RESUMO
Actin polymerization and assembly into stress fibers (SFs) is central to many cellular processes. However, how SFs form in response to the mechanical interaction of cells with their environment is not fully understood. Here we have identified Piezo2 mechanosensitive cationic channel as a transducer of environmental physical cues into mechanobiological responses. Piezo2 is needed by brain metastatic cells from breast cancer (MDA-MB-231-BrM2) to probe their physical environment as they anchor and pull on their surroundings or when confronted with confined migration through narrow pores. Piezo2-mediated Ca2+ influx activates RhoA to control the formation and orientation of SFs and focal adhesions (FAs). A possible mechanism for the Piezo2-mediated activation of RhoA involves the recruitment of the Fyn kinase to the cell leading edge as well as calpain activation. Knockdown of Piezo2 in BrM2 cells alters SFs, FAs, and nuclear translocation of YAP; a phenotype rescued by overexpression of dominant-positive RhoA or its downstream effector, mDia1. Consequently, hallmarks of cancer invasion and metastasis related to RhoA, actin cytoskeleton, and/or force transmission, such as migration, extracellular matrix degradation, and Serpin B2 secretion, were reduced in cells lacking Piezo2.
Assuntos
Citoesqueleto de Actina/metabolismo , Canais Iônicos/metabolismo , Mecanotransdução Celular/fisiologia , Proteína rhoA de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/genética , Cálcio/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Canais Iônicos/genética , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Most transient receptor potential (TRP) channels are regulated by phosphatidylinositol-4,5-biphosphate (PIP2), although the structural rearrangements occurring on PIP2 binding are currently far from clear. Here we report that activation of the TRP vanilloid 4 (TRPV4) channel by hypotonic and heat stimuli requires PIP2 binding to and rearrangement of the cytosolic tails. Neutralization of the positive charges within the sequence (121)KRWRK(125), which resembles a phosphoinositide-binding site, rendered the channel unresponsive to hypotonicity and heat but responsive to 4α-phorbol 12,13-didecanoate, an agonist that binds directly to transmembrane domains. Similar channel response was obtained by depletion of PIP2 from the plasma membrane with translocatable phosphatases in heterologous expression systems or by activation of phospholipase C in native ciliated epithelial cells. PIP2 facilitated TRPV4 activation by the osmotransducing cytosolic messenger 5'-6'-epoxyeicosatrienoic acid and allowed channel activation by heat in inside-out patches. Protease protection assays demonstrated a PIP2-binding site within the N-tail. The proximity of TRPV4 tails, analyzed by fluorescence resonance energy transfer, increased by depleting PIP2 mutations in the phosphoinositide site or by coexpression with protein kinase C and casein kinase substrate in neurons 3 (PACSIN3), a regulatory molecule that binds TRPV4 N-tails and abrogates activation by cell swelling and heat. PACSIN3 lacking the Bin-Amphiphysin-Rvs (F-BAR) domain interacted with TRPV4 without affecting channel activation or tail rearrangement. Thus, mutations weakening the TRPV4-PIP2 interacting site and conditions that deplete PIP2 or restrict access of TRPV4 to PIP2--in the case of PACSIN3--change tail conformation and negatively affect channel activation by hypotonicity and heat.
Assuntos
Fosfatidilinositol 4,5-Difosfato/metabolismo , Canais de Cátion TRPV/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Análise de Variância , Cálcio/metabolismo , Células Cultivadas , Clonagem Molecular , Citoplasma/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Técnicas de Patch-Clamp , Forbóis/metabolismo , Estrutura Terciária de ProteínaRESUMO
Calcium signaling participates in different cellular processes leading to cell migration. TRPV4, a non-selective cation channel that responds to mechano-osmotic stimulation and heat, is also involved in cell migration. However, the mechanistic involvement of TRPV4 in cell migration is currently unknown. We now report that expression of the mutant channel TRPV4-(121)AAWAA (lacking the phosphoinositide-binding site (121)KRWRK(125) and the response to physiological stimuli) altered HEK293 cell migration. Altered migration patterns included periods of fast and persistent motion followed by periods of stalling and turning, and the extension of multiple long cellular protrusions. TRPV4-WT overexpressing cells showed almost complete loss of directionality with frequent turns, no progression, and absence of long protrusions. Traction microscopy revealed higher tractions forces in the tail of TRPV4-(121)AAWAA than in TRPV4-WT expressing cells. These results are consistent with a defective and augmented tail retraction in TRPV4-(121)AAWAA- and TRPV4-WT-expressing cells, respectively. The activity of calpain, a protease implicated in focal adhesion (FA) disassembly, was decreased in TRPV4-(121)AAWAA compared with TRPV4-WT-expressing cells. Consistently, larger focal adhesions were seen in TRPV4-(121)AAWAA compared with TRPV4-WT-expressing HEK293 cells, a result that was also reproduced in T47D and U87 cells. Similarly, overexpression of the pore-dead mutant TRPV4-M680D resumed the TRPV4-(121)AAWAA phenotype presenting larger FA. The migratory phenotype obtained in HEK293 cells overexpressing TRPV4-(121)AAWAA was mimicked by knocking-down TRPC1, a cationic channel that participates in cell migration. Together, our results point to the participation of TRPV4 in the dynamics of trailing adhesions, a function that may require the interplay of TRPV4 with other cation channels or proteins present at the FA sites.
Assuntos
Estruturas da Membrana Celular/metabolismo , Movimento Celular , Canais de Cátion TRPV/metabolismo , Sítios de Ligação , Calpaína/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Mutação , Fosfatidilinositóis/metabolismo , Ligação Proteica , Canais de Cátion TRPV/química , Canais de Cátion TRPV/genéticaRESUMO
The widely distributed TRPV4 cationic channel participates in the transduction of both physical (osmotic, mechanical, and heat) and chemical (endogenous, plant-derived, and synthetic ligands) stimuli. In this chapter we will review TRPV4 expression, biophysics, structure, regulation, and interacting partners as well as physiological and pathological insights obtained in TRPV4 animal models and human genetic studies.
Assuntos
Canais de Cátion TRPV/metabolismo , Animais , Regulação da Expressão Gênica , Predisposição Genética para Doença , Genótipo , Humanos , Potenciais da Membrana , Camundongos Knockout , Conformação Proteica , Transdução de Sinais , Relação Estrutura-Atividade , Canais de Cátion TRPV/química , Canais de Cátion TRPV/deficiência , Canais de Cátion TRPV/genéticaRESUMO
Degradation of the extracellular matrix is a critical step of tumor cell invasion. Both protease-dependent and -independent mechanisms have been described as alternate processes in cancer cell motility. Interestingly, some effectors of protease-dependent degradation are focalized at invadosomes and are directly coupled with contractile and adhesive machineries composed of multiple mechanosensitive proteins. This review presents recent findings in protease-dependent mechanisms elucidating the ways the force affects extracellular matrix degradation by targeting protease expression and activity at invadosome. The aim is to highlight mechanosensing and mechanotransduction processes to direct the degradative activity at invadosomes, with the focus on membrane tension, proteases and mechanosensitive ion channels.
Assuntos
Matriz Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Metaloproteinases da Matriz/metabolismo , Mecanotransdução Celular , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Podossomos/metabolismo , Fenômenos Biomecânicos , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Adesão Celular , Membrana Celular/metabolismo , Membrana Celular/patologia , Movimento Celular , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Matriz Extracelular/patologia , Humanos , Metaloproteinases da Matriz/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Podossomos/genética , Podossomos/patologia , Canais de Sódio/genética , Canais de Sódio/metabolismo , Tensão SuperficialRESUMO
Functional transient receptor potential (TRP) channels result from the assembly of four subunits. Here, we show an interaction between the pre-S1, TRP, and the ankyrin repeat domain (ARD)-S1 linker domains of TRPV1 and TRPV4 that is essential for proper channel assembly. Neutralization of TRPV4 pre-S1 K462 resulted in protein retention in the ER, defective glycosylation and trafficking, and unresponsiveness to TRPV4-activating stimuli. Similar results were obtained with the equivalent mutation in TRPV1 pre-S1. Molecular dynamics simulations revealed that TRPV4-K462 generated an alternating hydrogen network with E745 (TRP box) and D425 (pre-S1 linker), and that K462Q mutation affected subunit folding. Consistently, single TRPV4-E745A or TRPV4-D425A mutations moderately affected TRPV4 biogenesis while double TRPV4-D425A/E745A mutation resumed the TRPV4-K462Q phenotype. Thus, the interaction between pre-S1, TRP, and linker domains is mandatory to generate a structural conformation that allows the contacts between adjacent subunits to promote correct assembly and trafficking to the plasma membrane.