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1.
Nat Cell Biol ; 21(6): 768-777, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061466

RESUMO

Controlling cellular processes with light can help elucidate their underlying mechanisms. Here we present zapalog, a small-molecule dimerizer that undergoes photolysis when exposed to blue light. Zapalog dimerizes any two proteins tagged with the FKBP and DHFR domains until exposure to light causes its photolysis. Dimerization can be repeatedly restored with uncleaved zapalog. We implement this method to investigate mitochondrial motility and positioning in cultured neurons. Using zapalog, we tether mitochondria to constitutively active kinesin motors, forcing them down the axon towards microtubule (+) ends until their instantaneous release via blue light, which results in full restoration of their endogenous motility. We find that one-third of stationary mitochondria cannot be pulled away from their position and that these firmly anchored mitochondria preferentially localize to VGLUT1-positive presynapses. Furthermore, inhibition of actin polymerization with latrunculin A reduces this firmly anchored pool. On release from exogenous motors, mitochondria are preferentially recaptured at presynapses.


Assuntos
Axônios/metabolismo , Mitocôndrias/genética , Fotólise , Multimerização Proteica/efeitos da radiação , Actinas/antagonistas & inibidores , Animais , Axônios/química , Axônios/efeitos da radiação , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Células COS , Cercopithecus aethiops , Cinesina/química , Luz , Microtúbulos/genética , Microtúbulos/efeitos da radiação , Mitocôndrias/química , Mitocôndrias/efeitos da radiação , Neurônios/química , Neurônios/efeitos da radiação , Polimerização/efeitos dos fármacos , Domínios Proteicos/genética , Domínios Proteicos/efeitos da radiação , Multimerização Proteica/genética , Sinapses/química , Sinapses/genética , Sinapses/efeitos da radiação , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/genética , Tiazolidinas/farmacologia , Proteína Vesicular 1 de Transporte de Glutamato/genética
2.
Nat Commun ; 10(1): 2129, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086189

RESUMO

De novo heterozygous missense variants in the γ-tubulin gene TUBG1 have been linked to human malformations of cortical development associated with intellectual disability and epilepsy. Here, we investigated through in-utero electroporation and in-vivo studies, how four of these variants affect cortical development. We show that TUBG1 mutants affect neuronal positioning, disrupting the locomotion of new-born neurons but without affecting progenitors' proliferation. We further demonstrate that pathogenic TUBG1 variants are linked to reduced microtubule dynamics but without major structural nor functional centrosome defects in subject-derived fibroblasts. Additionally, we developed a knock-in Tubg1Y92C/+ mouse model and assessed consequences of the mutation. Although centrosomal positioning in bipolar neurons is correct, they fail to initiate locomotion. Furthermore, Tubg1Y92C/+ animals show neuroanatomical and behavioral defects and increased epileptic cortical activity. We show that Tubg1Y92C/+ mice partially mimic the human phenotype and therefore represent a relevant model for further investigations of the physiopathology of cortical malformations.


Assuntos
Malformações do Desenvolvimento Cortical/genética , Microtúbulos/metabolismo , Neurogênese/genética , Neurônios/fisiologia , Tubulina (Proteína)/genética , Animais , Comportamento Animal , Movimento Celular/genética , Centrossomo/metabolismo , Córtex Cerebral/anormalidades , Córtex Cerebral/citologia , Córtex Cerebral/diagnóstico por imagem , Modelos Animais de Doenças , Embrião de Mamíferos , Epilepsia/genética , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Técnicas de Introdução de Genes , Predisposição Genética para Doença , Células HeLa , Humanos , Microscopia Intravital , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Microscopia Eletrônica , Microtúbulos/genética , Mutação de Sentido Incorreto
3.
Nat Commun ; 10(1): 682, 2019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30737408

RESUMO

Proper chromosome segregation depends upon kinetochore phosphorylation by the Chromosome Passenger Complex (CPC). Current models suggest the activity of the CPC decreases in response to the inter-kinetochore stretch that accompanies the formation of bi-oriented microtubule attachments, however little is known about tension-independent CPC phosphoregulation. Microtubule bundles initially lie in close proximity to inner centromeres and become depleted by metaphase. Here we find these microtubules control kinetochore phosphorylation by the CPC in a tension independent manner via a microtubule-binding site on the Borealin subunit. Disruption of Borealin-microtubule interactions generates reduced phosphorylation of prometaphase kinetochores, improper kinetochore-microtubule attachments and weakened spindle checkpoint signals. Experimental and modeling evidence suggests that kinetochore phosphorylation is greatly stimulated when the CPC binds microtubules that lie near the inner centromere, even if kinetochores have high inter-kinetochore stretch. We propose the CPC senses its local environment through microtubule structures to control phosphorylation of kinetochores.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromossomos/metabolismo , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Proteínas de Ciclo Celular/genética , Humanos , Microtúbulos/genética , Mitose/fisiologia , Fosforilação , Ligação Proteica
4.
Proc Natl Acad Sci U S A ; 116(9): 3584-3593, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30659149

RESUMO

Cilia are cell-surface, microtubule-based organelles that project into extracellular space. Motile cilia are conserved throughout eukaryotes, and their beat induces the flow of fluid, relative to cell surfaces. In mammals, the coordinated beat of motile cilia provides highly specialized functions associated with the movement of luminal contents, as seen with metachronal waves transporting mucus in the respiratory tract. Motile cilia are also present in the male and female reproductive tracts. In the female, wave-like motions of oviductal cilia transport oocytes and embryos toward the uterus. A similar function has been assumed for motile cilia in efferent ductules of the male-i.e., to transport immotile sperm from rete testis into the epididymis. However, we report here that efferent ductal cilia in the male do not display a uniform wave-like beat to transport sperm solely in one direction, but rather exert a centripetal force on luminal fluids through whip-like beating with continual changes in direction, generating turbulence, which maintains immotile spermatozoa in suspension within the lumen. Genetic ablation of two miRNA clusters (miR-34b/c and -449a/b/c) led to failure in multiciliogenesis in murine efferent ductules due to dysregulation of numerous genes, and this mouse model allowed us to demonstrate that loss of efferent duct motile cilia causes sperm aggregation and agglutination, luminal obstruction, and sperm granulomas, which, in turn, induce back-pressure atrophy of the testis and ultimately male infertility.


Assuntos
Cílios/genética , Infertilidade Masculina/genética , MicroRNAs/genética , Animais , Epididimo/crescimento & desenvolvimento , Epididimo/patologia , Feminino , Genitália Masculina/crescimento & desenvolvimento , Humanos , Infertilidade Masculina/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Microtúbulos/genética , Microtúbulos/metabolismo , Espermatozoides/crescimento & desenvolvimento , Espermatozoides/patologia , Testículo/crescimento & desenvolvimento , Testículo/metabolismo
5.
J Biol Chem ; 294(10): 3385-3396, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30602572

RESUMO

Mitochondria are organized as tubular networks in the cell and undergo fission and fusion. Although several of the molecular players involved in mediating mitochondrial dynamics have been identified, the precise cellular cues that initiate mitochondrial fission or fusion remain largely unknown. In fission yeast (Schizosaccharomyces pombe), mitochondria are organized along microtubule bundles. Here, we employed deletions of kinesin-like proteins to perturb microtubule dynamics and used high-resolution and time-lapse fluorescence microscopy, revealing that mitochondrial lengths mimic microtubule lengths. Furthermore, we determined that compared with WT cells, mutant cells with long microtubules exhibit fewer mitochondria, and mutant cells with short microtubules have an increased number of mitochondria because of reduced mitochondrial fission in the former and elevated fission in the latter. Correspondingly, upon onset of closed mitosis in fission yeast, wherein interphase microtubules assemble to form the spindle within the nucleus, we observed increased mitochondrial fission. We found that the consequent rise in the mitochondrial copy number is necessary to reduce partitioning errors during independent segregation of mitochondria between daughter cells. We also discovered that the association of mitochondria with microtubules physically impedes the assembly of the fission protein Dnm1 around mitochondria, resulting in inhibition of mitochondrial fission. Taken together, we demonstrate a mechanism for the regulation of mitochondrial fission that is dictated by the interaction between mitochondria and the microtubule cytoskeleton.


Assuntos
Dinaminas/metabolismo , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Dinaminas/genética , Microtúbulos/genética , Mitocôndrias/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética
6.
EMBO Rep ; 20(1)2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30552148

RESUMO

RNA-binding proteins (RBPs) determine spatiotemporal gene expression by mediating active transport and local translation of cargo mRNAs. Here, we cast a transcriptome-wide view on the transported mRNAs and cognate RBP binding sites during endosomal messenger ribonucleoprotein (mRNP) transport in Ustilago maydis Using individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP), we compare the key transport RBP Rrm4 and the newly identified endosomal mRNP component Grp1 that is crucial to coordinate hyphal growth. Both RBPs bind predominantly in the 3' untranslated region of thousands of shared cargo mRNAs, often in close proximity. Intriguingly, Rrm4 precisely binds at stop codons, which constitute landmark sites of translation, suggesting an intimate connection of mRNA transport and translation. Towards uncovering the code of recognition, we identify UAUG as specific binding motif of Rrm4 that is bound by its third RRM domain. Altogether, we provide first insights into the positional organisation of co-localising RBPs on individual cargo mRNAs.


Assuntos
Proteínas Fúngicas/genética , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genética , Ustilago/genética , Sítios de Ligação , Transporte Biológico/genética , Endossomos/genética , Regulação da Expressão Gênica , Microtúbulos/genética , Transporte de RNA/genética , RNA Mensageiro/genética , Transcriptoma/genética
7.
G3 (Bethesda) ; 8(8): 2663-2672, 2018 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-29930198

RESUMO

Idiopathic scoliosis (IS) is a structural lateral spinal curvature of ≥10° that affects up to 3% of otherwise healthy children and can lead to life-long problems in severe cases. It is well-established that IS is a genetic disorder. Previous studies have identified genes that may contribute to the IS phenotype, but the overall genetic etiology of IS is not well understood. We used exome sequencing to study five multigenerational families with IS. Bioinformatic analyses identified unique and low frequency variants (minor allele frequency ≤5%) that were present in all sequenced members of the family. Across the five families, we identified a total of 270 variants with predicted functional consequences in 246 genes, and found that eight genes were shared by two families. We performed GO term enrichment analyses, with the hypothesis that certain functional annotations or pathways would be enriched in the 246 genes identified in our IS families. Using three complementary programs to complete these analyses, we identified enriched categories that include stereocilia and other actin-based cellular projections, cilia and other microtubule-based cellular projections, and the extracellular matrix (ECM). Our results suggest that there are multiple paths to IS and provide a foundation for future studies of IS pathogenesis.


Assuntos
Citoesqueleto de Actina/genética , Matriz Extracelular/genética , Microtúbulos/genética , Escoliose/genética , Adulto , Criança , Matriz Extracelular/metabolismo , Feminino , Humanos , Masculino , Microtúbulos/metabolismo , Linhagem , Polimorfismo Genético , Escoliose/etiologia , Escoliose/patologia
8.
Int J Mol Sci ; 19(5)2018 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-29734651

RESUMO

Neuropathological aggregates of the intrinsically disordered microtubule-associated protein Tau are hallmarks of Alzheimer’s disease, with decades of research devoted to studying the protein’s aggregation properties both in vitro and in vivo. Recent demonstrations that Tau is capable of undergoing liquid-liquid phase separation (LLPS) reveal the possibility that protein-enriched phase separated compartments could serve as initiation sites for Tau aggregation, as shown for other amyloidogenic proteins, such as the Fused in Sarcoma protein (FUS) and TAR DNA-binding protein-43 (TDP-43). Although truncation, mutation, and hyperphosphorylation have been shown to enhance Tau LLPS and aggregation, the effect of hyperacetylation on Tau aggregation remains unclear. Here, we investigate how the acetylation of Tau affects its potential to undergo phase separation and aggregation. Our data show that the hyperacetylation of Tau by p300 histone acetyltransferase (HAT) disfavors LLPS, inhibits heparin-induced aggregation, and impedes access to LLPS-initiated microtubule assembly. We propose that Tau acetylation prevents the toxic effects of LLPS-dependent aggregation but, nevertheless, contributes to Tau loss-of-function pathology by inhibiting Tau LLPS-mediated microtubule assembly.


Assuntos
Doença de Alzheimer/metabolismo , Agregação Patológica de Proteínas/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Proteínas tau/metabolismo , Acetilação , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Heparina/química , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Extração Líquido-Líquido , Microtúbulos/genética , Microtúbulos/metabolismo , Fosforilação , Agregação Patológica de Proteínas/genética , Fatores de Transcrição de p300-CBP/genética , Proteínas tau/química , Proteínas tau/genética
9.
Development ; 145(11)2018 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-29739839

RESUMO

The shoot apical meristem of higher plants continuously generates new tissues and organs through complex changes in growth rates and directions of its individual cells. Cell growth, which is driven by turgor pressure, largely depends on the cell walls, which allow cell expansion through synthesis and structural changes. A previous study revealed a major contribution of wall isotropy in organ emergence, through the disorganization of cortical microtubules. We show here that this disorganization is coupled with the transcriptional control of genes involved in wall remodelling. Some of these genes are induced when microtubules are disorganized and cells shift to isotropic growth. Mechanical modelling shows that this coupling has the potential to compensate for reduced cell expansion rates induced by the shift to isotropic growth. Reciprocally, cell wall loosening induced by different treatments or altered cell wall composition promotes a disruption of microtubule alignment. Our data thus indicate the existence of a regulatory module activated during organ outgrowth, linking microtubule arrangements to cell wall remodelling.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Parede Celular/genética , Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Meristema/crescimento & desenvolvimento , Microtúbulos/metabolismo , Fenômenos Biomecânicos/fisiologia , Proliferação de Células/fisiologia , Ácidos Indolacéticos/metabolismo , Meristema/genética , Microtúbulos/genética
10.
Development ; 145(9)2018 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-29712642

RESUMO

Large-scale neurite pruning is an important specificity mechanism during neuronal morphogenesis. Drosophila sensory neurons prune their larval dendrites during metamorphosis. Pruning dendrites are severed in their proximal regions, but how this spatial information is encoded is not clear. Dendrite severing is preceded by local breakdown of dendritic microtubules through PAR-1-mediated inhibition of Tau. Here, we investigated spatial aspects of microtubule breakdown during dendrite pruning. Live imaging of fluorescently tagged tubulin shows that microtubule breakdown first occurs at proximal dendritic branchpoints, followed by breakdown at more distal branchpoints, suggesting that the process is triggered by a signal emanating from the soma. In fly dendrites, microtubules are arranged in uniformly oriented arrays where all plus ends face towards the soma. Mutants in kinesin-1 and -2, which are required for uniform microtubule orientation, show defects in microtubule breakdown and dendrite pruning. Our data suggest that the local microtubule organization at branchpoints determines where microtubule breakdown occurs. Local microtubule organization may therefore contribute spatial information for severing sites during dendrite pruning.


Assuntos
Dendritos/metabolismo , Proteínas de Drosophila/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Microtúbulos/metabolismo , Transdução de Sinais/fisiologia , Proteínas tau/metabolismo , Animais , Dendritos/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Quinase 3 da Glicogênio Sintase/genética , Cinesina/genética , Cinesina/metabolismo , Larva/genética , Larva/metabolismo , Microtúbulos/genética , Mutação , Proteínas tau/genética
11.
Proc Natl Acad Sci U S A ; 115(11): 2740-2745, 2018 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-29487209

RESUMO

Accurate segregation of chromosomes relies on the force-bearing capabilities of the kinetochore to robustly attach chromosomes to dynamic microtubule tips. The human Ska complex and Ndc80 complex are outer-kinetochore components that bind microtubules and are required to fully stabilize kinetochore-microtubule attachments in vivo. While purified Ska complex tracks with disassembling microtubule tips, it remains unclear whether the Ska complex-microtubule interaction is sufficiently strong to make a significant contribution to kinetochore-microtubule coupling. Alternatively, Ska complex might affect kinetochore coupling indirectly, through recruitment of phosphoregulatory factors. Using optical tweezers, we show that the Ska complex itself bears load on microtubule tips, strengthens Ndc80 complex-based tip attachments, and increases the switching dynamics of the attached microtubule tips. Cross-linking mass spectrometry suggests the Ska complex directly binds Ndc80 complex through interactions between the Ska3 unstructured C-terminal region and the coiled-coil regions of each Ndc80 complex subunit. Deletion of the Ska complex microtubule-binding domain or the Ska3 C terminus prevents Ska complex from strengthening Ndc80 complex-based attachments. Together, our results indicate that the Ska complex can directly strengthen the kinetochore-microtubule interface and regulate microtubule tip dynamics by forming an additional connection between the Ndc80 complex and the microtubule.


Assuntos
Cinetocoros/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Segregação de Cromossomos , Humanos , Cinetocoros/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/química , Microtúbulos/genética , Proteínas Nucleares/genética , Ligação Proteica
12.
Biochim Biophys Acta Mol Cell Res ; 1865(5): 734-748, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29499229

RESUMO

γ-Tubulin is essential for microtubule nucleation and also plays less understood roles in nuclear and cell-cycle-related functions. High abundancy of γ-tubulin in acentrosomal Arabidopsis cells facilitated purification and biochemical characterization of large molecular species of γ-tubulin. TEM, fluorescence, and atomic force microscopy of purified high molecular γ-tubulin forms revealed the presence of linear filaments with a double protofilament substructure, filament bundles and aggregates. Filament formation from highly purified γ-tubulin free of γ-tubulin complex proteins (GCPs) was demonstrated for both plant and human γ-tubulin. Moreover, γ-tubulin associated with porcine brain microtubules formed oligomers. Experimental evidence on the intrinsic ability of γ-tubulin to oligomerize/polymerize was supported by conservation of α- and ß-tubulin interfaces for longitudinal and lateral interactions for γ-tubulins. STED (stimulated emission depletion) microscopy of Arabidopsis cells revealed fine, short γ-tubulin fibrillar structures enriched on mitotic microtubular arrays that accumulated at polar regions of acentrosomal spindles and the outer nuclear envelope before mitosis, and were also present in nuclei. Fine fibrillar structures of γ-tubulin representing assemblies of higher order were localized in cell-cycle-dependent manner at sites of dispersed γ-tubulin location in acentrosomal plant cells as well as at sites of local γ-tubulin enrichment after drug treatment. Our findings that γ-tubulin preserves the capability of prokaryotic tubulins to self-organize into filaments assembling by lateral interaction into bundles/clusters help understanding of the relationship between structure and multiple cellular functions of this protein species and suggest that besides microtubule nucleation and organization, γ-tubulin may also have scaffolding or sequestration functions.


Assuntos
Citoesqueleto/genética , Proteínas Associadas aos Microtúbulos/genética , Agregados Proteicos/genética , Tubulina (Proteína)/genética , Citoesqueleto de Actina/química , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/ultraestrutura , Arabidopsis/química , Arabidopsis/genética , Citoesqueleto/química , Proteínas Associadas aos Microtúbulos/química , Microtúbulos/química , Microtúbulos/genética , Mitose/genética , Polimerização , Tubulina (Proteína)/química , Tubulina (Proteína)/ultraestrutura
13.
Biochim Biophys Acta Mol Basis Dis ; 1864(5 Pt A): 1634-1641, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29425932

RESUMO

Fibrosis is an undesirable consequence of injury and a critical problem in many diseases. Recent studies have demonstrated an association of C/EBP homologous protein (CHOP) with fibrosis. We investigated the mechanism of CHOP in kidney fibrosis progression after unilateral ureteral obstruction (UUO) using Chop gene-deleted (Chop-/-) mice and their wild-type littermates (Chop+/+). UUO-induced kidney fibrosis was reduced in the Chop-/- than Chop+/+ mice. After UUO, CHOP expression was detected in the cytosol and nucleus of distal tubule cells and collecting duct cells of the kidney. UUO formed the autophagosome and increased the expression of autophagy proteins, Beclin-1, LC3-I and II, and p62 in the kidneys. These UUO-induced changes were significantly reduced in Chop-/- mice. Furthermore, Chop gene deletion attenuated mitochondrial fragmentation with lower expression of Fis-1, a mitochondrial fission protein, but higher expression of Opa-1, a mitochondrial fusion protein, than that seen in the wild-type mice. UUO disrupted the microtubule, which is involved in autophagosome formation, and this disruption was milder in the Chop-/- than Chop+/+ mouse kidney, with less reduction of histone deacetylase 6 and α­tubulin acetyl transferase, which acetylates tubulin, a component of the microtubule. After UUO, apoptosis, a consequence of autophagy and mitochondrial damage, was reduced in the Chop-/- mouse kidney cells than in Chop+/+ mice. Thus, the ablation of Chop attenuates renal fibrosis, accompanied by reduced autophagy, mitochondrial fragmentation, microtubule disruption, and apoptosis. Overall, these results suggest that CHOP plays a critical role in the progression of kidney fibrosis, likely through regulation of autophagy and apoptosis.


Assuntos
Apoptose , Autofagia , Nefropatias , Microtúbulos/metabolismo , Fator de Transcrição CHOP , Obstrução Ureteral , Animais , Fibrose , Nefropatias/genética , Nefropatias/metabolismo , Nefropatias/patologia , Camundongos , Camundongos Knockout , Microtúbulos/genética , Microtúbulos/patologia , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Obstrução Ureteral/genética , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia
14.
Dev Biol ; 434(2): 249-266, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29287832

RESUMO

Control of microtubule dynamics is crucial for cell migration. We analyzed regulation of microtubule network dynamics in the zebrafish yolk cell during epiboly, the earliest coordinated gastrulation movement. We labeled microtubules with EMTB-3GFP and EB3-mCherry to visualize and measure microtubule dynamics by TIRF microscopy live imaging. Yolk cell microtubules dynamics is temporally modulated during epiboly progression. We used maternal zygotic Pou5f3 mutant (MZspg) embryos, which develop strong distortions of microtubule network organization and epiboly retardation, to investigate genetic control of microtubule dynamics. In MZspg embryos, microtubule plus-end growth tracks move slower and are less straight compared to wild-type. MZspg embryos have altered steroidogenic enzyme expression, resulting in increased pregnenolone and reduced progesterone levels. We show that progesterone positively affects microtubule plus-end growth and track straightness. Progesterone may thus act as a non-cell-autonomous regulator of microtubule dynamics across the large yolk cell, and may adjust differing demands on microtubule dynamics and stability during initiation and progression phases of epiboly.


Assuntos
Gástrula/embriologia , Gastrulação/efeitos dos fármacos , Microtúbulos/metabolismo , Progesterona/farmacologia , Peixe-Zebra/embriologia , Animais , Gastrulação/fisiologia , Microtúbulos/genética , Peixe-Zebra/genética
15.
Exp Cell Res ; 362(2): 477-488, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29253534

RESUMO

The process of tissue morphogenesis, especially for tissues reliant on the establishment of a specific cytoarchitecture for their functionality, depends a balanced interplay between cytoskeletal elements and their interactions with cell adhesion molecules. The microtubule cytoskeleton, which has many roles in the cell, is a determinant of directional cell migration, a process that underlies many aspects of development. We investigated the role of microtubules in development of the lens, a tissue where cell elongation underlies morphogenesis. Our studies with the microtubule depolymerizing agent nocodazole revealed an essential function for the acetylated population of stable microtubules in the elongation of lens fiber cells, which was linked to their regulation of the activation state of myosin. Suppressing myosin activation with the inhibitor blebbistatin could attenuate the loss of acetylated microtubules by nocodazole and rescue the effect of this microtubule depolymerization agent on both fiber cell elongation and lens integrity. Our results also suggest that acetylated microtubules impact lens morphogenesis through their interaction with N-cadherin junctions, with which they specifically associate in the region where lens fiber cell elongate. Disruption of the stable microtubule network increased N-cadherin junctional organization along lateral borders of differentiating lens fiber cells, which was prevented by suppression of myosin activity. These results reveal a role for the stable microtubule population in lens fiber cell elongation, acting in tandem with N-cadherin cell-cell junctions and the actomyosin network, giving insight into the cooperative role these systems play in tissue morphogenesis.


Assuntos
Caderinas/genética , Diferenciação Celular/genética , Cristalino/metabolismo , Morfogênese/genética , Acetilação/efeitos dos fármacos , Actomiosina/genética , Animais , Adesão Celular/genética , Movimento Celular/genética , Proliferação de Células/genética , Galinhas/genética , Citoesqueleto/genética , Humanos , Junções Intercelulares/efeitos dos fármacos , Junções Intercelulares/genética , Cristalino/crescimento & desenvolvimento , Microtúbulos/genética , Microtúbulos/metabolismo , Nocodazol/farmacologia
16.
Biochem Biophys Res Commun ; 495(1): 346-352, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29127012

RESUMO

Dystonia-1 (DYT1) is an autosomal dominant early-onset torsion form of dystonia, a neurological disease affecting movement. DYT1 is the prototypic hereditary dystonia and is caused by the mutation of the tor1a gene. The gene product has chaperone functions important for the control of protein folding and stability. Dystonia-4 (DYT4) is another autosomal dominant dystonia that is characterized by onset in the second to third decade of progressive laryngeal dysphonia. DYT4 is associated with the mutation of the tubb4a gene, although it remains to be understood how disease-associated mutation affects biochemical as well as cell biological properties of the gene product as the microtubule component (a tubulin beta subunit). Herein we demonstrate that DYT4-associated TUBB4A missense mutants (Arg2-to-Gly or Ala271-to-Thr) form disorganized tubulin networks in cells. Transfected mutants are indeed expressed in cytoplasmic regions, as observed in wild-type transfectants. However, mutant proteins do not exhibit typical radial tubulin networks. Rather, they have diminished ability to interact with tubulin alpha subunits. Processes do not form in sufficient amounts in cells of the N1E-115 neuronal cell line expressing each of these mutants as compared to parental cells. Together, DYT4-associated TUBB4A mutants themselves form aberrant tubulin networks and inhibit neuronal process growth, possibly explaining progress through the pathological states at cellular levels.


Assuntos
Distonia Muscular Deformante/metabolismo , Microtúbulos/metabolismo , Neuritos/metabolismo , Neuritos/patologia , Neurogênese , Tubulina (Proteína)/metabolismo , Células Cultivadas , Distonia Muscular Deformante/genética , Humanos , Microtúbulos/genética , Microtúbulos/patologia , Mutação/genética , Tubulina (Proteína)/genética
17.
Environ Mol Mutagen ; 59(1): 60-68, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28833460

RESUMO

Genotoxic effects of therapeutic ultrasound are poorly documented, when compared with the wide use of this physical agent. The aim of this work was to investigate the clastogenic and aneugenic potential of 1 MHz ultrasound, employing intensities (200 and 300 mW/cm2 ) above the cavitational threshold, but in the range of those normally used in therapeutics. Both normal fibroblasts (AG01522) and tumoral cells (MCF-7) were sonicated. While no effects on viability were noted, significant increases of CREST-negative micronuclei (indicative of clastogenesis) and CREST-positive micronuclei (indicative of aneuploidy) were detected. Clastogenesis was confirmed by increases of γ-H2AX foci, while increases of spindle anomalies confirmed the induction of aneuploidy. Our results confirm previous works that showed ultrasound-induced DNA breakage. Moreover, our experiments show that the known effect of ultrasound-induced damage to microtubules is also able to damage the mitotic spindle and induce aneuploidy. On the overall, this work highlights the importance to further investigate the potential risks related to therapeutics US. Environ. Mol. Mutagen. 59:60-68, 2018. © 2017 Wiley Periodicals, Inc.


Assuntos
Dano ao DNA/genética , Ondas Ultrassônicas/efeitos adversos , Aneugênicos/efeitos adversos , Aneuploidia , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Células Cultivadas , Fibroblastos/fisiologia , Genômica/métodos , Humanos , Células MCF-7 , Micronúcleos com Defeito Cromossômico , Testes para Micronúcleos/métodos , Microtúbulos/genética , Mutagênicos/efeitos adversos , Fuso Acromático/genética
18.
J Cell Biochem ; 119(1): 314-326, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28569402

RESUMO

Leucine-rich repeats and WD repeat domain containing protein 1 (LRWD1) is a testis-specific protein that mainly expressed in the sperm neck where centrosome is located. By using microarray analysis, LRWD1 is identified as a putative gene that involved in spermatogenesis. However, its role in human male germ cell development has not been extensively studied. When checking in the semen of patients with asthenozoospermia, teratozoospermia, and asthenoteratozoospermia, the level of LRWD1 in the sperm neck was significantly reduced with a defective neck or tail. When checking the sub-cellular localization of LRWD1 in the cells, we found that LRWD1 resided in the centrosome and its centrosomal residency was independent of microtubule transportation in NT2/D1, the human testicular embryonic carcinoma, cell line. Depletion of LRWD1 did not induce centrosome re-duplication but inhibited microtubule nucleation. In addition, the G1 arrest were observed in LRWD1 deficient NT2/D1 cells. Upon LRWD1 depletion, the levels of cyclin E, A, and phosphorylated CDK2, were reduced. Overexpression of LRWD1 promoted cell proliferation in NT2/D1, HeLa, and 239T cell lines. In addition, we also observed that autophagy was activated in LRWD1 deficient cells and inhibition of autophagy by chloroquine or bafilomycin A1 promoted cell death when LRWD1 was depleted. Thus, we found a novel function of LRWD1 in controlling microtubule nucleation and cell cycle progression in the human testicular embryonic carcinoma cells. J. Cell. Biochem. 119: 314-326, 2018. © 2017 Wiley Periodicals, Inc.


Assuntos
Carcinoma Embrionário/metabolismo , Ciclo Celular , Proteínas dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Testiculares/metabolismo , Carcinoma Embrionário/genética , Carcinoma Embrionário/patologia , Centrossomo/metabolismo , Centrossomo/patologia , Células HeLa , Humanos , Masculino , Proteínas dos Microtúbulos/genética , Microtúbulos/genética , Microtúbulos/patologia , Proteínas de Neoplasias/genética , Neoplasias Testiculares/genética , Neoplasias Testiculares/patologia
19.
Proc Natl Acad Sci U S A ; 114(52): 13738-13743, 2017 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-29229862

RESUMO

The microtubule motor kinesin-1 interacts via its cargo-binding domain with both microtubules and organelles, and hence plays an important role in controlling organelle transport and microtubule dynamics. In the absence of cargo, kinesin-1 is found in an autoinhibited conformation. The molecular basis of how cargo engagement affects the balance between kinesin-1's active and inactive conformations and roles in microtubule dynamics and organelle transport is not well understood. Here we describe the discovery of kinesore, a small molecule that in vitro inhibits kinesin-1 interactions with short linear peptide motifs found in organelle-specific cargo adaptors, yet activates kinesin-1's function of controlling microtubule dynamics in cells, demonstrating that these functions are mechanistically coupled. We establish a proof-of-concept that a microtubule motor-cargo interface and associated autoregulatory mechanism can be manipulated using a small molecule, and define a target for the modulation of microtubule dynamics.


Assuntos
Ativadores de Enzimas , Cinesina , Microtúbulos , Motivos de Aminoácidos , Ativadores de Enzimas/química , Ativadores de Enzimas/farmacologia , Células HeLa , Humanos , Cinesina/química , Cinesina/genética , Cinesina/metabolismo , Microtúbulos/química , Microtúbulos/genética , Microtúbulos/metabolismo
20.
Am J Hum Genet ; 101(6): 1006-1012, 2017 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-29198720

RESUMO

Leber congenital amaurosis (LCA) is a neurodegenerative disease of photoreceptor cells that causes blindness within the first year of life. It occasionally occurs in syndromic metabolic diseases and plurisystemic ciliopathies. Using exome sequencing in a multiplex family and three simplex case subjects with an atypical association of LCA with early-onset hearing loss, we identified two heterozygous mutations affecting Arg391 in ß-tubulin 4B isotype-encoding (TUBB4B). Inspection of the atomic structure of the microtubule (MT) protofilament reveals that the ß-tubulin Arg391 residue contributes to a binding pocket that interacts with α-tubulin contained in the longitudinally adjacent αß-heterodimer, consistent with a role in maintaining MT stability. Functional analysis in cultured cells overexpressing FLAG-tagged wild-type or mutant TUBB4B as well as in primary skin-derived fibroblasts showed that the mutant TUBB4B is able to fold, form αß-heterodimers, and co-assemble into the endogenous MT lattice. However, the dynamics of growing MTs were consistently altered, showing that the mutations have a significant dampening impact on normal MT growth. Our findings provide a link between sensorineural disease and anomalies in MT behavior and describe a syndromic LCA unrelated to ciliary dysfunction.


Assuntos
Amaurose Congênita de Leber/genética , Microtúbulos/genética , Tubulina (Proteína)/genética , Adulto , Sítios de Ligação/genética , Células Cultivadas , Criança , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Microtúbulos/metabolismo , Pessoa de Meia-Idade , Mutação de Sentido Incorreto/genética , Células Fotorreceptoras/metabolismo , Tubulina (Proteína)/metabolismo , Sequenciamento Completo do Exoma
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