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1.
Nat Commun ; 11(1): 4818, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968060

RESUMO

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


Assuntos
Movimento Celular/fisiologia , Extensões da Superfície Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Sistemas CRISPR-Cas , Adesão Celular , Linhagem Celular , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Técnicas de Inativação de Genes , Humanos , Cinética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/ultraestrutura , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/ultraestrutura , Miosinas/metabolismo , Pseudópodes/metabolismo , Receptor EphB2
2.
Nat Commun ; 11(1): 4479, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32900999

RESUMO

The giant protein titin is thought to be required for sarcomeric integrity in mature myocytes, but direct evidence for this hypothesis is limited. Here, we describe a mouse model in which Z-disc-anchored TTN is depleted in adult skeletal muscles. Inactivation of TTN causes sarcomere disassembly and Z-disc deformations, force impairment, myocyte de-stiffening, upregulation of TTN-binding mechanosensitive proteins and activation of protein quality-control pathways, concomitant with preferential loss of thick-filament proteins. Interestingly, expression of the myosin-bound Cronos-isoform of TTN, generated from an alternative promoter not affected by the targeting strategy, does not prevent deterioration of sarcomere formation and maintenance. Finally, we demonstrate that loss of Z-disc-anchored TTN recapitulates muscle remodeling in critical illness 'myosinopathy' patients, characterized by TTN-depletion and loss of thick filaments. We conclude that full-length TTN is required to integrate Z-disc and A-band proteins into the mature sarcomere, a function that is lost when TTN expression is pathologically lowered.


Assuntos
Fibras Musculares Esqueléticas/fisiologia , Proteínas Quinases/fisiologia , Sarcômeros/fisiologia , Animais , Fenômenos Biomecânicos , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Força Muscular/fisiologia , Atrofia Muscular/etiologia , Atrofia Muscular/patologia , Atrofia Muscular/fisiopatologia , Doenças Musculares/patologia , Doenças Musculares/fisiopatologia , Miosinas/metabolismo , Proteínas Quinases/deficiência , Proteínas Quinases/genética , Sarcômeros/patologia , Ubiquitinação
3.
Nat Commun ; 11(1): 3405, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636378

RESUMO

Omecamtiv mecarbil (OM) is a putative positive inotropic tool for treatment of systolic heart dysfunction, based on the finding that in vivo it increases the ejection fraction and in vitro it prolongs the actin-bond life time of the cardiac and slow-skeletal muscle isoforms of myosin. OM action in situ, however, is still poorly understood as the enhanced Ca2+-sensitivity of the myofilaments is at odds with the reduction of force and rate of force development observed at saturating Ca2+. Here we show, by combining fast sarcomere-level mechanics and ATPase measurements in single slow demembranated fibres from rabbit soleus, that the depressant effect of OM on the force per attached motor is reversed, without effect on the ATPase rate, by physiological concentrations of inorganic phosphate (Pi) (1-10 mM). This mechanism could underpin an energetically efficient reduction of systolic tension cost in OM-treated patients, whenever [Pi] increases with heart-beat frequency.


Assuntos
Miosinas Cardíacas/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Miosinas/metabolismo , Fosfatos/farmacologia , Ureia/análogos & derivados , Adenosina Trifosfatases/metabolismo , Animais , Cálcio/metabolismo , Sinergismo Farmacológico , Masculino , Músculo Esquelético/metabolismo , Coelhos , Sarcômeros/metabolismo , Estresse Mecânico , Ureia/farmacologia
4.
Life Sci ; 258: 118085, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32663578

RESUMO

BACKGROUND: An integral intestinal barrier is essential for intestinal homeostasis. Yet, as a side effect of cancer treatment, chemotherapeutic drugs have been reported to cause mucositis. In a recent study, we found that alginate oligosaccharides (AOS) prevent busulfan induced intestinal mucositis. However, it is not known if AOS improves small intestine epithelial cell integrity and migration, which are two essential processes for maintaining the mechanical barrier function of the small intestine. In the current investigation, we aimed to explore the effects of AOS on the integrity and migration of small intestine cells using swine intestinal epithelial IPEC-J2 cells. METHODS: Cell integrity was determined using the TEER assay. Cell migration capability was detected using a wound healing experiment. Small interfering RNA (siRNA) was used to inhibit mannose receptor (MR) expression. Western blotting and immunofluorescence staining were used to determine protein expression. RESULTS: Increasing levels of AOS improved cell integrity as measure by TEER. At the same time, AOS improved IPEC-J2 cell migration capacity as shown in the wound closure assay. It is interesting to note that AOS increased the expression of intestinal microvillus proteins and junction proteins to benefit cell integrity. MR siRNA blocked the action of AOS on cell integrity and cell migration and inhibited the expression of microvillus and cell junction proteins. CONCLUSION: We identified the underlying mechanisms by which AOS improved small intestinal mucositis. As a novel, natural food additive, AOS may be administered to prevent intestinal mucositis induced by chemotherapy or other issues.


Assuntos
Alginatos/farmacologia , Movimento Celular/efeitos dos fármacos , Intestino Delgado/citologia , Oligossacarídeos/farmacologia , Animais , Linhagem Celular , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microvilosidades/efeitos dos fármacos , Microvilosidades/metabolismo , Miosinas/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores de Superfície Celular/metabolismo , Suínos , Proteínas de Junções Íntimas/metabolismo , Cicatrização/efeitos dos fármacos
5.
Proc Natl Acad Sci U S A ; 117(31): 18511-18520, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690698

RESUMO

It is widely believed that cleavage-furrow formation during cytokinesis is driven by the contraction of a ring containing F-actin and type-II myosin. However, even in cells that have such rings, they are not always essential for furrow formation. Moreover, many taxonomically diverse eukaryotic cells divide by furrowing but have no type-II myosin, making it unlikely that an actomyosin ring drives furrowing. To explore this issue further, we have used one such organism, the green alga Chlamydomonas reinhardtii We found that although F-actin is associated with the furrow region, none of the three myosins (of types VIII and XI) is localized there. Moreover, when F-actin was eliminated through a combination of a mutation and a drug, furrows still formed and the cells divided, although somewhat less efficiently than normal. Unexpectedly, division of the large Chlamydomonas chloroplast was delayed in the cells lacking F-actin; as this organelle lies directly in the path of the cleavage furrow, this delay may explain, at least in part, the delay in cytokinesis itself. Earlier studies had shown an association of microtubules with the cleavage furrow, and we used a fluorescently tagged EB1 protein to show that microtubules are still associated with the furrows in the absence of F-actin, consistent with the possibility that the microtubules are important for furrow formation. We suggest that the actomyosin ring evolved as one way to improve the efficiency of a core process for furrow formation that was already present in ancestral eukaryotes.


Assuntos
Actinas/metabolismo , Chlamydomonas/citologia , Chlamydomonas/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Divisão Celular , Chlamydomonas/química , Citocinese , Microtúbulos/metabolismo , Miosinas/química , Miosinas/metabolismo , Ligação Proteica
6.
Gene ; 760: 144989, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32717307

RESUMO

Kinesin 14 family member KIFC1 is a mitotic kinesin which contains a C-terminal motor domain and plays a vital role for clustering the amplified centrosomes. Overexpression of KIFC1 in prostate cancer (PCa) cells showed resistance to docetaxel (DTX). The present study revealed that small KIFC1 inhibitor AZ82 suppresed the transcription and translation of KIFC1 significantly in PCa cells. AZ82 inhibited the KIFC1 expression both in the cytoplasm and nucleus of PCa cells. Inhibition of KIFC1 by AZ82 caused multipolar mitosis in PCa cells via de-clustering the amplified centrosomes and decreased the rate of cancer cell growth and proliferation. Moreover, depletion of KIFC1 reduced cells entering the cell cycle and caused PCa cells death through apoptosis by increasing the expression of Bax and Cytochrome C. Thereby, KIFC1 silencing and inhibition decreased the PCa cells survival by inducing multipolar mitosis as well as apoptosis, suggesting inhibition of KIFC1 using AZ82 might be a strategy to treat PCa by controlling the cancer cell proliferation.


Assuntos
Alanina/análogos & derivados , Centrossomo/efeitos dos fármacos , Cinesina/antagonistas & inibidores , Neoplasias da Próstata/tratamento farmacológico , Piridinas/farmacologia , Alanina/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Centrossomo/metabolismo , Dineínas/metabolismo , Humanos , Cinesina/genética , Cinesina/metabolismo , Masculino , Mitose/efeitos dos fármacos , Miosinas/metabolismo , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia
7.
Nat Commun ; 11(1): 3200, 2020 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-32581239

RESUMO

mTOR activation is essential and sufficient to cause polycystic kidneys in Tuberous Sclerosis Complex (TSC) and other genetic disorders. In disease models, a sharp increase of proliferation and cyst formation correlates with a dramatic loss of oriented cell division (OCD). We find that OCD distortion is intrinsically due to S6 kinase 1 (S6K1) activation. The concomitant loss of S6K1 in Tsc1-mutant mice restores OCD but does not decrease hyperproliferation, leading to non-cystic harmonious hyper growth of kidneys. Mass spectrometry-based phosphoproteomics for S6K1 substrates revealed Afadin, a known component of cell-cell junctions required to couple intercellular adhesions and cortical cues to spindle orientation. Afadin is directly phosphorylated by S6K1 and abnormally decorates the apical surface of Tsc1-mutant cells with E-cadherin and α-catenin. Our data reveal that S6K1 hyperactivity alters centrosome positioning in mitotic cells, affecting oriented cell division and promoting kidney cysts in conditions of mTOR hyperactivity.


Assuntos
Divisão Celular , Cinesina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Miosinas/metabolismo , Doenças Renais Policísticas/patologia , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Animais , Linhagem Celular , Cinesina/genética , Camundongos , Camundongos Mutantes , Mutação , Miosinas/genética , Fosforilação , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Transdução de Sinais , Esclerose Tuberosa/genética , Esclerose Tuberosa/metabolismo , Esclerose Tuberosa/patologia , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo
8.
Proc Natl Acad Sci U S A ; 117(23): 12817-12825, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32444491

RESUMO

Morphogenesis, tumor formation, and wound healing are regulated by tissue rigidity. Focal adhesion behavior is locally regulated by stiffness; however, how cells globally adapt, detect, and respond to rigidity remains unknown. Here, we studied the interplay between the rheological properties of the cytoskeleton and matrix rigidity. We seeded fibroblasts onto flexible microfabricated pillar arrays with varying stiffness and simultaneously measured the cytoskeleton organization, traction forces, and cell-rigidity responses at both the adhesion and cell scale. Cells adopted a rigidity-dependent phenotype whereby the actin cytoskeleton polarized on stiff substrates but not on soft. We further showed a crucial role of active and passive cross-linkers in rigidity-sensing responses. By reducing myosin II activity or knocking down α-actinin, we found that both promoted cell polarization on soft substrates, whereas α-actinin overexpression prevented polarization on stiff substrates. Atomic force microscopy indentation experiments showed that this polarization response correlated with cell stiffness, whereby cell stiffness decreased when active or passive cross-linking was reduced and softer cells polarized on softer matrices. Theoretical modeling of the actin network as an active gel suggests that adaptation to matrix rigidity is controlled by internal mechanical properties of the cytoskeleton and puts forward a universal scaling between nematic order of the actin cytoskeleton and the substrate-to-cell elastic modulus ratio. Altogether, our study demonstrates the implication of cell-scale mechanosensing through the internal stress within the actomyosin cytoskeleton and its coupling with local rigidity sensing at focal adhesions in the regulation of cell shape changes and polarity.


Assuntos
Citoesqueleto/metabolismo , Módulo de Elasticidade , Mecanotransdução Celular , Tecidos Suporte/química , Actinina/metabolismo , Polaridade Celular , Reagentes para Ligações Cruzadas/química , Citoesqueleto/ultraestrutura , Fibroblastos/metabolismo , Humanos , Modelos Teóricos , Miosinas/metabolismo
9.
Adv Exp Med Biol ; 1239: 7-19, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32451853

RESUMO

Directed movements on actin filaments within the cell are powered by molecular motors of the myosin superfamily. On actin filaments, myosin motors convert the energy from ATP into force and movement. Myosin motors power such diverse cellular functions as cytokinesis, membrane trafficking, organelle movements, and cellular migration. Myosin generates force and movement via a number of structural changes associated with hydrolysis of ATP, binding to actin, and release of the ATP hydrolysis products while bound to actin. Herein we provide an overview of those structural changes and how they relate to the actin-myosin ATPase cycle. These structural changes are the basis of chemo-mechanical transduction by myosin motors.


Assuntos
Miosinas/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Hidrólise , Movimento , Miosinas/metabolismo
10.
Adv Exp Med Biol ; 1239: 21-40, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32451854

RESUMO

Unconventional myosins are a large superfamily of actin-based molecular motors that use ATP as fuel to generate mechanical motions/forces. The distinct tails in different unconventional myosin subfamilies can recognize various cargoes including proteins and lipids. Thus, they can play diverse roles in many biological processes such as cellular trafficking, mechanical supports, force sensing, etc. This chapter focuses on some recent advances on the structural studies of how unconventional myosins specifically bind to cargoes with their cargo-binding domains.


Assuntos
Transporte Biológico , Miosinas/metabolismo , Actinas , Ligação Proteica
11.
Adv Exp Med Biol ; 1239: 61-84, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32451856

RESUMO

Several small molecule effectors of myosin function that target the motor domains of myosin classes I, II, V, and VI have been identified. Four distinct binding sites in the myosin motor domain have been reported with unique properties and mechanisms of action. This chapter describes the structural basis and activities of known small molecule effectors that allosterically target the myosin motor domain.


Assuntos
Miosinas/química , Miosinas/metabolismo , Regulação Alostérica/efeitos dos fármacos , Sítios de Ligação
12.
Adv Exp Med Biol ; 1239: 127-152, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32451858

RESUMO

High-speed atomic force microscopy (HS-AFM) is a unique tool that enables imaging of protein molecules during their functional activity at sub-100 ms temporal and submolecular spatial resolution. HS-AFM is suited for the study of highly dynamic proteins, including myosin motors. HS-AFM images of myosin V walking on actin filaments provide irrefutable evidence for the swinging lever arm motion propelling the molecule forward. Moreover, molecular behaviors that have not been noticed before are also displayed on the AFM movies. This chapter describes the principle, underlying techniques and performance of HS-AFM, filmed images of myosin V, and mechanistic insights into myosin motility provided from the filmed images.


Assuntos
Microscopia de Força Atômica , Movimento , Miosinas/metabolismo
13.
Adv Exp Med Biol ; 1239: 153-181, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32451859

RESUMO

Myosin 5a is a two-headed myosin that functions as a cargo transporter in cells. To accomplish this task it has evolved several unique structural and kinetic features that allow it to move processively as a single molecule along actin filaments. A plethora of biophysical techniques have been used to elucidate the detailed mechanism of its movement along actin filaments in vitro. This chapter describes how this mechanism was deduced.


Assuntos
Movimento , Miosinas , Imagem Individual de Molécula , Citoesqueleto de Actina , Actinas , Biofísica , Humanos , Cinética , Miosinas/metabolismo
14.
Adv Exp Med Biol ; 1239: 183-197, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32451860

RESUMO

Cellular organization through cytoskeletal trafficking is a process of fundamental importance. Highly specialized systems evolved that enable motors to identify and select the optimal tracks for motility. In this chapter, we examine the profound effect of actin filament networks on myosin motility patterns. We argue that the myosin classes have adaptations that allow them to detect local structural and chemical cues on actin. These cues are often arranged in a coherent manner on actin filament networks, allowing for directed transport over long distances. We identify a number of potentially important cues, ranging from the biochemical states of actin subunits all the way to multi-filament networks and bundles.


Assuntos
Actinas , Miosinas , Citoesqueleto de Actina , Actinas/metabolismo , Movimento , Miosinas/metabolismo
15.
Adv Exp Med Biol ; 1239: 199-231, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32451861

RESUMO

Although originally characterized as a cytoplasmic protein, myosin of various classes also performs key functions in the nucleus. We review the data concerning the nuclear localization, mechanism of entry, and functional interactions of myosin I, II, V, VI, X, XVI, and XVIII. To date, the first-characterized "nuclear myosin I" (or, in the prevailing nomenclature, myosin IC isoform B) remains the best-studied nuclear myosin, although results are rapidly accumulating that illuminate the roles of other myosin classes, and an outline of a unified picture of myosin functions in the nucleus is beginning to emerge. Reflecting the state of knowledge in this field, the review concentrates on the mechanisms mediating and regulating import of myosin IC into the nucleus and its role, alongside myosin V and VI, in transcription. Myosin functions in chromatin dynamics, epigenetic mechanisms, intranuclear motility, and nuclear export of RNA and protein are also addressed. Partners and regulators of myosin, such as nuclear actin, kinases, and phosphatases are briefly covered. Problem areas are identified and testable hypotheses are offered with an aim of focusing the research efforts on overcoming the gaps on the way toward a systems-level understanding of processes involving nuclear myosins and their place in cell physiology as a whole.


Assuntos
Núcleo Celular , Miosinas , Actinas , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Humanos , Miosinas/metabolismo , Monoéster Fosfórico Hidrolases , Fosfotransferases , Transporte Proteico
16.
Adv Exp Med Biol ; 1239: 381-389, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32451867

RESUMO

Class IX myosins are simultaneously motor and signaling molecules. In addition to myosin class-specific functions of the tail region, they feature unique motor properties. Within their motor region they contain a long insertion with a calmodulin- and a F-actin-binding site. The rate-limiting step in the ATPase cycle is ATP hydrolysis rather than, typical for other myosins, the release of either product. This means that class IX myosins spend a large fraction of their cycle time in the ATP-bound state, which is typically a low F-actin affinity state. Nevertheless, class IX myosins in the ATP-bound state stochastically switch between a low and a high F-actin affinity state. Single motor domains even show characteristics of processive movement towards the plus end of actin filaments. The insertion thereby acts as an actin tether. The motor domain transports as intramolecular cargo a signaling Rho GTPase-activating protein domain located in the tail region. Rho GTPase-activating proteins catalyze the conversion of active GTP-bound Rho to inactive GDP-bound Rho by stimulating GTP hydrolysis. In cells, Rho activity regulates actin cytoskeleton organization and actomyosin II contractility. Thus, class IX myosins regulate cell morphology, cell migration, cell-cell junctions and membrane trafficking. These cellular functions affect embryonic development, adult organ homeostasis and immune responses. Human diseases associated with mutations in the two class IX myosins, Myo9a and Myo9b, have been identified, including hydrocephalus and congenital myasthenic syndrome in connection with Myo9a and autoimmune diseases in connection with Myo9b.


Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Miosinas/metabolismo , Transdução de Sinais , Actinas/metabolismo , Humanos , Ligação Proteica
17.
Adv Exp Med Biol ; 1239: 391-403, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32451868

RESUMO

Myosin X (Myo10), an actin-based molecular motor, induces filopodia formation and controls cell migration in vitro. In the 25 years since Myo10 was first identified, it has been implicated in several different functions in different cell types including phagocytosis in macrophages, axon outgrowth in neurons, cell-cell adhesion in epithelial and endothelial cells, podosome formation in osteoclasts, spindle-pole positioning in meiosis and mitosis of cultured cells, migration of melanocytes and cranial neural crest cells, and invadopodia formation in cancer cells. Recently, the availability of Myo10-knockout (Myo10KO) mice has allowed for tremendous progress toward understanding the biological function of Myo10 in vivo.In this chapter, I address the structure of the Myo10 gene; the molecular structure of Myo10 protein with its multiple domains, e.g., PH, MyTH4, and FERM domains; the regulation of actin structures induced in cells by Myo10; the expression and function of Myo10 in vitro and in vivo; and the role of Myo10 in cancer. Previous reviews on Myo10 include Divito MM, Cheney RE, (Myosins: a superfamily of molecular motors chapter 14 MYOSIN X. In: Proteins and cell regulation, vol 7. Springer, Dordrecht, 2008) and Kerber ML, Cheney RE (J Cell Sci 124:3733-3741).


Assuntos
Miosinas/metabolismo , Actinas/metabolismo , Animais , Camundongos , Fagocitose
18.
Proc Natl Acad Sci U S A ; 117(22): 11865-11874, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32444484

RESUMO

Striated muscle contraction involves sliding of actin thin filaments along myosin thick filaments, controlled by calcium through thin filament activation. In relaxed muscle, the two heads of myosin interact with each other on the filament surface to form the interacting-heads motif (IHM). A key question is how both heads are released from the surface to approach actin and produce force. We used time-resolved synchrotron X-ray diffraction to study tarantula muscle before and after tetani. The patterns showed that the IHM is present in live relaxed muscle. Tetanic contraction produced only a very small backbone elongation, implying that mechanosensing-proposed in vertebrate muscle-is not of primary importance in tarantula. Rather, thick filament activation results from increases in myosin phosphorylation that release a fraction of heads to produce force, with the remainder staying in the ordered IHM configuration. After the tetanus, the released heads slowly recover toward the resting, helically ordered state. During this time the released heads remain close to actin and can quickly rebind, enhancing the force produced by posttetanic twitches, structurally explaining posttetanic potentiation. Taken together, these results suggest that, in addition to stretch activation in insects, two other mechanisms for thick filament activation have evolved to disrupt the interactions that establish the relaxed helices of IHMs: one in invertebrates, by either regulatory light-chain phosphorylation (as in arthropods) or Ca2+-binding (in mollusks, lacking phosphorylation), and another in vertebrates, by mechanosensing.


Assuntos
Músculo Estriado/fisiologia , Miosinas/metabolismo , Fosforilação/fisiologia , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Animais , Artrópodes/fisiologia , Evolução Molecular , Invertebrados/fisiologia , Modelos Moleculares , Contração Muscular , Relaxamento Muscular , Miosinas/química , Estrutura Secundária de Proteína , Aranhas/fisiologia , Vertebrados/fisiologia
19.
Proc Natl Acad Sci U S A ; 117(21): 11432-11443, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32381732

RESUMO

The structure and mechanics of many connective tissues are dictated by a collagen-rich extracellular matrix (ECM), where collagen fibers provide topological cues that direct cell migration. However, comparatively little is known about how cells navigate the hyaluronic acid (HA)-rich, nanoporous ECM of the brain, a problem with fundamental implications for development, inflammation, and tumor invasion. Here, we demonstrate that glioblastoma cells adhere to and invade HA-rich matrix using microtentacles (McTNs), which extend tens of micrometers from the cell body and are distinct from filopodia. We observe these structures in continuous culture models and primary patient-derived tumor cells, as well as in synthetic HA matrix and organotypic brain slices. High-magnification and superresolution imaging reveals McTNs are dynamic, CD44-coated tubular protrusions containing microtubules and actin filaments, which respectively drive McTN extension and retraction. Molecular mechanistic studies reveal that McTNs are stabilized by an interplay between microtubule-driven protrusion, actomyosin-driven retraction, and CD44-mediated adhesion, where adhesive and cytoskeletal components are mechanistically coupled by an IQGAP1-CLIP170 complex. McTNs represent a previously unappreciated mechanism through which cells engage nanoporous HA matrix and may represent an important molecular target in physiology and disease.


Assuntos
Glioblastoma/patologia , Receptores de Hialuronatos/metabolismo , Actinas/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Técnicas de Inativação de Genes , Glioblastoma/metabolismo , Humanos , Ácido Hialurônico/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Miosinas/metabolismo , Proteínas de Neoplasias/metabolismo , Oligopeptídeos/metabolismo , Técnicas de Cultura de Órgãos , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismo
20.
Proc Natl Acad Sci U S A ; 117(20): 10865-10875, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32366666

RESUMO

Cell-to-cell transmission of misfolding-prone α-synuclein (α-Syn) has emerged as a key pathological event in Parkinson's disease. This process is initiated when α-Syn-bearing fibrils enter cells via clathrin-mediated endocytosis, but the underlying mechanisms are unclear. Using a CRISPR-mediated knockout screen, we identify SLC35B2 and myosin-7B (MYO7B) as critical endocytosis regulators for α-Syn preformed fibrils (PFFs). We show that SLC35B2, as a key regulator of heparan sulfate proteoglycan (HSPG) biosynthesis, is essential for recruiting α-Syn PFFs to the cell surface because this process is mediated by interactions between negatively charged sugar moieties of HSPGs and clustered K-T-K motifs in α-Syn PFFs. By contrast, MYO7B regulates α-Syn PFF cell entry by maintaining a plasma membrane-associated actin network that controls membrane dynamics. Without MYO7B or actin filaments, many clathrin-coated pits fail to be severed from the membrane, causing accumulation of large clathrin-containing "scars" on the cell surface. Intriguingly, the requirement for MYO7B in endocytosis is restricted to α-Syn PFFs and other polycation-bearing cargos that enter cells via HSPGs. Thus, our study not only defines regulatory factors for α-Syn PFF endocytosis, but also reveals a previously unknown endocytosis mechanism for HSPG-binding cargos in general, which requires forces generated by MYO7B and actin filaments.


Assuntos
Endocitose/fisiologia , Miosinas/química , Miosinas/metabolismo , Polieletrólitos/metabolismo , alfa-Sinucleína/metabolismo , Linhagem Celular , Clatrina/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Modelos Moleculares , Doença de Parkinson/metabolismo , Conformação Proteica , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismo
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