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1.
J Cell Biol ; 223(9)2024 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-39013281

RESUMO

We previously identified talin rod domain-containing protein 1 (TLNRD1) as a potent actin-bundling protein in vitro. Here, we report that TLNRD1 is expressed in the vasculature in vivo. Its depletion leads to vascular abnormalities in vivo and modulation of endothelial cell monolayer integrity in vitro. We demonstrate that TLNRD1 is a component of the cerebral cavernous malformations (CCM) complex through its direct interaction with CCM2, which is mediated by a hydrophobic C-terminal helix in CCM2 that attaches to a hydrophobic groove on the four-helix domain of TLNRD1. Disruption of this binding interface leads to CCM2 and TLNRD1 accumulation in the nucleus and actin fibers. Our findings indicate that CCM2 controls TLNRD1 localization to the cytoplasm and inhibits its actin-bundling activity and that the CCM2-TLNRD1 interaction impacts endothelial actin stress fiber and focal adhesion formation. Based on these results, we propose a new pathway by which the CCM complex modulates the actin cytoskeleton and vascular integrity.


Assuntos
Hemangioma Cavernoso do Sistema Nervoso Central , Células Endoteliais da Veia Umbilical Humana , Humanos , Animais , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais/metabolismo , Adesões Focais/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Fibras de Estresse/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/metabolismo , Ligação Proteica , Camundongos , Núcleo Celular/metabolismo , Talina
2.
Small Methods ; 8(1): e2300719, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37926786

RESUMO

Cells are highly dynamic and adopt variable shapes and sizes. These variations are biologically important but challenging to investigate in a spatiotemporally controlled manner. Micropatterning, confining cells on microfabricated substrates with defined geometries and molecular compositions, is a powerful tool for controlling cell shape and interactions. However, conventional binary micropatterns are static and fail to address dynamic changes in cell polarity, spreading, and migration. Here, a method for dynamic micropatterning is reported, where the non-adhesive surface surrounding adhesive micropatterns is rapidly converted to support specific cell-matrix interactions while allowing simultaneous imaging of the cells. The technique is based on ultraviolet photopatterning of biotinylated polyethylene glycol-grafted poly-L-lysine, and it is simple, inexpensive, and compatible with a wide range of streptavidin-conjugated ligands. Experiments using biotinylation-based dynamic micropatterns reveal that distinct extracellular matrix ligands and bivalent integrin-clustering antibodies support different degrees of front-rear polarity in human glioblastoma cells, which correlates to altered directionality and persistence upon release and migration on fibronectin. Unexpectedly, however, neither an asymmetric cell shape nor centrosome orientation can fully predict the future direction of migration. Taken together, biotinylation-based dynamic micropatterns allow easily accessible and highly customizable control over cell morphology and motility.


Assuntos
Polaridade Celular , Centrossomo , Humanos , Polietilenoglicóis/química , Biotinilação , Comunicação Celular
3.
Proc Natl Acad Sci U S A ; 120(43): e2304288120, 2023 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-37844244

RESUMO

Integrin-dependent adhesion to the extracellular matrix (ECM) mediates mechanosensing and signaling in response to altered microenvironmental conditions. In order to provide tissue- and organ-specific cues, the ECM is composed of many different proteins that temper the mechanical properties and provide the necessary structural diversity. Despite most human tissues being soft, the prevailing view from predominantly in vitro studies is that increased stiffness triggers effective cell spreading and activation of mechanosensitive signaling pathways. To address the functional coupling of ECM composition and matrix rigidity on compliant substrates, we developed a matrix spot array system to screen cell phenotypes against different ECM mixtures on defined substrate stiffnesses at high resolution. We applied this system to both cancer and normal cells and surprisingly identified ECM mixtures that support stiffness-insensitive cell spreading on soft substrates. Employing the motor-clutch model to simulate cell adhesion on biochemically distinct soft substrates, with varying numbers of available ECM-integrin-cytoskeleton (clutch) connections, we identified conditions in which spreading would be supported on soft matrices. Combining simulations and experiments, we show that cell spreading on soft is supported by increased clutch engagement on specific ECM mixtures and even augmented by the partial inhibition of actomyosin contractility. Thus, "stiff-like" spreading on soft is determined by a balance of a cell's contractile and adhesive machinery. This provides a fundamental perspective for in vitro mechanobiology studies, identifying a mechanism through which cells spread, function, and signal effectively on soft substrates.


Assuntos
Matriz Extracelular , Integrinas , Humanos , Adesão Celular , Matriz Extracelular/metabolismo , Integrinas/metabolismo , Citoesqueleto/metabolismo , Transdução de Sinais
4.
Sci Adv ; 9(28): eadg1840, 2023 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-37436978

RESUMO

The progression of noninvasive ductal carcinoma in situ to invasive ductal carcinoma for patients with breast cancer results in a significantly poorer prognosis and is the precursor to metastatic disease. In this work, we have identified insulin-like growth factor-binding protein 2 (IGFBP2) as a potent adipocrine factor secreted by healthy breast adipocytes that acts as a barrier against invasive progression. In line with this role, adipocytes differentiated from patient-derived stromal cells were found to secrete IGFBP2, which significantly inhibited breast cancer invasion. This occurred through binding and sequestration of cancer-derived IGF-II. Moreover, depletion of IGF-II in invading cancer cells using small interfering RNAs or an IGF-II-neutralizing antibody ablated breast cancer invasion, highlighting the importance of IGF-II autocrine signaling for breast cancer invasive progression. Given the abundance of adipocytes in the healthy breast, this work exposes the important role they play in suppressing cancer progression and may help expound upon the link between increased mammary density and poorer prognosis.


Assuntos
Neoplasias da Mama , Feminino , Humanos , Adipócitos , Anticorpos Neutralizantes , Mama , Fator de Crescimento Insulin-Like II
5.
J Cell Sci ; 136(5)2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36861887

RESUMO

Myosin-X (MYO10), a molecular motor localizing to filopodia, is thought to transport various cargo to filopodia tips, modulating filopodia function. However, only a few MYO10 cargoes have been described. Here, using GFP-Trap and BioID approaches combined with mass spectrometry, we identified lamellipodin (RAPH1) as a novel MYO10 cargo. We report that the FERM domain of MYO10 is required for RAPH1 localization and accumulation at filopodia tips. Previous studies have mapped the RAPH1 interaction domain for adhesome components to its talin-binding and Ras-association domains. Surprisingly, we find that the RAPH1 MYO10-binding site is not within these domains. Instead, it comprises a conserved helix located just after the RAPH1 pleckstrin homology domain with previously unknown functions. Functionally, RAPH1 supports MYO10 filopodia formation and stability but is not required to activate integrins at filopodia tips. Taken together, our data indicate a feed-forward mechanism whereby MYO10 filopodia are positively regulated by MYO10-mediated transport of RAPH1 to the filopodium tip.


Assuntos
Integrinas , Pseudópodes , Sítios de Ligação , Espectrometria de Massas , Miosinas/genética
6.
Mol Oncol ; 17(6): 1007-1023, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-36461911

RESUMO

While organ-confined prostate cancer (PCa) is mostly therapeutically manageable, metastatic progression of PCa remains an unmet clinical challenge. Resistance to anoikis, a form of cell death initiated by cell detachment from the surrounding extracellular matrix, is one of the cellular processes critical for PCa progression towards aggressive disease. Therefore, further understanding of anoikis regulation in PCa might provide therapeutic opportunities. Here, we discover that PCa tumours with concomitant inhibition of two tumour suppressor phosphatases, PP2A and PTEN, are particularly aggressive, having < 50% 5-year secondary-therapy-free patient survival. Functionally, overexpression of PME-1, a methylesterase for the catalytic PP2A-C subunit, inhibits anoikis in PTEN-deficient PCa cells. In vivo, PME-1 inhibition increased apoptosis in in ovo PCa tumour xenografts, and attenuated PCa cell survival in zebrafish circulation. Molecularly, PME-1-deficient PC3 cells display increased trimethylation at lysines 9 and 27 of histone H3 (H3K9me3 and H3K27me3), a phenotype known to correlate with increased apoptosis sensitivity. In summary, our results demonstrate that PME-1 supports anoikis resistance in PTEN-deficient PCa cells. Clinically, these results identify PME-1 as a candidate biomarker for a subset of particularly aggressive PTEN-deficient PCa.


Assuntos
Anoikis , Hidrolases de Éster Carboxílico , Neoplasias da Próstata , Animais , Humanos , Masculino , Recidiva Local de Neoplasia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , PTEN Fosfo-Hidrolase/genética , Peixe-Zebra , Hidrolases de Éster Carboxílico/genética
7.
Nat Commun ; 13(1): 6953, 2022 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-36376313

RESUMO

The ErbB4 receptor isoforms JM-a and JM-b differ within their extracellular juxtamembrane (eJM) domains. Here, ErbB4 isoforms are used as a model to address the effect of structural variation in the eJM domain of receptor tyrosine kinases (RTK) on downstream signaling. A specific JM-a-like sequence motif is discovered, and its presence or absence (in JM-b-like RTKs) in the eJM domains of several RTKs is demonstrated to dictate selective STAT activation. STAT5a activation by RTKs including the JM-a like motif is shown to involve interaction with oligosaccharides of N-glycosylated cell surface proteins such as ß1 integrin, whereas STAT5b activation by JM-b is dependent on TYK2. ErbB4 JM-a- and JM-b-like RTKs are shown to associate with specific signaling complexes at different cell surface compartments using analyses of RTK interactomes and super-resolution imaging. These findings provide evidence for a conserved mechanism linking a ubiquitous extracellular motif in RTKs with selective intracellular STAT signaling.


Assuntos
Receptores Proteína Tirosina Quinases , Transdução de Sinais , Receptores Proteína Tirosina Quinases/metabolismo , Receptor ErbB-4/metabolismo , Isoformas de Proteínas/metabolismo , Membrana Celular/metabolismo , Fosforilação
8.
Dev Cell ; 57(20): 2350-2364.e7, 2022 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-36283390

RESUMO

Ductal carcinoma in situ (DCIS) is a pre-invasive stage of breast cancer. During invasion, the encapsulating DCIS basement membrane (BM) is compromised, and tumor cells invade the surrounding stroma. The mechanisms that regulate functional epithelial BMs in vivo are poorly understood. Myosin-X (MYO10) is a filopodia-inducing protein associated with metastasis and poor clinical outcome in invasive breast cancer (IBC). We identify elevated MYO10 expression in human DCIS and IBC, and this suggests links with disease progression. MYO10 promotes filopodia formation and cell invasion in vitro and cancer-cell dissemination from progressively invasive human DCIS xenografts. However, MYO10-depleted xenografts are more invasive. These lesions exhibit compromised BMs, poorly defined borders, and increased cancer-cell dispersal and EMT-marker-positive cells. In addition, cancer spheroids are dependent on MYO10-filopodia to generate a near-continuous extracellular matrix boundary. Thus, MYO10 is protective in early-stage breast cancer, correlating with tumor-limiting BMs, and pro-invasive at later stages, facilitating cancer-cell dissemination.


Assuntos
Neoplasias da Mama , Carcinoma Ductal de Mama , Carcinoma Intraductal não Infiltrante , Humanos , Feminino , Carcinoma Intraductal não Infiltrante/metabolismo , Carcinoma Intraductal não Infiltrante/patologia , Pseudópodes/metabolismo , Neoplasias da Mama/patologia , Miosinas/metabolismo , Membrana Basal/metabolismo , Carcinoma Ductal de Mama/metabolismo
9.
Nat Mater ; 21(9): 1081-1090, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35817964

RESUMO

How cells sense tissue stiffness to guide cell migration is a fundamental question in development, fibrosis and cancer. Although durotaxis-cell migration towards increasing substrate stiffness-is well established, it remains unknown whether individual cells can migrate towards softer environments. Here, using microfabricated stiffness gradients, we describe the directed migration of U-251MG glioma cells towards less stiff regions. This 'negative durotaxis' does not coincide with changes in canonical mechanosensitive signalling or actomyosin contractility. Instead, as predicted by the motor-clutch-based model, migration occurs towards areas of 'optimal stiffness', where cells can generate maximal traction. In agreement with this model, negative durotaxis is selectively disrupted and even reversed by the partial inhibition of actomyosin contractility. Conversely, positive durotaxis can be switched to negative by lowering the optimal stiffness by the downregulation of talin-a key clutch component. Our results identify the molecular mechanism driving context-dependent positive or negative durotaxis, determined by a cell's contractile and adhesive machinery.


Assuntos
Actomiosina , Fenômenos Biomecânicos , Movimento Celular
10.
iScience ; 25(6): 104459, 2022 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-35677646

RESUMO

MASTL is a mitotic accelerator with an emerging role in breast cancer progression. However, the mechanisms behind its oncogenicity remain largely unknown. Here, we identify a previously unknown role and eminent expression of MASTL in stem cells. MASTL staining from a large breast cancer patient cohort indicated a significant association with ß3 integrin, an established mediator of breast cancer stemness. MASTL silencing reduced OCT4 levels in human pluripotent stem cells and OCT1 in breast cancer cells. Analysis of the cell-surface proteome indicated a strong link between MASTL and the regulation of TGF-ß receptor II (TGFBR2), a key modulator of TGF-ß signaling. Overexpression of wild-type and kinase-dead MASTL in normal mammary epithelial cells elevated TGFBR2 levels. Conversely, MASTL depletion in breast cancer cells attenuated TGFBR2 levels and downstream signaling through SMAD3 and AKT pathways. Taken together, these results indicate that MASTL supports stemness regulators in pluripotent and cancerous stem cells.

11.
Adv Sci (Weinh) ; 9(6): e2104808, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34994086

RESUMO

A key behavior observed during morphogenesis, wound healing, and cancer invasion is that of collective and coordinated cellular motion. Hence, understanding the different aspects of such coordinated migration is fundamental for describing and treating cancer and other pathological defects. In general, individual cells exert forces on their environment in order to move, and collective motion is coordinated by cell-cell adhesion-based forces. However, this notion ignores other mechanisms that encourage cellular movement, such as pressure differences. Here, using model tumors, it is found that increased pressure drove coordinated cellular motion independent of cell-cell adhesion by triggering cell swelling in a soft extracellular matrix (ECM). In the resulting phenotype, a rapid burst-like stream of cervical cancer cells emerged from 3D aggregates embedded in soft collagen matrices (0.5 mg mL-1 ). This fluid-like pushing mechanism, recorded within 8 h after embedding, shows high cell velocities and super-diffusive motion. Because the swelling in this model system critically depends on integrin-mediated cell-ECM adhesions and cellular contractility, the swelling is likely triggered by unsustained mechanotransduction, providing new evidence that pressure-driven effects must be considered to more completely understand the mechanical forces involved in cell and tissue movement as well as invasion.


Assuntos
Movimento Celular/fisiologia , Mecanotransdução Celular/fisiologia , Modelos Biológicos , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/fisiopatologia , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Feminino , Humanos , Fenômenos Mecânicos , Pressão
12.
Blood Adv ; 6(8): 2595-2607, 2022 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-34991155

RESUMO

Platelets form hemostatic plugs to prevent blood loss, and they modulate immunity and inflammation in several ways. A key event during hemostasis is activation of integrin αIIbß3 through direct interactions of the ß3 cytoplasmic tail with talin and kindlin-3. Recently, we showed that human platelets express the adapter molecule Shank-associated RH domain interacting protein (SHARPIN), which can associate directly with the αIIb cytoplasmic tail and separately promote NF-κB pathway activation as a member of the Met-1 linear ubiquitination activation complex (LUBAC). Here we investigated the role of SHARPIN in platelets after crossing Sharpin flox/flox (fl/fl) mice with PF4-Cre or GPIbα-Cre mice to selectively delete SHARPIN in platelets. SHARPIN-null platelets adhered to immobilized fibrinogen through αIIbß3, and they spread more extensively than littermate control platelets in a manner dependent on feedback stimulation by platelet adenosine diphosphate (ADP) (P < .01). SHARPIN-null platelets showed increased colocalization of αIIbß3 with talin as assessed by super-resolution microscopy and increased binding of soluble fibrinogen in response to submaximal concentrations of ADP (P < .05). However, mice with SHARPIN-null platelets showed compromised thrombus growth on collagen and slightly prolonged tail bleeding times. Platelets lacking SHARPIN also showed reduced NF-κB activation and linear ubiquitination of protein substrates upon challenge with classic platelet agonists. Furthermore, the loss of platelet SHARPIN resulted in significant reduction in inflammation in murine models of colitis and peritonitis (P < .01). Thus, SHARPIN plays differential and context-dependent roles in platelets to regulate important inflammatory and integrin adhesive functions of these anucleate cells.


Assuntos
Plaquetas , Complexo Glicoproteico GPIIb-IIIa de Plaquetas , Difosfato de Adenosina , Animais , Plaquetas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Fibrinogênio/metabolismo , Inflamação , Camundongos , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Talina/metabolismo , Ubiquitinação
13.
Mol Oncol ; 16(1): 116-129, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34564954

RESUMO

In breast cancer, the currently approved anti-receptor tyrosine-protein kinase erbB-2 (HER2) therapies do not fully meet the expected clinical goals due to therapy resistance. Identifying alternative HER2-related therapeutic targets could offer a means to overcome these resistance mechanisms. We have previously demonstrated that an endosomal sorting protein, sortilin-related receptor (SorLA), regulates the traffic and signaling of HER2 and HER3, thus promoting resistance to HER2-targeted therapy in breast cancer. This study aims to assess the feasibility of targeting SorLA using a monoclonal antibody. Our results demonstrate that anti-SorLA antibody (SorLA ab) alters the resistance of breast cancer cells to HER2 monoclonal antibody trastuzumab in vitro and in ovo. We found that SorLA ab and trastuzumab combination therapy also inhibits tumor cell proliferation and tumor cell density in a mouse xenograft model of HER2-positive breast cancer. In addition, SorLA ab inhibits the proliferation of breast cancer patient-derived explant three-dimensional cultures. These results provide, for the first time, proof of principle that SorLA is a druggable target in breast cancer.


Assuntos
Antineoplásicos , Neoplasias da Mama , Proteínas Adaptadoras de Transporte Vesicular , Animais , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Camundongos , Receptor ErbB-2/metabolismo , Receptor ErbB-3 , Trastuzumab/farmacologia
14.
Nat Cell Biol ; 23(10): 1073-1084, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34616024

RESUMO

Spatially controlled, cargo-specific endocytosis is essential for development, tissue homeostasis and cancer invasion. Unlike cargo-specific clathrin-mediated endocytosis, the clathrin- and dynamin-independent endocytic pathway (CLIC-GEEC, CG pathway) is considered a bulk internalization route for the fluid phase, glycosylated membrane proteins and lipids. While the core molecular players of CG-endocytosis have been recently defined, evidence of cargo-specific adaptors or selective uptake of proteins for the pathway are lacking. Here we identify the actin-binding protein Swiprosin-1 (Swip1, EFHD2) as a cargo-specific adaptor for CG-endocytosis. Swip1 couples active Rab21-associated integrins with key components of the CG-endocytic machinery-Arf1, IRSp53 and actin-and is critical for integrin endocytosis. Through this function, Swip1 supports integrin-dependent cancer-cell migration and invasion, and is a negative prognostic marker in breast cancer. Our results demonstrate a previously unknown cargo selectivity for the CG pathway and a role for specific adaptors in recruitment into this endocytic route.


Assuntos
Neoplasias da Mama/patologia , Clatrina/metabolismo , Dinaminas/metabolismo , Endocitose , Integrina beta1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Actinas/metabolismo , Transporte Biológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Movimento Celular , Clatrina/genética , Dinaminas/genética , Feminino , Humanos , Integrina beta1/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas rab de Ligação ao GTP/genética
15.
Curr Biol ; 31(22): 4956-4970.e9, 2021 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-34610274

RESUMO

Actin-rich cellular protrusions direct versatile biological processes from cancer cell invasion to dendritic spine development. The stability, morphology, and specific biological functions of these protrusions are regulated by crosstalk between three main signaling axes: integrins, actin regulators, and small guanosine triphosphatases (GTPases). SHANK3 is a multifunctional scaffold protein, interacting with several actin-binding proteins and a well-established autism risk gene. Recently, SHANK3 was demonstrated to sequester integrin-activating small GTPases Rap1 and R-Ras to inhibit integrin activity via its Shank/ProSAP N-terminal (SPN) domain. Here, we demonstrate that, in addition to scaffolding actin regulators and actin-binding proteins, SHANK3 interacts directly with actin through its SPN domain. Molecular simulations and targeted mutagenesis of the SPN-ankyrin repeat region (ARR) interface reveal that actin binding is inhibited by an intramolecular closed conformation of SHANK3, where the adjacent ARR domain covers the actin-binding interface of the SPN domain. Actin and Rap1 compete with each other for binding to SHANK3, and mutation of SHANK3, resulting in reduced actin binding, augments inhibition of Rap1-mediated integrin activity. This dynamic crosstalk has functional implications for cell morphology and integrin activity in cancer cells. In addition, SHANK3-actin interaction regulates dendritic spine morphology in neurons and autism-linked phenotypes in vivo.


Assuntos
Actinas , Fenômenos Biológicos , Actinas/metabolismo , Integrinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas rap1 de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/metabolismo
16.
Cell Rep ; 36(11): 109716, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34525374

RESUMO

Filopodia assemble unique integrin-adhesion complexes to sense the extracellular matrix. However, the mechanisms of integrin regulation in filopodia are poorly defined. Here, we report that active integrins accumulate at the tip of myosin-X (MYO10)-positive filopodia, while inactive integrins are uniformly distributed. We identify talin and MYO10 as the principal integrin activators in filopodia. In addition, deletion of MYO10's FERM domain, or mutation of its ß1-integrin-binding residues, reveals MYO10 as facilitating integrin activation, but not transport, in filopodia. However, MYO10's isolated FERM domain alone cannot activate integrins, potentially because of binding to both integrin tails. Finally, because a chimera construct generated by swapping MYO10-FERM by talin-FERM enables integrin activation in filopodia, our data indicate that an integrin-binding FERM domain coupled to a myosin motor is a core requirement for integrin activation in filopodia. Therefore, we propose a two-step integrin activation model in filopodia: receptor tethering by MYO10 followed by talin-mediated integrin activation.


Assuntos
Integrina beta1/metabolismo , Miosinas/metabolismo , Pseudópodes/metabolismo , Talina/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Adesões Focais/metabolismo , Humanos , Integrina beta1/química , Integrina beta1/genética , Miosinas/antagonistas & inibidores , Miosinas/genética , Ligação Proteica , Domínios Proteicos , Interferência de RNA , RNA Interferente Pequeno/metabolismo
17.
J Cell Biol ; 220(9)2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34264272

RESUMO

Talin is a mechanosensitive adapter protein that couples integrins to the cytoskeleton. Talin rod domain-containing protein 1 (TLNRD1) shares 22% homology with the talin R7R8 rod domains, and is highly conserved throughout vertebrate evolution, although little is known about its function. Here we show that TLNRD1 is an α-helical protein structurally homologous to talin R7R8. Like talin R7R8, TLNRD1 binds F-actin, but because it forms a novel antiparallel dimer, it also bundles F-actin. In addition, it binds the same LD motif-containing proteins, RIAM and KANK, as talin R7R8. In cells, TLNRD1 localizes to actin bundles as well as to filopodia. Increasing TLNRD1 expression enhances filopodia formation and cell migration on 2D substrates, while TLNRD1 down-regulation has the opposite effect. Together, our results suggest that TLNRD1 has retained the diverse interactions of talin R7R8, but has developed distinct functionality as an actin-bundling protein that promotes filopodia assembly.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Chaperonas Moleculares/metabolismo , Pseudópodes/metabolismo , Talina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actinas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Movimento Celular , Clonagem Molecular , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Osteoblastos/citologia , Osteoblastos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Multimerização Proteica , Pseudópodes/ultraestrutura , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Talina/genética
18.
Oncogene ; 40(7): 1300-1317, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33420373

RESUMO

Current evidence indicates that resistance to the tyrosine kinase-type cell surface receptor (HER2)-targeted therapies is frequently associated with HER3 and active signaling via HER2-HER3 dimers, particularly in the context of breast cancer. Thus, understanding the response to HER2-HER3 signaling and the regulation of the dimer is essential to decipher therapy relapse mechanisms. Here, we investigate a bidirectional relationship between HER2-HER3 signaling and a type-1 transmembrane sorting receptor, sortilin-related receptor (SorLA; SORL1). We demonstrate that heregulin-mediated signaling supports SorLA transcription downstream of the mitogen-activated protein kinase pathway. In addition, we demonstrate that SorLA interacts directly with HER3, forming a trimeric complex with HER2 and HER3 to attenuate lysosomal degradation of the dimer in a Ras-related protein Rab4-dependent manner. In line with a role for SorLA in supporting the stability of the HER2 and HER3 receptors, loss of SorLA compromised heregulin-induced cell proliferation and sensitized metastatic anti-HER2 therapy-resistant breast cancer cells to neratinib in cancer spheroids in vitro and in vivo in a zebrafish brain xenograft model.


Assuntos
Neoplasias da Mama/genética , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas de Membrana Transportadoras/genética , Receptor ErbB-2/genética , Receptor ErbB-3/genética , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Xenoenxertos , Humanos , Camundongos , Neuregulina-1/farmacologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Peixe-Zebra , Proteínas rab4 de Ligação ao GTP/genética
19.
Cells ; 9(7)2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32640605

RESUMO

Microtubule-associated serine/threonine kinase-like (MASTL; Greatwall) is a well-characterized kinase, whose catalytic role has been extensively studied in relation to cell-cycle acceleration. Importantly, MASTL has been implicated to play a substantial role in cancer progression and subsequent studies have shown that MASTL is a significant regulator of the cellular actomyosin cytoskeleton. Several kinases have non-catalytic properties, which are essential or even sufficient for their functions. Likewise, MASTL functions have been attributed both to kinase-dependent phosphorylation of downstream substrates, but also to kinase-independent regulation of the actomyosin contractile machinery. In this review, we aimed to highlight the catalytic and non-catalytic roles of MASTL in proliferation, migration, and invasion. Further, we discussed the implications of this dual role for therapeutic design.


Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Neoplasias/enzimologia , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Actinas/metabolismo , Animais , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Humanos , Proteínas Associadas aos Microtúbulos/genética , Neoplasias/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética
20.
J Cell Biol ; 219(6)2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32311005

RESUMO

Microtubule-associated serine/threonine-protein kinase-like (MASTL) is a mitosis-accelerating kinase with emerging roles in cancer progression. However, possible cell cycle-independent mechanisms behind its oncogenicity remain ambiguous. Here, we identify MASTL as an activator of cell contractility and MRTF-A/SRF (myocardin-related transcription factor A/serum response factor) signaling. Depletion of MASTL increased cell spreading while reducing contractile actin stress fibers in normal and breast cancer cells and strongly impairing breast cancer cell motility and invasion. Transcriptome and proteome profiling revealed MASTL-regulated genes implicated in cell movement and actomyosin contraction, including Rho guanine nucleotide exchange factor 2 (GEF-H1, ARHGEF2) and MRTF-A target genes tropomyosin 4.2 (TPM4), vinculin (VCL), and nonmuscle myosin IIB (NM-2B, MYH10). Mechanistically, MASTL associated with MRTF-A and increased its nuclear retention and transcriptional activity. Importantly, MASTL kinase activity was not required for regulation of cell spreading or MRTF-A/SRF transcriptional activity. Taken together, we present a previously unknown kinase-independent role for MASTL as a regulator of cell adhesion, contractility, and MRTF-A/SRF activity.


Assuntos
Citoesqueleto de Actina/enzimologia , Adesão Celular/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais/genética , Transativadores/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Núcleo Celular/metabolismo , Perfilação da Expressão Gênica , Humanos , Integrinas/genética , Integrinas/metabolismo , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Miosina não Muscular Tipo IIB/genética , Miosina não Muscular Tipo IIB/metabolismo , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteoma/metabolismo , RNA Interferente Pequeno , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fibras de Estresse/genética , Fibras de Estresse/metabolismo , Transativadores/genética , Transcriptoma/genética , Tropomiosina/genética , Tropomiosina/metabolismo , Vinculina/genética , Vinculina/metabolismo
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