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1.
Nat Commun ; 12(1): 2058, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33824332

RESUMO

Wnt signaling regulates cell proliferation and cell differentiation as well as migration and polarity during development. However, it is still unclear how the Wnt ligand distribution is precisely controlled to fulfil these functions. Here, we show that the planar cell polarity protein Vangl2 regulates the distribution of Wnt by cytonemes. In zebrafish epiblast cells, mouse intestinal telocytes and human gastric cancer cells, Vangl2 activation generates extremely long cytonemes, which branch and deliver Wnt protein to multiple cells. The Vangl2-activated cytonemes increase Wnt/ß-catenin signaling in the surrounding cells. Concordantly, Vangl2 inhibition causes fewer and shorter cytonemes to be formed and reduces paracrine Wnt/ß-catenin signaling. A mathematical model simulating these Vangl2 functions on cytonemes in zebrafish gastrulation predicts a shift of the signaling gradient, altered tissue patterning, and a loss of tissue domain sharpness. We confirmed these predictions during anteroposterior patterning in the zebrafish neural plate. In summary, we demonstrate that Vangl2 is fundamental to paracrine Wnt/ß-catenin signaling by controlling cytoneme behaviour.


Assuntos
Proteínas de Membrana/metabolismo , Pseudópodes/metabolismo , Via de Sinalização Wnt , Animais , Animais Geneticamente Modificados , Padronização Corporal , Embrião não Mamífero/metabolismo , Ativação Enzimática , Fibroblastos/metabolismo , Gastrulação , Células HEK293 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos Endogâmicos C57BL , Placa Neural/embriologia , Placa Neural/metabolismo , Neurogênese , Comunicação Parácrina , Análise de Sistemas , Telócitos/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo
2.
Nat Cell Biol ; 23(2): 147-159, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33558729

RESUMO

Coordinated polymerization of actin filaments provides force for cell migration, morphogenesis and endocytosis. Capping protein (CP) is a central regulator of actin dynamics in all eukaryotes. It binds to actin filament (F-actin) barbed ends with high affinity and slow dissociation kinetics to prevent filament polymerization and depolymerization. However, in cells, CP displays remarkably rapid dynamics within F-actin networks, but the underlying mechanism remains unclear. Here, we report that the conserved cytoskeletal regulator twinfilin is responsible for CP's rapid dynamics and specific localization in cells. Depletion of twinfilin led to stable association between CP and cellular F-actin arrays, as well as to its retrograde movement throughout leading-edge lamellipodia. These were accompanied by diminished F-actin turnover rates. In vitro single-filament imaging approaches revealed that twinfilin directly promotes dissociation of CP from filament barbed ends, while enabling subsequent filament depolymerization. These results uncover a bipartite mechanism that controls how actin cytoskeleton-mediated forces are generated in cells.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Pseudópodes/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Mutação/genética , Polimerização
3.
Nat Commun ; 12(1): 791, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542237

RESUMO

Cells migrate collectively to form tissues and organs during morphogenesis. Contact inhibition of locomotion (CIL) drives collective migration by inhibiting lamellipodial protrusions at cell-cell contacts and promoting polarization at the leading edge. Here, we report a CIL-related collective cell behavior of myotubes that lack lamellipodial protrusions, but instead use filopodia to move as a cohesive cluster in a formin-dependent manner. We perform genetic, pharmacological and mechanical perturbation analyses to reveal the essential roles of Rac2, Cdc42 and Rho1 in myotube migration. These factors differentially control protrusion dynamics and cell-matrix adhesion formation. We also show that active Rho1 GTPase localizes at retracting free edge filopodia and that Rok-dependent actomyosin contractility does not mediate a contraction of protrusions at cell-cell contacts, but likely plays an important role in the constriction of supracellular actin cables. Based on these findings, we propose that contact-dependent asymmetry of cell-matrix adhesion drives directional movement, whereas contractile actin cables contribute to the integrity of the migrating cell cluster.


Assuntos
Movimento Celular/fisiologia , Morfogênese/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Pseudópodes/metabolismo , Citoesqueleto de Actina/metabolismo , Actomiosina/metabolismo , Animais , Caderinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Proteínas de Ligação ao GTP/metabolismo , Microscopia Intravital , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
4.
Nat Cell Biol ; 23(2): 198-208, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33526902

RESUMO

Cells achieve highly efficient and accurate communication through cellular projections such as neurites and filopodia, yet there is a lack of genetically encoded tools that can selectively manipulate their composition and dynamics. Here, we present a versatile optogenetic toolbox of artificial multi-headed myosin motors that can move bidirectionally within long cellular extensions and allow for the selective transport of GFP-tagged cargo with light. Utilizing these engineered motors, we could transport bulky transmembrane receptors and organelles as well as actin remodellers to control the dynamics of both filopodia and neurites. Using an optimized in vivo imaging scheme, we further demonstrate that, upon limb amputation in axolotls, a complex array of filopodial extensions is formed. We selectively modulated these filopodial extensions and showed that they re-establish a Sonic Hedgehog signalling gradient during regeneration. Considering the ubiquitous existence of actin-based extensions, this toolbox shows the potential to manipulate cellular communication with unprecedented accuracy.


Assuntos
Comunicação Celular , Miosinas/metabolismo , Optogenética , Engenharia de Proteínas , Citoesqueleto de Actina/metabolismo , Ambystoma mexicanum/fisiologia , Animais , Transporte Biológico , Linhagem Celular , Sobrevivência Celular/efeitos da radiação , Extremidades/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Proteínas Hedgehog/metabolismo , Cinética , Luz , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Neuritos/metabolismo , Pseudópodes/metabolismo , Regeneração/fisiologia , Transdução de Sinais , Vesículas Transportadoras/metabolismo
5.
Cell Prolif ; 54(3): e12994, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33458919

RESUMO

OBJECTIVES: Cancer cell migration to secondary organs remains an essential cause of death among breast cancer (BrCa) patients. Cell motility mainly relies on actin dynamics. Our previous reports verified that dishevelled-associated activator of morphogenesis 1 (Daam1) regulates invadopodia extension and BrCa cell motility. However, how Daam1 is involved in actin filament assembly and promotes pseudopodia formation in BrCa cells remains unclear. MATERIALS AND METHODS: One hundred human BrCa samples were collected at Women's Hospital of Nanjing Medical University. Immunohistochemistry (IHC) was used to examine Daam1 and Fascin expression. Wound healing and Boyden chamber assays were used to explore cell migration and pseudopodia extension of BrCa cells. Co-IP/pull down and Western blotting were performed to study the physical interaction between Daam1 and Fascin. Immunofluorescence assays were performed to observe whether Daam1 and Fascin were colocalized and mediated actin filament assembly. RESULTS: Fascin was upregulated in BrCa tissues compared with that in paracarcinoma tissues. The downregulation of Fascin caused a decline in pseudopodia formation and cell motility. Moreover, we found that Daam1 interacted with Fascin via formin homology (FH) domains, especially the FH2 domain. Immunofluorescence assays showed that Daam1 and Fascin partially colocalized to actin filaments, and the knockdown of Daam1 or Fascin failed to colocalize to short and curved actin filaments. CONCLUSIONS: Daam1 specifically binds to Fascin via FH domains and cooperatively facilitates pseudopodia formation and cell migration by promoting actin filament assembly in BrCa.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/patologia , Movimento Celular/fisiologia , Forminas/metabolismo , Pseudópodes/patologia , Citoesqueleto de Actina/metabolismo , Neoplasias da Mama/metabolismo , Forminas/farmacologia , Humanos , Pseudópodes/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
6.
Biochem Biophys Res Commun ; 535: 54-59, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33341673

RESUMO

Filopodia are slender actin-rich plasma membrane protrusions that function to drive cell migration and invasion. Despite the observation of defective filopodia formation in many malignant tumors, the regulation mechanism remained unknown to date. In the present study, for the first time, we demonstrate that RAB5A, a Rab GTPase family protein, is a potent regulator of filopodia formation in pancreatic cancer cells. High expression of RAB5A was associated with filopodia formation and migration in pancreatic cancer cells. Overexpression of RAB5A promoted filopodia formation and migration in CF Pac-1 cells. In contrast, down-regulation of RAB5A expression in SW1990 cells with a high endogenous RAB5A expression level impeded the formation of filopodia. Further analysis indicated that RAB5A was required for cdc42 activation in CF Pac-1 and SW1990 cells. Moreover, to investigate the underlying mechanism by which the activation of cdc42 mediates RAB5A-induced filopodia formation, the active state of ß1-integrin was examined in cells with different expression levels of RAB5A. We observed that RAB5A regulated the accumulation of the active ß1-integrin. We demonstrated that down-regulation of the expression of ß1-integrin strongly suppressed filopodia formation and cdc42 activation mediated by RAB5A. These results indicate the important role of RAB5A in the regulation of filopodia formation in pancreatic cancer cells, which is dependent on the activation of cdc42 and ß1-integrin.


Assuntos
Integrina beta1/metabolismo , Neoplasias Pancreáticas/metabolismo , Pseudópodes/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos
7.
Dev Biol ; 469: 125-134, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33096063

RESUMO

Collective cell migration is a process where cohorts of cells exhibit coordinated migratory behavior. During individual and collective cellular migration, cells must extend protrusions to interact with the extracellular environment, sense chemotactic cues, and act as points of attachment. The mechanisms and regulators of protrusive behavior have been widely studied in individually migrating cells; however, how this behavior is regulated throughout collectives is not well understood. To address this, we used the zebrafish posterior lateral line primordium (pLLP) as a model. The pLLP is a cluster of ~150 â€‹cells that migrates along the zebrafish trunk, depositing groups of cells that will become sensory organs. To define protrusive behavior, we performed mosaic analysis to sparsely label pLLP cells with a transgene marking filamentous actin. This approach revealed an abundance of brush-like protrusions throughout the pLLP that orient in the direction of migration. Formation of these protrusions depends on the Arp2/3 complex, a regulator of dendritic actin. This argues that these brush-like protrusions are an in vivo example of lamellipodia. Mosaic analysis demonstrated that these lamellipodia-like protrusions are located in a close proximity to the overlying skin. Immunostaining revealed an abundance of focal adhesion complexes surrounding the pLLP. Disruption of these complexes specifically in pLLP cells led to impaired pLLP migration. Finally, we show that Erk signaling, a known regulator of focal adhesions, is required for proper formation of lamellipodia-like protrusions and pLLP migration. Altogether, our results suggest a model where the coordinated dynamics of lamellipodia-like protrusions, making contact with either the overlying skin or the extracellular matrix through focal adhesions, promotes migration of pLLP cells.


Assuntos
Movimento Celular , Adesões Focais/fisiologia , Pseudópodes/fisiologia , Peixe-Zebra/embriologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/análise , Animais , Sistema de Sinalização das MAP Quinases , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Pseudópodes/enzimologia , Pseudópodes/metabolismo , Peixe-Zebra/fisiologia
8.
Methods Mol Biol ; 2217: 85-113, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33215379

RESUMO

Cell migration is a complex biophysical process which involves the coordination of molecular assemblies including integrin-dependent adhesions, signaling networks and force-generating cytoskeletal structures incorporating both actin polymerization and myosin activity. During the last decades, proteomic studies have generated impressive protein-protein interaction maps, although the subcellular location, duration, strength, sequence, and nature of these interactions are still concealed. In this chapter we describe how recent developments in superresolution microscopy (SRM) and single-protein tracking (SPT) start to unravel protein interactions and actions in subcellular molecular assemblies driving cell migration.


Assuntos
Movimento Celular , Integrinas/metabolismo , Microscopia/métodos , Optogenética/métodos , Mapeamento de Interação de Proteínas/métodos , Imagem Individual de Molécula/métodos , Actinas/genética , Actinas/metabolismo , Animais , Adesão Celular , Linhagem Celular Transformada , Criptocromos/genética , Criptocromos/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Expressão Gênica , Integrinas/genética , Camundongos , Microscopia/instrumentação , Miosinas/genética , Miosinas/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Ligação Proteica , Pseudópodes/metabolismo , Pseudópodes/ultraestrutura , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T/genética , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
9.
Nat Commun ; 11(1): 6437, 2020 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-33353942

RESUMO

The actin-related protein (Arp)2/3 complex nucleates branched actin filament networks pivotal for cell migration, endocytosis and pathogen infection. Its activation is tightly regulated and involves complex structural rearrangements and actin filament binding, which are yet to be understood. Here, we report a 9.0 Å resolution structure of the actin filament Arp2/3 complex branch junction in cells using cryo-electron tomography and subtomogram averaging. This allows us to generate an accurate model of the active Arp2/3 complex in the branch junction and its interaction with actin filaments. Notably, our model reveals a previously undescribed set of interactions of the Arp2/3 complex with the mother filament, significantly different to the previous branch junction model. Our structure also indicates a central role for the ArpC3 subunit in stabilizing the active conformation.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/química , Complexo 2-3 de Proteínas Relacionadas à Actina/ultraestrutura , Tomografia com Microscopia Eletrônica , Fibroblastos/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Camundongos , Modelos Moleculares , Células NIH 3T3 , Conformação Proteica , Pseudópodes/metabolismo
10.
Nat Commun ; 11(1): 5778, 2020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-33188196

RESUMO

Breakdown of vascular barriers is a major complication of inflammatory diseases. Anucleate platelets form blood-clots during thrombosis, but also play a crucial role in inflammation. While spatio-temporal dynamics of clot formation are well characterized, the cell-biological mechanisms of platelet recruitment to inflammatory micro-environments remain incompletely understood. Here we identify Arp2/3-dependent lamellipodia formation as a prominent morphological feature of immune-responsive platelets. Platelets use lamellipodia to scan for fibrin(ogen) deposited on the inflamed vasculature and to directionally spread, to polarize and to govern haptotactic migration along gradients of the adhesive ligand. Platelet-specific abrogation of Arp2/3 interferes with haptotactic repositioning of platelets to microlesions, thus impairing vascular sealing and provoking inflammatory microbleeding. During infection, haptotaxis promotes capture of bacteria and prevents hematogenic dissemination, rendering platelets gate-keepers of the inflamed microvasculature. Consequently, these findings identify haptotaxis as a key effector function of immune-responsive platelets.


Assuntos
Plaquetas/patologia , Vasos Sanguíneos/patologia , Quimiotaxia , Inflamação/patologia , Pneumonia/sangue , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Adulto , Animais , Movimento Celular , Microambiente Celular , Modelos Animais de Doenças , Fibrinogênio/metabolismo , Humanos , Lipopolissacarídeos , Lesão Pulmonar/microbiologia , Lesão Pulmonar/patologia , Staphylococcus aureus Resistente à Meticilina/fisiologia , Camundongos Endogâmicos C57BL , Microvasos/patologia , Pneumonia/microbiologia , Pseudópodes/metabolismo
11.
Nat Commun ; 11(1): 5397, 2020 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-33106478

RESUMO

The migration of many cell types relies on the formation of actomyosin-dependent protrusions called blebs, but the mechanisms responsible for focusing this kind of protrusive activity to the cell front are largely unknown. Here, we employ zebrafish primordial germ cells (PGCs) as a model to study the role of cell-cell adhesion in bleb-driven single-cell migration in vivo. Utilizing a range of genetic, reverse genetic and mathematical tools, we define a previously unknown role for E-cadherin in confining bleb-type protrusions to the leading edge of the cell. We show that E-cadherin-mediated frictional forces impede the backwards flow of actomyosin-rich structures that define the domain where protrusions are preferentially generated. In this way, E-cadherin confines the bleb-forming region to a restricted area at the cell front and reinforces the front-rear axis of migrating cells. Accordingly, when E-cadherin activity is reduced, the bleb-forming area expands, thus compromising the directional persistence of the cells.


Assuntos
Actinas/metabolismo , Caderinas/metabolismo , Movimento Celular , Células Germinativas/citologia , Pseudópodes/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Actinas/genética , Actomiosina/genética , Actomiosina/metabolismo , Animais , Caderinas/genética , Feminino , Células Germinativas/metabolismo , Masculino , Pseudópodes/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
12.
Nat Commun ; 11(1): 4818, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968060

RESUMO

Migrating cells move across diverse assemblies of extracellular matrix (ECM) that can be separated by micron-scale gaps. For membranes to protrude and reattach across a gap, actin filaments, which are relatively weak as single filaments, must polymerize outward from adhesion sites to push membranes towards distant sites of new adhesion. Here, using micropatterned ECMs, we identify T-Plastin, one of the most ancient actin bundling proteins, as an actin stabilizer that promotes membrane protrusions and enables bridging of ECM gaps. We show that T-Plastin widens and lengthens protrusions and is specifically enriched in active protrusions where F-actin is devoid of non-muscle myosin II activity. Together, our study uncovers critical roles of the actin bundler T-Plastin to promote protrusions and migration when adhesion is spatially-gapped.


Assuntos
Movimento Celular/fisiologia , Extensões da Superfície Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Sistemas CRISPR-Cas , Adesão Celular , Linhagem Celular , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Técnicas de Inativação de Genes , Humanos , Cinética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/ultraestrutura , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/ultraestrutura , Miosinas/metabolismo , Pseudópodes/metabolismo , Receptor EphB2
13.
PLoS Pathog ; 16(9): e1008878, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32946535

RESUMO

As an obligate intracellular pathogen, host cell invasion is paramount to Chlamydia trachomatis proliferation. While the mechanistic underpinnings of this essential process remain ill-defined, it is predicted to involve delivery of prepackaged effector proteins into the host cell that trigger plasma membrane remodeling and cytoskeletal reorganization. The secreted effector proteins TmeA and TarP, have risen to prominence as putative key regulators of cellular invasion and bacterial pathogenesis. Although several studies have begun to unravel molecular details underlying the putative function of TarP, the physiological function of TmeA during host cell invasion is unknown. Here, we show that TmeA employs molecular mimicry to bind to the GTPase binding domain of N-WASP, which results in recruitment of the actin branching ARP2/3 complex to the site of chlamydial entry. Electron microscopy revealed that TmeA mutants are deficient in filopodia capture, suggesting that TmeA/N-WASP interactions ultimately modulate host cell plasma membrane remodeling events necessary for chlamydial entry. Importantly, while both TmeA and TarP are necessary for effective host cell invasion, we show that these effectors target distinct pathways that ultimately converge on activation of the ARP2/3 complex. In line with this observation, we show that a double mutant suffers from a severe entry defect nearly identical to that observed when ARP3 is chemically inhibited or knocked down. Collectively, our study highlights both TmeA and TarP as essential regulators of chlamydial invasion that modulate the ARP2/3 complex through distinct signaling platforms, resulting in plasma membrane remodeling events that are essential for pathogen uptake.


Assuntos
Proteínas de Bactérias , Membrana Celular/metabolismo , Chlamydia trachomatis , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/genética , Membrana Celular/patologia , Chlamydia trachomatis/genética , Chlamydia trachomatis/metabolismo , Chlamydia trachomatis/patogenicidade , Células HeLa , Humanos , Mutação , Domínios Proteicos , Pseudópodes/genética , Pseudópodes/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética
14.
PLoS Biol ; 18(8): e3000774, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32745097

RESUMO

The Scar/WAVE complex is the principal catalyst of pseudopod and lamellipod formation. Here we show that Scar/WAVE's proline-rich domain is polyphosphorylated after the complex is activated. Blocking Scar/WAVE activation stops phosphorylation in both Dictyostelium and mammalian cells, implying that phosphorylation modulates pseudopods after they have been formed, rather than controlling whether they are initiated. Unexpectedly, phosphorylation is not promoted by chemotactic signaling but is greatly stimulated by cell:substrate adhesion and diminished when cells deadhere. Phosphorylation-deficient or phosphomimetic Scar/WAVE mutants are both normally functional and rescue the phenotype of knockout cells, demonstrating that phosphorylation is dispensable for activation and actin regulation. However, pseudopods and patches of phosphorylation-deficient Scar/WAVE last substantially longer in mutants, altering the dynamics and size of pseudopods and lamellipods and thus changing migration speed. Scar/WAVE phosphorylation does not require ERK2 in Dictyostelium or mammalian cells. However, the MAPKKK homologue SepA contributes substantially-sepA mutants have less steady-state phosphorylation, which does not increase in response to adhesion. The mutants also behave similarly to cells expressing phosphorylation-deficient Scar, with longer-lived pseudopods and patches of Scar recruitment. We conclude that pseudopod engagement with substratum is more important than extracellular signals at regulating Scar/WAVE's activity and that phosphorylation acts as a pseudopod timer by promoting Scar/WAVE turnover.


Assuntos
Dictyostelium/genética , MAP Quinase Quinase Quinase 3/genética , Proteínas de Protozoários/genética , Pseudópodes/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Animais , Sistemas CRISPR-Cas , Adesão Celular , Linhagem Celular Tumoral , Quimiotaxia/genética , Dictyostelium/metabolismo , Dictyostelium/ultraestrutura , Edição de Genes/métodos , Regulação da Expressão Gênica , MAP Quinase Quinase Quinase 3/metabolismo , Melanócitos/metabolismo , Melanócitos/ultraestrutura , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Mutação , Células NIH 3T3 , Fenótipo , Fosforilação , Ploidias , Proteínas de Protozoários/metabolismo , Pseudópodes/genética , Pseudópodes/ultraestrutura , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo
15.
PLoS Pathog ; 16(5): e1008489, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32365140

RESUMO

Remodelling of the actin cytoskeleton in response to external stimuli is obligatory for many cellular processes in the amoebic cell. A rapid and local rearrangement of the actin cytoskeleton is required for the development of the cellular protrusions during phagocytosis, trogocytosis, migration, and invasion. Here, we demonstrated that EhC2B, a C2 domain-containing protein, is an actin modulator. EhC2B was first identified as an effector of EhRab21 from E. histolytica. In vitro interaction studies including GST pull-down, fluorescence-based assay and ITC also corroborated with our observation. In the amoebic trophozoites, EhC2B accumulates at the pseudopods and the tips of phagocytic cups. FRAP based studies confirmed the recruitment and dynamics of EhC2B at the phagocytic cup. Moreover, we have shown the role of EhC2B in erythrophagocytosis. It is well known that calcium-dependent signal transduction is essential for the cytoskeletal dynamics during phagocytosis in the amoebic parasite. Using liposome pelleting assay, we demonstrated that EhC2B preferentially binds to the phosphatidylserine in the presence of calcium. The EhC2B mutants defective in calcium or lipid-binding failed to localise beneath the plasma membrane. The cells overexpressing these mutants have also shown a significant reduction in erythrophagocytosis. The role of EhC2B in erythrophagocytosis and pseudopod formation was also validated by siRNA-based gene knockdown approach. Finally, with the help of in vitro nucleation assay using fluorescence spectroscopy and total internal reflection fluorescence microscopy, we have established that EhC2B is an actin nucleator. Collectively, based on the results from the study, we propose that EhC2B acts like a molecular bridge which promotes membrane deformation via its actin nucleation activity during the progression of the phagocytic cup in a calcium-dependent manner.


Assuntos
Actinas/metabolismo , Citofagocitose , Entamoeba histolytica/metabolismo , Eritrócitos , Proteínas de Protozoários/metabolismo , Pseudópodes/metabolismo , Actinas/genética , Domínios C2 , Entamoeba histolytica/genética , Humanos , Proteínas de Protozoários/genética , Pseudópodes/genética
16.
Adv Exp Med Biol ; 1246: 129-151, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32399829

RESUMO

Although the cytosolic Ca2+ signalling event in phagocytosis is well established, and the mechanism for generating such signals also understood, the target for the Ca2+ signal and how this relates to the phagocytic outcome is less clear. In this chapter, we present the evidence for a role of the Ca2+ activated protease, calpain, in phagocytosis. The abundant evidence for Ca2+ changes and calpain activation during cell shape changes is extended to include the specific cell shape change which accompanies phagocytosis. The discussion therefore includes a brief description of the domain structure of calpain and their functions. Also the mechanism by which calpain activation is limited at the cell periphery subdomains, and how this would allow phagocytic pseudopodia to form locally.


Assuntos
Cálcio/metabolismo , Calpaína/metabolismo , Fagocitose , Animais , Forma Celular , Citosol/metabolismo , Ativação Enzimática , Humanos , Pseudópodes/metabolismo
17.
Nat Commun ; 11(1): 1921, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32317641

RESUMO

Actomyosin supracellular networks emerge during development and tissue repair. These cytoskeletal structures are able to generate large scale forces that can extensively remodel epithelia driving tissue buckling, closure and extension. How supracellular networks emerge, are controlled and mechanically work still remain elusive. During Drosophila oogenesis, the egg chamber elongates along the anterior-posterior axis. Here we show that a dorsal-ventral polarized supracellular F-actin network, running around the egg chamber on the basal side of follicle cells, emerges from polarized intercellular filopodia that radiate from basal stress fibers and extend penetrating neighboring cell cortexes. Filopodia can be mechanosensitive and function as cell-cell anchoring sites. The small GTPase Cdc42 governs the formation and distribution of intercellular filopodia and stress fibers in follicle cells. Finally, our study shows that a Cdc42-dependent supracellular cytoskeletal network provides a scaffold integrating local oscillatory actomyosin contractions at the tissue scale to drive global polarized forces and tissue elongation.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Oogênese , Actinas/metabolismo , Actomiosina/metabolismo , Animais , Anisotropia , Adesão Celular , Polaridade Celular , Citoesqueleto/metabolismo , Epitélio/metabolismo , Feminino , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Miosina Tipo II/metabolismo , Optogenética , Pseudópodes/metabolismo , Interferência de RNA
18.
Sci Rep ; 10(1): 6679, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32317647

RESUMO

Substrate topographic patterning is a powerful tool that can be used to manipulate cell shape and orientation. To gain a better understanding of the relationship between surface topography and keratocyte behavior, surface patterns consisting of linear aligned or orthogonally aligned microchannels were used. Photolithography and polymer molding techniques were used to fabricate micropatterns on the surface of polydimethylsiloxane (PDMS). Cells on linear aligned substrates were elongated and aligned in the channel direction, while cells on orthogonal substrates had a more spread morphology. Both linear and orthogonal topographies induced chromatin condensation and resulted in higher expressions of keratocyte specific genes and sulfated glycosaminoglycans (sGAG), compared with non-patterned substrates. However, despite differences in cell morphology and focal adhesions, many genes associated with a native keratocyte phenotype, such as keratocan and ALDH3A1, remain unchanged on the different patterned substrates. This information could be used to optimize substrates for keratocyte culture and to develop scaffolds for corneal regeneration.


Assuntos
Ceratócitos da Córnea/citologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Cromatina/metabolismo , Ceratócitos da Córnea/efeitos dos fármacos , Ceratócitos da Córnea/metabolismo , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Dimetilpolisiloxanos/farmacologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Adesões Focais/efeitos dos fármacos , Adesões Focais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosaminoglicanos/metabolismo , Humanos , Fenótipo , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Pseudópodes/ultraestrutura
19.
Sci Rep ; 10(1): 6580, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313105

RESUMO

Tumor invasion requires efficient cell migration, which is achieved by the generation of persistent and polarized lamellipodia. The generation of lamellipodia is supported by actin dynamics at the leading edge where a complex of proteins known as the WAVE regulatory complex (WRC) promotes the required assembly of actin filaments to push the front of the cell ahead. By using an U2OS osteosarcoma cell line with high metastatic potential, proven by a xenotransplant in zebrafish larvae, we have studied the role of the plasma membrane Ca2+ channel ORAI1 in this process. We have found that epidermal growth factor (EGF) triggered an enrichment of ORAI1 at the leading edge, where colocalized with cortactin (CTTN) and other members of the WRC, such as CYFIP1 and ARP2/3. ORAI1-CTTN co-precipitation was sensitive to the inhibition of the small GTPase RAC1, an upstream activator of the WRC. RAC1 potentiated ORAI1 translocation to the leading edge, increasing the availability of surface ORAI1 and increasing the plasma membrane ruffling. The role of ORAI1 at the leading edge was studied in genetically engineered U2OS cells lacking ORAI1 expression that helped us to prove the key role of this Ca2+ channel on lamellipodia formation, lamellipodial persistence, and cell directness, which are required for tumor cell invasiveness in vivo.


Assuntos
Cortactina/genética , Proteína ORAI1/genética , Osteossarcoma/genética , Pseudópodes/genética , Proteínas rac1 de Ligação ao GTP/genética , Citoesqueleto de Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular/genética , Humanos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Pseudópodes/metabolismo
20.
Artigo em Inglês | MEDLINE | ID: mdl-32112978

RESUMO

Ceramide kinase (CerK) phosphorylates ceramide to ceramide-1-phosphate (C1P), a bioactive sphingolipid. Since the mechanisms responsible for regulating the proliferation and migration/metastasis of cancer cells by the CerK/C1P pathway remain unclear, we conducted the present study. The knockdown of CerK in A549 lung and MCF-7 breast cancer cells (shCerK cells) increased the formation of lamellipodia, which are membrane protrusions coupled with cell migration. Mouse embryonic fibroblasts prepared from CerK-null mice also showed an enhanced formation of lamellipodia. The overexpression of CerK inhibited lamellipodium formation in A549 cells. The knockdown of CerK increased the number of cells having lamellipodia with Rac1 and the levels of active Rac1-GTP form, whereas the overexpression of CerK decreased them. CerK was located in lamellipodia after the epidermal growth factor treatment, indicating that CerK functioned there to inhibit Rac1. The migration of A549 cells was negatively regulated by CerK. An intravenous injection of A549-shCerK cells into nude mice resulted in markedly stronger metastatic responses in the lungs than an injection of control cells. The in vitro growth of A549 cells and in vivo expansion after the injection into mouse flanks were not affected by the CerK knockdown. These results suggest that the activation of CerK/C1P pathway has inhibitory roles on lamellipodium formation, migration, and metastasis of A549 lung cancer cells.


Assuntos
Neoplasias Pulmonares/patologia , Metástase Neoplásica/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Pseudópodes/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Células A549 , Animais , Movimento Celular , Ceramidas/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Células MCF-7 , Masculino , Camundongos , Invasividade Neoplásica/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto
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