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1.
Invest Ophthalmol Vis Sci ; 60(14): 4583-4595, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31675075

RESUMO

Purpose: Trabecular meshwork (TM) cells detect and coordinate responses to intraocular pressure (IOP) in the eye. TM cells become dysfunctional in glaucoma where IOP is often elevated. Recently, we showed that normal TM (NTM) cells communicate by forming tubular connections called tunneling nanotubes (TNTs). Here, we investigated TNTs in glaucomatous TM (GTM) cells. Methods: Primary GTM and NTM cells were established from cadaver eyes. Transfer of Vybrant DiO and DiD-labeled vesicles via TNT connections was measured. Imaris software measured the number and length of cell protrusions from immunofluorescent confocal images. Live-cell imaging of the actin cytoskeleton was performed. The distribution of myosin-X, a regulator of TNTs/filopodia, was investigated in TM cells and tissue. Results: GTM cells contained significantly more transferred fluorescent vesicles than NTM cells (49.6% vs. 35%). Although NTM cells had more protrusions at the cell surface than GTM cells (7.61 vs. 4.65 protrusions/cell), GTM protrusions were significantly longer (12.1 µm vs. 9.76 µm). Live-cell imaging demonstrated that the GTM actin cytoskeleton was less dynamic, and vesicle transfer between cells was significantly slower than NTM cells. Furthermore, rearrangement of the actin cortex adjacent to the TNT may influence TNT formation. Myosin-X immunostaining was punctate and disorganized in GTM cells and tissue compared to age-matched NTM controls. Conclusions: Together, our data demonstrate that GTM cells have phenotypic and functional differences in their TNTs. Significantly slower vesicle transfer via TNTs in GTM cells may delay the timely propagation of cellular signals when pressures become elevated in glaucoma.


Assuntos
Citoesqueleto de Actina/metabolismo , Glaucoma de Ângulo Aberto/patologia , Miosinas/metabolismo , Nanotubos , Pseudópodes/metabolismo , Malha Trabecular/patologia , Western Blotting , Tamanho Celular , Células Cultivadas , Senescência Celular/fisiologia , Densitometria , Glaucoma de Ângulo Aberto/metabolismo , Humanos , Microscopia Confocal , Fagocitose/fisiologia , Fenótipo , Transdução de Sinais/fisiologia , Malha Trabecular/metabolismo
2.
Nat Commun ; 10(1): 4183, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519913

RESUMO

The obligate intracellular parasite Toxoplasma gondii replicates in an unusual process, described as internal budding. Multiple dausghter parasites are formed sequentially within a single mother cell, requiring replication and distribution of essential organelles such as micronemes. These organelles are thought to be formed de novo in the developing daughter cells. Using dual labelling of a microneme protein MIC2 and super-resolution microscopy, we show that micronemes are recycled from the mother to the forming daughter parasites using a highly dynamic F-actin network. While this recycling pathway is F-actin dependent, de novo synthesis of micronemes appears to be F-actin independent. The F-actin network connects individual parasites, supports long, multidirectional vesicular transport, and regulates transport, density and localisation of micronemal vesicles. The residual body acts as a storage and sorting station for these organelles. Our data describe an F-actin dependent mechanism in apicomplexans for transport and recycling of maternal organelles during intracellular development.


Assuntos
Actinas/metabolismo , Toxoplasma/metabolismo , Citoesqueleto de Actina/metabolismo , Transporte Proteico/fisiologia , Proteínas de Protozoários/metabolismo , Vacúolos/metabolismo
3.
Cell Physiol Biochem ; 53(4): 623-637, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31550089

RESUMO

BACKGROUND/AIMS: In articular cartilage, chondrocytes are the predominant cell type. A long-term stay in space can lead to bone loss and cartilage breakdown. Due to the poor regenerative capacity of cartilage, this may impair the crewmembers' mobility and influence mission activities. Beside microgravity other factors such as cosmic radiation and vibration might be important for cartilage degeneration. Vibration at different frequencies showed various effects on cartilage in vivo, but knowledge about its impact on chondrocytes in vitro is sparse. METHODS: Human chondrocytes were exposed to a vibration device, simulating the vibration profile occurring during parabolic flights, for 24 h (VIB) and compared to static controls. Phase-contrast microscopy, immunofluorescence, F-actin and TUNEL staining as well as quantitative real-time PCR were performed to examine effects on morphology, cell viability and shape as well as gene expression. The results were compared to earlier studies using semantic analyses. RESULTS: No morphological changes or cytoskeletal alterations were observed in VIB and no apoptotic cells were found. A reorganization and increase in fibronectin were detected in VIB samples by immunofluorescence technique. PXN, VCL, ANXA1, ANXA2, BAX, and BCL2 revealed differential regulations. CONCLUSION: Long-term VIB did not damage human chondrocytes in vitro. The reduction of ANXA2, and up-regulation of ANXA1, PXN and VCL mRNAs suggest that long-term vibration might even positively influence cultured chondrocytes.


Assuntos
Condrócitos/metabolismo , Vibração , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Anexina A2/genética , Anexina A2/metabolismo , Linhagem Celular , Condrócitos/citologia , Condrócitos/patologia , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Redes Reguladoras de Genes , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Vimentina/genética , Vimentina/metabolismo
4.
Nat Cell Biol ; 21(9): 1068-1077, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31481797

RESUMO

Recent development of innovative tools for live imaging of actin filaments (F-actin) enabled the detection of surprising nuclear structures responding to various stimuli, challenging previous models that actin is substantially monomeric in the nucleus. We review these discoveries, focusing on double-strand break (DSB) repair responses. These studies revealed a remarkable network of nuclear filaments and regulatory mechanisms coordinating chromatin dynamics with repair progression and led to a paradigm shift by uncovering the directed movement of repair sites.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Núcleo Celular/metabolismo , Reparo do DNA/fisiologia , Animais , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , Humanos
5.
Nat Commun ; 10(1): 3940, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477736

RESUMO

Collective cell migration is involved in development, wound healing and metastasis. In the Drosophila ovary, border cells (BC) form a small cluster that migrates collectively through the egg chamber. To achieve directed motility, the BC cluster coordinates the formation of protrusions in its leader cell and contractility at the rear. Restricting protrusions to leader cells requires the actin and plasma membrane linker Moesin. Herein, we show that the Ste20-like kinase Misshapen phosphorylates Moesin in vitro and in BC. Depletion of Misshapen disrupts protrusion restriction, thereby allowing other cells within the cluster to protrude. In addition, we show that Misshapen is critical to generate contractile forces both at the rear of the cluster and at the base of protrusions. Together, our results indicate that Misshapen is a key regulator of BC migration as it coordinates two independent pathways that restrict protrusion formation to the leader cells and induces contractile forces.


Assuntos
Actomiosina/genética , Movimento Celular/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Oogênese/genética , Proteínas Serina-Treonina Quinases/genética , Citoesqueleto de Actina/metabolismo , Actomiosina/metabolismo , Algoritmos , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Feminino , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Modelos Genéticos , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA
6.
Biol Cell ; 111(10): 245-261, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31403697

RESUMO

BACKGROUND INFORMATION: Metastatic disease is caused by the ability of cancer cells to reach distant organs and form secondary lesions at new locations. Dissemination of cancer cells depends on their migration plasticity - an ability to switch between motility modes driven by distinct molecular machineries. One of such switches is mesenchymal-to-amoeboid transition. Although mesenchymal migration of individual cells requires Arp2/3-dependent actin polymerisation, amoeboid migration is characterised by a high level of actomyosin contractility and often involves the formation of membrane blebs. The acquisition of amoeboid motility by mesenchymal cells is often associated with enhanced metastasis. RESULTS: We studied the ability of mesenchymal HT1080 fibrosarcoma cells to switch to amoeboid motility. We induced the transition from lamellipodium-rich to blebbing phenotype either by down-regulating the Arp2/3 complex, pharmacologically or by RNAi, or by decreasing substrate adhesiveness. Each of these treatments induced blebbing in a subset of fibrosarcoma cells, but not in normal subcutaneous fibroblasts. A significant fraction of HT1080 cells that switched to blebbing behaviour exhibited stem cell-like features, such as expression of the stem cell marker CD133, an increased efflux of Hoechst-33342 and positive staining for Oct4, Sox2 and Nanog. Furthermore, the isolated CD133+ cells demonstrated an increased ability to switch to bleb-rich amoeboid phenotype both under inhibitor's treatment and in 3D collagen gels. CONCLUSIONS: Together, our data show a significant correlation between the increased ability of cells to switch between migration modes and their stem-like features, suggesting that migration plasticity is an additional property of stem-like population of fibrosarcoma cells. This combination of features could facilitate both dissemination of these cells to distant locations, and their establishment self-renewal in a new microenvironment, as required for metastasis formation. SIGNIFICANCE: These data suggest that migration plasticity is a new feature of cancer stem-like cells that can significantly facilitate their dissemination to a secondary location by allowing them to adapt quickly to challenging microenvironments. Moreover, it complements their resistance to apoptosis and self-renewal potential, thus enabling them not only to disseminate efficiently, but also to survive and colonise new niches.


Assuntos
Movimento Celular , Fibrossarcoma/patologia , Células-Tronco Neoplásicas/patologia , Antígeno AC133/metabolismo , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Humanos , Microambiente Tumoral
7.
Nat Commun ; 10(1): 3459, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31371777

RESUMO

Myosin VI plays crucial roles in diverse cellular processes. In autophagy, Myosin VI can facilitate the maturation of autophagosomes through interactions with Tom1 and the autophagy receptors, Optineurin, NDP52 and TAX1BP1. Here, we report the high-resolution crystal structure of the C-terminal cargo-binding domain (CBD) of Myosin VI in complex with Tom1, which elucidates the mechanistic basis underpinning the specific interaction between Myosin VI and Tom1, and uncovers that the C-terminal CBD of Myosin VI adopts a unique cargo recognition mode to interact with Tom1 for tethering. Furthermore, we show that Myosin VI can serve as a bridging adaptor to simultaneously interact with Tom1 and autophagy receptors through two distinct interfaces. In all, our findings provide mechanistic insights into the interactions of Myosin VI with Tom1 and relevant autophagy receptors, and are valuable for further understanding the functions of these proteins in autophagy and the cargo recognition modes of Myosin VI.


Assuntos
Citoesqueleto de Actina/metabolismo , Cadeias Pesadas de Miosina/química , Domínios e Motivos de Interação entre Proteínas , Proteínas/química , Autofagossomos/metabolismo , Autofagia/fisiologia , Cristalografia por Raios X , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Modelos Moleculares , Proteínas de Neoplasias , Proteínas Nucleares , Ligação Proteica , Fator de Transcrição TFIIIA
8.
Results Probl Cell Differ ; 67: 27-48, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31435791

RESUMO

Formin homology proteins (formins) are a highly conserved family of cytoskeletal remodeling proteins that are involved in a diverse array of cellular functions. Formins are best known for their ability to regulate actin dynamics, but the same functional domains also govern stability and organization of microtubules. It is thought that this dual activity allows them to coordinate the activity of these two major cytoskeletal networks and thereby influence cellular architecture. Golgi ribbon assembly is dependent upon cooperative interactions between actin filaments and cytoplasmic microtubules originating both at the Golgi itself and from the centrosome. Similarly, centrosome assembly, centriole duplication, and centrosome positioning are also reliant on a dialogue between both cytoskeletal networks. As presented in this chapter, a growing body of evidence suggests that multiple formin proteins play essential roles in these central cellular processes.


Assuntos
Centríolos/metabolismo , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Complexo de Golgi/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Microtúbulos/metabolismo
9.
Results Probl Cell Differ ; 67: 81-93, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31435793

RESUMO

In vertebrate cells, the Golgi apparatus is located in close proximity to the centriole. The architecture of the Golgi/centriole complex depends on a multitude of factors, including the actin filament cytoskeleton. In turn, both the Golgi and centriole act as the actin nucleation centers. Actin organization and polymerization also depend on the small GTPase RhoA pathway. In this chapter, we summarize the most current knowledge on how the genetic, magnetic, or pharmacologic interference with RhoA pathway and actin cytoskeleton directly or indirectly affects architecture, structure, and function of the Golgi/centriole complex.


Assuntos
Actinas/metabolismo , Centríolos/metabolismo , Complexo de Golgi/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo
10.
Soft Matter ; 15(31): 6300-6307, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31342050

RESUMO

Networks of filamentous actin (F-actin) are important for the mechanics of most animal cells. These cytoskeletal networks are highly dynamic, with a variety of actin-associated proteins that control cross-linking, polymerization and force generation in the cytoskeleton. Inspired by recent rheological experiments on reconstituted solutions of dynamic actin filaments, we report a theoretical model that describes stress relaxation behavior of these solutions in the presence of severing proteins. We show that depending on the kinetic rates of assembly, disassembly, and severing, one can observe both length-dependent and length-independent relaxation behavior.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , Modelos Biológicos
11.
Biochim Biophys Acta Mol Cell Res ; 1866(10): 1634-1649, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31255721

RESUMO

Ligand-induced activation of Exchange Protein Activated by cAMP-1 (EPAC1) is implicated in numerous physiological and pathological processes, including cardiac fibrosis where changes in EPAC1 expression have been detected. However, little is known about how EPAC1 expression is regulated. Therefore, we investigated regulation of EPAC1 expression by cAMP in cardiac fibroblasts. Elevation of cAMP using forskolin, cAMP-analogues or adenosine A2B-receptor activation significantly reduced EPAC1 mRNA and protein levels and inhibited formation of F-actin stress fibres. Inhibition of actin polymerisation with cytochalasin-D, latrunculin-B or the ROCK inhibitor, Y-27632, mimicked effects of cAMP on EPAC1 mRNA and protein levels. Elevated cAMP also inhibited activity of an EPAC1 promoter-reporter gene, which contained a consensus binding element for TEAD, which is a target for inhibition by cAMP. Inhibition of TEAD activity using siRNA-silencing of its co-factors YAP and TAZ, expression of dominant-negative TEAD or treatment with YAP-TEAD inhibitors, significantly inhibited EPAC1 expression. However, whereas expression of constitutively-active YAP completely reversed forskolin inhibition of EPAC1-promoter activity it did not rescue EPAC1 mRNA levels. Chromatin-immunoprecipitation detected a significant reduction in histone3-lysine27-acetylation at the EPAC1 proximal promoter in response to forskolin stimulation. HDAC1/3 inhibition partially reversed forskolin inhibition of EPAC1 expression, which was completely rescued by simultaneously expressing constitutively active YAP. Taken together, these data demonstrate that cAMP downregulates EPAC1 gene expression via disrupting the actin cytoskeleton, which inhibits YAP/TAZ-TEAD activity in concert with HDAC-mediated histone deacetylation at the EPAC1 proximal promoter. This represents a novel negative feedback mechanism controlling EPAC1 levels in response to cAMP elevation.


Assuntos
AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Processamento de Proteína Pós-Traducional , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Amidas , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Técnicas de Cultura de Células , Linhagem Celular , Citocalasina D/metabolismo , Fibroblastos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Histonas/metabolismo , Humanos , Masculino , Piridinas , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Tiazolidinas/metabolismo
12.
Int J Mol Sci ; 20(14)2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31336621

RESUMO

The retinal pigment epithelium (RPE) is a unique epithelium, with major roles which are essential in the visual cycle and homeostasis of the outer retina. The RPE is a monolayer of polygonal and pigmented cells strategically placed between the neuroretina and Bruch membrane, adjacent to the fenestrated capillaries of the choriocapillaris. It shows strong apical (towards photoreceptors) to basal/basolateral (towards Bruch membrane) polarization. Multiple functions are bound to a complex structure of highly organized and polarized intracellular components: the cytoskeleton. A strong connection between the intracellular cytoskeleton and extracellular matrix is indispensable to maintaining the function of the RPE and thus, the photoreceptors. Impairments of these intracellular structures and the regular architecture they maintain often result in a disrupted cytoskeleton, which can be found in many retinal diseases, including age-related macular degeneration (AMD). This review article will give an overview of current knowledge on the molecules and proteins involved in cytoskeleton formation in cells, including RPE and how the cytoskeleton is affected under stress conditions-especially in AMD.


Assuntos
Citoesqueleto/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Biomarcadores , Matriz Extracelular/metabolismo , Humanos , Degeneração Macular/etiologia , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Microtúbulos/metabolismo
13.
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
14.
Elife ; 82019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31348003

RESUMO

Atxn7, a subunit of SAGA chromatin remodeling complex, is subject to polyglutamine expansion at the amino terminus, causing spinocerebellar ataxia type 7 (SCA7), a progressive retinal and neurodegenerative disease. Within SAGA, the Atxn7 amino terminus anchors Non-stop, a deubiquitinase, to the complex. To understand the scope of Atxn7-dependent regulation of Non-stop, substrates of the deubiquitinase were sought. This revealed Non-stop, dissociated from Atxn7, interacts with Arp2/3 and WAVE regulatory complexes (WRC), which control actin cytoskeleton assembly. There, Non-stop countered polyubiquitination and proteasomal degradation of WRC subunit SCAR. Dependent on conserved WRC interacting receptor sequences (WIRS), Non-stop augmentation increased protein levels, and directed subcellular localization, of SCAR, decreasing cell area and number of protrusions. In vivo, heterozygous mutation of SCAR did not significantly rescue knockdown of Atxn7, but heterozygous mutation of Atxn7 rescued haploinsufficiency of SCAR.


Assuntos
Citoesqueleto de Actina/metabolismo , Ataxina-7/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Endopeptidases/metabolismo , Proteínas dos Microfilamentos/metabolismo , Animais , Regulação da Expressão Gênica , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas
15.
Nat Cell Biol ; 21(8): 933-939, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31358965

RESUMO

Actomyosin networks, the cell's major force production machineries, remodel cellular membranes during myriad dynamic processes1,2 by assembling into various architectures with distinct force generation properties3,4. While linear and branched actomyosin architectures are well characterized in cell-culture and cell-free systems3, it is not known how actin and myosin networks form and function to remodel membranes in complex three-dimensional mammalian tissues. Here, we use four-dimensional spinning-disc confocal microscopy with image deconvolution to acquire macromolecular-scale detail of dynamic actomyosin networks in exocrine glands of live mice. We address how actin and myosin organize around large membrane-bound secretory vesicles and generate the forces required to complete exocytosis5-7. We find that actin and non-muscle myosin II (NMII) assemble into previously undescribed polyhedral-like lattices around the vesicle membrane. The NMII lattice comprises bipolar minifilaments8-10 as well as non-canonical three-legged configurations. Using photobleaching and pharmacological perturbations in vivo, we show that actomyosin contractility and actin polymerization together push on the underlying vesicle membrane to overcome the energy barrier and complete exocytosis7. Our imaging approach thus unveils a force-generating actomyosin lattice that regulates secretion in the exocrine organs of live animals.


Assuntos
Actomiosina/metabolismo , Exocitose/fisiologia , Contração Muscular/fisiologia , Miosinas/metabolismo , Citoesqueleto de Actina/metabolismo , Actomiosina/genética , Animais , Membrana Celular/metabolismo , Exocitose/genética , Camundongos Transgênicos , Microscopia Confocal/métodos , Miosinas/genética , Vesículas Secretórias/metabolismo
16.
Genes Dev ; 33(13-14): 828-843, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171701

RESUMO

Adenovirus transformed cells have a dedifferentiated phenotype. Eliminating E1A in transformed human embryonic kidney cells derepressed ∼2600 genes, generating a gene expression profile closely resembling mesenchymal stem cells (MSCs). This was associated with a dramatic change in cell morphology from one with scant cytoplasm and a globular nucleus to one with increased cytoplasm, extensive actin stress fibers, and actomyosin-dependent flattening against the substratum. E1A-induced hypoacetylation at histone H3 Lys27 and Lys18 (H3K27/18) was reversed. Most of the increase in H3K27/18ac was in enhancers near TEAD transcription factors bound by Hippo signaling-regulated coactivators YAP and TAZ. E1A causes YAP/TAZ cytoplasmic sequestration. After eliminating E1A, YAP/TAZ were transported into nuclei, where they associated with poised enhancers with DNA-bound TEAD4 and H3K4me1. This activation of YAP/TAZ required RHO family GTPase signaling and caused histone acetylation by p300/CBP, chromatin remodeling, and cohesin loading to establish MSC-associated enhancers and then superenhancers. Consistent results were also observed in primary rat embryo kidney cells, human fibroblasts, and human respiratory tract epithelial cells. These results together with earlier studies suggest that YAP/TAZ function in a developmental checkpoint controlled by signaling from the actin cytoskeleton that prevents differentiation of a progenitor cell until it is in the correct cellular and tissue environment.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas E1A de Adenovirus/metabolismo , Diferenciação Celular/genética , Inativação Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosfoproteínas/genética , Citoesqueleto de Actina/metabolismo , Adenoviridae , Animais , Células Cultivadas , Células HEK293 , Humanos , Ratos , Transdução de Sinais , Fatores de Transcrição
17.
Mol Biol Cell ; 30(16): 2025-2036, 2019 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-31216217

RESUMO

Contractile arrays of actin and myosin II filaments drive many essential processes in nonmuscle cells, including migration and adhesion. Sequential organization of actin and myosin along one dimension is followed by expansion into a two-dimensional network of parallel actomyosin fibers, in which myosin filaments are aligned to form stacks. The process of stack formation has been studied in detail. However, factors that oppose myosin stack formation have not yet been described. Here, we show that tropomyosins act as negative regulators of myosin stack formation. Knockdown of any or all tropomyosin isoforms in rat embryonic fibroblasts resulted in longer and more numerous myosin stacks and a highly ordered actomyosin organization. The molecular basis for this, we found, is the competition between tropomyosin and alpha-actinin for binding actin. Surprisingly, excessive order in the actomyosin network resulted in smaller focal adhesions, lower tension within the network, and smaller traction forces. Conversely, disordered actomyosin bundles induced by alpha-actinin knockdown led to higher than normal tension and traction forces. Thus, tropomyosin acts as a check on alpha-actinin to achieve intermediate levels of myosin stacks matching the force requirements of the cell.


Assuntos
Actinina/metabolismo , Actomiosina/metabolismo , Contração Muscular , Tropomiosina/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Fenômenos Biomecânicos , Fibroblastos/metabolismo , Adesões Focais/metabolismo , Modelos Biológicos , Miosina Tipo II/metabolismo , Ratos , Fibras de Estresse/metabolismo
18.
Int J Mol Sci ; 20(11)2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31195605

RESUMO

The actin cytoskeleton is involved in regulating stomatal movement, which forms distinct actin arrays within guard cells of stomata with different apertures. How those actin arrays are formed and maintained remains largely unexplored. Elucidation of the dynamic behavior of differently oriented actin filaments in guard cells will enhance our understanding in this regard. Here, we initially developed a program called 'guard cell microfilament analyzer' (GCMA) that enables the selection of individual actin filaments and analysis of their orientations semiautomatically in guard cells. We next traced the dynamics of individual actin filaments and performed careful quantification in open and closed stomata. We found that de novo nucleation of actin filaments occurs at both dorsal and ventral sides of guard cells from open and closed stomata. Interestingly, most of the nucleated actin filaments elongate radially and longitudinally in open and closed stomata, respectively. Strikingly, radial filaments tend to form bundles whereas longitudinal filaments tend to be removed by severing and depolymerization in open stomata. By contrast, longitudinal filaments tend to form bundles that are severed less frequently in closed stomata. These observations provide insights into the formation and maintenance of distinct actin arrays in guard cells in stomata of different apertures.


Assuntos
Citoesqueleto de Actina/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Estômatos de Plantas/citologia , Estômatos de Plantas/metabolismo , Actinas/metabolismo , Ritmo Circadiano/fisiologia , Polimerização
19.
Mol Biol Cell ; 30(16): 2037-2052, 2019 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-31166831

RESUMO

The epithelial junctional complex, composed of tight junctions, adherens junctions, desmosomes, and an associated actomyosin cytoskeleton, forms the apical junctional ring (AJR), which must maintain its continuity in the face of external mechanical forces that accompany normal physiological functions. The AJR of umbrella cells, which line the luminal surface of the bladder, expands during bladder filling and contracts upon voiding; however, the mechanisms that drive these events are unknown. Using native umbrella cells as a model, we observed that the umbrella cell's AJR assumed a nonsarcomeric organization in which filamentous actin and ACTN4 formed unbroken continuous rings, while nonmuscle myosin II (NMMII) formed linear tracts along the actin ring. Expansion of the umbrella cell AJR required formin-dependent actin assembly, but was independent of NMMII ATPase function. AJR expansion also required membrane traffic, RAB13-dependent exocytosis, specifically, but not trafficking events regulated by RAB8A or RAB11A. In contrast, the voiding-induced contraction of the AJR depended on NMMII and actin dynamics, RHOA, and dynamin-dependent endocytosis. Taken together, our studies indicate that a mechanism by which the umbrella cells retain continuity during cyclical changes in volume is the expansion and contraction of their AJR, processes regulated by the actomyosin cytoskeleton and membrane trafficking events.


Assuntos
Polaridade Celular , Bexiga Urinária/citologia , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Junções Aderentes/metabolismo , Animais , Dinaminas/metabolismo , Feminino , GTP Fosfo-Hidrolases/metabolismo , Miosina Tipo II/metabolismo , Ratos Sprague-Dawley , Sarcômeros/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
20.
Int J Mol Sci ; 20(11)2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31151140

RESUMO

As a conserved actin-regulating protein, CAP (adenylyl Cyclase-Associated Protein) functions to facilitate the rearrangement of the actin cytoskeleton. The ubiquitously expressed isoform CAP1 drives mammalian cell migration, and accordingly, most studies on the involvement of CAP1 in human cancers have largely been based on the rationale that up-regulated CAP1 will stimulate cancer cell migration and invasiveness. While findings from some studies reported so far support this case, lines of evidence largely from our recent studies point to a more complex and profound role for CAP1 in the invasiveness of cancer cells, where the potential activation of cell adhesion signaling is believed to play a key role. Moreover, CAP1 was also found to control proliferation in breast cancer cells, through the regulation of ERK (External signal-Regulated Kinase). Alterations in the activities of FAK (Focal Adhesion Kinase) and ERK from CAP1 depletion that are consistent to the opposite adhesion and proliferation phenotypes were detected in the metastatic and non-metastatic breast cancer cells. In this review, we begin with the overview of the literature on CAP, by highlighting the molecular functions of mammalian CAP1 in regulating the actin cytoskeleton and cell adhesion. We will next discuss the role of the FAK/ERK axis, and possibly Rap1, in mediating CAP1 signals to control breast cancer cell adhesion, invasiveness, and proliferation, largely based on our latest findings. Finally, we will discuss the relevance of these novel mechanistic insights to ultimately realizing the translational potential of CAP1 in targeted therapeutics for breast cancer.


Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Transdução de Sinais , Citoesqueleto de Actina/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Adesão Celular/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Movimento Celular/genética , Proliferação de Células , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Ligação Proteica , Relação Estrutura-Atividade , Pesquisa Médica Translacional
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