RESUMO
Levodopa is the most used drug to treat motor symptoms in Parkinson's disease (PD). However, dopaminergic side effects such as nausea and vomiting may occur. Several evidences indicate a major role for dopamine receptors D2 (DRD2) and D3 (DRD3) in emetic activity. The aim of this study was to investigate the relationship of DRD2 rs1799732 and DRD3 rs6280 gene polymorphisms with gastrointestinal (GI) symptoms induced by levodopa in PD patients. Two hundred and seventeen PD patients on levodopa therapy were investigated. DRD2 rs1799732 and DRD3 rs6280 polymorphisms were genotyped by PCR-based methods. Multiple Poisson regression method with robust variance estimators was performed to assess the association between polymorphisms and gastrointestinal symptoms. The analyses showed that DRD2 Ins/Ins (prevalence ratio (PR)=2.374, 95% confidence interval (CI): 1.105-5.100; P=0.027) and DRD3 Ser/Ser genotypes (PR=1.677, 95% CI 1.077-2.611; P=0.022) were independent and predictors of gastrointestinal symptoms associated with levodopa therapy. Despite all the efforts to alleviate GI symptoms, this adverse effect still occurs in PD patients. Pharmacogenetic studies of GI symptoms induced by levodopa therapy have the potential to display new ways to better understand the molecular mechanisms involved in these side effects.
Assuntos
Gastroenteropatias/induzido quimicamente , Gastroenteropatias/genética , Levodopa/efeitos adversos , Levodopa/uso terapêutico , Doença de Parkinson/tratamento farmacológico , Polimorfismo Genético/genética , Receptores de Dopamina D2/genética , Receptores de Dopamina D3/genética , Idoso , Feminino , Predisposição Genética para Doença/genética , Genótipo , Humanos , Masculino , Doença de Parkinson/genéticaRESUMO
We compare the effects of Nordic walking training (NW) and Free walk (FW) on functional parameters (motor symptoms, balance) and functional mobility (Timed Up and Go at Self-selected Speed - TUGSS, and at forced speed, TUGFS; Self-selected Walking Speed, SSW; locomotor rehabilitation index, LRI) of Parkinson's disease (PD) patients. The study included 33 patients with clinical diagnosis of idiopathic PD, and staging between 1 and 4 in the Hoehn and Yahr scale (H&Y) randomized into two groups: NW (N = 16) and FW (N = 17) for 6 weeks. Baseline characteristics were compared trough a one-way ANOVA. Outcomes were analyzed using the Generalized Estimation Equations (GEE) with a Bonferroni post-hoc. Data were analyzed using SPSS v.20.0. Improvements in UPDRS III (P < 0.001), balance scores (P < 0.035), TUGSS distance (P < 0.001), TUGFS distance (P < 0.001), SSW (P < 0.001), and LRI (P < 0.001) were found for both groups. However, the NW group showed significant differences (P < 0.001) when compared to the FW group for the functional mobility. We conclude the NW improves functional parameters and walking mobility demonstrating that NW is as effective as the FW, including benefits for FW on the functional mobility of people with PD.
Assuntos
Terapia por Exercício/métodos , Doença de Parkinson/reabilitação , Caminhada , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/fisiopatologia , Índice de Gravidade de DoençaRESUMO
Levodopa is the most effective symptomatic therapy for Parkinson's disease, but its chronic use could lead to chronic adverse outcomes, such as motor fluctuations, dyskinesia and visual hallucinations. HOMER1 is a protein with pivotal function in glutamate transmission, which has been related to the pathogenesis of these complications. This study investigates whether polymorphisms in the HOMER1 gene promoter region are associated with the occurrence of the chronic complications of levodopa therapy. A total of 205 patients with idiopathic Parkinson's disease were investigated. Patients were genotyped for rs4704559, rs10942891 and rs4704560 by allelic discrimination with Taqman assays. The rs4704559 G allele was associated with a lower prevalence of dyskinesia (prevalence ratio (PR)=0.615, 95% confidence interval (CI) 0.426-0.887, P=0.009) and visual hallucinations (PR=0.515, 95% CI 0.295-0.899, P=0.020). Our data suggest that HOMER1 rs4704559 G allele has a protective role for the development of levodopa adverse effects.
Assuntos
Proteínas de Transporte/genética , Levodopa/efeitos adversos , Doença de Parkinson/tratamento farmacológico , Feminino , Proteínas de Arcabouço Homer , Humanos , Levodopa/uso terapêutico , MasculinoRESUMO
The aim of this study was to identify the relative frequency of Huntington's disease (HD) and HD-like (HDL) disorders HDL1, HDL2, spinocerebellar ataxia type 2 (SCA2), SCA17, dentatorubral-pallidoluysian degeneration (DRPLA), benign hereditary chorea, neuroferritinopathy and chorea-acanthocytosis (CHAC), in a series of Brazilian families. Patients were recruited in seven centers if they or their relatives presented at least chorea, besides other findings. Molecular studies of HTT, ATXN2, TBP, ATN1, JPH3, FTL, NKX2-1/TITF1 and VPS13A genes were performed. A total of 104 families were ascertained from 2001 to 2012: 71 families from South, 25 from Southeast and 8 from Northeast Brazil. There were 93 HD, 4 HDL2 and 1 SCA2 families. Eleven of 104 index cases did not have a family history: 10 with HD. Clinical characteristics were similar between HD and non-HD cases. In HD, the median expanded (CAG)n (range) was 44 (40-81) units; R(2) between expanded HTT and age-at-onset (AO) was 0.55 (p=0.0001, Pearson). HDL2 was found in Rio de Janeiro (2 of 9 families) and Rio Grande do Sul states (2 of 68 families). We detected HD in 89.4%, HDL2 in 3.8% and SCA2 in 1% of 104 Brazilian families. There were no cases of HDL1, SCA17, DRPLA, neuroferritinopathy, benign hereditary chorea or CHAC. Only six families (5.8%) remained without diagnosis.
Assuntos
Coreia/genética , Demência/genética , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Doença de Huntington/genética , Ataxias Espinocerebelares/genética , Adulto , Brasil , Coreia/diagnóstico , Coreia/epidemiologia , Coreia/patologia , Transtornos Cognitivos/diagnóstico , Transtornos Cognitivos/epidemiologia , Transtornos Cognitivos/genética , Transtornos Cognitivos/patologia , Demência/diagnóstico , Demência/epidemiologia , Demência/patologia , Feminino , Transtornos Heredodegenerativos do Sistema Nervoso/diagnóstico , Transtornos Heredodegenerativos do Sistema Nervoso/epidemiologia , Transtornos Heredodegenerativos do Sistema Nervoso/patologia , Humanos , Doença de Huntington/diagnóstico , Doença de Huntington/epidemiologia , Doença de Huntington/patologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Ataxias Espinocerebelares/diagnóstico , Ataxias Espinocerebelares/epidemiologia , Ataxias Espinocerebelares/patologia , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
The aim of this study was a three-dimensional analysis of vascular cooling effects on microwave ablation (MWA) in an ex vivo porcine model. A glass tube, placed in parallel to the microwave antenna at distances of 2.5, 5.0 and 10.0 mm (A-V distance), simulated a natural liver vessel. Seven flow rates (0, 1, 2, 5, 10, 100, 500 ml/min) were evaluated. Ablations were segmented into 2 mm slices for a 3D-reconstruction. A qualitative and quantitative analysis was performed. 126 experiments were carried out. Cooling effects occurred in all test series with flow rates ≥ 2 ml/min in the ablation periphery. These cooling effects had no impact on the total ablation volume (p > 0.05) but led to changes in ablation shape at A-V distances of 5.0 mm and 10.0 mm. Contrary, at a A-V distance of 2.5 mm only flow rates of ≥ 10 ml/min led to relevant cooling effects in the ablation centre. These cooling effects influenced the ablation shape, whereas the total ablation volume was reduced only at a maximal flow rate of 500 ml/min (p = 0.002). Relevant cooling effects exist in MWA. They mainly depend on the distance of the vessel to the ablation centre.
Assuntos
Técnicas de Ablação , Ablação por Cateter , Ablação por Radiofrequência , Técnicas de Ablação/métodos , Animais , Ablação por Cateter/métodos , Fígado/irrigação sanguínea , Fígado/cirurgia , Micro-Ondas/uso terapêutico , SuínosRESUMO
The past year has seen important contributions made to resolving how chromosomes attach to and move on the mitotic spindle of animal cells. These include the findings that: kinetochore microtubules are derived from the asters (i.e. centrosomes); poleward chromosome motion need not be coupled to kinetochore microtubules disassembly; the motor for poleward chromosome motion is associated with the kinetochore; and immunological evidence that this motor is cytoplasmic dynein.
Assuntos
Cromossomos/fisiologia , Mitose/fisiologia , Anáfase/fisiologia , Animais , Fuso Acromático/fisiologiaRESUMO
During the past year important progress has been made in refining our understanding of how chromosomes become equally distributed to daughter cells during mitosis. Unlike the situation in diatoms and yeast, it now appears that spindle pole (centrosome) separation during spindle formation and anaphase B is mediated in vertebrates primarily by an astral pulling, and not a pushing, mechanism. Kinetochore motility is directionally unstable, which has important consequences for how chromosomes move to the equator of the forming spindle. Finally, the observation that sister chromatid disjunction occurs even in the presence of high levels of maturation promoting factor reveals that the series of biochemical events responsible for this phenomenon is not an obligatory part of the pathway by which the cell exits mitosis.
Assuntos
Cromossomos Fúngicos/fisiologia , Cromossomos/fisiologia , Kluyveromyces/fisiologia , Mitose/fisiologia , Saccharomyces cerevisiae/fisiologia , Schizosaccharomyces/fisiologia , Anáfase , Sequência de Bases , Cromátides/fisiologia , Sequência Conservada , Genes Fúngicos , Kluyveromyces/ultraestrutura , Saccharomyces cerevisiae/ultraestrutura , Schizosaccharomyces/ultraestrutura , Fuso Acromático/fisiologiaRESUMO
The process of cell division, or mitosis, has fascinated biologists since its discovery in the late 1870s. Progress through mitosis is traditionally divided into stages that were defined over 100 years ago from analyses of fixed material from higher plants and animals. However, this terminology often leads to ambiguity, especially when comparing different systems. We therefore suggest that mitosis can be re-staged to reflect more accurately the molecular pathways that underlie key transitions.
Assuntos
Biologia/história , Mitose/fisiologia , Animais , Fase G2/fisiologia , História do Século XIX , Humanos , Interfase/fisiologiaRESUMO
Here we show that the rate of poleward chromosome motion in zw10-null mutants is greatly attenuated throughout the division process, and that chromosome disjunction at anaphase is highly asynchronous. Our results show that ZW10 protein, together with Rod, is involved in production and/or regulation of the force responsible for poleward chromosome motion.
Assuntos
Proteínas de Ciclo Celular , Cromossomos/fisiologia , Proteínas de Drosophila , Drosophila/genética , Drosophila/fisiologia , Proteínas de Insetos/genética , Proteínas de Insetos/fisiologia , Mutação , Animais , Drosophila/citologia , Genes de Insetos , Masculino , Meiose/genética , Meiose/fisiologia , Microscopia de Vídeo , Proteínas Motores Moleculares/genética , Proteínas Motores Moleculares/fisiologia , Movimento/fisiologia , Espermatócitos/citologia , Espermatócitos/fisiologiaRESUMO
OBJECTIVES: Systemic lupus erythematosus (SLE) shows a strong sex bias, preferentially affecting females, and B cells are thought to play a pivotal role in its pathogenesis. Here, we compared the splenic B-cell compartments, their autoreactivity and activation threshold of female and male NZB/W F1, a murine lupus model reflecting the sex bias observed in patients with SLE. METHODS: Autoantibody levels and the amount of autoantibody secreting cells were determined using ELISA and ELISPOT. Flow cytometry and immunofluorescence were applied to analyse the composition of the splenic B-cell pool. Purified follicular (FO) and marginal zone (MZ) B cells were stimulated and the frequency of autoreactive cells was determined. Finally, the proliferative response of FO and MZ B cells upon stimulation was assessed using CFSE dilution and [(3)H]-Thymidin incorporation. RESULTS: Higher autoantibody titres were detected in female NZB/W F1 mice, which were mainly produced in the spleen. Analysing the composition of the splenic B-cell subsets, no differences were found prior to disease development. Autoreactive dsDNA-specific B cells were mostly found in the MZ compartment, while SmD1((83-119))-reactive cells were more evenly distributed. Equal frequencies of autoreactive B cells were found in female and malemice, and no difference in the response to polyclonal stimuli of the cells of both sexes was detected. CONCLUSIONS: No differences in the composition or functionality of splenic B cells were observed that account for the different disease course in both sexes.
Assuntos
Subpopulações de Linfócitos B/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Envelhecimento/imunologia , Envelhecimento/patologia , Animais , Anticorpos Antinucleares/sangue , Autoimunidade , Subpopulações de Linfócitos B/patologia , Proliferação de Células , Modelos Animais de Doenças , Feminino , Humanos , Lúpus Eritematoso Sistêmico/patologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos NZB , Fragmentos de Peptídeos/imunologia , Ribonucleoproteínas Nucleares Pequenas/imunologia , Caracteres Sexuais , Baço/imunologia , Baço/patologia , Proteínas Centrais de snRNPRESUMO
The somatic cells of all higher animals contain a single minute organelle called the centrosome. For years, the functions of the centrosome were thought to revolve around its ability to nucleate and organize the various microtubule arrays seen in interphase and mitosis. But the centrosome is more than just a microtubule-organizing center. Recent work reveals that this organelle is essential for cell-cycle progression and that this requirement is independent of its ability to organize microtubules. Here, we review the various functions attributed to the centrosome and ask which are essential for the survival and reproduction of the cell, the organism, or both.
Assuntos
Centríolos/fisiologia , Centrossomo/fisiologia , Fuso Acromático/fisiologia , Animais , Centríolos/ultraestrutura , Centrossomo/ultraestrutura , Cílios/ultraestrutura , Fase G1/fisiologia , Humanos , Masculino , Microscopia Eletrônica , Microtúbulos/fisiologia , Microtúbulos/ultraestrutura , Fuso Acromático/ultraestruturaRESUMO
A replicated chromosome possesses two discrete, complex, dynamic, macromolecular assemblies, known as kinetochores, that are positioned on opposite sides of the primary constriction of the chromosome. Here, the authors review how kinetochores control chromosome segregation during mitosis in vertebrates. They attach the chromosome to the opposing spindle poles by trapping the dynamic plus-ends of microtubules growing from the poles. They then produce much of the force for chromosome poleward motion, regulate when this force is applied, and act as a site for microtubule assembly and disassembly. Finally, they control the metaphase-anaphase transition by inhibiting chromatid separation until the chromatids are properly attached.
Assuntos
Cinetocoros/fisiologia , Mitose/fisiologia , Animais , Cromátides , Humanos , Transdução de Sinais , Fuso Acromático , VertebradosRESUMO
The distribution of ribonucleoprotein (RNP) within the mitotic spindle of newt lung epithelial cells was studied with the high voltage electron microscope (HVEM) using Bernhard's uranyl-EDTA-lead staining of thick sections in conjunction with the ribonuclease digestion of fixed cells. The results indicate that aside from ribosomes, the major RNP-containing components of the spindle are the kinetochores and centrioles, both of which stain electron-opaque after EDTA treatment. In both cases, the electron-opaque material associated with these microtubule organizing centers (MTOC's) can be removed by RNAse digestion and cold perchloric acid (PCA) extraction under conditions which leave the spindle microtubules (Mts) centrioles, and kinetochores intact. The staining reaction is not abolished by cold PCA extraction alone or by substituting other positively charged proteins (i.e., cytochrome c or lysozyme) for RNAse. The RNP component of the kinetochore is closely associated with the bases of the kinetochore microtubules. The RNP component of the centriole can be seen to surround the microtubules of the triplet blades. No evidence was found to indicate the presence of RNP in the pericentriolar material. The possible function of both kinetochore and centriolar RNP is discussed.
Assuntos
Centríolos/análise , Centrômero/análise , Cromossomos/análise , Mitose , Nucleoproteínas/análise , Organoides/análise , Ribonucleoproteínas/análise , Animais , Células Cultivadas , Ácido Edético/farmacologia , Pulmão , Microtúbulos/análise , Percloratos/farmacologia , Ribonucleases/farmacologia , Salamandridae , Coloração e RotulagemRESUMO
The reproduction of spindle poles is a key event in the cell's preparation for mitosis. To gain further insight into how this process is controlled, we systematically characterized the ultrastructure of spindle poles whose reproductive capacity had been experimentally altered. In particular, we wanted to determine if the ability of a pole to reproduce before the next division is related to the number of centrioles it contains. We used mercaptoethanol to indirectly induce the formation of monopolar spindles in sea urchin eggs. We followed individually treated eggs in vivo with a polarizing microscope during the induction and development of monopolar spindles. We then fixed each egg at one of three predetermined key stages and serially semithick sectioned it for observation in a high-voltage electron microscope. We thus know the history of each egg before fixation and, from earlier studies, what that cell would have done had it not been fixed. We found that spindle poles that would have given rise to monopolar spindles at the next mitosis have only one centriole whereas spindle poles that would have formed bipolar spindles at the next division have two centrioles. By serially sectioning each egg, we were able to count all centrioles present. In the twelve cells examined, we found no cases of acentriolar spindle poles or centriole reduplication. Thus, the reproductive capacity of a spindle pole is linked to the number of centrioles it contains. Our experimental results also show, contrary to existing reports, that the daughter centriole of a centrosome can acquire pericentriolar material without first becoming a parent. Furthermore, our results demonstrate that the splitting apart of mother and daughter centrioles is an event that is distinct from, and not dependent on, centriole duplication.
Assuntos
Centríolos/ultraestrutura , Fuso Acromático/ultraestrutura , Animais , Feminino , Microscopia Eletrônica , Mitose , Óvulo/ultraestrutura , Ouriços-do-MarRESUMO
We tested the ability of chromosomes in a mitotic cytoplasm to organize a bipolar spindle in the absence of centrosomes. Sea urchin eggs were treated with 5 X 10(-6) colcemid for 7-9 min before fertilization to block future microtubule assembly. Fertilization events were normal except that a sperm aster was not formed and the pronuclei remained up to 70 microns apart. After nuclear envelope breakdown, individual eggs were irradiated with 366-nm light to inactivate photochemically the colcemid. A functional haploid bipolar spindle was immediately assembled in association with the male chromosomes. In contrast to the male pronucleus, the female pronucleus in most of these eggs remained as a small nonbirefringent hyaline area throughout mitosis. High-voltage electron microscopy of serial semithick sections from individual eggs, previously followed in vivo, revealed that the female chromosomes were randomly distributed within the remnants of the nuclear envelope. No microtubules were found in these pronuclear areas even though the chromosomes were well-condensed and had prominent kinetochores with well-developed coronas. In the remaining eggs, a weakly birefringent monaster was assembled in the female pronuclear area. These observations demonstrate that chromosomes in a mitotic cytoplasm cannot organize a bipolar spindle in the absence of a spindle pole or even in the presence of a monaster. In fact, chromosomes do not even assemble kinetochore microtubules in the absence of a spindle pole, and kinetochore microtubules form only on kinetochores facing the pole when a monaster is present. This study also provides direct experimental proof for the longstanding paradigm that the sperm provides the centrosomes used in the development of the sea urchin zygote.
Assuntos
Núcleo Celular/ultraestrutura , Cromossomos/ultraestrutura , Fuso Acromático/ultraestrutura , Zigoto/ultraestrutura , Animais , Citoplasma/ultraestrutura , Feminino , Microscopia Eletrônica , Mitose , Ouriços-do-MarRESUMO
When centrosomes are destroyed during prophase by laser microsurgery, vertebrate somatic cells form bipolar acentrosomal mitotic spindles (Khodjakov, A., R.W. Cole, B.R. Oakley, and C.L. Rieder. 2000. Curr. Biol. 10:59-67), but the fate of these cells is unknown. Here, we show that, although these cells lack the radial arrays of astral microtubules normally associated with each spindle pole, they undergo a normal anaphase and usually produce two acentrosomal daughter cells. Relative to controls, however, these cells exhibit a significantly higher (30-50%) failure rate in cytokinesis. This failure correlates with the inability of the spindle to properly reposition itself as the cell changes shape. Also, we destroyed just one centrosome during metaphase and followed the fate of the resultant acentrosomal and centrosomal daughter cells. Within 72 h, 100% of the centrosome-containing cells had either entered DNA synthesis or divided. By contrast, during this period, none of the acentrosomal cells had entered S phase. These data reveal that the primary role of the centrosome in somatic cells is not to form the spindle but instead to ensure cytokinesis and subsequent cell cycle progression.
Assuntos
Centrômero/fisiologia , Anáfase/fisiologia , Animais , Ciclo Celular , Divisão Celular , Linhagem Celular , Fase G1/fisiologia , Metáfase/fisiologia , Microtúbulos , Fatores de Tempo , VertebradosRESUMO
We used video-light microscopy and laser microsurgery to test the hypothesis that as a bioriented prometaphase chromosome changes position in PtK1 cells, the kinetochore moving away from its associated pole (AP) exerts a pushing force on the centromere. When we rapidly severed congressing chromosomes near the spindle equator between the sister kinetochores, the kinetochore that was originally "leading" the motion towards a pole (P) always (17/17 cells) continued moving P whereas the "trailing" kinetochore moving AP always stopped moving as soon as the operation was completed. This trailing kinetochore then initiated motion towards the pole it was originally moving away from up to 50 s later. The same result was observed (15/15 cells) when we selectively destroyed the leading (P moving) kinetochore on a congressing chromosome positioned > or = 3 microns from the pole it was moving away from. When we conducted this experiment on congressing chromosomes positioned within 3 microns of the pole, the centromere region either stopped moving, before switching into motion towards the near pole (2/4 cells), or it continued to move AP for 30-44 s (2/4 cells) before switching into P motion. Finally, kinetochore-free chromosome fragments, generated in the polar regions of PtK1 spindles, were ejected AP and often towards the spindle equator at approximately 2 microns/min. From these data we conclude that the kinetochore moving AP on a moving chromosome does not exert a significant pushing force on the chromosome. Instead, our results reveal that, when not generating a P force, kinetochores are in a "neutral" state that allows them to remain stationary or to coast AP in response to external forces sufficient to allow their K-fiber to elongate.
Assuntos
Cromossomos/fisiologia , Cinetocoros/fisiologia , Cinetocoros/ultraestrutura , Animais , Ciclo Celular , Linhagem Celular , Centrômero/fisiologia , Centrômero/ultraestrutura , Cromossomos/ultraestrutura , Lasers , Microscopia de Vídeo , Microcirurgia , Fatores de TempoRESUMO
Epithelial cells cultured from the lung of the Northwest rough-skinned newt (Taricha granulosa granulosa) were subjected to brief (10-15 min) elevated temperature shocks of 33 degrees-36 degrees C during metaphase. Electron microscope studies on these cells reveal that the spindle microtubules (Mts) are differentially stable to heat treatment. The great majority of nonkinetochore Mts are destroyed within the first few minutes of the shock while kinetochore and adjacent Mts rearrange to form hexagonal closely packed structures before disassembling, the latter occurring only after prolonged heat treatment. The significance and theoretical implications of the formation of hexagonal closely packed Mt structures and of the differential stability of spindle Mts to heating are discussed. The data suggest the existence of one or more heat-sensitive structural component(s) which maintain the individual minimum spacing seen between spindle Mts. To our knowledge, this is the first reported instance of the experimental rearrangement of kinetochore Mts into reversible, hexagonal closely packed bundles.
Assuntos
Microtúbulos/ultraestrutura , Mitose , Animais , Temperatura Alta , Pulmão/ultraestrutura , Organoides/ultraestrutura , SalamandridaeRESUMO
The ultrastructure of spindle formation during the first meiotic division in oocytes of the Strepsipteran insect Xenos peckii Kirby (Acroschismus wheeleri Pierce) was examined in serial thick (0.25-micron) and thin sections. During late prophase the nuclear envelope became extremely convoluted and fenestrated. At this time vesicular and tubular membrane elements permeated the nucleoplasm and formed a thin fusiform sheath, 5-7 micron in length, around each of the randomly oriented and condensing tetrads. These membrane elements appeared to arise from the nuclear envelope and/or in association with annulate lamellae in the nuclear region. All of the individual tetrads and their associated fusiform sheaths became aligned within the nucleus subsequent to the breakdown of the nuclear envelope. Microtubules (MTs) were found associated with membranes of the meiotic apparatus only after the nuclear envelope had broken down. Kinetochores, with associated MTs, were first recognizable as electron-opaque patches on the chromosomes at this time. The fully formed metaphase arrested Xenos oocyte meiotic apparatus contained an abundance of membranes and had diffuse poles that lacked distinct polar MT organizing centers. From these observations we conclude that the apparent individual chromosomal spindles--seen in the light microscope to form around each Xenos tetrad during "intranuclear prometaphase" (Hughes-Schrader, S., 1924, J. Morphol. 39:157-197)--actually form during late prophase, lack MTs, and are therefore not complete miniature bipolar spindles, as had been commonly assumed. Thus, the unique mode of spindle formation in Xenos oocytes cannot be used to support the hypothesis that chromosomes (kinetochores) induce the polymerization of their associated MTs. Our observation that MTs appeared in association with and parallel to tubular membrane components of the Xenos meiotic apparatus after these membranes became oriented with respect to the tetrads, is consistent with the notion that membranes associated with the spindle determine the orientation of spindle MTs and also play a part in regulating their formation.
Assuntos
Insetos/citologia , Meiose , Oócitos/ultraestrutura , Animais , Núcleo Celular/ultraestrutura , Feminino , Interfase , Membranas Intracelulares/ultraestrutura , Metáfase , Microscopia Eletrônica , Microtúbulos/ultraestrutura , Membrana Nuclear/ultraestrutura , PrófaseRESUMO
gamma-Tubulin is a centrosomal component involved in microtubule nucleation. To determine how this molecule behaves during the cell cycle, we have established several vertebrate somatic cell lines that constitutively express a gamma-tubulin/green fluorescent protein fusion protein. Near simultaneous fluorescence and DIC light microscopy reveals that the amount of gamma-tubulin associated with the centrosome remains relatively constant throughout interphase, suddenly increases during prophase, and then decreases to interphase levels as the cell exits mitosis. This mitosis-specific recruitment of gamma-tubulin does not require microtubules. Fluorescence recovery after photobleaching (FRAP) studies reveal that the centrosome possesses two populations of gamma-tubulin: one that turns over rapidly and another that is more tightly bound. The dynamic exchange of centrosome-associated gamma-tubulin occurs throughout the cell cycle, including mitosis, and it does not require microtubules. These data are the first to characterize the dynamics of centrosome-associated gamma-tubulin in vertebrate cells in vivo and to demonstrate the microtubule-independent nature of these dynamics. They reveal that the additional gamma-tubulin required for spindle formation does not accumulate progressively at the centrosome during interphase. Rather, at the onset of mitosis, the centrosome suddenly gains the ability to bind greater than three times the amount of gamma-tubulin than during interphase.