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1.
Nat Cell Biol ; 22(3): 297-309, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32066907

RESUMO

Non-centrosomal microtubule-organizing centres (ncMTOCs) have a variety of roles that are presumed to serve the diverse functions of the range of cell types in which they are found. ncMTOCs are diverse in their composition, subcellular localization and function. Here we report a perinuclear MTOC in Drosophila fat body cells that is anchored by the Nesprin homologue Msp300 at the cytoplasmic surface of the nucleus. Msp300 recruits the microtubule minus-end protein Patronin, a calmodulin-regulated spectrin-associated protein (CAMSAP) homologue, which functions redundantly with Ninein to further recruit the microtubule polymerase Msps-a member of the XMAP215 family-to assemble non-centrosomal microtubules and does so independently of the widespread microtubule nucleation factor γ-Tubulin. Functionally, the fat body ncMTOC and the radial microtubule arrays that it organizes are essential for nuclear positioning and for secretion of basement membrane components via retrograde dynein-dependent endosomal trafficking that restricts plasma membrane growth. Together, this study identifies a perinuclear ncMTOC with unique architecture that regulates microtubules, serving vital functions.


Assuntos
Membrana Basal/metabolismo , Núcleo Celular , Centro Organizador dos Microtúbulos/fisiologia , Actinas/fisiologia , Animais , Membrana Celular , Núcleo Celular/ultraestrutura , Centrossomo , Drosophila/metabolismo , Drosophila/ultraestrutura , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Dineínas/fisiologia , Endossomos/metabolismo , Corpo Adiposo/metabolismo , Corpo Adiposo/ultraestrutura , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Centro Organizador dos Microtúbulos/ultraestrutura , Microtúbulos/fisiologia , Proteínas Musculares/metabolismo , Tubulina (Proteína)/fisiologia
2.
PLoS Comput Biol ; 16(1): e1007250, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31929522

RESUMO

Actin protrusion dynamics plays an important role in the regulation of three-dimensional (3D) cell migration. Cells form protrusions that adhere to the surrounding extracellular matrix (ECM), mechanically probe the ECM and contract in order to displace the cell body. This results in cell migration that can be directed by the mechanical anisotropy of the ECM. However, the subcellular processes that regulate protrusion dynamics in 3D cell migration are difficult to investigate experimentally and therefore not well understood. Here, we present a computational model of cell migration through a degradable viscoelastic ECM. This model is a 2D representation of 3D cell migration. The cell is modeled as an active deformable object that captures the viscoelastic behavior of the actin cortex and the subcellular processes underlying 3D cell migration. The ECM is regarded as a viscoelastic material, with or without anisotropy due to fibrillar strain stiffening, and modeled by means of the meshless Lagrangian smoothed particle hydrodynamics (SPH) method. ECM degradation is captured by local fluidization of the material and permits cell migration through the ECM. We demonstrate that changes in ECM stiffness and cell strength affect cell migration and are accompanied by changes in number, lifetime and length of protrusions. Interestingly, directly changing the total protrusion number or the average lifetime or length of protrusions does not affect cell migration. A stochastic variability in protrusion lifetime proves to be enough to explain differences in cell migration velocity. Force-dependent adhesion disassembly does not result in faster migration, but can make migration more efficient. We also demonstrate that when a number of simultaneous protrusions is enforced, the optimal number of simultaneous protrusions is one or two, depending on ECM anisotropy. Together, the model provides non-trivial new insights in the role of protrusions in 3D cell migration and can be a valuable contribution to increase the understanding of 3D cell migration mechanics.


Assuntos
Actinas , Movimento Celular/fisiologia , Matriz Extracelular , Modelos Biológicos , Actinas/química , Actinas/metabolismo , Actinas/fisiologia , Biologia Computacional , Simulação por Computador , Elasticidade/fisiologia , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Viscosidade
3.
J Orthop Res ; 38(1): 105-116, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31228280

RESUMO

Traditional tendon-to-bone repair where the tendon is reattached to bone via suture anchors often results in disorganized scar production rather than the formation of a zonal insertion. In contrast, ligament reconstructions where tendon grafts are passed through bone tunnels can yield zonal tendon-to-bone attachments between the graft and adjacent bone. Therefore, ligament reconstructions can be used to study mechanisms that regulate zonal tendon-to-bone repair in the adult. Anterior cruciate ligament (ACL) reconstructions are one of the most common reconstruction procedures and while we know that cells from outside the graft produce the attachments, we have not yet established specific cell populations that give rise to this tissue. To address this knowledge gap, we performed ACL reconstructions in lineage tracing mice where α-smooth muscle actin (αSMACreERT2) was used to label αSMA-expressing progenitors within the bone marrow that produced zonal attachments. Expression of αSMA was increased during early stages of the repair process such that the contribution of SMA-labeled cells to the tunnel integration was highest when tamoxifen was delivered in the first week post-surgery. The zonal attachments shared features with normal entheses, including tidemarks oriented perpendicularly to collagen fibers, Col1a1-expressing cells, alkaline phosphatase activity, and proteoglycan-rich staining. Finally, the integration strength increased with time, requiring 112% greater force to remove the graft from the tunnel at 28 days compared with 14 days post-surgery. Future studies will target these progenitor cells to define the pathways that regulate zonal tendon-to-bone repair in the adult. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:105-116, 2020.


Assuntos
Actinas/análise , Reconstrução do Ligamento Cruzado Anterior/métodos , Células da Medula Óssea/metabolismo , Osso e Ossos/cirurgia , Células-Tronco/metabolismo , Tendões/cirurgia , Actinas/fisiologia , Animais , Células da Medula Óssea/química , Camundongos , Cicatrização
4.
Int J Mol Sci ; 20(23)2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31801239

RESUMO

Many biological processes are triggered or driven by mechanical forces in the cytoskeletal network, but these transducing forces have rarely been assessed. Striated muscle, with its well-organized structure provides an opportunity to assess intracellular forces using small-angle X-ray fiber diffraction. We present a new methodology using Monte Carlo simulations of muscle contraction in an explicit 3D sarcomere lattice to predict the fiber deformations and length changes along thin filaments during contraction. Comparison of predicted diffraction patterns to experimental meridional X-ray reflection profiles allows assessment of the stepwise changes in intermonomer spacings and forces in the myofilaments within living muscle cells. These changes along the filament length reflect the effect of forces from randomly attached crossbridges. This approach enables correlation of the molecular events, such as the current number of attached crossbridges and the distributions of crossbridge forces to macroscopic measurements of force and length changes during muscle contraction. In addition, assessments of fluctuations in local forces in the myofilaments may reveal how variations in the filament forces acting on signaling proteins in the sarcomere M-bands and Z-discs modulate gene expression, protein synthesis and degradation, and as well to mechanisms of adaptation of muscle in response to changes in mechanical loading.


Assuntos
Citoesqueleto de Actina/fisiologia , Actinas/fisiologia , Contração Isométrica/fisiologia , Músculo Estriado/fisiologia , Miosinas/fisiologia , Sarcômeros/fisiologia , Citoesqueleto de Actina/ultraestrutura , Actinas/ultraestrutura , Animais , Simulação por Computador , Conectina/fisiologia , Conectina/ultraestrutura , Modelos Biológicos , Método de Monte Carlo , Músculo Estriado/diagnóstico por imagem , Miosinas/ultraestrutura , Rana catesbeiana/fisiologia , Sarcômeros/ultraestrutura , Espalhamento a Baixo Ângulo , Técnicas de Cultura de Tecidos , Difração de Raios X
5.
Proc Natl Acad Sci U S A ; 116(48): 23894-23900, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31719206

RESUMO

In this work, we explore fundamental energy requirements during mammalian cell movement. Starting with the conservation of mass and momentum for the cell cytosol and the actin-network phase, we develop useful identities that compute dissipated energies during extensions of the cell boundary. We analyze 2 complementary mechanisms of cell movement: actin-driven and water-driven. The former mechanism occurs on 2-dimensional cell-culture substrate without appreciable external hydraulic resistance, while the latter mechanism is prominent in confined channels where external hydraulic resistance is high. By considering various forms of energy input and dissipation, we find that the water-driven cell-migration mechanism is inefficient and requires more energy. However, in environments with sufficiently high hydraulic resistance, the efficiency of actin-polymerization-driven cell migration decreases considerably, and the water-based mechanism becomes more efficient. Hence, the most efficient way for cells to move depends on the physical environment. This work can be extended to higher dimensions and has implication for understanding energetics of morphogenesis in early embryonic development and cancer-cell metastasis and provides a physical basis for understanding changing metabolic requirements for cell movement in different conditions.


Assuntos
Movimento Celular , Actinas/metabolismo , Actinas/fisiologia , Permeabilidade da Membrana Celular , Forma Celular , Metabolismo Energético , Modelos Biológicos , Polimerização , Água/metabolismo
6.
EMBO Rep ; 20(12): e48896, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31584242

RESUMO

The obligate intracellular parasites Toxoplasma gondii and Plasmodium spp. invade host cells by injecting a protein complex into the membrane of the targeted cell that bridges the two cells through the assembly of a ring-like junction. This circular junction stretches while the parasites apply a traction force to pass through, a step that typically concurs with transient constriction of the parasite body. Here we analyse F-actin dynamics during host cell invasion. Super-resolution microscopy and real-time imaging highlighted an F-actin pool at the apex of pre-invading parasite, an F-actin ring at the junction area during invasion but also networks of perinuclear and posteriorly localised F-actin. Mutant parasites with dysfunctional acto-myosin showed significant decrease of junctional and perinuclear F-actin and are coincidently affected in nuclear passage through the junction. We propose that the F-actin machinery eases nuclear passage by stabilising the junction and pushing the nucleus through the constriction. Our analysis suggests that the junction opposes resistance to the passage of the parasite's nucleus and provides the first evidence for a dual contribution of actin-forces during host cell invasion by apicomplexan parasites.


Assuntos
Actinas/fisiologia , Interações Hospedeiro-Parasita/fisiologia , Plasmodium falciparum/fisiologia , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/fisiologia , Toxoplasma/parasitologia , Toxoplasma/patogenicidade , Actinas/genética , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Núcleo Celular/parasitologia , Núcleo Celular/fisiologia , Células Cultivadas , Técnicas de Inativação de Genes , Humanos , Merozoítos/genética , Merozoítos/patogenicidade , Merozoítos/fisiologia , Modelos Biológicos , Mutação , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Transdução de Sinais , Toxoplasma/genética , Virulência/fisiologia
7.
Nat Commun ; 10(1): 4651, 2019 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-31604948

RESUMO

Mammalian oocytes assemble a bipolar acentriolar microtubule spindle to segregate chromosomes during asymmetric division. There is increasing evidence that actin in the spindle interior not only participates in spindle migration and positioning but also protects oocytes from chromosome segregation errors leading to aneuploidy. Here we show that actin is an integral component of the meiotic machinery that closely interacts with microtubules during all major events of human oocyte maturation from the time point of spindle assembly till polar body extrusion and metaphase arrest. With the aid of drugs selectively affecting cytoskeleton dynamics and transiently disturbing the integrity of the two cytoskeleton systems, we identify interdependent structural rearrangements indicative of a close communication between actin and microtubules as fundamental feature of human oocytes. Our data support a model of actin-microtubule interplay that is essential for bipolar spindle assembly and correct partitioning of the nuclear genome in human oocyte meiosis.


Assuntos
Actinas/fisiologia , Segregação de Cromossomos/fisiologia , Oócitos/citologia , Fuso Acromático/metabolismo , Feminino , Humanos , Meiose , Microtúbulos/fisiologia , Oócitos/ultraestrutura , Corpos Polares/citologia , Corpos Polares/metabolismo , Corpos Polares/ultraestrutura , Fuso Acromático/ultraestrutura , Tubulina (Proteína)/metabolismo
8.
Am J Physiol Cell Physiol ; 317(6): C1304-C1312, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31553646

RESUMO

Skeletal muscle weakness is associated with oxidative stress and oxidative posttranslational modifications on contractile proteins. There is indirect evidence that reactive oxygen/nitrogen species (ROS/RNS) affect skeletal muscle myofibrillar function, although the details of the acute effects of ROS/RNS on myosin-actin interactions are not known. In this study, we examined the effects of peroxynitrite (ONOO-) on the contractile properties of individual skeletal muscle myofibrils by monitoring myofibril-induced displacements of an atomic force cantilever upon activation and relaxation. The isometric force decreased by ~50% in myofibrils treated with the ONOO- donor (SIN-1) or directly with ONOO-, which was independent of the cross-bridge abundancy condition (i.e., rigor or relaxing condition) during SIN-1 or ONOO- treatment. The force decrease was attributed to an increase in the cross-bridge detachment rate (gapp) in combination with a conservation of the force redevelopment rate (kTr) and hence, an increase in the population of cross-bridges transitioning from force-generating to non-force-generating cross-bridges during steady-state. Taken together, the results of this study provide important information on how ROS/RNS affect myofibrillar force production which may be of importance for conditions where increased oxidative stress is part of the pathophysiology.


Assuntos
Contração Isométrica/efeitos dos fármacos , Molsidomina/análogos & derivados , Miofibrilas/efeitos dos fármacos , Miosinas/antagonistas & inibidores , Doadores de Óxido Nítrico/farmacologia , Oxidantes/farmacologia , Ácido Peroxinitroso/farmacologia , Actinas/antagonistas & inibidores , Actinas/química , Actinas/fisiologia , Animais , Contração Isométrica/fisiologia , Molsidomina/química , Molsidomina/farmacologia , Miofibrilas/fisiologia , Miofibrilas/ultraestrutura , Miosinas/química , Miosinas/fisiologia , Doadores de Óxido Nítrico/química , Estresse Oxidativo , Músculos Psoas/efeitos dos fármacos , Músculos Psoas/fisiologia , Músculos Psoas/ultraestrutura , Coelhos , Técnicas de Cultura de Tecidos
9.
mSphere ; 4(5)2019 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-31484739

RESUMO

Urgency urinary incontinence (UUI) and overactive bladder (OAB) can both potentially be influenced by commensal and urinary tract infection-associated bacteria. The sensing of bladder filling involves interplay between various components of the nervous system, eventually resulting in contraction of the detrusor muscle during micturition. This study models host responses to various urogenital bacteria, first by using urothelial bladder cell lines and then with myofibroblast contraction assays. To measure responses, we examined Ca2+ influx, gene expression, and alpha smooth muscle actin deposition assays. Organisms such as Escherichia coli and Gardnerella vaginalis were found to strongly induce Ca2+ influx and contraction, whereas Lactobacillus crispatus and L. gasseri did not induce this response. Additionally, supernatants from lactobacilli impeded Ca2+ influx and contraction induced by uropathogens. Upon further investigation of factors associated with purinergic signaling pathways, the Ca2+ influx and contraction of cells correlated with the amount of extracellular ATP produced by E. coli Certain lactobacilli appear to mitigate this response by utilizing extracellular ATP or producing inhibitory compounds that may act as a receptor agonist or Ca2+ channel blocker. These findings suggest that members of the urinary microbiota may be influencing UUI or OAB.IMPORTANCE The ability of uropathogenic bacteria to release excitatory compounds, such as ATP, may act as a virulence factor to stimulate signaling pathways that could have profound effects on the urothelium, perhaps extending to the vagina. This may be countered by the ability of certain commensal urinary microbiota constituents, such as lactobacilli. Further understanding of these interactions is important for the treatment and prevention of UUI and OAB. The clinical implications may require a more targeted approach to enhance the commensal bacteria and reduce ATP release by pathogens.


Assuntos
Trifosfato de Adenosina/metabolismo , Bactérias/metabolismo , Cálcio/metabolismo , Miofibroblastos/citologia , Bexiga Urinária/microbiologia , Actinas/fisiologia , Bactérias/patogenicidade , Linhagem Celular , Colágeno/fisiologia , Humanos , Lactobacillales , Microbiota , Contração Muscular , Miofibroblastos/microbiologia , Simbiose , Bexiga Urinária/fisiologia , Urotélio/citologia
10.
PLoS One ; 14(8): e0220810, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31437196

RESUMO

Freely crawling cells are often viewed as randomly moving Brownian particles but they generally exhibit some directional persistence. This property is often related to their zigzag motile behaviors that can be described as a noisy but temporally structured sequence of "runs" and "turns." However, its underlying biophysical mechanism is largely unexplored. Here, we carefully investigate the collective actin wave dynamics associated with the zigzag-crawling movements of microglia (as primary brain immune cells) employing a number of different quantitative imaging modalities including synthetic aperture microscopy and optical diffraction tomography, as well as conventional fluorescence imaging and scanning electron microscopy. Interestingly, we find that microglia exhibit two distinct types of actin waves working at two quite different time scales and locations, and they seem to serve different purposes. One type of actin waves is fast "peripheral ruffles" arising spontaneously with an oscillating period of about 6 seconds at some portion of the leading edge of crawling microglia, where the vigorously biased peripheral ruffles seem to set the direction of a new turn (in 2-D free space). When the cell turning events are inhibited with a physical confinement (in 1-D track), the peripheral ruffles still exist at the leading edge with no bias but showing phase coherence in the cell crawling direction. The other type is "dorsal actin waves" which also exhibits an oscillatory behavior but with a much longer period of around 2 minutes compared to the fast "peripheral ruffles". Dorsal actin waves (whether the cell turning events are inhibited or not) initiate in the lamellipodium just behind the leading edge, travelling down toward the core region of the cell and disappear. Such dorsal wave propagations seem to be correlated with migration of the cell. Thus, we may view the dorsal actin waves are connected with the "run" stage of cell body, whereas the fast ruffles at the leading front are involved in the "turn" stage.


Assuntos
Actinas/fisiologia , Movimento Celular/fisiologia , Microglia/fisiologia , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Estruturas da Membrana Celular/metabolismo , Fibroblastos/metabolismo , Microglia/metabolismo , Pseudópodes/metabolismo , Ratos , Ratos Sprague-Dawley
11.
Nat Commun ; 10(1): 2850, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253801

RESUMO

Cancer cells secrete matrix metalloproteinases to remodel the extracellular matrix, which enables them to overcome tissue barriers and form metastases. The membrane-bound matrix metalloproteinase MT1-MMP (MMP14) is internalized by endocytosis and recycled in endosomal compartments. It is largely unknown how endosomal sorting and recycling of MT1-MMP are controlled. Here, we show that the endosomal protein WDFY2 controls the recycling of MT1-MMP. WDFY2 localizes to endosomal tubules by binding to membranes enriched in phosphatidylinositol 3-phosphate (PtdIns3P). We identify the v-SNARE VAMP3 as an interaction partner of WDFY2. WDFY2 knockout causes a strong redistribution of VAMP3 into small vesicles near the plasma membrane. This is accompanied by increased, VAMP3-dependent secretion of MT1-MMP, enhanced degradation of extracellular matrix, and increased cell invasion. WDFY2 is frequently lost in metastatic cancers, most predominantly in ovarian and prostate cancer. We propose that WDFY2 acts as a tumor suppressor by serving as a gatekeeper for VAMP3 recycling.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metaloproteinases da Matriz/metabolismo , Invasividade Neoplásica , Proteína 3 Associada à Membrana da Vesícula/metabolismo , Actinas/fisiologia , Linhagem Celular Tumoral , Membrana Celular , Exocitose/fisiologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Metaloproteinases da Matriz/genética , Microtúbulos , Fosfatos de Fosfatidilinositol/fisiologia , Transporte Proteico , Proteína 3 Associada à Membrana da Vesícula/genética , Proteínas rab4 de Ligação ao GTP/genética , Proteínas rab4 de Ligação ao GTP/metabolismo
12.
Biochemistry (Mosc) ; 84(6): 583-592, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31238858

RESUMO

Actin plays an important role in cellular adhesion, muscle and non-muscle contractility, migration, polarization, mitosis, and meiosis. Investigation of specific mechanisms underlying these processes is essential not only for fundamental research but also for clinical applications, since modulations of actin isoforms are directly or indirectly correlate with severe pathologies. In this review we summarize the isoform-specific functions of actin associated with adhesion structures, motility and division of normal and tumor cells; alterations of the expression and structural organization of actin isoforms in normal and tumor cells. Selective regulation of cytoplasmic ß- or γ-actin expression determines functional diversity between isoforms: ß-actin plays the predominant role in contraction and intercellular adhesion, and γ-actin is responsible for the cellular plasticity and motility. Similar data were obtained in different epithelial and mesenchymal neoplastic cell cultures, as well as in immunomorphological comparison of normal human tissues with tumor analogues. Reorganization of the actin cytoskeleton and cell-cell contacts is essential for proliferation control and acquisition of invasiveness in epithelial tumors.


Assuntos
Actinas/fisiologia , Isoformas de Proteínas/fisiologia , Actinas/química , Animais , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Transformação Celular Neoplásica , Citoplasma/metabolismo , Citosol/metabolismo , Humanos , Mamíferos , Isoformas de Proteínas/química , Relação Estrutura-Atividade
13.
Cell ; 177(6): 1463-1479.e18, 2019 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-31080065

RESUMO

Segregation of maternal determinants within the oocyte constitutes the first step in embryo patterning. In zebrafish oocytes, extensive ooplasmic streaming leads to the segregation of ooplasm from yolk granules along the animal-vegetal axis of the oocyte. Here, we show that this process does not rely on cortical actin reorganization, as previously thought, but instead on a cell-cycle-dependent bulk actin polymerization wave traveling from the animal to the vegetal pole of the oocyte. This wave functions in segregation by both pulling ooplasm animally and pushing yolk granules vegetally. Using biophysical experimentation and theory, we show that ooplasm pulling is mediated by bulk actin network flows exerting friction forces on the ooplasm, while yolk granule pushing is achieved by a mechanism closely resembling actin comet formation on yolk granules. Our study defines a novel role of cell-cycle-controlled bulk actin polymerization waves in oocyte polarization via ooplasmic segregation.


Assuntos
Actinas/metabolismo , Ciclo Celular/fisiologia , Oócitos/metabolismo , Actinas/fisiologia , Animais , Polaridade Celular/fisiologia , Citoplasma/metabolismo , Gema de Ovo/fisiologia , Polimerização , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Zigoto
14.
Cells ; 8(4)2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-31003495

RESUMO

The cytoskeleton of animal cells is one of the most complicated and functionally versatile structures, involved in processes such as endocytosis, cell division, intra-cellular transport, motility, force transmission, reaction to external forces, adhesion and preservation, and adaptation of cell shape. These functions are mediated by three classical cytoskeletal filament types, as follows: Actin, microtubules, and intermediate filaments. The named filaments form a network that is highly structured and dynamic, responding to external and internal cues with a quick reorganization that is orchestrated on the time scale of minutes and has to be tightly regulated. Especially in brain tumors, the cytoskeleton plays an important role in spreading and migration of tumor cells. As the cytoskeletal organization and regulation is complex and many-faceted, this review aims to summarize the findings about cytoskeletal filament types, including substructures formed by them, such as lamellipodia, stress fibers, and interactions between intermediate filaments, microtubules and actin. Additionally, crucial regulatory aspects of the cytoskeletal filaments and the formed substructures are discussed and integrated into the concepts of cell motility. Even though little is known about the impact of cytoskeletal alterations on the progress of glioma, a final point discussed will be the impact of established cytoskeletal alterations in the cellular behavior and invasion of glioma.


Assuntos
Citoesqueleto/metabolismo , Citoesqueleto/fisiologia , Actinas/metabolismo , Actinas/fisiologia , Animais , Transporte Biológico , Divisão Celular , Movimento Celular , Forma Celular , Glioma/metabolismo , Humanos , Filamentos Intermediários/metabolismo , Filamentos Intermediários/fisiologia , Microtúbulos/metabolismo , Microtúbulos/fisiologia , Transdução de Sinais
15.
Mol Biol Cell ; 30(7): 838-850, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30865564

RESUMO

Integrin α6ß4 is an essential, dynamic adhesion receptor for laminin 332 found on epithelial cells, required for formation of strong cell-extracellular matrix (ECM) adhesion and induced migration, and coordinated by regions of the ß4C cytoplasmic domain. ß4E, a unique splice variant of ß4 expressed in normal tissue, contains a cytoplasmic domain of 231 amino acids with a unique sequence of 114 amino acids instead of ß4C's canonical 1089 amino acids. We determined the distribution of α6ß4E within normal human glandular epithelium and its regulation and effect on cellular biophysical properties. Canonical α6ß4C expressed in all basal cells, as expected, while α6ß4E expressed within a subset of luminal cells. α6ß4E expression was induced by three-dimensional culture conditions, activated Src, was reversible, and was stabilized by bortezomib, a proteasome inhibitor. α6ß4C expressed in all cells during induced migration, whereas α6ß4E was restricted to a subset of cells with increased kinetics of cell-cell and cell-ECM resistance properties. Interestingly, α6ß4E presented in "ringlike" patterns measuring ∼1.75 × 0.72 microns and containing actin and CD9 at cell-ECM locations. In contrast, α6ß4C expressed only within hemidesmosome-like structures containing BP180. Integrin α6ß4E is an inducible adhesion isoform in normal epithelial cells that can alter biophysical properties of cell-cell and cell-ECM interactions.


Assuntos
Adesão Celular/fisiologia , Movimento Celular/fisiologia , Integrina alfa6beta4/fisiologia , Actinas/metabolismo , Actinas/fisiologia , Linhagem Celular Tumoral , Desmossomos/metabolismo , Células Epiteliais/metabolismo , Matriz Extracelular/metabolismo , Hemidesmossomos/metabolismo , Humanos , Integrina alfa6beta4/genética , Integrina alfa6beta4/metabolismo , Integrinas/metabolismo , Laminina/metabolismo , Isoformas de Proteínas , Tetraspanina 29/metabolismo , Tetraspanina 29/fisiologia
16.
Mol Biol Cell ; 30(11): 1298-1313, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30893012

RESUMO

Fibroblasts transformed by the proto-oncogene Src form individual invadopodia that can spontaneously self-organize into large matrix-degrading superstructures called rosettes. However, the mechanisms by which the invadopodia can spatiotemporally reorganize their architecture is not well understood. Here, we show that Hic-5, a close relative of the scaffold protein paxillin, is essential for the formation and organization of rosettes in active Src-transfected NIH3T3 fibroblasts and cancer-associated fibroblasts. Live cell imaging, combined with domain-mapping analysis of Hic-5, identified critical motifs as well as phosphorylation sites that are required for the formation and dynamics of rosettes. Using pharmacological inhibition and mutant expression, we show that FAK kinase activity, along with its proximity to and potential interaction with the LD2,3 motifs of Hic-5, is necessary for rosette formation. Invadopodia dynamics and their coalescence into rosettes were also dependent on Rac1, formin, and myosin II activity. Superresolution microscopy revealed the presence of formin FHOD1 and INF2-mediated unbranched radial F-actin fibers emanating from invadopodia and rosettes, which may facilitate rosette formation. Collectively, our data highlight a novel role for Hic-5 in orchestrating the organization of invadopodia into higher-order rosettes, which may promote the localized matrix degradation necessary for tumor cell invasion.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteínas com Domínio LIM/metabolismo , Podossomos/metabolismo , Processamento de Proteína Pós-Traducional , Quinases da Família src/genética , Actinas/metabolismo , Actinas/fisiologia , Animais , Linhagem Celular Transformada , Proteínas do Citoesqueleto/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas Fetais/metabolismo , Proteínas Fetais/fisiologia , Fibroblastos/fisiologia , Proteína-Tirosina Quinases de Adesão Focal/fisiologia , Forminas/metabolismo , Forminas/fisiologia , Proteínas com Domínio LIM/fisiologia , Camundongos , Miosina Tipo II/metabolismo , Miosina Tipo II/fisiologia , Células NIH 3T3 , Neuropeptídeos/metabolismo , Neuropeptídeos/fisiologia , Fosforilação , Podossomos/fisiologia , Formação de Roseta , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/fisiologia
17.
J Pediatr Surg ; 54(10): 2028-2031, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30885557

RESUMO

BACKGROUND: Hirschsprung disease is a functional obstruction of the gastrointestinal tract due to the congenital absence of ganglion cells in the intermyenteric plexuses of the distal bowel. Gastrointestinal motility requires intact muscular layers as well as neural network connection to function properly. The Actin G2 gene is the main gene encoding actin gamma 2; a smooth muscle actin found in enteric tissues. AIM: This study of the Actin G2 gene in patients with Hirschsprung disease explores a possible molecular basis abnormal muscle function and post-surgical pseudo-obstruction in a group of patients. As far as the authors are aware, this is the first report confirming structural muscle deficits in Hirschsprung disease. PATIENTS AND METHODS: Ethical permission and informed consent were obtained. DNA was extracted from whole blood samples in 10 patients with histologically proven HSCR patients. PCR amplification of the ACTG2 gene, were subjected to semi-automated bi-directional sequencing analysis. Sequencing results were analyzed using FinchTV Sequence Alignment Software (http:/en.biosoft.net) to read chromatogram files. Further predicting bioinformatic investigation was obtained by PolyPhen 2 software to evaluate the significance of the observed amino acid changes. RESULTS: Ten new patients with similar HSCR phenotypes were prospectively investigated for variation in the Actin G2 gamma gene (ACTG2) variations. The results of ACTG2 gene analysis showing variation in exons 5, 8 and 10 of the ACTG2 gene in 7 of them (64%). The c.109C > G S345 L was the most frequent occurring in 6 of the 10 patients (54%), the c.171 A > A K119E in 2 and the significant c.108 T > G W357G variation in exon 10 (1 patient) Four patients had a combination of different variants in different exons which were less significant. Allele frequency on a control sample of the South African population showed no comparable pathology link scores (http://gnomad.broadinstitute.org/). Bioinformatic in silico modeling showed that the residue replacements in both variants (Lys to Glu and Trp to Gly) are highly non-conservative and variation can alter interactions within the protein conformation. CONCLUSIONS: The Actin smooth muscle gene showed variation in 64% of samples, indicating a reason for abnormal functioning muscle in many HSCR patients. Hirschsprung disease is part of a complex spectrum which also includes smooth muscle. LEVEL OF EVIDENCE: VI.


Assuntos
Actinas/genética , Doença de Hirschsprung , Actinas/fisiologia , Doença de Hirschsprung/genética , Doença de Hirschsprung/fisiopatologia , Humanos , Músculo Liso/fisiopatologia , Fenótipo , Estudos Prospectivos
18.
Mol Biol Cell ; 30(7): 851-862, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30601697

RESUMO

Ena/VASP tetramers are processive actin elongation factors that localize to diverse F-actin networks composed of filaments bundled by different cross-linking proteins, such as filopodia (fascin), lamellipodia (fimbrin), and stress fibers (α-actinin). Previously, we found that Ena takes approximately threefold longer processive runs on trailing barbed ends of fascin-bundled F-actin. Here, we used single-molecule TIRFM (total internal reflection fluorescence microscopy) and developed a kinetic model to further dissect Ena/VASP's processive mechanism on bundled filaments. We discovered that Ena's enhanced processivity on trailing barbed ends is specific to fascin bundles, with no enhancement on fimbrin or α-actinin bundles. Notably, Ena/VASP's processive run length increases with the number of both fascin-bundled filaments and Ena "arms," revealing avidity facilitates enhanced processivity. Consistently, Ena tetramers form more filopodia than mutant dimer and trimers in Drosophila culture cells. Moreover, enhanced processivity on trailing barbed ends of fascin-bundled filaments is an evolutionarily conserved property of Ena/VASP homologues, including human VASP and Caenorhabditis elegans UNC-34. These results demonstrate that Ena tetramers are tailored for enhanced processivity on fascin bundles and that avidity of multiple arms associating with multiple filaments is critical for this process. Furthermore, we discovered a novel regulatory process whereby bundle size and bundling protein specificity control activities of a processive assembly factor.


Assuntos
Actinas/metabolismo , Proteínas de Transporte/fisiologia , Proteínas do Citoesqueleto/metabolismo , Proteínas dos Microfilamentos/fisiologia , Citoesqueleto de Actina/metabolismo , Actinina/metabolismo , Actinas/fisiologia , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Citoesqueleto/metabolismo , Cinética , Camundongos , Proteínas dos Microfilamentos/metabolismo , Microscopia de Fluorescência/métodos , Elongação Traducional da Cadeia Peptídica/fisiologia , Ligação Proteica , Pseudópodes/fisiologia
19.
Nat Commun ; 10(1): 52, 2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30604763

RESUMO

The presence of aberrant number of centrioles is a recognized cause of aneuploidy and hallmark of cancer. Hence, centriole duplication needs to be tightly regulated. It has been proposed that centriole separation limits centrosome duplication. The mechanism driving centriole separation is poorly understood and little is known on how this is linked to centriole duplication. Here, we propose that actin-generated forces regulate centriole separation. By imposing geometric constraints via micropatterns, we were able to prove that precise acto-myosin force arrangements control direction, distance and time of centriole separation. Accordingly, inhibition of acto-myosin contractility impairs centriole separation. Alongside, we observed that organization of acto-myosin force modulates specifically the length of S-G2 phases of the cell cycle, PLK4 recruitment at the centrosome and centriole fidelity. These discoveries led us to suggest that acto-myosin forces might act in fundamental mechanisms of aneuploidy prevention.


Assuntos
Actinas/metabolismo , Ciclo Celular/fisiologia , Centríolos/metabolismo , Miosinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Actinas/fisiologia , Aneuploidia , Ciclo Celular/efeitos dos fármacos , Centríolos/fisiologia , Células HeLa , Humanos , Microscopia Intravital/métodos , Microscopia Confocal , Miosinas/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Timidina/farmacologia , Imagem com Lapso de Tempo/métodos
20.
Mol Biol Rep ; 46(1): 309-315, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30515694

RESUMO

The selection of a suitable reference gene is an important prerequisite for the precise analysis of target gene expression by real-time quantitative PCR (qPCR). The present study aims to explore the expression pattern of the Macrobrachium nipponense (M. nipponense) ß-actin gene under Aeromonas hydrophila bacterial infection conditions. The complete sequence of the ß-actin gene from M. nipponense was cloned by PCR. Identified and named ß-actin genes were searched in the NCBI database, and the characteristics of the ß-actin gene were analyzed using bioinformatics methods. The expression profiles of ß-actin under stresses challenged by bacteria after 3, 6, 12, 24 and 48 h were investigated by measuring Ct values by qPCR. The prokaryotic expression vector pET-30a-actin was constructed by PCR and recombinant DNA techniques. Fused protein was induced by IPTG in the transformed Escherichia coli BL21 (DE3). Recombinant rActin was purified by nickel column. The bioinformatics analysis result revealed that the deduced protein encoded by the ß-actin gene from M. nipponense had the highest homology with other prawns in the homologous assay (99%). The phylogenetic tree indicates that the ß-actin from M. nipponense and other crustaceans have a single cluster. The qPCR results revealed that a stable expression of ß-actin was observed in response to the A. hydrophila challenge for 3-48 h, and the Ct value was 22 ± 1.5. ß-actin was ranked as a stable gene after the bacterial challenge, which was selected as the appropriate reference gene in M. nipponense.


Assuntos
Actinas/genética , Perfilação da Expressão Gênica/métodos , Palaemonidae/genética , Actinas/fisiologia , Aeromonas hydrophila/patogenicidade , Animais , Infecções Bacterianas/genética , Infecções Bacterianas/metabolismo , Clonagem Molecular/métodos , Biologia Computacional/métodos , Perfilação da Expressão Gênica/normas , Palaemonidae/microbiologia , Palaemonidae/fisiologia , Filogenia , Reação em Cadeia da Polimerase em Tempo Real/métodos
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