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1.
Cell Prolif ; 53(1): e12670, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31568631

RESUMO

OBJECTIVES: To investigate the heterogeneous feature of actin filaments (ACFs) associated with the cellular membrane in HeLa and HCT-116 cells at the nanoscale level. MATERIALS AND METHODS: Fluorescence microscopy coupled with atomic force microscopy (AFM) was used to identify and characterize ACFs of cells. The distribution of ACFs was detected by Fluor-488-phalloidin-labelled actin. The morphology of the ACFs was probed by AFM images. The spatial correlation of the microvilli and ACFs was explored with different forces of AFM loading on cells. RESULTS: Intricate but ordered structures of the actin cytoskeletons associated with cellular membrane were characterized and revealed. Two different layers of ACFs with distinct structural organizations were directly observed in HCT-116 and HeLa cells. Bundle-shaped ACFs protruding the cellular membrane forming the microvilli, and the network ACFs underneath the cellular membrane were resolved with high resolution under near-physiological conditions. Approximately 14 nm lateral resolution was achieved when imaging single ACF beneath the cellular membrane. On the basis of the observed spatial distribution of the ultrastructure of the ACF organization, a model for this organization of ACFs was proposed. CONCLUSIONS: We revealed the two layers of the ACF organization in Hela and HCT-116 cells. The resolved heterogeneous structures at the nanoscale level provide a spatial view of the ACFs, which would contribute to the understanding of the essential biological functions of the actin cytoskeleton.


Assuntos
Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Microscopia de Força Atômica , Coloração e Rotulagem , Células HCT116 , Células HeLa , Humanos
2.
Nat Cell Biol ; 21(11): 1357-1369, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31659275

RESUMO

αMß2 integrin (complement receptor 3) is a major receptor for phagocytosis in macrophages. In other contexts, integrins' activities and functions are mechanically linked to actin dynamics through focal adhesions. We asked whether mechanical coupling of αMß2 integrin to the actin cytoskeleton mediates phagocytosis. We found that particle internalization was driven by formation of Arp2/3 and formin-dependent actin protrusions that wrapped around the particle. Focal complex-like adhesions formed in the phagocytic cup that contained ß2 integrins, focal adhesion proteins and tyrosine kinases. Perturbation of talin and Syk demonstrated that a talin-dependent link between integrin and actin and Syk-mediated recruitment of vinculin enable force transmission to target particles and promote phagocytosis. Altering target mechanical properties demonstrated more efficient phagocytosis of stiffer targets. Thus, macrophages use tyrosine kinase signalling to build a mechanosensitive, talin- and vinculin-mediated, focal adhesion-like molecular clutch, which couples integrins to cytoskeletal forces to drive particle engulfment.


Assuntos
Macrófagos/imunologia , Mecanotransdução Celular , Fagocitose/imunologia , Quinase Syk/genética , Talina/genética , Vinculina/genética , Citoesqueleto de Actina/imunologia , Citoesqueleto de Actina/ultraestrutura , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/imunologia , Actinas/genética , Actinas/imunologia , Animais , Matriz Extracelular/imunologia , Matriz Extracelular/metabolismo , Adesões Focais/imunologia , Adesões Focais/ultraestrutura , /imunologia , Regulação da Expressão Gênica , Humanos , Antígeno de Macrófago 1/genética , Antígeno de Macrófago 1/imunologia , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Microesferas , Fagossomos/imunologia , Fagossomos/ultraestrutura , Poliestirenos , Cultura Primária de Células , Células RAW 264.7 , Quinase Syk/imunologia , Células THP-1 , Talina/imunologia , Vinculina/imunologia
3.
PLoS Biol ; 17(10): e3000500, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31652255

RESUMO

Clathrin-mediated endocytosis involves the sequential assembly of more than 60 proteins at the plasma membrane. An important fraction of these proteins regulates the assembly of an actin-related protein 2/3 (Arp2/3)-branched actin network, which is essential to generate the force during membrane invagination. We performed, on wild-type (WT) yeast and mutant strains lacking putative actin crosslinkers, a side-by-side comparison of in vivo endocytic phenotypes and in vitro rigidity measurements of reconstituted actin patches. We found a clear correlation between softer actin networks and a decreased efficiency of endocytosis. Our observations support a chain-of-consequences model in which loss of actin crosslinking softens Arp2/3-branched actin networks, directly limiting the transmission of the force. Additionally, the lifetime of failed endocytic patches increases, leading to a larger number of patches and a reduced pool of polymerizable actin, which slows down actin assembly and further impairs endocytosis.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/genética , Endocitose/genética , Regulação Fúngica da Expressão Gênica , Mecanotransdução Celular , Saccharomyces cerevisiae/genética , Citoesqueleto de Actina/ultraestrutura , Complexo 2-3 de Proteínas Relacionadas à Actina/deficiência , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Actinas/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/genética , Fenômenos Biomecânicos , Clatrina/deficiência , Clatrina/genética , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética
4.
Zygote ; 27(4): 241-249, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31397235

RESUMO

During sea urchins fertilization, the activating spermatozoon triggers a series of physiological changes that transforms the quiescent egg into a dynamic zygote. It has been suggested that several of these egg activation events, e.g. sperm-induced plasma membrane depolarization and the Ca2+-linked cortical reaction, play additional roles to prevent the entry of supernumerary spermatozoa. In particular, the abrupt shift in egg membrane potential at fertilization, which is sustained by a Na+ influx, has been considered as a fast mechanism to block polyspermy. To test the relevance of the Na+-mediated fast electrical block to polyspermy, we fertilized sea urchin eggs in artificial seawater with a low concentration of Na+; nearly all the eggs were still monospermic, as judged by the number of Hoechst 33422-stained sperm. When fertilized in normal seawater, eggs that were pre-incubated in the low Na+ medium exhibited impaired elevation of the fertilization envelope. Nevertheless, these eggs manifested entry of a single spermatozoon, suggesting that the fertilization envelope was not the primary determinant of the block to polyspermy. Furthermore, we showed that the abnormal cleavage patterns displayed by eggs pre-incubated in low Na+, which were often considered a hallmark of polyspermy, were due to the alterations in the cortical actin filaments dynamics following fertilization, and not to the formation of multipolar spindles associated with supernumerary sperm centrosomes. Hence, our results suggested that Paracentrotus lividus eggs do not utilize Na+ to rapidly prevent additional spermatozoa from entering the egg, at variance with the hypothesis of an electrical fast block to polyspermy.


Assuntos
Fertilização/fisiologia , Óvulo/fisiologia , Paracentrotus/fisiologia , Sódio/metabolismo , Interações Espermatozoide-Óvulo/fisiologia , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Feminino , Masculino , Potenciais da Membrana/fisiologia , Microscopia Confocal , Microscopia Eletrônica de Varredura , Óvulo/metabolismo , Óvulo/ultraestrutura , Espermatozoides/fisiologia , Zigoto/fisiologia
5.
Acta Trop ; 199: 105122, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31398313

RESUMO

Echinococcus granulosus is a cestode parasite whose cytoskeleton plasticity allows it to enter and develop inside its hosts, completing thus its life cycle. We focused our attention on F-actin organization and distribution in E. granulosus protoscoleces (PSC) in order to contribute to the knowledge of the parasite cytoskeleton. In particular, we addressed some aspects of F-actin rearrangements in PSC at different stages of the evagination/invagination process. The use of light microscopy allowed us to identify different PSC structures and phalloidin staining displayed a parasite's highly organized F-actin cytoskeleton. Suckers exhibit an important musculature composed of a set of radial fibers. At the rostellum, the F-actin filaments are arranged in a bulbar shape with perforations that appear to be the attachment places for the hooks. Also, "circular" structures of F-actin were identified, which remind the flame cells. Furthermore, parasite F-actin filaments, unevenly distributed, seem to have remained substantially unchanged during the evagination/invagination process. Finally, we showed that the scolex of an evaginated E. granulosus PSC reinvaginates in vitro without any treatment.


Assuntos
Citoesqueleto de Actina/fisiologia , Actinas/análise , Echinococcus granulosus/anatomia & histologia , Echinococcus granulosus/fisiologia , Matadouros , Citoesqueleto de Actina/ultraestrutura , Animais , Bovinos , Echinococcus granulosus/crescimento & desenvolvimento , Estágios do Ciclo de Vida , Pulmão/parasitologia , Microscopia de Fluorescência , Faloidina , Coloração e Rotulagem , Uruguai
6.
Anat Histol Embryol ; 48(5): 505-507, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31389074

RESUMO

The distribution of actin filaments was examined in the seminiferous epithelium of the Habu (Trimeresurus flavoviridis; snake), by transmission electron microscopy and fluorescence histochemistry. By transmission electron microscopy, actin filaments were clearly found only at the site between Sertoli cell and spermatid without a lattice-like structure. Fluorescence histochemistry showed a weak labelling of actin filaments in the seminiferous epithelium, whereas these findings seem to be common among reptiles, they are different from those in mammals. Additionally, the bundles of actin filaments adjacent to the plasma membrane of Sertoli cells, appeared in other reptiles, were not observed in the Habu.


Assuntos
Citoesqueleto de Actina/ultraestrutura , Epitélio Seminífero/citologia , Animais , Masculino , Epitélio Seminífero/ultraestrutura , Células de Sertoli/citologia , Espermátides/ultraestrutura , Testículo/citologia , Trimeresurus
7.
Int J Mol Sci ; 20(14)2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31340471

RESUMO

The side effects induced by nanoparticle exposure at a cellular level are one of the priority research topics due to the steady increase in the use of nanoparticles (NPs). Recently, the focus on cellular morphology and mechanical behavior is gaining relevance in order to fully understand the cytotoxic mechanisms. In this regard, we have evaluated the morphomechanical alteration in human breast adenocarcinoma cell line (MCF-7) exposed to TiO2NPs at two different concentrations (25 and 50 µg/mL) and two time points (24 and 48 h). By using confocal and atomic force microscopy, we demonstrated that TiO2NP exposure induces significant alterations in cellular membrane elasticity, due to actin proteins rearrangement in cytoskeleton, as calculated in correspondence to nuclear and cytoplasmic compartments. In this work, we have emphasized the alteration in mechanical properties of the cellular membrane, induced by nanoparticle exposure.


Assuntos
Núcleo Celular/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citotoxinas/toxicidade , Nanopartículas/toxicidade , Titânio/toxicidade , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/ultraestrutura , Fenômenos Biomecânicos , Membrana Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Sobrevivência Celular/efeitos dos fármacos , Citosol/ultraestrutura , Citotoxinas/química , Elasticidade/efeitos dos fármacos , Humanos , Células MCF-7 , Microscopia de Força Atômica , Nanopartículas/ultraestrutura , Titânio/química
8.
Int J Mol Sci ; 20(14)2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31340547

RESUMO

BACKGROUND: Lung cancer cells are known to change proliferation and migration under simulated microgravity. In this study, we sought to evaluate cell adherence, apoptosis, cytoskeleton arrangement, and gene expression under simulated microgravity. METHODS: Human lung cancer cells were exposed to simulated microgravity in a random-positioning machine (RPM). Cell morphology and adherence were observed under phase-contrast microscopy, cytoskeleton staining was performed, apoptosis rate was determined, and changes in gene and protein expression were detected by real-time PCR with western blot confirmation. RESULTS: Three-dimensional (3D)-spheroid formation was observed under simulated microgravity. Cell viability was not impaired. Actin filaments showed a shift in alignment from longitudinal to spherical. Apoptosis rate was significantly increased in the spheroids compared to the control. TP53, CDKN2A, PTEN, and RB1 gene expression was significantly upregulated in the adherent cells under simulated microgravity with an increase in corresponding protein production for p14 and RB1. SOX2 expression was significantly upregulated in the adherent cells, but protein was not. Gene expressions of AKT3, PIK3CA, and NFE2L2 remained unaltered. CONCLUSION: Simulated microgravity induces alteration in cell adherence, increases apoptosis rate, and leads to upregulation of tumor suppressor genes in human lung cancer cells.


Assuntos
Apoptose/genética , Adesão Celular/genética , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Ausência de Peso , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Células Epiteliais/ultraestrutura , Humanos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Simulação de Ausência de Peso/instrumentação , Simulação de Ausência de Peso/métodos
9.
Cell Mol Life Sci ; 76(20): 4103-4115, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31250032

RESUMO

Cardiovascular diseases (CVDs) are among the leading threats to human health. The advanced glycation end product (AGE) and receptor for AGE (RAGE) signaling pathway regulates the pathogenesis of CVDs, through its effects on arterial stiffness, atherosclerosis, mitochondrial dysfunction, oxidative stress, calcium homeostasis, and cytoskeletal function. Targeting the AGE/RAGE pathway is a potential therapeutic strategy for ameliorating CVDs. Vitamin D has several beneficial effects on the cardiovascular system. Experimental findings have shown that vitamin D regulates AGE/RAGE signaling and its downstream effects. This article provides a comprehensive review of the mechanistic insights into AGE/RAGE involvement in CVDs and the modulation of the AGE/RAGE signaling pathways by vitamin D.


Assuntos
Cardiomiopatias Diabéticas/prevenção & controle , Produtos Finais de Glicação Avançada/genética , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocardite/prevenção & controle , Receptor para Produtos Finais de Glicação Avançada/genética , Vitamina D/uso terapêutico , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Cálcio/metabolismo , Cardiotônicos/uso terapêutico , Cardiomiopatias Diabéticas/sangue , Cardiomiopatias Diabéticas/genética , Cardiomiopatias Diabéticas/patologia , Regulação da Expressão Gênica , Produtos Finais de Glicação Avançada/sangue , Guanidinas/uso terapêutico , Humanos , Traumatismo por Reperfusão Miocárdica/sangue , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Miocardite/sangue , Miocardite/genética , Miocardite/patologia , Estresse Oxidativo/efeitos dos fármacos , Receptor para Produtos Finais de Glicação Avançada/sangue , Transdução de Sinais , Tiazóis/uso terapêutico , Rigidez Vascular/efeitos dos fármacos , Vitamina D/sangue
10.
Nat Cell Biol ; 21(5): 603-613, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30988424

RESUMO

Mitochondrial fission involves the preconstriction of an organelle followed by scission by dynamin-related protein Drp1. Preconstriction is facilitated by actin and non-muscle myosin II through a mechanism that remains unclear, largely due to the unknown cytoskeletal ultrastructure at mitochondrial constrictions. Here, using platinum replica electron microscopy, we show that mitochondria in cells are embedded in an interstitial cytoskeletal network that contains abundant unbranched actin filaments. Both spontaneous and induced mitochondrial constrictions typically associate with a criss-cross array of long actin filaments that comprise part of this interstitial network. Non-muscle myosin II is found adjacent to mitochondria but is not specifically enriched at the constriction sites. During ionomycin-induced mitochondrial fission, F-actin clouds colocalize with mitochondrial constriction sites, whereas dynamic myosin II clouds are present in the vicinity of constrictions. We propose that myosin II promotes mitochondrial constriction by inducing stochastic deformations of the interstitial actin network, which applies pressure on the mitochondrial surface and thus initiates curvature-sensing mechanisms that complete mitochondrial constriction.


Assuntos
Actinas/genética , Citoesqueleto/ultraestrutura , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial/genética , Miosina Tipo II/genética , Citoesqueleto de Actina/química , Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Animais , Células COS , Constrição , Citoesqueleto/metabolismo , Ionomicina/farmacologia , Mitocôndrias/genética , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Miosina Tipo II/química , Miosina Tipo II/metabolismo
11.
J Cell Sci ; 132(4)2019 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-30700498

RESUMO

Cell polarity - the morphological and functional differentiation of cellular compartments in a directional manner - is required for processes such as orientation of cell division, directed cellular growth and motility. How the interplay of components within the complexity of a cell leads to cell polarity is still heavily debated. In this Review, we focus on one specific aspect of cell polarity: the non-uniform accumulation of proteins on the cell membrane. In cells, this is achieved through reaction-diffusion and/or cytoskeleton-based mechanisms. In reaction-diffusion systems, components are transformed into each other by chemical reactions and are moving through space by diffusion. In cytoskeleton-based processes, cellular components (i.e. proteins) are actively transported by microtubules (MTs) and actin filaments to specific locations in the cell. We examine how minimal systems - in vitro reconstitutions of a particular cellular function with a minimal number of components - are designed, how they contribute to our understanding of cell polarity (i.e. protein accumulation), and how they complement in vivo investigations. We start by discussing the Min protein system from Escherichia coli, which represents a reaction-diffusion system with a well-established minimal system. This is followed by a discussion of MT-based directed transport for cell polarity markers as an example of a cytoskeleton-based mechanism. To conclude, we discuss, as an example, the interplay of reaction-diffusion and cytoskeleton-based mechanisms during polarity establishment in budding yeast.


Assuntos
Citoesqueleto de Actina/metabolismo , Membrana Celular/metabolismo , Polaridade Celular , Escherichia coli/metabolismo , Microtúbulos/metabolismo , Saccharomyces cerevisiae/metabolismo , Citoesqueleto de Actina/ultraestrutura , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Transporte Biológico , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Membrana Celular/ultraestrutura , Proteínas do Citoesqueleto/metabolismo , Difusão , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/metabolismo , Microtúbulos/ultraestrutura , Modelos Biológicos , Saccharomyces cerevisiae/ultraestrutura , Termodinâmica , Proteína cdc42 de Ligação ao GTP/metabolismo
12.
J Cell Sci ; 132(3)2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30659115

RESUMO

The pathological significance of Tau (encoded by MAPT) in mechanisms driving cell migration in glioblastoma is unclear. By using an shRNA approach to deplete microtubule-stabilizing Tau in U87 cells, we determined its impact on cytoskeletal coordination during migration. We demonstrated here that the motility of these Tau-knockdown cells (shTau cells) was significantly (36%) lower than that of control cells. The shTau cells displayed a slightly changed motility in the presence of nocodazole, which inhibits microtubule formation. Such reduced motility of shTau cells was characterized by a 28% lower number of microtubule bundles at the non-adhesive edges of the tails. In accordance with Tau-stabilized microtubules being required for cell movement, measurements of the front, body and rear section displacements of cells showed inefficient tail retraction in shTau cells. The tail retraction was restored by treatment with Y27632, an inhibitor of Rho-ROCK signaling. Moreover, we clearly identified that shTau cells displayed relocation of the active phosphorylated form of p190-RhoGAP (also known as ARHGAP35), which inhibits Rho-ROCK signaling, and focal adhesion kinase (FAK, also known as PTK2) in cell bodies. In conclusion, our findings indicate that Tau governs the remodeling of microtubule and actin networks for the retraction of the tail of cells, which is necessary for effective migration.


Assuntos
Citoesqueleto de Actina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Microtúbulos/metabolismo , Neuroglia/metabolismo , Proteínas Repressoras/genética , Quinases Associadas a rho/genética , Proteínas tau/genética , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/ultraestrutura , Actinas/genética , Actinas/metabolismo , Amidas/farmacologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Neuroglia/efeitos dos fármacos , Neuroglia/patologia , Nocodazol/farmacologia , Piridinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Transdução de Sinais , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo , Proteínas tau/antagonistas & inibidores , Proteínas tau/metabolismo
13.
J Cell Sci ; 132(3)2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30659121

RESUMO

The linker of nucleoskeleton to cytoskeleton (LINC) complex is an essential multi-protein structure spanning the nuclear envelope. It connects the cytoplasm to the nucleoplasm, functions to maintain nuclear shape and architecture and regulates chromosome dynamics during cell division. Knowledge of LINC complex composition and function in the plant kingdom is primarily limited to Arabidopsis, but critically missing from the evolutionarily distant monocots, which include grasses, the most important agronomic crops worldwide. To fill this knowledge gap, we identified and characterized 22 maize genes, including a new grass-specific KASH gene family. By using bioinformatic, biochemical and cell biological approaches, we provide evidence that representative KASH candidates localize to the nuclear periphery and interact with Zea mays (Zm)SUN2 in vivo FRAP experiments using domain deletion constructs verified that this SUN-KASH interaction was dependent on the SUN but not the coiled-coil domain of ZmSUN2. A summary working model is proposed for the entire maize LINC complex encoded by conserved and divergent gene families. These findings expand our knowledge of the plant nuclear envelope in a model grass species, with implications for both basic and applied cellular research.This article has an associated First Person interview with the first author of the paper.


Assuntos
Proteínas Associadas aos Microtúbulos/genética , Membrana Nuclear/metabolismo , Matriz Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas de Plantas/genética , Zea mays/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Divisão Celular , Cromatina/metabolismo , Cromatina/ultraestrutura , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Ontologia Genética , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Anotação de Sequência Molecular , Família Multigênica , Membrana Nuclear/ultraestrutura , Matriz Nuclear/ultraestrutura , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Células Vegetais/metabolismo , Células Vegetais/ultraestrutura , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Zea mays/metabolismo
14.
J Cell Sci ; 132(3)2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30630894

RESUMO

The junctional complexes that couple cardiomyocytes must transmit the mechanical forces of contraction while maintaining adhesive homeostasis. The adherens junction (AJ) connects the actomyosin networks of neighboring cardiomyocytes and is required for proper heart function. Yet little is known about the molecular composition of the cardiomyocyte AJ or how it is organized to function under mechanical load. Here, we define the architecture, dynamics and proteome of the cardiomyocyte AJ. Mouse neonatal cardiomyocytes assemble stable AJs along intercellular contacts with organizational and structural hallmarks similar to mature contacts. We combine quantitative mass spectrometry with proximity labeling to identify the N-cadherin (CDH2) interactome. We define over 350 proteins in this interactome, nearly 200 of which are unique to CDH2 and not part of the E-cadherin (CDH1) interactome. CDH2-specific interactors comprise primarily adaptor and adhesion proteins that promote junction specialization. Our results provide novel insight into the cardiomyocyte AJ and offer a proteomic atlas for defining the molecular complexes that regulate cardiomyocyte intercellular adhesion. This article has an associated First Person interview with the first authors of the paper.


Assuntos
Citoesqueleto de Actina/metabolismo , Actomiosina/genética , Junções Aderentes/metabolismo , Caderinas/genética , Mecanotransdução Celular , Miócitos Cardíacos/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actomiosina/metabolismo , Junções Aderentes/ultraestrutura , Animais , Animais Recém-Nascidos , Caderinas/metabolismo , Adesão Celular , Comunicação Celular , Regulação da Expressão Gênica , Ontologia Genética , Camundongos , Anotação de Sequência Molecular , Miócitos Cardíacos/ultraestrutura , Cultura Primária de Células , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteômica/métodos
15.
Environ Toxicol ; 34(4): 469-475, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30614199

RESUMO

Cadmium (Cd) is considered a possible etiological factor in neurodegenerative diseases. However, the exact mechanism by which Cd induces neurotoxicity is not well elucidated. In this study, Neuro-2a cells were treated with 0, 10, 20, and 40 µM cadmium chloride for 24 hours to investigate the effects of Cd on the cytoskeleton of nerve cells. MTT assay and ELISA assay were used to examine cell viability and release of lactate dehydrogenase (LDH) from cells, respectively. Results showed that Cd reduced cell viability and increased the release of LDH in a dose-dependent manner (P < 0.05). The morphology of treated cell was damaged as indicated by cell collapse and dimensionality reduction. Moreover, the axonal spines and normal features of Cd-treated neurons disappeared. We checked the ultrastructure of Neuro-2a cells and found that Cd-induced swelling, membrane damage, overflow of cytoplasm contents, and cell fragmentation. Damaged mitochondria, expanded endoplasmic reticulum, and abnormal microfilaments were found in Cd-treated cells rather than in untreated cells. Compared with the control group, the relative release of glutamate in the supernatant after Cd treatment was reduced, indicating that Cd exposure could reduce the release of glutamate by inhibiting the function of nerve-2a cells. Cd decreased the mRNA and protein expression levels of cytoskeletal proteins including DBN, SYP, and TAU, which might promote cytoskeleton alterations in Cd-treated cells. In conclusion, Cd-induced actin cytoskeleton alterations and dysfunction of cultured neurons. The results of the present study provide new insights for the investigation of Cd-induced neurotoxicity.


Assuntos
Citoesqueleto de Actina/efeitos dos fármacos , Cádmio/toxicidade , Poluentes Ambientais/toxicidade , Neurônios/efeitos dos fármacos , Citoesqueleto de Actina/ultraestrutura , Animais , Axônios/efeitos dos fármacos , Axônios/ultraestrutura , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Camundongos , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Neurônios/ultraestrutura , Síndromes Neurotóxicas/patologia
16.
J Cell Sci ; 132(3)2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30635445

RESUMO

During plant cytokinesis a radially expanding membrane-enclosed cell plate is formed from fusing vesicles that compartmentalizes the cell in two. How fusion is spatially restricted to the site of cell plate formation is unknown. Aggregation of cell-plate membrane starts near regions of microtubule overlap within the bipolar phragmoplast apparatus of the moss Physcomitrella patens Since vesicle fusion generally requires coordination of vesicle tethering and subsequent fusion activity, we analyzed the subcellular localization of several subunits of the exocyst, a tethering complex active during plant cytokinesis. We found that the exocyst complex subunit Sec6 but not the Sec3 or Sec5 subunits localized to microtubule overlap regions in advance of cell plate construction in moss. Moreover, Sec6 exhibited a conserved physical interaction with an ortholog of the Sec1/Munc18 protein KEULE, an important regulator for cell-plate membrane vesicle fusion in Arabidopsis Recruitment of the P. patens protein KEULE and vesicles to the early cell plate was delayed upon Sec6 gene silencing. Our findings, thus, suggest that vesicle-vesicle fusion is, in part, enabled by a pool of exocyst subunits at microtubule overlaps, which is recruited independently of vesicle delivery.


Assuntos
Bryopsida/genética , Citocinese/genética , Regulação da Expressão Gênica de Plantas , Microtúbulos/metabolismo , Proteínas de Plantas/genética , Proteínas de Transporte Vesicular/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Bryopsida/metabolismo , Bryopsida/ultraestrutura , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Inativação Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Células Vegetais/metabolismo , Células Vegetais/ultraestrutura , Proteínas de Plantas/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Transporte Vesicular/metabolismo
17.
J Cell Sci ; 132(4)2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30670475

RESUMO

The endoplasmic reticulum (ER) is a major membrane-bound organelle in all eukaryotic cells. This organelle comprises morphologically distinct domains, including the nuclear envelope and peripheral sheets and tubules. The tubules are connected by three-way junctions into a network. Several membrane proteins have been implicated in network formation; curvature-stabilizing proteins generate the tubules themselves, and membrane-anchored GTPases fuse tubules into a network. Recent experiments have shown that a tubular network can be formed with reconstituted proteoliposomes containing the yeast membrane-fusing GTPase Sey1 and a curvature-stabilizing protein of either the reticulon or REEP protein families. The network forms in the presence of GTP and is rapidly disassembled when GTP hydrolysis of Sey1 is inhibited, indicating that continuous membrane fusion is required for its maintenance. Atlastin, the ortholog of Sey1 in metazoans, forms a network on its own, serving both as a fusion and curvature-stabilizing protein. These results show that the reticular ER can be generated by a surprisingly small set of proteins, and represents an energy-dependent steady state between formation and disassembly. Models for the molecular mechanism by which curvature-stabilizing proteins cooperate with fusion GTPases to form a reticular network have been proposed, but many aspects remain speculative, including the function of additional proteins, such as the lunapark protein, and the mechanism by which the ER interacts with the cytoskeleton. How the nuclear envelope and peripheral ER sheets are formed remain major unresolved questions in the field. Here, we review reconstitution experiments with purified curvature-stabilizing proteins and fusion GTPases, discuss mechanistic implications and point out open questions.


Assuntos
Citoesqueleto de Actina/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Microtúbulos/metabolismo , Membrana Nuclear/metabolismo , Proteolipídeos/metabolismo , Citoesqueleto de Actina/ultraestrutura , Fenômenos Biomecânicos , Membrana Celular/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Fusão de Membrana , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Microtúbulos/ultraestrutura , Modelos Biológicos , Membrana Nuclear/ultraestrutura , Proteolipídeos/ultraestrutura , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo
18.
Genes Cells ; 24(3): 202-213, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30664308

RESUMO

Fascin, an actin-bundling protein, is present in the filopodia and lamellipodia of growth cones. However, few studies have examined lamellipodial fascin because it is difficult to observe. In this study, we evaluated lamellipodial fascin. We visualized the actin meshwork of lamellipodia in live growth cones by super-resolution microscopy. Fascin was colocalized with the actin meshwork in lamellipodia. Ser39 of fascin is a well-known phosphorylation site that controls the binding of fascin to actin filaments. Fluorescence recovery after photobleaching experiments with confocal microscopy showed that binding of fascin was controlled by phosphorylation of Ser39 in lamellipodia. Moreover, TPA, an agonist of protein kinase C, induced phosphorylation of fascin and dissociation from actin filaments in lamellipodia. Time series images showed that dissociation of fascin from the actin meshwork was induced by TPA. As fascin dissociated from actin filaments, the orientation of the actin filaments became parallel to the leading edge. The angle of actin filaments against the leading edge was changed from 73° to 15°. This decreased the elasticity of the lamellipodia by 40%, as measured by atomic force microscopy. These data suggest that actin bundles made by fascin contribute to elasticity of the growth cone.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Transporte/metabolismo , Proteínas dos Microfilamentos/metabolismo , Pseudópodes/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Proteínas de Transporte/química , Linhagem Celular , Elasticidade , Recuperação de Fluorescência Após Fotodegradação , Camundongos , Proteínas dos Microfilamentos/química , Fosforilação , Pseudópodes/ultraestrutura
19.
ACS Nano ; 13(1): 346-356, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30485065

RESUMO

The mechanical properties of cells affect their function, in sensing, development, and motility. However, the rigidity of the cell surface and its correlation to its local topography remain poorly understood. Here, we applied quantitative imaging AFM to capture high-resolution force maps at the surface of nonadherent T cells. Using this method, we found a positive topography-rigidity correlation at the cells' surface, as opposed to a negative correlation at the surface of adherent cells. We then used 3D single-molecule localization microscopy of the membrane and cortical actin and an actin-perturbing drug to implicate actin involvement in the positive rigidity-topography correlation in T cells. Our results clearly reveal the variability of cell-surface rigidity and its underlying mechanism, showing a functional role for cortical actin in the PM protrusions of T cells, since they are locally more rigid than their surroundings. These findings suggest the possible functional role of membrane protrusions as mechanosensors.


Assuntos
Membrana Celular/ultraestrutura , Imagem Individual de Molécula , Citoesqueleto de Actina/química , Citoesqueleto de Actina/ultraestrutura , Membrana Celular/química , Elasticidade , Células HEK293 , Humanos , Imageamento Tridimensional , Células Jurkat
20.
J Cell Biol ; 218(1): 251-266, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30420355

RESUMO

During autophagy, actin filament networks move and remodel cellular membranes to form autophagosomes that enclose and metabolize cytoplasmic contents. Two actin regulators, WHAMM and JMY, participate in autophagosome formation, but the signals linking autophagy to actin assembly are poorly understood. We show that, in nonstarved cells, cytoplasmic JMY colocalizes with STRAP, a regulator of JMY's nuclear functions, on nonmotile vesicles with no associated actin networks. Upon starvation, JMY shifts to motile, LC3-containing membranes that move on actin comet tails. LC3 enhances JMY's de novo actin nucleation activity via a cryptic actin-binding sequence near JMY's N terminus, and STRAP inhibits JMY's ability to nucleate actin and activate the Arp2/3 complex. Cytoplasmic STRAP negatively regulates autophagy. Finally, we use purified proteins to reconstitute LC3- and JMY-dependent actin network formation on membranes and inhibition of network formation by STRAP. We conclude that LC3 and STRAP regulate JMY's actin assembly activities in trans during autophagy.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Autofagossomos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/genética , Transativadores/genética , Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Autofagossomos/ultraestrutura , Autofagia/genética , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Osteoblastos/metabolismo , Osteoblastos/ultraestrutura , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Células Sf9 , Transdução de Sinais , Spodoptera , Transativadores/metabolismo
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