, actin-independent roles for formins have recently been discovered. In this mini-review, we summarize these findings and highlight the novel nuclear and perinulcear functions of formins. In light of the emerging new biology of formins, we also discuss the fundamental principles governing the versatile activity and multimodal regulation of these proteins.
Assuntos
Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Proteínas Fetais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Actinas/metabolismo , Animais , Forminas , Humanos , Modelos Biológicos , Transdução de SinaisRESUMO
Dendritic cells (DCs) play a pivotal role in immune surveillance, acting as sentinels that coordinate immune responses within tissues. Although differences in the identity and functional states of DC subpopulations have been identified through multiparametric flow cytometry and single-cell RNA sequencing, these methods do not provide information about the spatial context in which the cells are located. This knowledge is crucial for understanding tissue organisation and cellular cross-talk. Recent developments in multiplex imaging techniques can now offer insights into this complex spatial and functional landscape. This review provides a concise overview of these imaging methodologies, emphasising their application in identifying DCs to delineate their tissue-specific functions and aiding newcomers in navigating this field.
RESUMO
The cortex controls cell shape. In mouse oocytes, the cortex thickens in an Arp2/3-complex-dependent manner, ensuring chromosome positioning and segregation. Surprisingly, we identify that mouse oocytes lacking the Arp2/3 complex undergo cortical actin remodeling upon division, followed by cortical contractions that are unprecedented in mammalian oocytes. Using genetics, imaging, and machine learning, we show that these contractions stir the cytoplasm, resulting in impaired organelle organization and activity. Oocyte capacity to avoid polyspermy is impacted, leading to a reduced female fertility. We could diminish contractions and rescue cytoplasmic anomalies. Similar contractions were observed in human oocytes collected as byproducts during IVF (in vitro fertilization) procedures. These contractions correlate with increased cytoplasmic motion, but not with defects in spindle assembly or aneuploidy in mice or humans. Our study highlights a multiscale effect connecting cortical F-actin, contractions, and cytoplasmic organization and affecting oocyte quality, with implications for female fertility.
Assuntos
Oócitos , Fuso Acromático , Humanos , Feminino , Animais , Camundongos , Citoplasma , Citoesqueleto de Actina , Complexo 2-3 de Proteínas Relacionadas à Actina , Actinas , Meiose , MamíferosRESUMO
Clathrin-mediated endocytosis (CME) involves spatially and temporally restricted molecular dynamics, to which protein kinases and actin contribute. However, whether and how these two elements merge to properly execute CME remains unknown. Here, we show that neural Wiskott-Aldrich syndrome protein (N-WASP) and casein kinase 2 (CK2) form a complex and localize to clathrin-coated vesicles. N-WASP binds to and is phosphorylated by CK2, thereby reducing the kinase activity of CK2. By contrast, N-WASP-promoted actin polymerization is decreased upon both phosphorylation and binding of CK2. Knockdown of CK2 and N-WASP, either alone or in combination, causes a similar inhibition in the initial rate of CME of epidermal growth factor receptor (EGFR) and its accumulation at the plasma membrane. Increased levels of EGFR at the cell surface can only be efficiently rescued by reconstituting the N-WASP-CK2 complex with either wild-type or phosphorylation-mimicking N-WASP and wild-type CK2. Notably, perturbation of N-WASP-CK2 complex function showed that N-WASP controls the presence of F-actin at clathrin-coated structures. In summary, the N-WASP-CK2 complex integrates in a single circuit different activities contributing to CME.
Assuntos
Caseína Quinase II/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Actinas/metabolismo , Clatrina/metabolismo , Endocitose , Receptores ErbB/metabolismo , Células HeLa , Humanos , Imunoprecipitação , FosforilaçãoRESUMO
The interaction between tumor cells and activated fibroblasts determines malignant features of desmoplastic carcinomas such as rapid growth, progression towards a metastatic phenotype, and resistance to chemotherapy. On one hand, tumor cells can activate normal fibroblasts and even reprogram them into CAFs through complex mechanisms that also involve soluble factors. Among them, transforming growth factor beta (TGF-ß) and Platelet-Derived Growth Factor (PDGF) have an established role in the acquisition of pro-tumorigenic phenotypes by fibroblasts. On the other hand, activated fibroblasts release Interleukin-6 (IL-6), which increases tumor-cell invasiveness and chemoresistance. However, the interplay between breast cancer cells and fibroblasts, as well as the modes of action of TGF-ß, PDGF, and IL-6, are difficult to investigate in vivo. Here, we validated the usage of advanced cell culture models as tools to study the interplay between mammary tumor cells and fibroblasts, taking mouse and human triple-negative tumor cells and fibroblasts as a case study. We employed two different settings, one permitting only paracrine signaling, the other both paracrine and cell-contact-based signaling. These co-culture systems allowed us to unmask how TGF-ß, PDGF and IL-6 mediate the interplay between mammary tumor cells and fibroblasts. We found that the fibroblasts underwent activation induced by the TGF-ß and the PDGF produced by the tumor cells, which increased their proliferation and IL-6 secretion. The IL-6 secreted by activated fibroblasts enhanced tumor-cell proliferation and chemoresistance. These results show that these breast cancer avatars possess an unexpected high level of complexity, which resembles that observed in vivo. As such, advanced co-cultures provide a pathologically relevant tractable system to study the role of the TME in breast cancer progression with a reductionist approach.
RESUMO
Neural Wiskott-Aldrich syndrome protein (N-WASP) and WAVE are members of a family of proteins that use the Arp2/3 complex to stimulate actin assembly in actin-based motile processes. By entering into distinct macromolecular complexes, they act as convergent nodes of different signalling pathways. The role of WAVE in generating lamellipodial protrusion during cell migration is well established. Conversely, the precise cellular functions of N-WASP have remained elusive. Here, we report that Abi1, an essential component of the WAVE protein complex, also has a critical role in regulating N-WASP-dependent function. Consistently, Abi1 binds to N-WASP with nanomolar affinity and, cooperating with Cdc42, potently induces N-WASP activity in vitro. Molecular genetic approaches demonstrate that Abi1 and WAVE, but not N-WASP, are essential for Rac-dependent membrane protrusion and macropinocytosis. Conversely, Abi1 and N-WASP, but not WAVE, regulate actin-based vesicular transport, epidermal growth factor receptor (EGFR) endocytosis, and EGFR and transferrin receptor (TfR) cell-surface distribution. Thus, Abi1 is a dual regulator of WAVE and N-WASP activities in specific processes that are dependent on actin dynamics.
Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Membrana Celular/metabolismo , Proteínas do Citoesqueleto , Receptores ErbB/metabolismo , Células HeLa , Humanos , Receptores da Transferrina/metabolismo , Vesículas Transportadoras/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Proteína Neuronal da Síndrome de Wiskott-Aldrich/fisiologia , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Cancer-associated fibroblasts (CAF) are key regulators of tumorigenesis. Further insights into the tumor-promoting mechanisms of action of CAFs could help improve cancer diagnosis and treatment. Here we show that the formin mDia2 regulates the positioning and function of mitochondria in dermal fibroblasts, thereby promoting a protumorigenic CAF phenotype. Mechanistically, mDia2 stabilized the mitochondrial trafficking protein MIRO1. Loss of mDia2 or MIRO1 in fibroblasts or CAFs reduced the presence of mitochondria and ATP levels near the plasma membrane and at CAF-tumor cell contact sites, caused metabolic alterations characteristic of mitochondrial dysfunction, and suppressed the secretion of protumorigenic proteins. In mouse models of squamous carcinogenesis, genetic or pharmacologic inhibition of mDia2, MIRO1, or their common upstream regulator activin A inhibited tumor formation. Consistently, co-upregulation of mDia2 and MIRO1 in the stroma of various human cancers negatively correlated with survival. This work unveils a key role of mitochondria in the protumorigenic CAF phenotype and identifies an activin A-mDia2-MIRO1 signaling axis in CAFs with diagnostic and therapeutic potential. SIGNIFICANCE: Inhibition of mDia2/MIRO1-mediated mitochondrial positioning in CAFs induces mitochondrial dysfunction and suppresses tumor growth, revealing a promising therapeutic strategy to target tumor-stroma cross-talk.
Assuntos
Fibroblastos Associados a Câncer , Animais , Humanos , Camundongos , Trifosfato de Adenosina/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Carcinogênese/patologia , Fibroblastos/metabolismo , Forminas , Mitocôndrias/fisiologia , Membranas MitocondriaisRESUMO
WAVE2 belongs to a family of proteins that mediates actin reorganization by relaying signals from Rac to the Arp2/3 complex, resulting in lamellipodia protrusion. WAVE2 displays Arp2/3-dependent actin nucleation activity in vitro, and does not bind directly to Rac. Instead, it forms macromolecular complexes that have been reported to exert both positive and negative modes of regulation. How these complexes are assembled, localized and activated in vivo remains to be established. Here we use tandem mass spectrometry to identify an Abi1-based complex containing WAVE2, Nap1 (Nck-associated protein) and PIR121. Abi1 interacts directly with the WHD domain of WAVE2, increases WAVE2 actin polymerization activity and mediates the assembly of a WAVE2-Abi1-Nap1-PIR121 complex. The WAVE2-Abi1-Nap1-PIR121 complex is as active as the WAVE2-Abi1 sub-complex in stimulating Arp2/3, and after Rac activation it is re-localized to the leading edge of ruffles in vivo. Consistently, inhibition of Abi1 by RNA interference (RNAi) abrogates Rac-dependent lamellipodia protrusion. Thus, Abi1 orchestrates the proper assembly of the WAVE2 complex and mediates its activation at the leading edge in vivo.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Movimento Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Proteínas dos Microfilamentos/metabolismo , Pseudópodes/metabolismo , Proteína 2 Relacionada a Actina , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/genética , Células HeLa , Humanos , Substâncias Macromoleculares , Estrutura Terciária de Proteína/fisiologia , Pseudópodes/ultraestrutura , Interferência de RNA/fisiologia , Transdução de Sinais/fisiologia , Família de Proteínas da Síndrome de Wiskott-Aldrich , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
Direct cell-to-cell spreading of Listeria monocytogenes requires the bacteria to induce actin-based finger-like membrane protrusions in donor host cells that are endocytosed through caveolin-rich membrane invaginations by adjacent receiving cells. An actin shell surrounds these endocytic sites; however, its structure, composition, and functional significance remain elusive. Here, we show that the formin mDia1, but surprisingly not the Arp2/3 complex, is enriched at the membrane invaginations generated by L. monocytogenes during HeLa and Jeg-3 cell infections. Electron microscopy reveals a band of linear actin filaments that run along the longitudinal axis of the invagination membrane. Mechanistically, mDia1 expression is vital for the assembly of this F-actin shell. mDia1 is also required for the recruitment of Filamin A, a caveola-associated F-actin cross-linking protein, and caveolin-1 to the invaginations. Importantly, mixed-cell infection assays show that optimal caveolin-based L. monocytogenes cell-to-cell spreading correlates with the formation of the linear actin filament-containing shell by mDia1. IMPORTANCE Listeria monocytogenes spreads from one cell to another to colonize tissues. This cell-to-cell movement requires the propulsive force of an actin-rich comet tail behind the advancing bacterium, which ultimately distends the host plasma membrane into a slender bacterium-containing membrane protrusion. These membrane protrusions induce a corresponding invagination in the membrane of the adjacent host cell. The host cell that receives the protrusion utilizes caveolin-based endocytosis to internalize the structures, and filamentous actin lines these membrane invaginations. Here, we set out to determine the structure and function of this filamentous actin "shell." We demonstrate that the formin mDia1, but not the Arp2/3 complex, localizes to the invaginations. Morphologically, we show that this actin is organized into linear arrays and not branched dendritic networks. Mechanistically, we show that the actin shell is assembled by mDia1 and that mDia1 is required for efficient cell-to-cell transfer of L. monocytogenes.
Assuntos
Actinas/metabolismo , Membrana Celular/microbiologia , Forminas/metabolismo , Listeria monocytogenes/fisiologia , Listeriose/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/microbiologia , Caveolina 1/genética , Caveolina 1/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Filaminas/genética , Filaminas/metabolismo , Forminas/genética , Células HeLa , Humanos , Listeria monocytogenes/genética , Listeriose/genética , Listeriose/microbiologiaRESUMO
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.
Assuntos
Canais de Cloreto/metabolismo , Forminas/genética , Forminas/metabolismo , Mutação , Pseudópodes/metabolismo , Actinas/metabolismo , Canais de Cloreto/deficiência , Canais de Cloreto/genética , Forminas/química , Células HeLa , HumanosRESUMO
Cancer-associated fibroblasts (CAFs) are key regulators of tumorigenesis and promising targets for next-generation therapies. We discovered that cancer cell-derived activin A reprograms fibroblasts into pro-tumorigenic CAFs. Mechanistically, this occurs via Smad2-mediated transcriptional regulation of the formin mDia2, which directly promotes filopodia formation and cell migration. mDia2 also induces expression of CAF marker genes through prevention of p53 nuclear accumulation, resulting in the production of a pro-tumorigenic matrisome and secretome. The translational relevance of this finding is reflected by activin A overexpression in tumor cells and of mDia2 in the stroma of skin cancer and other malignancies and the correlation of high activin A/mDia2 levels with poor patient survival. Blockade of this signaling axis using inhibitors of activin, activin receptors, or mDia2 suppressed cancer cell malignancy and squamous carcinogenesis in 3D organotypic cultures, ex vivo, and in vivo, providing a rationale for pharmacological inhibition of activin A-mDia2 signaling in stratified cancer patients.
Assuntos
Ativinas/metabolismo , Carcinogênese , Carcinoma de Células Escamosas , Proteínas Associadas aos Microtúbulos/metabolismo , NADPH Desidrogenase/metabolismo , Animais , Fibroblastos , Forminas , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Signaling from receptor tyrosine kinases (RTKs)* requires the sequential activation of the small GTPases Ras and Rac. Son of sevenless (Sos-1), a bifunctional guanine nucleotide exchange factor (GEF), activates Ras in vivo and displays Rac-GEF activity in vitro, when engaged in a tricomplex with Eps8 and E3b1-Abi-1, a RTK substrate and an adaptor protein, respectively. A mechanistic understanding of how Sos-1 coordinates Ras and Rac activity is, however, still missing. Here, we demonstrate that (a) Sos-1, E3b1, and Eps8 assemble into a tricomplex in vivo under physiological conditions; (b) Grb2 and E3b1 bind through their SH3 domains to the same binding site on Sos-1, thus determining the formation of either a Sos-1-Grb2 (S/G) or a Sos-1-E3b1-Eps8 (S/E/E8) complex, endowed with Ras- and Rac-specific GEF activities, respectively; (c) the Sos-1-Grb2 complex is disrupted upon RTKs activation, whereas the S/E/E8 complex is not; and (d) in keeping with the previous result, the activation of Ras by growth factors is short-lived, whereas the activation of Rac is sustained. Thus, the involvement of Sos-1 at two distinct and differentially regulated steps of the signaling cascade allows for coordinated activation of Ras and Rac and different duration of their signaling within the cell.