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1.
Semin Cell Dev Biol ; 129: 63-74, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35577698

RESUMO

Cellular protrusions generated by the actin cytoskeleton are central to the process of building the body of the embryo. Problems with cellular protrusions underlie human diseases and syndromes, including implantation defects and pregnancy loss, congenital birth defects, and cancer. Cells use protrusive activity together with actin-myosin contractility to create an ordered body shape of the embryo. Here, I review how actin-rich protrusions are used by two major morphological cell types, epithelial and mesenchymal cells, during collective cell migration to sculpt the mouse embryo body. Pre-gastrulation epithelial collective migration of the anterior visceral endoderm is essential for establishing the anterior-posterior body axis. Gastrulation mesenchymal collective migration of the mesoderm wings is crucial for body elongation, and somite and heart formation. Analysis of mouse mutants with disrupted cellular protrusions revealed the key role of protrusions in embryonic morphogenesis and embryo survival. Recent technical approaches have allowed examination of the mechanisms that control cell and tissue movements in vivo in the complex 3D microenvironment of living mouse embryos. Advancing our understanding of protrusion-driven morphogenesis should provide novel insights into human developmental disorders and cancer metastasis.


Assuntos
Actinas , Desenvolvimento Embrionário , Actinas/metabolismo , Animais , Movimento Celular , Extensões da Superfície Celular/metabolismo , Endoderma , Feminino , Gastrulação , Humanos , Mesoderma , Camundongos , Gravidez
2.
J Cell Sci ; 135(10)2022 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-35621127

RESUMO

Podosomes are mechanosensitive protrusive actin structures that are prominent in myeloid cells, and they have been linked to vascular extravasation. Recent studies have suggested that podosomes are hierarchically organized and have coordinated dynamics on the cell scale, which implies that the local force generation by single podosomes can be different from their global combined action. Complementary to previous studies focusing on individual podosomes, here we investigated the cell-wide force generation of podosome-bearing ER-Hoxb8 monocytes. We found that the occurrence of focal tractions accompanied by a cell-wide substrate indentation cannot be explained by summing the forces of single podosomes. Instead, our findings suggest that superimposed contraction on the cell scale gives rise to a buckling mechanism that can explain the measured cell-scale indentation. Specifically, the actomyosin network contraction causes peripheral in-plane substrate tractions, while the accumulated internal stress results in out-of-plane deformation in the central cell region via a buckling instability, producing the cell-scale indentation. Hence, we propose that contraction of the actomyosin network, which connects the podosomes, leads to a substrate indentation that acts in addition to the protrusion forces of individual podosomes. This article has an associated First Person interview with the first author of the paper.


Assuntos
Podossomos , Actomiosina , Extensões da Superfície Celular , Humanos , Monócitos , Tração
3.
Semin Cell Dev Biol ; 129: 93-102, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35370088

RESUMO

Gamete fusion is of considerable importance in reproductive events, as it determines the gamete pairs or chromosomes that the next generation will inherit. To preserve species specificity with an appropriate karyotype, the fusion between gametes requires regulatory mechanisms to ensure limited fusion competency. In many organisms, gamete surfaces are not smooth, but present constitutive or transient cellular protrusions suggested to be involved in gamete fusion. However, the molecular mechanisms and the factors essential for the membrane-membrane fusion process and cellular protrusion involvement have remained unclear. Recent advances in the identification and functional analysis of the essential factors for gamete interaction have revealed the molecular mechanisms underlying their activity regulation and dynamics. In homogametic fertilization, dynamic regulation of the fusion core machinery on cellular protrusions was precisely uncovered. In heterogametic fertilization, oocyte fusion competency was suggested to correlate with the compartmentalization of the fusion essential factor and protrusion formation. These findings shed light on the significance of cellular protrusions in gamete fusion as a physically and functionally specialized site for cellular fusion. In this review, we consider the developments in gamete interaction research in various species with different fertilization modes, highlighting the commonalities in the relationship between gamete fusion and cellular protrusions.


Assuntos
Fertilização , Interações Espermatozoide-Óvulo , Extensões da Superfície Celular , Células Germinativas , Oócitos , Interações Espermatozoide-Óvulo/fisiologia
4.
Elife ; 112022 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-35467525

RESUMO

In addition to diffusive signals, cells in tissue also communicate via long, thin cellular protrusions, such as airinemes in zebrafish. Before establishing communication, cellular protrusions must find their target cell. Here, we demonstrate that the shapes of airinemes in zebrafish are consistent with a finite persistent random walk model. The probability of contacting the target cell is maximized for a balance between ballistic search (straight) and diffusive search (highly curved, random). We find that the curvature of airinemes in zebrafish, extracted from live-cell microscopy, is approximately the same value as the optimum in the simple persistent random walk model. We also explore the ability of the target cell to infer direction of the airineme's source, finding that there is a theoretical trade-off between search optimality and directional information. This provides a framework to characterize the shape, and performance objectives, of non-canonical cellular protrusions in general.


Assuntos
Extensões da Superfície Celular , Peixe-Zebra , Animais , Difusão
5.
Semin Cell Dev Biol ; 129: 126-134, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35260295

RESUMO

Cell-cell communications are central to a variety of physiological and pathological processes in multicellular organisms. Cells often rely on cellular protrusions to communicate with one another, which enable highly selective and efficient signaling within complex tissues. Owing to significant improvements in imaging techniques, identification of signaling protrusions has increased in recent years. These protrusions are structurally specialized for signaling and facilitate interactions between cells. Therefore, physical regulation of these structures must be key for the appropriate strength and pattern of signaling outcomes. However, the typical approaches for understanding signaling regulation tend to focus solely on changes in signaling molecules, such as gene expression, protein-protein interaction, and degradation. In this short review, we summarize the studies proposing the removal of different types of signaling protrusions-including cilia, neurites, MT (microtubule based)-nanotubes and microvilli-and discuss their mechanisms and significance in signaling regulation.


Assuntos
Comunicação Celular , Extensões da Superfície Celular , Extensões da Superfície Celular/metabolismo , Microtúbulos/metabolismo , Neuritos , Transdução de Sinais
6.
J Cell Biol ; 221(3)2022 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-35180289

RESUMO

Chemotactic migration is a fundamental cellular behavior relying on the coordinated flux of lipids and cargo proteins toward the leading edge. We found here that the core autophagy protein ATG9A plays a critical role in the chemotactic migration of several human cell lines, including highly invasive glioma cells. Depletion of ATG9A protein altered the formation of large and persistent filamentous actin (F-actin)-rich lamellipodia that normally drive directional migration. Using live-cell TIRF microscopy, we demonstrated that ATG9A-positive vesicles are targeted toward the migration front of polarized cells, where their exocytosis correlates with protrusive activity. Finally, we found that ATG9A was critical for efficient delivery of ß1 integrin to the leading edge and normal adhesion dynamics. Collectively, our data uncover a new function for ATG9A protein and indicate that ATG9A-positive vesicles are mobilized during chemotactic stimulation to facilitate expansion of the lamellipodium and its anchorage to the extracellular matrix.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Movimento Celular , Extensões da Superfície Celular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Actinas/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Quimiotaxia , Exocitose , Proteínas de Fluorescência Verde , Humanos , Integrina beta1/metabolismo , Glicoproteínas de Membrana/metabolismo , Pseudópodes/metabolismo , Reprodutibilidade dos Testes
7.
PLoS Pathog ; 18(2): e1010324, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35130324

RESUMO

The bacterial pathogen Shigella flexneri causes 270 million cases of bacillary dysentery worldwide every year, resulting in more than 200,000 deaths. S. flexneri pathogenic properties rely on its ability to invade epithelial cells and spread from cell to cell within the colonic epithelium. This dissemination process relies on actin-based motility in the cytosol of infected cells and formation of membrane protrusions that project into adjacent cells and resolve into double-membrane vacuoles (DMVs) from which the pathogen escapes, thereby achieving cell-to-cell spread. S. flexneri dissemination is facilitated by the type 3 secretion system (T3SS) through poorly understood mechanisms. Here, we show that the T3SS effector IpgD facilitates the resolution of membrane protrusions into DMVs during S. flexneri dissemination. The phosphatidylinositol 4-phosphatase activity of IpgD decreases PtdIns(4,5)P2 levels in membrane protrusions, thereby counteracting de novo cortical actin formation in protrusions, a process that restricts the resolution of protrusions into DMVs. Finally, using an infant rabbit model of shigellosis, we show that IpgD is required for efficient cell-to-cell spread in vivo and contributes to the severity of dysentery.


Assuntos
Proteínas de Bactérias/metabolismo , Extensões da Superfície Celular/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Shigella flexneri/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Actinas/metabolismo , Animais , Proteínas de Bactérias/genética , Extensões da Superfície Celular/microbiologia , Colo/microbiologia , Modelos Animais de Doenças , Disenteria Bacilar/microbiologia , Células HT29 , Interações Hospedeiro-Patógeno , Humanos , Monoéster Fosfórico Hidrolases/genética , Coelhos , Shigella flexneri/genética
8.
J Cell Sci ; 135(4)2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35099014

RESUMO

Cell migration is a complex process underlying physiological and pathological processes such as brain development and cancer metastasis. The autophagy-linked FYVE protein (ALFY; also known as WDFY3), an autophagy adaptor protein known to promote clearance of protein aggregates, has been implicated in brain development and neural migration during cerebral cortical neurogenesis in mice. However, a specific role of ALFY in cell motility and extracellular matrix adhesion during migration has not been investigated. Here, we reveal a novel role for ALFY in the endocytic pathway and in cell migration. We show that ALFY localizes to RAB5- and EEA1-positive early endosomes in a PtdIns(3)P-dependent manner and is highly enriched in cellular protrusions at the leading and lagging edge of migrating cells. We find that cells lacking ALFY have reduced attachment and altered protein levels and glycosylation of integrins, resulting in the inability to form a proper leading edge and loss of directional cell motility.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Extensões da Superfície Celular , Animais , Movimento Celular , Extensões da Superfície Celular/metabolismo , Endossomos/metabolismo , Células HeLa , Humanos , Camundongos
9.
Int J Mol Sci ; 23(2)2022 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-35054941

RESUMO

Clostridium botulinum produces the botulinum neurotoxin that causes botulism, a rare but potentially lethal paralysis. Endospores play an important role in the survival, transmission, and pathogenesis of C. botulinum. C. botulinum strains are very diverse, both genetically and ecologically. Group I strains are terrestrial, mesophilic, and produce highly heat-resistant spores, while Group II strains can be terrestrial (type B) or aquatic (type E) and are generally psychrotrophic and produce spores of moderate heat resistance. Group III strains are either terrestrial or aquatic, mesophilic or slightly thermophilic, and the heat resistance properties of their spores are poorly characterized. Here, we analyzed the sporulation dynamics in population, spore morphology, and other spore properties of 10 C. botulinum strains belonging to Groups I-III. We propose two distinct sporulation strategies used by C. botulinum Groups I-III strains, report their spore properties, and suggest a putative role for the exosporium in conferring high heat resistance. Strains within each physiological group produced spores with similar characteristics, likely reflecting adaptation to respective environmental habitats. Our work provides new information on the spores and on the population and single-cell level strategies in the sporulation of C. botulinum.


Assuntos
Botulismo/microbiologia , Extensões da Superfície Celular/fisiologia , Clostridium botulinum/fisiologia , Viabilidade Microbiana , Esporos Bacterianos/fisiologia , Extensões da Superfície Celular/ultraestrutura , Clostridium botulinum/ultraestrutura , Esporos Bacterianos/ultraestrutura
10.
Biophys J ; 121(1): 102-118, 2022 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-34861242

RESUMO

Orchestration of cell migration is essential for development, tissue regeneration, and the immune response. This dynamic process integrates adhesion, signaling, and cytoskeletal subprocesses across spatial and temporal scales. In mesenchymal cells, adhesion complexes bound to extracellular matrix mediate both biochemical signal transduction and physical interaction with the F-actin cytoskeleton. Here, we present a mathematical model that offers insight into both aspects, considering spatiotemporal dynamics of nascent adhesions, active signaling molecules, mechanical clutching, actin treadmilling, and nonmuscle myosin II contractility. At the core of the model is a positive feedback loop, whereby adhesion-based signaling promotes generation of barbed ends at, and protrusion of, the cell's leading edge, which in turn promotes formation and stabilization of nascent adhesions. The model predicts a switch-like transition and optimality of membrane protrusion, determined by the balance of actin polymerization and retrograde flow, with respect to extracellular matrix density. The model, together with new experimental measurements, explains how protrusion can be modulated by mechanical effects (nonmuscle myosin II contractility and adhesive bond stiffness) and F-actin turnover.


Assuntos
Actinas , Miosina Tipo II , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Extensões da Superfície Celular , Miosina Tipo II/metabolismo , Transdução de Sinais
11.
Curr Top Membr ; 88: 205-234, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34862027

RESUMO

Membrane protrusions are a critical facet of cell function. Mediating fundamental processes such as cell migration, cell-cell interactions, phagocytosis, as well as assessment and remodeling of the cell environment. Different protrusion types and morphologies can promote different cellular functions and occur downstream of distinct signaling pathways. As such, techniques to quantify and understand the inner workings of protrusion dynamics are critical for a comprehensive understanding of cell biology. In this chapter, we describe approaches to analyze cellular protrusions and correlate physical changes in cell morphology with biochemical signaling processes. We address methods to quantify and characterize protrusion types and velocity, mathematical approaches to predictive models of cytoskeletal changes, and implementation of protein engineering and biosensor design to dissect cell signaling driving protrusive activity. Combining these approaches allows cell biologists to develop a comprehensive understanding of the dynamics of membrane protrusions.


Assuntos
Extensões da Superfície Celular , Pseudópodes , Actinas , Movimento Celular , Citoesqueleto , Endocitose
12.
Science ; 374(6568): 717-723, 2021 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-34735222

RESUMO

The evolutionary origin of metazoan cell types such as neurons and muscles is not known. Using whole-body single-cell RNA sequencing in a sponge, an animal without nervous system and musculature, we identified 18 distinct cell types. These include nitric oxide­sensitive contractile pinacocytes, amoeboid phagocytes, and secretory neuroid cells that reside in close contact with digestive choanocytes that express scaffolding and receptor proteins. Visualizing neuroid cells by correlative x-ray and electron microscopy revealed secretory vesicles and cellular projections enwrapping choanocyte microvilli and cilia. Our data show a communication system that is organized around sponge digestive chambers, using conserved modules that became incorporated into the pre- and postsynapse in the nervous systems of other animals.


Assuntos
Evolução Biológica , Poríferos/citologia , Animais , Comunicação Celular , Extensões da Superfície Celular/ultraestrutura , Cílios/fisiologia , Cílios/ultraestrutura , Sistema Digestório/citologia , Mesoderma/citologia , Sistema Nervoso/citologia , Fenômenos Fisiológicos do Sistema Nervoso , Óxido Nítrico/metabolismo , Poríferos/genética , Poríferos/metabolismo , RNA-Seq , Vesículas Secretórias/ultraestrutura , Transdução de Sinais , Análise de Célula Única , Transcriptoma
13.
Cell Rep ; 37(7): 110008, 2021 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-34788623

RESUMO

Clathrin-mediated endocytosis (CME) is critical for cellular signal transduction, receptor recycling, and membrane homeostasis in mammalian cells. Acute depletion of cholesterol disrupts CME, motivating analysis of CME dynamics in the context of human disorders of cholesterol metabolism. We report that inhibition of post-squalene cholesterol biosynthesis impairs CME. Imaging of membrane bending dynamics and the CME pit ultrastructure reveals prolonged clathrin pit lifetimes and shallow clathrin-coated structures, suggesting progressive impairment of curvature generation correlates with diminishing sterol abundance. Sterol structural requirements for efficient CME include 3' polar head group and B-ring conformation, resembling the sterol structural prerequisites for tight lipid packing and polarity. Furthermore, Smith-Lemli-Opitz fibroblasts with low cholesterol abundance exhibit deficits in CME-mediated transferrin internalization. We conclude that sterols lower the energetic costs of membrane bending during pit formation and vesicular scission during CME and suggest that reduced CME activity may contribute to cellular phenotypes observed within disorders of cholesterol metabolism.


Assuntos
Vesículas Revestidas por Clatrina/metabolismo , Endocitose/fisiologia , Esteróis/farmacologia , Extensões da Superfície Celular/metabolismo , Extensões da Superfície Celular/fisiologia , Colesterol/metabolismo , Clatrina/metabolismo , Fibroblastos/metabolismo , Células HEK293 , Humanos , Metabolismo dos Lipídeos/fisiologia , Lipídeos/fisiologia , Proteínas de Membrana/metabolismo , Receptores da Transferrina/metabolismo , Esteróis/metabolismo
14.
Cells ; 10(11)2021 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-34831465

RESUMO

Currently, breast cancer patients are classified uniquely according to the expression level of hormone receptors, and human epidermal growth factor receptor 2 (HER2). This coarse classification is insufficient to capture the phenotypic complexity and heterogeneity of the disease. A methodology was developed for absolute quantification of receptor surface density ρR, and molecular interaction (dimerization), as well as the associated heterogeneities, of HER2 and its family member, the epidermal growth factor receptor (EGFR) in the plasma membrane of HER2 overexpressing breast cancer cells. Quantitative, correlative light microscopy (LM) and liquid-phase electron microscopy (LPEM) were combined with quantum dot (QD) labeling. Single-molecule position data of receptors were obtained from scanning transmission electron microscopy (STEM) images of intact cancer cells. Over 280,000 receptor positions were detected and statistically analyzed. An important finding was the subcellular heterogeneity in heterodimer shares with respect to plasma membrane regions with different dynamic properties. Deriving quantitative information about EGFR and HER2 ρR, as well as their dimer percentages, and the heterogeneities thereof, in single cancer cells, is potentially relevant for early identification of patients with HER2 overexpressing tumors comprising an enhanced share of EGFR dimers, likely increasing the risk for drug resistance, and thus requiring additional targeted therapeutic strategies.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/ultraestrutura , Microscopia Eletrônica , Multimerização Proteica , Receptor ErbB-2/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Extensões da Superfície Celular/metabolismo , Receptores ErbB/metabolismo , Feminino , Humanos , Modelos Biológicos , Pontos Quânticos
15.
PLoS Comput Biol ; 17(11): e1009576, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34748539

RESUMO

Advances in genetic engineering technologies have allowed the construction of artificial genetic circuits, which have been used to generate spatial patterns of differential gene expression. However, the question of how cells can be programmed, and how complex the rules need to be, to achieve a desired tissue morphology has received less attention. Here, we address these questions by developing a mathematical model to study how cells can collectively grow into clusters with different structural morphologies by secreting diffusible signals that can influence cellular growth rates. We formulate how growth regulators can be used to control the formation of cellular protrusions and how the range of achievable structures scales with the number of distinct signals. We show that a single growth inhibitor is insufficient for the formation of multiple protrusions but may be achieved with multiple growth inhibitors, and that other types of signals can regulate the shape of protrusion tips. These examples illustrate how our approach could potentially be used to guide the design of regulatory circuits for achieving a desired target structure.


Assuntos
Proliferação de Células/fisiologia , Forma Celular/fisiologia , Técnicas de Reprogramação Celular/métodos , Modelos Biológicos , Animais , Agregação Celular/fisiologia , Comunicação Celular/fisiologia , Extensões da Superfície Celular/fisiologia , Técnicas de Reprogramação Celular/estatística & dados numéricos , Biologia Computacional , Simulação por Computador , Redes Reguladoras de Genes , Engenharia Genética/métodos , Engenharia Genética/estatística & dados numéricos , Inibidores do Crescimento/fisiologia , Humanos , Morfogênese/fisiologia , Biologia Sintética
16.
J Vis Exp ; (177)2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34779432

RESUMO

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the construction and regeneration of tissues, and is critical in embryonic development, immunological responses, and wound healing. Dysregulation of cell motility contributes to pathological disorders, such as chronic inflammation and cancer metastasis. Cell migration, tissue invasion, axon, and dendrite outgrowth all initiate with actin polymerization-mediated cell-edge protrusions. Here, we describe a simple, efficient, time-saving method for the imaging and quantitative analysis of cell-edge protrusion dynamics during spreading. This method measures discrete features of cell-edge membrane dynamics, such as protrusions, retractions, and ruffles, and can be used to assess how manipulations of key actin regulators impact cell-edge protrusions in diverse contexts.


Assuntos
Extensões da Superfície Celular , Microscopia , Actinas/metabolismo , Membrana Celular/metabolismo , Movimento Celular/fisiologia
17.
Appl Opt ; 60(25): G10-G18, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34613190

RESUMO

Understanding biological responses to directed energy (DE) is critical to ensure the safety of personnel within the Department of Defense. At the Air Force Research Laboratory, we have developed or adapted advanced optical imaging systems that quantify biophysical responses to DE. One notable cellular response to DE exposure is the formation of blebs, or semi-spherical protrusions of the plasma membrane in living cells. In this work, we demonstrate the capacity of quantitative phase imaging (QPI) to both visualize and quantify the formation of membrane blebs following DE exposure. QPI is an interferometric imaging tool that uses optical path length as a label-free contrast mechanism and is sensitive to the non-aqueous mass density, or dry mass, of living cells. Blebs from both CHO-K1 and U937 cells were generated after exposure to a series of 600 ns, 21.2 kV/cm electric pulses. These blebs were visualized in real time, and their dry mass relative to the rest of the cell body was quantified as a function of time. It is our hope that this system will lead to an improved understanding of both DE-induced and apoptotic blebbing.


Assuntos
Fenômenos Biofísicos/fisiologia , Membrana Celular , Extensões da Superfície Celular , Microscopia de Interferência/métodos , Imagem Óptica/métodos , Animais , Células CHO , Extensões da Superfície Celular/fisiologia , Extensões da Superfície Celular/ultraestrutura , Cricetulus , Estimulação Elétrica/métodos , Desenho de Equipamento , Humanos , Microscopia de Interferência/instrumentação , Imagem Óptica/instrumentação , Tamanho das Organelas , Células U937
18.
Mol Microbiol ; 116(5): 1328-1346, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34608697

RESUMO

Shigella flexneri is a gram-negative bacterial pathogen that causes dysentery. Critical for disease is the ability of Shigella to use an actin-based motility (ABM) process to spread between cells of the colonic epithelium. ABM transports bacteria to the periphery of host cells, allowing the formation of plasma membrane protrusions that mediate spread to adjacent cells. Here we demonstrate that efficient protrusion formation and cell-to-cell spread of Shigella involves bacterial stimulation of host polarized exocytosis. Using an exocytic probe, we found that exocytosis is locally upregulated in bacterial protrusions in a manner that depends on the Shigella type III secretion system. Experiments involving RNA interference (RNAi) indicate that efficient bacterial protrusion formation and spread require the exocyst, a mammalian multi-protein complex known to mediate polarized exocytosis. In addition, the exocyst component Exo70 and the exocyst regulator RalA were recruited to Shigella protrusions, suggesting that bacteria manipulate exocyst function. Importantly, RNAi-mediated depletion of exocyst proteins or RalA reduced the frequency of protrusion formation and also the lengths of protrusions, demonstrating that the exocyst controls both the initiation and elongation of protrusions. Collectively, our results reveal that Shigella co-opts the exocyst complex to disseminate efficiently in host cell monolayers.


Assuntos
Extensões da Superfície Celular/metabolismo , Disenteria Bacilar/microbiologia , Exocitose , Shigella flexneri/fisiologia , Sistemas de Secreção Tipo III/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas ral de Ligação ao GTP/metabolismo , Actinas/metabolismo , Proteínas de Bactérias/metabolismo , Células CACO-2 , Extensões da Superfície Celular/microbiologia , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Interferência de RNA
19.
Exp Cell Res ; 408(1): 112852, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34599931

RESUMO

There is a strong association between arsenic exposure and lung cancer development, however, the mechanism by which arsenic exposure leads to carcinogenesis is not clear. In our previous study, we observed that when BEAS-2B cells are chronically exposed to arsenic, there is an increase in secreted TGFα, as well as an increase in EGFR expression and activity. Further, these changes were broadly accompanied with an increase in cell migration. The overarching goal of this study was to acquire finer resolution of the arsenic-dependent changes in cell migration, as well as to understand the role of increased EGFR expression and activity levels in the underlying mechanisms of cell migration. To do this, we used a combination of biochemical and single cell assays, and observed chronic arsenic treatment enhancing cell migration by increasing cell speed, cell persistence and cell protrusion length. All three parameters were further increased by the addition of TGFα, indicating EGFR activity is sufficient to enhance those aspects of cell migration. In contrast, EGFR activity was necessary for the increase in cell speed, as it was reversed with an EGFR inhibitor, AG1478, but was not necessary to enhance persistence and protrusion length. From these data, we were able to isolate both EGFR-dependent and -independent features of cell migration that were enhanced by chronic arsenic exposure.


Assuntos
Movimento Celular/fisiologia , Extensões da Superfície Celular/metabolismo , Transformação Celular Neoplásica/metabolismo , Células Epiteliais/metabolismo , Carcinogênese/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Transdução de Sinais/fisiologia
20.
J Neuroimmunol ; 361: 577752, 2021 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-34715591

RESUMO

Neuromyelitis Optica (NMO) is an autoimmune inflammatory disease that affects the optic nerves and spinal cord. The autoantibody is generated against the abundant water channel protein of the brain, Aquaporin 4 (AQP4). Of the two isoforms of AQP4, the shorter one (M23) often exists as a supramolecular assembly known as an orthogonal array of particles (OAPs). There have been debates about the fate of these AQP4 clusters upon binding to the antibody, the exact mechanism of its turnover, and the proteins associated with the process. Recently several clinical cases of NMO were reported delineating the effect of Rituximab (RTX) therapy. Extending these reports at the cell signaling level, we developed a glioma based cellular model that mimicked antibody binding and helped us track the subsequent events including a variation of AQP4 levels, alterations in cellular morphology, and the changes in downstream signaling cascades. Our results revealed the extent of perturbations in the signaling pathways related to stress involving ERK, JNK, and AKT1 together with markers for cell death. We could also decipher the possible routes of degradation of AQP4, post-exposure to antibody. We further investigated the effect of autoantibody on AQP4 transcriptional level and involvement of FOXO3a and miRNA-145 in the regulation of transcription. This study highlights the differential outcome at the cellular level when treated with the serum of the same patient pre and post RTX therapy and for the first time mechanistically describes the effect of RTX.


Assuntos
Aquaporina 4/metabolismo , Autoanticorpos/sangue , Autoantígenos/metabolismo , Imunoglobulina G/sangue , Neuromielite Óptica/metabolismo , Rituximab/uso terapêutico , Adulto , Aquaporina 4/genética , Aquaporina 4/imunologia , Autoantígenos/genética , Autoantígenos/imunologia , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/ultraestrutura , Forma Celular , Extensões da Superfície Celular/ultraestrutura , Feminino , Proteína Forkhead Box O3/fisiologia , Glioblastoma , Humanos , Leupeptinas/farmacologia , Masculino , MicroRNAs/genética , Microscopia Confocal , Neuromielite Óptica/tratamento farmacológico , Neuromielite Óptica/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais , Análise de Célula Única , Transcrição Genética , Adulto Jovem
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