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1.
PLoS Biol ; 18(12): e3001003, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33315855

RESUMO

Stem-cell niche signaling is short-range in nature, such that only stem cells but not their differentiating progeny receive self-renewing signals. At the apical tip of the Drosophila testis, 8 to 10 germline stem cells (GSCs) surround the hub, a cluster of somatic cells that organize the stem-cell niche. We have previously shown that GSCs form microtubule-based nanotubes (MT-nanotubes) that project into the hub cells, serving as the platform for niche signal reception; this spatial arrangement ensures the reception of the niche signal specifically by stem cells but not by differentiating cells. The receptor Thickveins (Tkv) is expressed by GSCs and localizes to the surface of MT-nanotubes, where it receives the hub-derived ligand Decapentaplegic (Dpp). The fate of Tkv receptor after engaging in signaling on the MT-nanotubes has been unclear. Here we demonstrate that the Tkv receptor is internalized into hub cells from the MT-nanotube surface and subsequently degraded in the hub cell lysosomes. Perturbation of MT-nanotube formation and Tkv internalization from MT-nanotubes into hub cells both resulted in an overabundance of Tkv protein in GSCs and hyperactivation of a downstream signal, suggesting that the MT-nanotubes also serve a second purpose to dampen the niche signaling. Together, our results demonstrate that MT-nanotubes play dual roles to ensure the short-range nature of niche signaling by (1) providing an exclusive interface for the niche ligand-receptor interaction; and (2) limiting the amount of stem cell receptors available for niche signal reception.


Assuntos
Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/fisiologia , Nicho de Células-Tronco/fisiologia , Células-Tronco/metabolismo , Células-Tronco Germinativas Adultas/metabolismo , Células-Tronco Germinativas Adultas/fisiologia , Animais , Diferenciação Celular/fisiologia , Drosophila melanogaster/metabolismo , Células Germinativas/citologia , Células Germinativas/metabolismo , Ligantes , Masculino , Microtúbulos/metabolismo , Microtúbulos/fisiologia , Transdução de Sinais/fisiologia , Células-Tronco/citologia , Testículo/metabolismo
2.
PLoS Comput Biol ; 16(9): e1008132, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32877399

RESUMO

Tubulin dimers associate longitudinally and laterally to form metastable microtubules (MTs). MT disassembly is preceded by subtle structural changes in tubulin fueled by GTP hydrolysis. These changes render the MT lattice unstable, but it is unclear exactly how they affect lattice energetics and strain. We performed long-time atomistic simulations to interrogate the impacts of GTP hydrolysis on tubulin lattice conformation, lateral inter-dimer interactions, and (non-)local lateral coordination of dimer motions. The simulations suggest that most of the hydrolysis energy is stored in the lattice in the form of longitudinal strain. While not significantly affecting lateral bond stability, the stored elastic energy results in more strongly confined and correlated dynamics of GDP-tubulins, thereby entropically destabilizing the MT lattice.


Assuntos
Microtúbulos , Tubulina (Proteína) , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Hidrólise , Microtúbulos/química , Microtúbulos/metabolismo , Microtúbulos/fisiologia , Simulação de Dinâmica Molecular , Conformação Proteica , Termodinâmica , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/fisiologia
3.
Proc Natl Acad Sci U S A ; 117(36): 22193-22203, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32839317

RESUMO

The establishment of axon/dendrite polarity is fundamental for neurons to integrate into functional circuits, and this process is critically dependent on microtubules (MTs). In the early stages of the establishment process, MTs in axons change dramatically with the morphological building of neurons; however, how the MT network changes are triggered is unclear. Here we show that CAMSAP1 plays a decisive role in the neuronal axon identification process by regulating the number of MTs. Neurons lacking CAMSAP1 form a multiple axon phenotype in vitro, while the multipolar-bipolar transition and radial migration are blocked in vivo. We demonstrate that the polarity regulator MARK2 kinase phosphorylates CAMSAP1 and affects its ability to bind to MTs, which in turn changes the protection of MT minus-ends and also triggers asymmetric distribution of MTs. Our results indicate that the polarized MT network in neurons is a decisive factor in establishing axon/dendritic polarity and is initially triggered by polarized signals.


Assuntos
Polaridade Celular/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/fisiologia , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Imunoprecipitação , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Neurônios , Paclitaxel , Ligação Proteica
4.
PLoS Negl Trop Dis ; 14(8): e0008469, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32764759

RESUMO

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus, which has rapidly spread around the globe thereby causing millions of infections. CHIKV is an enveloped virus belonging to the Togaviridae family and enters its host cell primarily via clathrin-mediated endocytosis. Upon internalization, the endocytic vesicle containing the virus particle moves through the cell and delivers the virus to early endosomes where membrane fusion is observed. Thereafter, the nucleocapsid dissociates and the viral RNA is translated into proteins. In this study, we examined the importance of the microtubule network during the early steps of infection and dissected the intracellular trafficking behavior of CHIKV particles during cell entry. We observed two distinct CHIKV intracellular trafficking patterns prior to membrane hemifusion. Whereas half of the CHIKV virions remained static during cell entry and fused in the cell periphery, the other half showed fast-directed microtubule-dependent movement prior to delivery to Rab5-positive early endosomes and predominantly fused in the perinuclear region of the cell. Disruption of the microtubule network reduced the number of infected cells. At these conditions, membrane hemifusion activity was not affected yet fusion was restricted to the cell periphery. Furthermore, follow-up experiments revealed that disruption of the microtubule network impairs the delivery of the viral genome to the cell cytosol. We therefore hypothesize that microtubules may direct the particle to a cellular location that is beneficial for establishing infection or aids in nucleocapsid uncoating.


Assuntos
Vírus Chikungunya/fisiologia , Genoma Viral , Microtúbulos/fisiologia , Replicação Viral/fisiologia , Animais , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Humanos , Nocodazol/farmacologia , Moduladores de Tubulina/farmacologia
5.
Circ Heart Fail ; 13(7): e006935, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32635769

RESUMO

BACKGROUND: NEXN (nexilin) is a protein of the junctional membrane complex required for development of cardiac T-tubules. Global and cardiomyocyte-specific loss of Nexn in mice leads to a rapidly progressive dilated cardiomyopathy and premature death. Therefore, little is known as to the role of NEXN in adult cardiomyocytes. Transverse-axial tubular system remodeling are well-known features in heart failure. Although NEXN is required during development for T-tubule formation, its role, if any, in mature T-tubules remains to be addressed. METHODS: Nexn inducible adult cardiomyocyte-specific KO mice were generated. Comprehensive morphological and functional analyses were performed. Heart samples (n>3) were analyzed by molecular, biochemical, and electron microscopy analyses. Isolated single adult cardiomyocytes were analyzed by confocal microscopy, and myocyte shortening/re-lengthening and Ca2+ transient studies were conducted. RESULTS: Inducible cardiomyocyte-specific loss of Nexn in adult mice resulted in a dilated cardiomyopathy with reduced cardiac function (13% reduction in percentage fractional shortening; P<0.05). In vivo and in vitro analyses of adult mouse heart samples revealed that NEXN was essential for optimal contraction and calcium handling and was required for maintenance of T-tubule network organization (transverse tubular component in Nexn inducible adult cardiomyocyte-specific KO mice reduced by 40% with respect to controls, P<0.05). CONCLUSIONS: Results here reported reveal NEXN to be a pivotal component of adult junctional membrane complexes required for maintenance of transverse-axial tubular architecture. These results demonstrate that NEXN plays an essential role in the adult cardiomyocyte and give further understanding of pathological mechanisms responsible for cardiomyopathy in patients carrying mutations in the NEXN gene.


Assuntos
Cardiomiopatia Dilatada/fisiopatologia , Proteínas dos Microfilamentos/fisiologia , Microtúbulos/fisiologia , Miócitos Cardíacos/fisiologia , Disfunção Ventricular Esquerda/fisiopatologia , Fatores Etários , Animais , Cálcio/metabolismo , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Modelos Animais de Doenças , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microtúbulos/metabolismo , Miócitos Cardíacos/metabolismo , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/metabolismo
6.
Proc Natl Acad Sci U S A ; 117(28): 16154-16159, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601228

RESUMO

The metaphase spindle is a dynamic structure orchestrating chromosome segregation during cell division. Recently, soft matter approaches have shown that the spindle behaves as an active liquid crystal. Still, it remains unclear how active force generation contributes to its characteristic spindle-like shape. Here we combine theory and experiments to show that molecular motor-driven forces shape the structure through a barreling-type instability. We test our physical model by titrating dynein activity in Xenopus egg extract spindles and quantifying the shape and microtubule orientation. We conclude that spindles are shaped by the interplay between surface tension, nematic elasticity, and motor-driven active forces. Our study reveals how motor proteins can mold liquid crystalline droplets and has implications for the design of active soft materials.


Assuntos
Metáfase/fisiologia , Fuso Acromático/fisiologia , Animais , Fenômenos Biomecânicos , Dineínas/antagonistas & inibidores , Dineínas/metabolismo , Elasticidade , Cristais Líquidos , Metáfase/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Microtúbulos/fisiologia , Mitose , Fuso Acromático/química , Fuso Acromático/efeitos dos fármacos , Tensão Superficial , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/metabolismo , Xenopus laevis
7.
Curr Opin Cell Biol ; 63: 204-211, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32361559

RESUMO

Mechanical forces are known to influence cellular processes with consequences at the cellular and physiological level. The cell nucleus is the largest and stiffest organelle, and it is connected to the cytoskeleton for proper cellular function. The connection between the nucleus and the cytoskeleton is in most cases mediated by the linker of nucleoskeleton and cytoskeleton (LINC) complex. Not surprisingly, the nucleus and the associated cytoskeleton are implicated in multiple mechanotransduction pathways important for cellular activities. Herein, we review recent advances describing how the LINC complex, the nuclear lamina, and nuclear pore complexes are involved in nuclear mechanotransduction. We will also discuss how the perinuclear actin cytoskeleton is important for the regulation of nuclear mechanotransduction. Additionally, we discuss the relevance of nuclear mechanotransduction for cell migration, development, and how nuclear mechanotransduction impairment leads to multiple disorders.


Assuntos
Núcleo Celular/fisiologia , Mecanotransdução Celular/fisiologia , Animais , Movimento Celular/fisiologia , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Humanos , Microtúbulos/metabolismo , Microtúbulos/fisiologia , Lâmina Nuclear/fisiologia , Poro Nuclear/metabolismo , Poro Nuclear/fisiologia
8.
Sci Rep ; 10(1): 7696, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32376876

RESUMO

When stretched, cells cultured on 2D substrates share a universal softening and fluidization response that arises from poorly understood remodeling of well-conserved cytoskeletal elements. It is known, however, that the structure and distribution of the cytoskeleton is profoundly influenced by the dimensionality of a cell's environment. Therefore, in this study we aimed to determine whether cells cultured in a 3D matrix share this softening behavior and to link it to cytoskeletal remodeling. To achieve this, we developed a high-throughput approach to measure the dynamic mechanical properties of cells and allow for sub-cellular imaging within physiologically relevant 3D microtissues. We found that fibroblast, smooth muscle and skeletal muscle microtissues strain softened but did not fluidize, and upon loading cessation, they regained their initial mechanical properties. Furthermore, microtissue prestress decreased with the strain amplitude to maintain a constant mean tension. This adaptation under an auxotonic condition resulted in lengthening. A filamentous actin cytoskeleton was required, and responses were mirrored by changes to actin remodeling rates and visual evidence of stretch-induced actin depolymerization. Our new approach for assessing cell mechanics has linked behaviors seen in 2D cultures to a 3D matrix, and connected remodeling of the cytoskeleton to homeostatic mechanical regulation of tissues.


Assuntos
Citoesqueleto/fisiologia , Homeostase/fisiologia , Citoesqueleto de Actina/fisiologia , Animais , Linhagem Celular , Fibroblastos/fisiologia , Camundongos , Microtúbulos/fisiologia , Modelos Biológicos , Fibras Musculares Esqueléticas/fisiologia , Miócitos de Músculo Liso/fisiologia , Estresse Mecânico
9.
Proc Natl Acad Sci U S A ; 117(22): 12155-12163, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32430325

RESUMO

Microtubule polarity in axons and dendrites defines the direction of intracellular transport in neurons. Axons contain arrays of uniformly polarized microtubules with plus-ends facing the tips of the processes (plus-end-out), while dendrites contain microtubules with a minus-end-out orientation. It has been shown that cytoplasmic dynein, targeted to cortical actin, removes minus-end-out microtubules from axons. Here we have identified Spindly, a protein known for recruitment of dynein to kinetochores in mitosis, as a key factor required for dynein-dependent microtubule sorting in axons of Drosophila neurons. Depletion of Spindly affects polarity of axonal microtubules in vivo and in primary neuronal cultures. In addition to these defects, depletion of Spindly in neurons causes major collapse of axonal patterning in the third-instar larval brain as well as severe coordination impairment in adult flies. These defects can be fully rescued by full-length Spindly, but not by variants with mutations in its dynein-binding site. Biochemical analysis demonstrated that Spindly binds F-actin, suggesting that Spindly serves as a link between dynein and cortical actin in axons. Therefore, Spindly plays a critical role during neurodevelopment by mediating dynein-driven sorting of axonal microtubules.


Assuntos
Axônios/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Dineínas/metabolismo , Microtúbulos/fisiologia , Neurônios/fisiologia , Actinas/metabolismo , Animais , Transporte Biológico , Proteínas de Ciclo Celular/genética , Células Cultivadas , Córtex Cerebral/metabolismo , Dendritos/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/citologia
10.
Plant Sci ; 292: 110405, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32005401

RESUMO

As a group of plant-specific proteins, OVATE family protein (OFP) members have been shown to function as transcriptional repressors and involve in plant growth regulation in Arabidopsis and rice. It has also been shown that OFPs can interact with TONNEAU1 Recruiting Motif (TRM) proteins to regulate tomato fruit shape. In this study, we show that mutant plants with knock-down expression of OFP1, OFP2, OFP3, and OFP5 exhibit longer hypocotyls and cotyledons due to enhanced cell elongation. Overexpression of OFPs disturb the arrangement of cortical microtubule arrays in pavement cells and promote abnormal pavement cell expansion perpendicular to the direction of petiole growth, resulting in the kidney-shaped cotyledons in transgenic plants. OFP2 and OFP5 interact directly with the microtubule regulating protein TONNEAU2 (TON2), and genetic analysis suggests TON2 is required for the function of OFPs. We also show that altering the expression of OFPs affects light and BR regulated microtubule reorientation. BR treatment reduce the protein accumulation of OFP2, suggesting OFP2 mediates BR regulated microtubule reorientation. Taken together, our study provides evidences showing that OFP family proteins negatively regulate cell expansion by modulating microtubule reorganization, which requires the function of TON2.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Crescimento Celular , Microtúbulos/fisiologia , Fosfoproteínas Fosfatases/genética , Proteínas Repressoras/genética , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Repressoras/metabolismo
11.
Int J Mol Sci ; 21(3)2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32033476

RESUMO

Oligodendrocytes are specialized cells that myelinate axons in the central nervous system. Defects in oligodendrocyte function and failure to form or maintain myelin sheaths can cause a number of neurological disorders. Oligodendrocytes are differentiated from oligodendrocyte progenitor cells (OPCs), which extend several processes that contact, elaborate, and eventually wrap axonal segments to form multilayered myelin sheaths. These processes require extensive changes in the cytoarchitecture and must be regulated by reorganization of the cytoskeleton. Here, we established a simple protocol to isolate and differentiate mouse OPCs, and by using this method, we investigated a role of microtubules (MTs) in oligodendrocyte differentiation. Oligodendrocytes developed a complex network of MTs during differentiation, and treatment of differentiating oligodendrocytes with nanomolar concentrations of MT-targeting agents (MTAs) markedly affected oligodendrocyte survival and differentiation. We found that acute exposure to vincristine and nocodazole at early stages of oligodendrocyte differentiation markedly increased MT arborization and enhanced differentiation, whereas taxol and epothilone B treatment produced opposing outcomes. Furthermore, treatment of myelinating co-cultures of oligodendrocytes and neurons with nanomolar concentrations of MTAs at late stages of oligodendrocyte differentiation induced dysmyelination. Together, these results suggest that MTs play an important role in the survival, differentiation, and myelination of oligodendrocytes.


Assuntos
Diferenciação Celular/fisiologia , Microtúbulos/fisiologia , Oligodendroglia/fisiologia , Animais , Axônios/metabolismo , Axônios/fisiologia , Células Cultivadas , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/fisiologia , Técnicas de Cocultura/métodos , Citoesqueleto/metabolismo , Citoesqueleto/fisiologia , Camundongos , Camundongos Endogâmicos ICR , Microtúbulos/metabolismo , Proteína Básica da Mielina/metabolismo , Bainha de Mielina/metabolismo , Bainha de Mielina/fisiologia , Neurogênese/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Células Precursoras de Oligodendrócitos/metabolismo , Células Precursoras de Oligodendrócitos/fisiologia , Oligodendroglia/metabolismo
12.
PLoS Comput Biol ; 16(2): e1007649, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32084125

RESUMO

In multi-ciliated cells, directed and synchronous ciliary beating in the apical membrane occurs through appropriate configuration of basal bodies (BBs, roots of cilia). Although it has been experimentally shown that the position and orientation of BBs are coordinated by apical cytoskeletons (CSKs), such as microtubules (MTs), and planar cell polarity (PCP), the underlying mechanism for achieving the patterning of BBs is not yet understood. In this study, we propose that polarity in bundles of apical MTs play a crucial role in the patterning of BBs. First, the necessity of the polarity was discussed by theoretical consideration on the symmetry of the system. The existence of the polarity was investigated by measuring relative angles between the MTs and BBs using published experimental data. Next, a mathematical model for BB patterning was derived by combining the polarity and self-organizational ability of CSKs. In the model, BBs were treated as finite-size particles in the medium of CSKs and excluded volume effects between BBs and CSKs were taken into account. The model reproduces the various experimental observations, including normal and drug-treated phenotypes. Our model with polarity provides a coherent and testable mechanism for apical BB pattern formation. We have also discussed the implication of our study on cell chirality.


Assuntos
Corpos Basais/fisiologia , Cílios/fisiologia , Citoesqueleto/fisiologia , Animais , Membrana Celular , Polaridade Celular , Simulação por Computador , Elasticidade , Células Epiteliais/citologia , Camundongos , Microtúbulos/fisiologia , Modelos Teóricos , Nocodazol/farmacologia , Fenótipo , Traqueia/fisiologia
13.
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
14.
Circulation ; 141(11): 902-915, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-31941365

RESUMO

BACKGROUND: Diastolic dysfunction is a prevalent and therapeutically intractable feature of heart failure (HF). Increasing ventricular compliance can improve diastolic performance, but the viscoelastic forces that resist diastolic filling and become elevated in human HF are poorly defined. Having recently identified posttranslationally detyrosinated microtubules as a source of viscoelasticity in cardiomyocytes, we sought to test whether microtubules contribute meaningful viscoelastic resistance to diastolic stretch in human myocardium. METHODS: Experiments were conducted in isolated human cardiomyocytes and trabeculae. First, slow and rapid (diastolic) stretch was applied to intact cardiomyocytes from nonfailing and HF hearts and viscoelasticity was characterized after interventions targeting microtubules. Next, intact left ventricular trabeculae from HF patient hearts were incubated with colchicine or vehicle and subject to pre- and posttreatment mechanical testing, which consisted of a staircase protocol and rapid stretches from slack length to increasing strains. RESULTS: Viscoelasticity was increased during diastolic stretch of HF cardiomyocytes compared with nonfailing counterparts. Reducing either microtubule density or detyrosination reduced myocyte stiffness, particularly at diastolic strain rates, indicating reduced viscous forces. In myocardial tissue, we found microtubule depolymerization reduced myocardial viscoelasticity, with an effect that decreased with increasing strain. Colchicine reduced viscoelasticity at strains below, but not above, 15%, with a 2-fold reduction in energy dissipation upon microtubule depolymerization. Post hoc subgroup analysis revealed that myocardium from patients with HF with reduced ejection fraction were more fibrotic and elastic than myocardium from patients with HF with preserved ejection fraction, which were relatively more viscous. Colchicine reduced viscoelasticity in both HF with preserved ejection fraction and HF with reduced ejection fraction myocardium. CONCLUSIONS: Failing cardiomyocytes exhibit elevated viscosity and reducing microtubule density or detyrosination lowers viscoelastic resistance to diastolic stretch in human myocytes and myocardium. In failing myocardium, microtubules elevate stiffness over the typical working range of strains and strain rates, but exhibited diminishing effects with increasing length, consistent with an increasing contribution of the extracellular matrix or myofilament proteins at larger excursions. These studies indicate that a stabilized microtubule network provides a viscous impediment to diastolic stretch, particularly in HF.


Assuntos
Insuficiência Cardíaca/patologia , Microtúbulos/fisiologia , Miocárdio/ultraestrutura , Miócitos Cardíacos/ultraestrutura , Adulto , Idoso , Colchicina/farmacologia , Diástole , Elasticidade , Feminino , Humanos , Masculino , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Pessoa de Meia-Idade , Contração Miocárdica , Miócitos Cardíacos/efeitos dos fármacos , Processamento de Proteína Pós-Traducional , Sesquiterpenos/farmacologia , Estresse Mecânico , Volume Sistólico , Tirosina/metabolismo , Disfunção Ventricular Esquerda/patologia , Viscosidade
15.
Sci Rep ; 10(1): 918, 2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31969604

RESUMO

Brain penetrant microtubule stabilising agents (MSAs) are being increasingly validated as potential therapeutic strategies for neurodegenerative diseases and traumatic injuries of the nervous system. MSAs are historically used to treat malignancies to great effect. However, this treatment strategy can also cause adverse off-target impacts, such as the generation of debilitating neuropathy and axonal loss. Understanding of the effects that individual MSAs have on neurons of the central nervous system is still incomplete. Previous research has revealed that aberrant microtubule stabilisation can perturb many neuronal functions, such as neuronal polarity, neurite outgrowth, microtubule dependant transport and overall neuronal viability. In the current study, we evaluate the dose dependant impact of epothilone D, a brain penetrant MSA, on both immature and relatively mature mouse cortical neurons in vitro. We show that epothilone D reduces the viability, growth and complexity of immature cortical neurons in a dose dependant manner. Furthermore, in relatively mature cortical neurons, we demonstrate that while cellularly lethal doses of epothilone D cause cellular demise, low sub lethal doses can also affect mitochondrial transport over time. Our results reveal an underappreciated mitochondrial disruption over a wide range of epothilone D doses and reiterate the importance of understanding the dosage, timing and intended outcome of MSAs, with particular emphasis on brain penetrant MSAs being considered to target neurons in disease and trauma.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Córtex Cerebral/citologia , Epotilonas/efeitos adversos , Microtúbulos/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Epotilonas/administração & dosagem , Técnicas In Vitro , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microtúbulos/fisiologia , Mitocôndrias/metabolismo , Terapia de Alvo Molecular , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/etiologia , Neurônios/fisiologia
16.
Physiol Plant ; 168(3): 709-724, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31381165

RESUMO

5-aminolevulinic acid (ALA), a plant growth regulator with great application potential in agriculture and horticulture, induces stomatal opening and inhibits stomatal closure by decreasing guard cell H2 O2 . However, the mechanisms behind ALA-decreased H2 O2 in guard cells are not fully understood. Here, using type 2A protein phosphatase (PP2A) inhibitors, microtubule-stabilizing/disrupting drugs and green fluorescent protein-tagged α-tubulin 6 transgenic Arabidopsis (GFP-TUA6), we find that PP2A and cortical microtubules (MTs) are involved in ALA-regulated stomatal movement. Then, we analyze stomatal responses of Arabidopsis overexpressing C2 catalytic subunit of PP2A (PP2A-C2) and pp2a-c2 mutant to ALA and abscisic acid (ABA) under both light and dark conditions, and show that PP2A-C2 participates in ALA-induced stomatal movement. Furthermore, using pharmacological methods and confocal studies, we reveal that PP2A and MTs function upstream and downstream, respectively, of H2 O2 in guard cell signaling. Finally, we demonstrate the role of H2 O2 -mediated microtubule arrangement in ALA inhibiting ABA-induced stomatal closure. Our findings indicate that MTs regulated by PP2A-mediated H2 O2 decreasing play an important role in ALA guard cell signaling, revealing new insights into stomatal movement regulation.


Assuntos
Ácido Aminolevulínico/farmacologia , Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Peróxido de Hidrogênio/metabolismo , Microtúbulos/fisiologia , Fosfoproteínas Fosfatases/fisiologia , Estômatos de Plantas/fisiologia , Ácido Abscísico , Estômatos de Plantas/citologia , Transdução de Sinais
17.
J Cell Physiol ; 235(1): 26-30, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31219174

RESUMO

Microtubule-interfering agents have been very useful both as biological tools in studying mitosis and as chemotherapeutic agents against cancer. It remains poorly understood how these agents converge on the spindle assembly checkpoint (SAC) to halt mitotic progression, while inhibiting microtubule dynamics by different mechanisms. Cells arrested at mitosis by various microtubule-interfering agents exhibit strikingly different defects in the mitotic spindle. However, all the arrested cells possess the 3F3/2 phosphoepitope at the sister kinetochores of chromosomes, indicating the decrease of tension across the paired kinetochores. In addition, microtubule-interfering agents result in a comparable reduction in the distance between sister kinetochores, suggesting that these agents decrease interkinetochore tension to similar degrees. Here, we discuss recent progress that suggests impairment of kinetochore-microtubule attachment and reduction of interkinetochore tension as common mechanisms underlying the persistent SAC activation in response to diverse microtubule-interfering agents.


Assuntos
Cinetocoros/fisiologia , Microtúbulos/fisiologia , Humanos , Mitose/fisiologia , Fuso Acromático/genética , Fuso Acromático/fisiologia
18.
J Neurosci ; 40(1): 220-236, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31685653

RESUMO

Tau is a microtubule-associated protein that becomes dysregulated in a group of neurodegenerative diseases called tauopathies. Differential tau isoforms, expression levels, promoters, and disruption of endogenous genes in transgenic mouse models of tauopathy make it difficult to draw definitive conclusions about the biological role of tau in these models. We addressed this shortcoming by characterizing the molecular and cognitive phenotypes associated with the pathogenic P301L tau mutation (rT2 mice) in relation to a genetically matched transgenic mouse overexpressing nonmutant (NM) 4-repeat (4R) human tau (rT1 mice). Both male and female mice were included in this study. Unexpectedly, we found that 4R NM human tau (hTau) exhibited abnormal dynamics in young mice that were lost with the P301L mutation, including elevated protein stability and hyperphosphorylation, which were associated with cognitive impairment in 5-month-old rT1 mice. Hyperphosphorylation of NM hTau was observed as early as 4 weeks of age, and transgene suppression for the first 4 or 12 weeks of life prevented abnormal molecular and cognitive phenotypes in rT1, demonstrating that NM hTau pathogenicity is specific to postnatal development. We also show that NM hTau exhibits stronger binding to microtubules than P301L hTau, and is associated with mitochondrial abnormalities. Overall, our genetically matched mice have revealed that 4R NM hTau overexpression is pathogenic in a manner distinct from classical aging-related tauopathy, underlining the importance of assaying the effects of transgenic disease-related proteins at appropriate stages in life.SIGNIFICANCE STATEMENT Due to differences in creation of transgenic lines, the pathological properties of the P301L mutation confers to the tau protein in vivo have remained elusive, perhaps contributing to the lack of disease-modifying therapies for tauopathies. In an attempt to characterize P301L-specific effects on tau biology and cognition in novel genetically matched transgenic mouse models, we surprisingly found that nonmutant human tau has development-specific pathogenic properties of its own. Our findings indicate that overexpression of 4-repeat human tau during postnatal development is associated with excessive microtubule binding, which may disrupt important cellular processes, such as mitochondrial dynamics, leading to elevated stability and hyperphosphorylation of tau, and eventual cognitive impairments.


Assuntos
Transtornos da Memória/genética , Doenças Mitocondriais/genética , Proteínas tau/genética , Animais , Células Cultivadas , Feminino , Genes Sintéticos , Hipocampo/citologia , Humanos , Mutação INDEL , Masculino , Aprendizagem em Labirinto , Transtornos da Memória/fisiopatologia , Camundongos , Camundongos Transgênicos , Microtúbulos/fisiologia , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Doenças Mitocondriais/fisiopatologia , Mutação de Sentido Incorreto , Estresse Oxidativo , Fenótipo , Fosforilação , Mutação Puntual , Prosencéfalo/fisiologia , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes , Sequências Repetitivas de Aminoácidos , Especificidade da Espécie , Regulação para Cima , Proteínas tau/biossíntese
19.
Biochim Biophys Acta Mol Cell Res ; 1867(1): 118572, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31678117

RESUMO

Microtubule-dependent motors usually work together to transport organelles through the crowded intracellular milieu. Thus, transport performance depends on how motors organize on the cargo. Unfortunately, the lack of methodologies capable of measuring this organization in cells determines that many aspects of the collective action of motors remain elusive. Here, we combined fluorescence fluctuations and single particle tracking techniques to address how kinesins organize on rod-like mitochondria moving along microtubules in cells. This methodology simultaneously provides mitochondria trajectories and EGFP-tagged kinesin-1 intensity at different mitochondrial positions with millisecond resolution. We show that kinesin exchange at the mitochondrion surface is within ~100 ms and depends on the organelle speed. During anterograde transport, the mitochondrial leading tip presents slower motor exchange in comparison to the rear tip. In contrast, retrograde mitochondria show similar exchange rates of kinesins at both tips. Numerical simulations provide theoretical support to these results and evidence that motors do not share the load equally during intracellular transport.


Assuntos
Cinesina/metabolismo , Microtúbulos/fisiologia , Organelas/metabolismo , Animais , Transporte Biológico , Células Cultivadas , Drosophila , Fluorescência , Cinética , Microtúbulos/metabolismo , Espectrometria de Fluorescência
20.
EMBO J ; 39(2): e102378, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31782546

RESUMO

In most animal cells, mitotic spindle formation is mediated by coordination of centrosomal and acentrosomal pathways. At the onset of mitosis, centrosomes promote spindle bipolarization. However, the mechanism through which the acentrosomal pathways facilitate the establishment of spindle bipolarity in early mitosis is not completely understood. In this study, we show the critical roles of nuclear mitotic apparatus protein (NuMA) in the generation of spindle bipolarity in acentrosomal human cells. In acentrosomal human cells, we found that small microtubule asters containing NuMA formed at the time of nuclear envelope breakdown. In addition, these asters were assembled by dynein and the clustering activity of NuMA. Subsequently, NuMA organized the radial array of microtubules, which incorporates Eg5, and thus facilitated spindle bipolarization. Importantly, in cells with centrosomes, we also found that NuMA promoted the initial step of spindle bipolarization in early mitosis. Overall, these data suggest that canonical centrosomal and NuMA-mediated acentrosomal pathways redundantly promote spindle bipolarity in human cells.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cinesina/metabolismo , Microtúbulos/fisiologia , Mitose/fisiologia , Fuso Acromático/fisiologia , Células HeLa , Humanos
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