Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 15.161
Filtrar
1.
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
2.
PLoS Biol ; 18(8): e3000820, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866173

RESUMO

Mutations in the gene encoding the microtubule-severing protein spastin (spastic paraplegia 4 [SPG4]) cause hereditary spastic paraplegia (HSP), associated with neurodegeneration, spasticity, and motor impairment. Complicated forms (complicated HSP [cHSP]) further include cognitive deficits and dementia; however, the etiology and dysfunctional mechanisms of cHSP have remained unknown. Here, we report specific working and associative memory deficits upon spastin depletion in mice. Loss of spastin-mediated severing leads to reduced synapse numbers, accompanied by lower miniature excitatory postsynaptic current (mEPSC) frequencies. At the subcellular level, mutant neurons are characterized by longer microtubules with increased tubulin polyglutamylation levels. Notably, these conditions reduce kinesin-microtubule binding, impair the processivity of kinesin family protein (KIF) 5, and reduce the delivery of presynaptic vesicles and postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Rescue experiments confirm the specificity of these results by showing that wild-type spastin, but not the severing-deficient and disease-associated K388R mutant, normalizes the effects at the synaptic, microtubule, and transport levels. In addition, short hairpin RNA (shRNA)-mediated reduction of tubulin polyglutamylation on spastin knockout background normalizes KIF5 transport deficits and attenuates the loss of excitatory synapses. Our data provide a mechanism that connects spastin dysfunction with the regulation of kinesin-mediated cargo transport, synapse integrity, and cognition.


Assuntos
Ácido Glutâmico/metabolismo , Cinesina/metabolismo , Transtornos da Memória/metabolismo , Transtornos da Memória/fisiopatologia , Memória de Curto Prazo , Neurônios/metabolismo , Espastina/deficiência , Tubulina (Proteína)/metabolismo , Potenciais de Ação , Animais , Membrana Celular/metabolismo , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Potenciais Pós-Sinápticos Excitadores , Hipocampo/patologia , Hipocampo/fisiopatologia , Camundongos Knockout , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Atividade Motora , Neurônios/patologia , Neurônios/ultraestrutura , Transporte Proteico , Espastina/metabolismo , Sinapses/metabolismo , Sinapses/ultraestrutura , Vesículas Sinápticas/metabolismo
3.
Nat Commun ; 11(1): 4640, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934232

RESUMO

Small molecule inhibitors are prime reagents for studies in microtubule cytoskeleton research, being applicable across a range of biological models and not requiring genetic engineering. However, traditional chemical inhibitors cannot be experimentally applied with spatiotemporal precision suiting the length and time scales inherent to microtubule-dependent cellular processes. We have synthesised photoswitchable paclitaxel-based microtubule stabilisers, whose binding is induced by photoisomerisation to their metastable state. Photoisomerising these reagents in living cells allows optical control over microtubule network integrity and dynamics, cell division and survival, with biological response on the timescale of seconds and spatial precision to the level of individual cells within a population. In primary neurons, they enable regulation of microtubule dynamics resolved to subcellular regions within individual neurites. These azobenzene-based microtubule stabilisers thus enable non-invasive, spatiotemporally precise modulation of the microtubule cytoskeleton in living cells, and promise new possibilities for studying intracellular transport, cell motility, and neuronal physiology.


Assuntos
Microtúbulos/química , Paclitaxel/química , Linhagem Celular Tumoral , Citoesqueleto/química , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Humanos , Isomerismo , Microtúbulos/metabolismo , Neurônios/química , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Paclitaxel/farmacologia
4.
Phys Rev Lett ; 125(5): 058101, 2020 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-32794890

RESUMO

Diffusion of tracer particles in the cytoplasm of mammalian cells is often anomalous with a marked heterogeneity even within individual particle trajectories. Despite considerable efforts, the mechanisms behind these observations have remained largely elusive. To tackle this problem, we performed extensive single-particle tracking experiments on quantum dots in the cytoplasm of living mammalian cells at varying conditions. Analyses of the trajectories reveal a strong, microtubule-dependent subdiffusion with antipersistent increments and a substantial heterogeneity. Furthermore, particles stochastically switch between different mobility states, most likely due to transient associations with the cytoskeleton-shaken endoplasmic reticulum network. Comparison to simulations highlight that all experimental observations can be fully described by an intermittent fractional Brownian motion, alternating between two states of different mobility.


Assuntos
Citoplasma/metabolismo , Modelos Biológicos , Citoesqueleto de Actina/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Simulação por Computador , Citocalasina D/farmacologia , Citoplasma/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Difusão , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Nocodazol/farmacologia , Pontos Quânticos , Processos Estocásticos , Tiazolidinas/farmacologia
5.
Phys Rev Lett ; 125(7): 078101, 2020 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-32857554

RESUMO

The friction between cytoskeletal filaments is of central importance for the formation of cellular structures such as the mitotic spindle and the cytokinetic ring. This friction is caused by passive cross-linkers, yet the underlying mechanism and the dependence on cross-linker density are poorly understood. Here, we use theory and computer simulations to study the friction between two filaments that are cross-linked by passive proteins, which can hop between discrete binding sites while physically excluding each other. The simulations reveal that filaments move via rare discrete jumps, which are associated with free-energy barrier crossings. We identify the reaction coordinate that governs the relative microtubule movement and derive an exact analytical expression for the free-energy barrier and the friction coefficient. Our analysis not only elucidates the molecular mechanism underlying cross-linker-induced filament friction, but also predicts that the friction coefficient scales superexponentially with the density of cross-linkers.


Assuntos
Citoesqueleto/química , Citoesqueleto/fisiologia , Modelos Biológicos , Modelos Químicos , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/fisiologia , Sítios de Ligação , Citoesqueleto/metabolismo , Fricção , Microtúbulos/química , Microtúbulos/metabolismo , Proteínas Motores Moleculares/metabolismo , Termodinâmica
6.
Nat Commun ; 11(1): 3945, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32770028

RESUMO

TP53 missense mutations leading to the expression of mutant p53 oncoproteins are frequent driver events during tumorigenesis. p53 mutants promote tumor growth, metastasis and chemoresistance by affecting fundamental cellular pathways and functions. Here, we demonstrate that p53 mutants modify structure and function of the Golgi apparatus, culminating in the increased release of a pro-malignant secretome by tumor cells and primary fibroblasts from patients with Li-Fraumeni cancer predisposition syndrome. Mechanistically, interacting with the hypoxia responsive factor HIF1α, mutant p53 induces the expression of miR-30d, which in turn causes tubulo-vesiculation of the Golgi apparatus, leading to enhanced vesicular trafficking and secretion. The mut-p53/HIF1α/miR-30d axis potentiates the release of soluble factors and the deposition and remodeling of the ECM, affecting mechano-signaling and stromal cells activation within the tumor microenvironment, thereby enhancing tumor growth and metastatic colonization.


Assuntos
Neoplasias da Mama/genética , Transformação Celular Neoplásica/genética , Complexo de Golgi/patologia , Síndrome de Li-Fraumeni/genética , MicroRNAs/metabolismo , Proteína Supressora de Tumor p53/genética , Animais , Biópsia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Feminino , Fibroblastos , Regulação Neoplásica da Expressão Gênica , Complexo de Golgi/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Síndrome de Li-Fraumeni/patologia , Camundongos , Microtúbulos/metabolismo , Microtúbulos/patologia , Mutação , Cultura Primária de Células , Vesículas Secretórias/metabolismo , Vesículas Secretórias/patologia , Transdução de Sinais/genética , Pele/citologia , Pele/patologia , Microambiente Tumoral/genética , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Mol Cell ; 80(1): 9-20, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32860741

RESUMO

Cell division requires the assembly and organization of a microtubule spindle for the proper separation of chromosomes in mitosis and meiosis. Phase separation is an emerging paradigm for understanding spatial and temporal regulation of a variety of cellular processes, including cell division. Phase-separated condensates have been recently discovered at many structures during cell division as a possible mechanism for properly localizing, organizing, and activating proteins involved in cell division. Here, we review how these condensates play roles in regulating microtubule density and organization and spindle assembly and function and in activating some of the key players in cell division. We conclude with perspectives on areas of future research for this exciting and rapidly advancing field.


Assuntos
Divisão Celular , Animais , Cromossomos/metabolismo , Humanos , Meiose , Microtúbulos/metabolismo , Fuso Acromático/metabolismo
8.
PLoS One ; 15(8): e0236293, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32760074

RESUMO

To divide replicated chromosomes equally between daughter cells, kinetochores must attach to microtubules emanating from opposite poles of the mitotic spindle (biorientation). An error correction mechanism facilitates this process by destabilizing erroneous kinetochore-microtubule attachments. Here we present a stochastic model of kinetochore-microtubule attachments, via an essential protein Ndc80 in budding yeast, Saccharomyces cerevisiae. Using the model, we calculate the stochastic dynamics of a pair of sister kinetochores as they transition among different attachment states. First of all, we determine the kinase-to-phosphatase balance point that maximizes the probability of biorientation, while starting from an erroneous attachment state. We find that the balance point is sensitive to the rates of microtubule-Ndc80 dissociation and derive an approximate analytical formula that defines the balance point. Secondly, we determine the probability of transition from low-tension amphitelic to monotelic attachment and find that, despite this probability being approximately 33%, biorientation can be achieved with high probability. Thirdly, we calculate the contribution of the geometrical orientation of sister kinetochores to the probability of biorientation and show that, in the absence of geometrical orientation, the biorientation error rate is much larger than that observed in experiments. Finally, we study the coupling of the error correction mechanism to the spindle assembly checkpoint by calculating the average binding of checkpoint-related proteins to the kinetochore during the error correction process.


Assuntos
Segregação de Cromossomos , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Modelos Genéticos , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Pontos de Checagem da Fase M do Ciclo Celular/genética , Processos Estocásticos
9.
Nat Commun ; 11(1): 3388, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636396

RESUMO

Expansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining ExM with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.


Assuntos
Corantes Fluorescentes/química , Hidrogéis/química , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Animais , Tampões (Química) , Células COS , Centríolos/metabolismo , Chlamydomonas reinhardtii/metabolismo , Chlorocebus aethiops , Simulação por Computador , Eletrólitos , Epitopos , Imageamento Tridimensional , Microtúbulos/metabolismo , Distribuição Normal , Fotoquímica
10.
Nat Commun ; 11(1): 3495, 2020 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-32661310

RESUMO

Cell biologists generally consider that microtubules and actin play complementary roles in long- and short-distance transport in animal cells. On the contrary, using melanosomes of melanocytes as a model, we recently discovered that the motor protein myosin-Va works with dynamic actin tracks to drive long-range organelle dispersion in opposition to microtubules. This suggests that in animals, as in yeast and plants, myosin/actin can drive long-range transport. Here, we show that the SPIRE-type actin nucleators (predominantly SPIRE1) are Rab27a effectors that co-operate with formin-1 to generate actin tracks required for myosin-Va-dependent transport in melanocytes. Thus, in addition to melanophilin/myosin-Va, Rab27a can recruit SPIREs to melanosomes, thereby integrating motor and track assembly activity at the organelle membrane. Based on this, we suggest a model in which organelles and force generators (motors and track assemblers) are linked, forming an organelle-based, cell-wide network that allows their collective activity to rapidly disperse the population of organelles long-distance throughout the cytoplasm.


Assuntos
Actinas/metabolismo , Proteínas rab27 de Ligação ao GTP/metabolismo , Biologia Celular , Citoesqueleto/metabolismo , Células HEK293 , Humanos , Microtúbulos/metabolismo , Organelas , Filogenia , Proteínas rab27 de Ligação ao GTP/genética
11.
Nat Commun ; 11(1): 3521, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665556

RESUMO

Microtubules (MTs) mediate mitosis, directional signaling, and are therapeutic targets in cancer. Yet in vivo analysis of cancer cell MT behavior within the tumor microenvironment remains challenging. Here we developed an imaging pipeline using plus-end tip tracking and intravital microscopy to quantify MT dynamics in live xenograft tumor models. Among analyzed features, cancer cells in vivo displayed higher coherent orientation of MT dynamics along their cell major axes compared with 2D in vitro cultures, and distinct from 3D collagen gel cultures. This in vivo MT phenotype was reproduced in vitro when cells were co-cultured with IL4-polarized MΦ. MΦ depletion, MT disruption, targeted kinase inhibition, and altered MΦ polarization via IL10R blockade all reduced MT coherence and/or tumor cell elongation. We show that MT coherence is a defining feature for in vivo tumor cell dynamics and migration, modulated by local signaling from pro-tumor macrophages.


Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animais , Ciclo Celular/genética , Ciclo Celular/fisiologia , Movimento Celular/genética , Movimento Celular/fisiologia , Feminino , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Mitose/genética , Mitose/fisiologia , Análise de Componente Principal , Células RAW 264.7
12.
PLoS Negl Trop Dis ; 14(7): e0008396, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32722702

RESUMO

The parasitophorous vacuoles (PVs) that insulate Leishmania spp. in host macrophages are vacuolar compartments wherein promastigote forms differentiate into amastigote that are the replicative form of the parasite and are also more resistant to host responses. We revisited the biogenesis of tight-fitting PVs that insulate L. infantum in promastigote-infected macrophage-like RAW 264.7 cells by time-dependent confocal laser multidimensional imaging analysis. Pharmacological disassembly of the cellular microtubule network and silencing of the dynein gene led to an impaired interaction of L. infantum-containing phagosomes with late endosomes and lysosomes, resulting in the tight-fitting parasite-containing phagosomes never transforming into mature PVs. Analysis of the shape of the L. infantum parasite within PVs, showed that factors that impair promastigote-amastigote differentiation can also result in PVs whose maturation is arrested. These findings highlight the importance of the MT-dependent interaction of L. infantum-containing phagosomes with the host macrophage endolysosomal pathway to secure the intracellular fate of the parasite.


Assuntos
Leishmania infantum/fisiologia , Leishmaniose Visceral/parasitologia , Macrófagos/parasitologia , Microtúbulos/parasitologia , Animais , Endossomos/metabolismo , Humanos , Leishmania infantum/crescimento & desenvolvimento , Leishmaniose Visceral/metabolismo , Camundongos , Microtúbulos/metabolismo , Fagossomos/metabolismo , Células RAW 264.7
13.
Nat Commun ; 11(1): 3765, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32724196

RESUMO

Microtubules are dynamic tubulin polymers responsible for many cellular processes, including the capture and segregation of chromosomes during mitosis. In contrast to textbook models of tubulin self-assembly, we have recently demonstrated that microtubules elongate by addition of bent guanosine triphosphate tubulin to the tips of curving protofilaments. Here we explore this mechanism of microtubule growth using Brownian dynamics modeling and electron cryotomography. The previously described flaring shapes of growing microtubule tips are remarkably consistent under various assembly conditions, including different tubulin concentrations, the presence or absence of a polymerization catalyst or tubulin-binding drugs. Simulations indicate that development of substantial forces during microtubule growth and shortening requires a high activation energy barrier in lateral tubulin-tubulin interactions. Modeling offers a mechanism to explain kinetochore coupling to growing microtubule tips under assisting force, and it predicts a load-dependent acceleration of microtubule assembly, providing a role for the flared morphology of growing microtubule ends.


Assuntos
Microtúbulos/metabolismo , Modelos Biológicos , Tubulina (Proteína)/metabolismo , Animais , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Simulação de Dinâmica Molecular , Polimerização/efeitos dos fármacos , Suínos , Tubulina (Proteína)/isolamento & purificação , Tubulina (Proteína)/ultraestrutura , Moduladores de Tubulina/farmacologia
14.
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
15.
Proc Natl Acad Sci U S A ; 117(29): 17399-17408, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32641513

RESUMO

The cytoskeleton plays a key role in establishing robust cell shape. In animals, it is well established that cell shape can also influence cytoskeletal organization. Cytoskeletal proteins are well conserved between animal and plant kingdoms; nevertheless, because plant cells exhibit major structural differences to animal cells, the question arises whether the plant cytoskeleton also responds to geometrical cues. Recent numerical simulations predicted that a geometry-based rule is sufficient to explain the microtubule (MT) organization observed in cells. Due to their high flexural rigidity and persistence length of the order of a few millimeters, MTs are rigid over cellular dimensions and are thus expected to align along their long axis if constrained in specific geometries. This hypothesis remains to be tested in cellulo Here, we explore the relative contribution of geometry to the final organization of actin and MT cytoskeletons in single plant cells of Arabidopsis thaliana We show that the cytoskeleton aligns with the long axis of the cells. We find that actin organization relies on MTs but not the opposite. We develop a model of self-organizing MTs in three dimensions, which predicts the importance of MT severing, which we confirm experimentally. This work is a first step toward assessing quantitatively how cellular geometry contributes to the control of cytoskeletal organization in living plant cells.


Assuntos
Fenômenos Fisiológicos Celulares , Forma Celular/fisiologia , Citoesqueleto/fisiologia , Células Vegetais/fisiologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actinas , Arabidopsis/metabolismo , Citocalasina D/farmacologia , Microtúbulos/metabolismo , Células Vegetais/efeitos dos fármacos , Células Vegetais/ultraestrutura , Protoplastos
16.
PLoS Pathog ; 16(6): e1008597, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32511265

RESUMO

During infection of neurons by alphaherpesviruses including Pseudorabies virus (PRV) and Herpes simplex virus type 1 (HSV-1) viral nucleocapsids assemble in the cell nucleus, become enveloped in the cell body then traffic into and down axons to nerve termini for spread to adjacent epithelia. The viral membrane protein US9p and the membrane glycoprotein heterodimer gE/gI play critical roles in anterograde spread of both HSV-1 and PRV, and several models exist to explain their function. Biochemical studies suggest that PRV US9p associates with the kinesin-3 motor KIF1A in a gE/gI-stimulated manner, and the gE/gI-US9p complex has been proposed to recruit KIF1A to PRV for microtubule-mediated anterograde trafficking into or along the axon. However, as loss of gE/gI-US9p essentially abolishes delivery of alphaherpesviruses to the axon it is difficult to determine the microtubule-dependent trafficking properties and motor-composition of Δ(gE/gI-US9p) particles. Alternatively, studies in HSV-1 have suggested that gE/gI and US9p are required for the appearance of virions in the axon because they act upstream, to help assemble enveloped virions in the cell body. We prepared Δ(gE/gI-US9p) mutant, and control parental PRV particles from differentiated cultured neuronal or porcine kidney epithelial cells and quantitated the efficiency of virion assembly, the properties of microtubule-dependent transport and the ability of viral particles to recruit kinesin motors. We find that loss of gE/gI-US9p has no significant effect upon PRV particle assembly but leads to greatly diminished plus end-directed traffic, and enhanced minus end-directed and bidirectional movement along microtubules. PRV particles prepared from infected differentiated mouse CAD neurons were found to be associated with either kinesin KIF1A or kinesin KIF5C, but not both. Loss of gE/gI-US9p resulted in failure to recruit KIF1A and KF5C, but did not affect dynein binding. Unexpectedly, while KIF5C was expressed in undifferentiated and differentiated CAD neurons it was only found associated with PRV particles prepared from differentiated cells.


Assuntos
Herpesvirus Suídeo 1 , Peptídeos e Proteínas de Sinalização Intracelular , Cinesina/metabolismo , Lipoproteínas , Microtúbulos/metabolismo , Pseudorraiva , Proteínas do Envelope Viral , Proteínas Virais , Liberação de Vírus , Animais , Transporte Biológico Ativo , Linhagem Celular , Deleção de Genes , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinesina/genética , Lipoproteínas/genética , Lipoproteínas/metabolismo , Microtúbulos/genética , Microtúbulos/virologia , Pseudorraiva/genética , Pseudorraiva/metabolismo , Pseudorraiva/patologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
17.
Nature ; 582(7810): 115-118, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32494070

RESUMO

During cell division, remodelling of the nuclear envelope enables chromosome segregation by the mitotic spindle1. The reformation of sealed nuclei requires ESCRTs (endosomal sorting complexes required for transport) and LEM2, a transmembrane ESCRT adaptor2-4. Here we show how the ability of LEM2 to condense on microtubules governs the activation of ESCRTs and coordinated spindle disassembly. The LEM motif of LEM2 binds BAF, conferring on LEM2 an affinity for chromatin5,6, while an adjacent low-complexity domain (LCD) promotes LEM2 phase separation. A proline-arginine-rich sequence within the LCD binds to microtubules and targets condensation of LEM2 to spindle microtubules that traverse the nascent nuclear envelope. Furthermore, the winged-helix domain of LEM2 activates the ESCRT-II/ESCRT-III hybrid protein CHMP7 to form co-oligomeric rings. Disruption of these events in human cells prevented the recruitment of downstream ESCRTs, compromised spindle disassembly, and led to defects in nuclear integrity and DNA damage. We propose that during nuclear reassembly LEM2 condenses into a liquid-like phase and coassembles with CHMP7 to form a macromolecular O-ring seal at the confluence between membranes, chromatin and the spindle. The properties of LEM2 described here, and the homologous architectures of related inner nuclear membrane proteins7,8, suggest that phase separation may contribute to other critical envelope functions, including interphase repair8-13 and chromatin organization14-17.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Anáfase , Cromatina/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Microtúbulos/química , Microtúbulos/metabolismo , Membrana Nuclear/química , Fuso Acromático/metabolismo
18.
Nat Commun ; 11(1): 3123, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561740

RESUMO

Intracellular trafficking of organelles, driven by kinesin-1 stepping along microtubules, underpins essential cellular processes. In absence of other proteins on the microtubule surface, kinesin-1 performs micron-long runs. Under crowding conditions, however, kinesin-1 motility is drastically impeded. It is thus unclear how kinesin-1 acts as an efficient transporter in intracellular environments. Here, we demonstrate that TRAK1 (Milton), an adaptor protein essential for mitochondrial trafficking, activates kinesin-1 and increases robustness of kinesin-1 stepping on crowded microtubule surfaces. Interaction with TRAK1 i) facilitates kinesin-1 navigation around obstacles, ii) increases the probability of kinesin-1 passing through cohesive islands of tau and iii) increases the run length of kinesin-1 in cell lysate. We explain the enhanced motility by the observed direct interaction of TRAK1 with microtubules, providing an additional anchor for the kinesin-1-TRAK1 complex. Furthermore, TRAK1 enables mitochondrial transport in vitro. We propose adaptor-mediated tethering as a mechanism regulating kinesin-1 motility in various cellular environments.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Cinesina/metabolismo , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/isolamento & purificação , Animais , Linhagem Celular Tumoral , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Cinesina/genética , Cinesina/isolamento & purificação , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia de Fluorescência , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismo
19.
J Vis Exp ; (159)2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32478717

RESUMO

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. Furthermore, despite progress in understanding postsynaptic MTs, much less is known about the contributions of presynaptic MTs to neuronal morphogenesis. In particular, studies of in vivo MT dynamics in Drosophila sensory dendrites yielded significant insights into polymer-level behavior. However, the technical and analytical challenges associated with live imaging of the fly neuromuscular junction (NMJ) have limited comparable studies of presynaptic MT dynamics. Moreover, while there are many highly effective software strategies for automated analysis of MT dynamics in vitro and ex vivo, in vivo data often necessitate significant operator input or entirely manual analysis due to inherently inferior signal-to-noise ratio in images and complex cellular morphology.  To address this, this study optimized a new software platform for automated and unbiased in vivo particle detection. Multiparametric analysis of live time-lapse confocal images of EB1-GFP labeled MTs was performed in both dendrites and the NMJ of Drosophila larvae and found striking differences in MT behaviors. MT dynamics were furthermore analyzed following knockdown of the MT-associated protein (MAP) dTACC, a key regulator of Drosophila synapse development, and identified statistically significant changes in MT dynamics compared to wild type. These results demonstrate that this novel strategy for the automated multiparametric analysis of both pre- and postsynaptic MT dynamics at the polymer-level significantly reduces human-in-the-loop criteria. The study furthermore shows the utility of this method in detecting distinct MT behaviors upon dTACC-knockdown, indicating a possible future application for functional screens of factors that regulate MT dynamics in vivo. Future applications of this method may also focus on elucidating cell type and/or compartment-specific MT behaviors, and multicolor correlative imaging of EB1-GFP with other cellular and subcellular markers of interest.


Assuntos
Dendritos/metabolismo , Drosophila melanogaster/metabolismo , Imageamento Tridimensional , Microtúbulos/metabolismo , Junção Neuromuscular/metabolismo , Imagem Individual de Molécula , Sinapses/metabolismo , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Larva/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Interferência de RNA , Software
20.
J Vis Exp ; (159)2020 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-32510485

RESUMO

Understanding cell and tissue level regulation of growth and morphogenesis has been at the forefront of biological research for many decades. Advances in molecular and imaging technologies allowed us to gain insights into how biochemical signals influence morphogenetic events. However, it is increasingly evident that apart from biochemical signals, mechanical cues also impact several aspects of cell and tissue growth. The Arabidopsis shoot apical meristem (SAM) is a dome-shaped structure responsible for the generation of all aboveground organs. The organization of the cortical microtubule cytoskeleton that mediates apoplastic cellulose deposition in plant cells is spatially distinct. Visualization and quantitative assessment of patterns of cortical microtubules are necessary for understanding the biophysical nature of cells at the SAM, as cellulose is the stiffest component of the plant cell wall. The stereotypical form of cortical microtubule organization is also a consequence of tissue-wide physical forces existing at the SAM. Perturbation of these physical forces and subsequent monitoring of cortical microtubule organization allows for the identification of candidate proteins involved in mediating mechano-perception and transduction. Here we describe a protocol that helps investigate such processes.


Assuntos
Arabidopsis/citologia , Citoesqueleto/metabolismo , Fenômenos Mecânicos , Microtúbulos/metabolismo , Imagem Molecular , Arabidopsis/crescimento & desenvolvimento , Fenômenos Biomecânicos , Sobrevivência Celular , Parede Celular/metabolismo , Celulose/metabolismo , Meristema/citologia , Meristema/crescimento & desenvolvimento , Morfogênese
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA