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1.
PLoS Comput Biol ; 20(9): e1012330, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39236069

RESUMO

How can inter-individual variability be quantified? Measuring many features per experiment raises the question of choosing them to recapitulate high-dimensional data. Tackling this challenge on spindle elongation phenotypes, we showed that only three typical elongation patterns describe spindle elongation in C. elegans one-cell embryo. These archetypes, automatically extracted from the experimental data using principal component analysis (PCA), accounted for more than 95% of inter-individual variability of more than 1600 experiments across more than 100 different conditions. The two first archetypes were related to spindle average length and anaphasic elongation rate. The third archetype, accounting for 6% of the variability, was novel and corresponded to a transient spindle shortening in late metaphase, reminiscent of kinetochore function-defect phenotypes. Importantly, these three archetypes were robust to the choice of the dataset and were found even considering only non-treated conditions. Thus, the inter-individual differences between genetically perturbed embryos have the same underlying nature as natural inter-individual differences between wild-type embryos, independently of the temperatures. We thus propose that beyond the apparent complexity of the spindle, only three independent mechanisms account for spindle elongation, weighted differently in the various conditions. Interestingly, the spindle-length archetypes covered both metaphase and anaphase, suggesting that spindle elongation in late metaphase is sufficient to predict the late anaphase length. We validated this idea using a machine-learning approach. Finally, given amounts of these three archetypes could represent a quantitative phenotype. To take advantage of this, we set out to predict interacting genes from a seed based on the PCA coefficients. We exemplified this firstly on the role of tpxl-1 whose homolog tpx2 is involved in spindle microtubule branching, secondly the mechanism regulating metaphase length, and thirdly the central spindle players which set the length at anaphase. We found novel interactors not in public databases but supported by recent experimental publications.


Assuntos
Caenorhabditis elegans , Fenótipo , Fuso Acromático , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/genética , Fuso Acromático/fisiologia , Animais , Análise de Componente Principal , Biologia Computacional , Embrião não Mamífero/embriologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo
2.
EMBO Rep ; 22(5): e50770, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33900015

RESUMO

In Caenorhabditis elegans zygote, astral microtubules generate forces essential to position the mitotic spindle, by pushing against and pulling from the cortex. Measuring microtubule dynamics there, we revealed the presence of two populations, corresponding to pulling and pushing events. It offers a unique opportunity to study, under physiological conditions, the variations of both spindle-positioning forces along space and time. We propose a threefold control of pulling force, by polarity, spindle position and mitotic progression. We showed that the sole anteroposterior asymmetry in dynein on-rate, encoding pulling force imbalance, is sufficient to cause posterior spindle displacement. The positional regulation, reflecting the number of microtubule contacts in the posterior-most region, reinforces this imbalance only in late anaphase. Furthermore, we exhibited the first direct proof that dynein processivity increases along mitosis. It reflects the temporal control of pulling forces, which strengthens at anaphase onset following mitotic progression and independently from chromatid separation. In contrast, the pushing force remains constant and symmetric and contributes to maintaining the spindle at the cell centre during metaphase.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Microtúbulos , Fuso Acromático , Zigoto
3.
Cell ; 134(5): 843-53, 2008 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-18775316

RESUMO

oskar mRNA localization to the posterior of the Drosophila oocyte defines where the abdomen and germ cells form in the embryo. Although this localization requires microtubules and the plus end-directed motor, kinesin, its mechanism is controversial and has been proposed to involve active transport to the posterior, diffusion and trapping, or exclusion from the anterior and lateral cortex. By following oskar mRNA particles in living oocytes, we show that the mRNA is actively transported along microtubules in all directions, with a slight bias toward the posterior. This bias is sufficient to localize the mRNA and is reversed in mago, barentsz, and Tropomyosin II mutants, which mislocalize the mRNA anteriorly. Since almost all transport is mediated by kinesin, oskar mRNA localizes by a biased random walk along a weakly polarized cytoskeleton. We also show that each component of the oskar mRNA complex plays a distinct role in particle formation and transport.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Transporte de RNA , RNA Mensageiro/metabolismo , Animais , Padronização Corporal , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oócitos/química , RNA Mensageiro/análise , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/análise , Ribonucleoproteínas/metabolismo , Tropomiosina/genética , Tropomiosina/metabolismo
4.
J Microsc ; 285(1): 3-19, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34623634

RESUMO

Artificial intelligence is nowadays used for cell detection and classification in optical microscopy during post-acquisition analysis. The microscopes are now fully automated and next expected to be smart by making acquisition decisions based on the images. It calls for analysing them on the fly. Biology further imposes training on a reduced data set due to cost and time to prepare the samples and have the data sets annotated by experts. We propose a real-time image processing compliant with these specifications by balancing accurate detection and execution performance. We characterised the images using a generic, high-dimensional feature extractor. We then classified the images using machine learning to understand the contribution of each feature in decision and execution time. We found that the non-linear-classifier random forests outperformed Fisher's linear discriminant. More importantly, the most discriminant and time-consuming features could be excluded without significant accuracy loss, offering a substantial gain in execution time. It suggests a feature-group redundancy likely related to the biology of the observed cells. We offer a method to select fast and discriminant features. In our assay, a 79.6 ± 2.4% accurate classification of a cell took 68.7 ± 3.5 ms (mean ± SD, 5-fold cross-validation nested in 10 bootstrap repeats), corresponding to 14 cells per second, dispatched into eight phases of the cell cycle, using 12 feature groups and operating a consumer market ARM-based embedded system. A simple neural network offered similar performances paving the way to faster training and classification, using parallel execution on a general-purpose graphic processing unit. Finally, this strategy is also usable for deep neural networks paving the way to optimizing these algorithms for smart microscopy.

5.
Biophys J ; 115(11): 2189-2205, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30447992

RESUMO

During asymmetric division of the Caenorhabditis elegans zygote, to properly distribute cell fate determinants, the mitotic spindle is asymmetrically localized by a combination of centering and cortical-pulling microtubule-mediated forces, the dynamics of the latter being regulated by mitotic progression. Here, we show a, to our knowledge, novel and additional regulation of these forces by spindle position itself. For that, we observed the onset of transverse spindle oscillations, which reflects the burst of anaphase pulling forces. After delaying anaphase onset, we found that the position at which the spindle starts to oscillate was unchanged compared to control embryos and uncorrelated to anaphase onset. In mapping the cortical microtubule dynamics, we measured a steep increase in microtubule contact density after the posterior centrosome reached the critical position of 70% of embryo length, strongly suggesting the presence of a positional switch for spindle oscillations. Expanding a previous model based on a force-generator temporal control, we implemented this positional switch and observed that the large increase in microtubule density accounted for the pulling force burst. Thus, we propose that the spindle position influences the cortical availability of microtubules on which the active force generators, controlled by cell cycle progression, can pull. Importantly, we found that this positional control relies on the polarity-dependent LET-99 cortical band, the boundary of which could be probed by microtubules. This dual positional and temporal control well accounted for our observation that the oscillation onset position resists changes in cellular geometry and moderate variations in the active force generator number. Finally, our model suggests that spindle position at mitosis end is more sensitive to the polarity factor LET-99, which restricts the region of active force generators to a posterior-most region, than to microtubule number or force generator number/activity. Overall, we show that robustness in spindle positioning originates in cell mechanics rather than biochemical networks.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Embrião não Mamífero/metabolismo , Retroalimentação Fisiológica , Microtúbulos/fisiologia , Mitose , Fuso Acromático/fisiologia , Animais , Caenorhabditis elegans/embriologia , Proteínas de Caenorhabditis elegans/genética , Centrossomo/fisiologia , Embrião não Mamífero/citologia
6.
Development ; 142(9): 1684-94, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25858456

RESUMO

E-cadherin (E-cad) is the main component of epithelial junctions in multicellular organisms, where it is essential for cell-cell adhesion. The localisation of E-cad is often strongly polarised in the apico-basal axis. However, the mechanisms required for its polarised distribution are still largely unknown. We performed a systematic RNAi screen in vivo to identify genes required for the strict E-cad apical localisation in C. elegans epithelial epidermal cells. We found that the loss of clathrin, its adaptor AP-1 and the AP-1 interactor SOAP-1 induced a basolateral localisation of E-cad without affecting the apico-basal diffusion barrier. We further found that SOAP-1 controls AP-1 localisation, and that AP-1 is required for clathrin recruitment. Finally, we also show that AP-1 controls E-cad apical delivery and actin organisation during embryonic elongation, the final morphogenetic step of embryogenesis. We therefore propose that a molecular pathway, containing SOAP-1, AP-1 and clathrin, controls the apical delivery of E-cad and morphogenesis.


Assuntos
Proteínas do Domínio Armadillo/metabolismo , Caderinas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriologia , Adesão Celular/fisiologia , Polaridade Celular/fisiologia , Desenvolvimento Embrionário/fisiologia , Epiderme/fisiologia , Animais , Clatrina/metabolismo , Epiderme/metabolismo , Microscopia Confocal , Microscopia Eletrônica , Interferência de RNA , Fator de Transcrição AP-1/metabolismo
7.
J Cell Sci ; 128(20): 3720-30, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26359299

RESUMO

In higher eukaryotes, efficient chromosome congression relies, among other players, on the activity of chromokinesins. Here, we provide a quantitative analysis of kinetochore oscillations and positioning in Schizosaccharomyces pombe, a model organism lacking chromokinesins. In wild-type cells, chromosomes align during prophase and, while oscillating, maintain this alignment throughout metaphase. Chromosome oscillations are dispensable both for kinetochore congression and stable kinetochore alignment during metaphase. In higher eukaryotes, kinesin-8 family members control chromosome congression by regulating their oscillations. By contrast, here, we demonstrate that fission yeast kinesin-8 controls chromosome congression by an alternative mechanism. We propose that kinesin-8 aligns chromosomes by controlling pulling forces in a length-dependent manner. A coarse-grained model of chromosome segregation implemented with a length-dependent process that controls the force at kinetochores is necessary and sufficient to mimic kinetochore alignment, and prevents the appearance of lagging chromosomes. Taken together, these data illustrate how the local action of a motor protein at kinetochores provides spatial cues within the spindle to align chromosomes and to prevent aneuploidy.


Assuntos
Relógios Biológicos/fisiologia , Segregação de Cromossomos/fisiologia , Cromossomos Fúngicos/metabolismo , Cinesinas/metabolismo , Modelos Biológicos , Schizosaccharomyces/metabolismo , Cromossomos Fúngicos/genética , Cinesinas/genética , Schizosaccharomyces/genética
8.
Development ; 141(6): 1381-91, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24595291

RESUMO

How signaling gradients supply positional information in a field of moving cells is an unsolved question in patterning and morphogenesis. Here, we ask how a Wnt signaling gradient regulates the dynamics of a wavefront of cellular change in a flow of cells during somitogenesis. Using time-controlled perturbations of Wnt signaling in the zebrafish embryo, we changed segment length without altering the rate of somite formation or embryonic elongation. This result implies specific Wnt regulation of the wavefront velocity. The observed Wnt signaling gradient dynamics and timing of downstream events support a model for wavefront regulation in which cell flow plays a dominant role in transporting positional information.


Assuntos
Somitos/embriologia , Somitos/metabolismo , Via de Sinalização Wnt/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Padronização Corporal/genética , Padronização Corporal/fisiologia , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Resposta ao Choque Térmico/genética , Resposta ao Choque Térmico/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Modelos Biológicos , Proteínas com Domínio T/genética , Proteínas com Domínio T/fisiologia , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/genética , Proteínas Wnt/fisiologia , Via de Sinalização Wnt/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética
9.
Biophys J ; 111(8): 1773-1784, 2016 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-27760363

RESUMO

Precise positioning of the mitotic spindle is important for specifying the plane of cell division, which in turn determines how the cytoplasmic contents of the mother cell are partitioned into the daughter cells, and how the daughters are positioned within the tissue. During metaphase in the early Caenorhabditis elegans embryo, the spindle is aligned and centered on the anterior-posterior axis by a microtubule-dependent machinery that exerts restoring forces when the spindle is displaced from the center. To investigate the accuracy and stability of centering, we tracked the position and orientation of the mitotic spindle during the first cell division with high temporal and spatial resolution. We found that the precision is remarkably high: the cell-to-cell variation in the transverse position of the center of the spindle during metaphase, as measured by the standard deviation, was only 1.5% of the length of the short axis of the cell. Spindle position is also very stable: the standard deviation of the fluctuations in transverse spindle position during metaphase was only 0.5% of the short axis of the cell. Assuming that stability is limited by fluctuations in the number of independent motor elements such as microtubules or dyneins underlying the centering machinery, we infer that the number is ∼1000, consistent with the several thousand of astral microtubules in these cells. Astral microtubules grow out from the two spindle poles, make contact with the cell cortex, and then shrink back shortly thereafter. The high stability of centering can be accounted for quantitatively if, while making contact with the cortex, the astral microtubules buckle as they exert compressive, pushing forces. We thus propose that the large number of microtubules in the asters provides a highly precise mechanism for positioning the spindle during metaphase while assembly is completed before the onset of anaphase.


Assuntos
Caenorhabditis elegans/embriologia , Embrião não Mamífero/citologia , Fuso Acromático/metabolismo , Animais , Microtúbulos/metabolismo
10.
bioRxiv ; 2023 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-37961478

RESUMO

Recent studies have highlighted the significance of the spindle midzone - the region positioned between chromosomes - in ensuring proper chromosome segregation. By combining advanced 3D electron tomography and cutting-edge light microscopy we have discovered a previously unknown role of the regulation of microtubule dynamics within the spindle midzone of C. elegans. Using Fluorescence recovery after photobleaching and a combination of second harmonic generation and two-photon fluorescence microscopy, we found that the length of the antiparallel microtubule overlap zone in the spindle midzone is constant throughout anaphase, and independent of cortical pulling forces as well as the presence of the microtubule bundling protein SPD-1. Further investigations of SPD-1 and the chromokinesin KLP-19 in C. elegans suggest that KLP-19 regulates the overlap length and functions independently of SPD-1. Our data shows that KLP-19 plays an active role in regulating the length and turn-over of microtubules within the midzone as well as the size of the antiparallel overlap region throughout mitosis. Depletion of KLP-19 in mitosis leads to an increase in microtubule length in the spindle midzone, which also leads to increased microtubule - microtubule interaction, thus building up a more robust microtubule network. The spindle is globally stiffer and more stable, which has implications for the transmission of forces within the spindle affecting chromosome segregation dynamics. Our data shows that by localizing KLP-19 to the spindle midzone in anaphase microtubule dynamics can be locally controlled allowing the formation of a functional midzone.

11.
Methods Appl Fluoresc ; 8(2): 024006, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-32032967

RESUMO

Fluorescence Lifetime Imaging Microscopy (FLIM) is a robust tool to measure Förster Resonance Energy Transfer (FRET) between two fluorescent proteins, mainly when using genetically-encoded FRET biosensors. It is then possible to monitor biological processes such as kinase activity with a good spatiotemporal resolution and accuracy. Therefore, it is of interest to improve this methodology for future high content screening purposes. We here implement a time-gated FLIM microscope that can image and quantify fluorescence lifetime with a higher speed than conventional techniques such as Time-Correlated Single Photon Counting (TCSPC). We then improve our system to perform automatic screen analysis in a 96-well plate format. Moreover, we use a FRET biosensor of AURKA activity, a mitotic kinase involved in several epithelial cancers. Our results show that our system is suitable to measure FRET within our biosensor paving the way to the screening of novel compounds, potentially allowing to find new inhibitors of AURKA activity.


Assuntos
Aurora Quinase A/análise , Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia de Fluorescência/métodos , Aurora Quinase A/metabolismo , Linhagem Celular Tumoral , Humanos
12.
Curr Biol ; 16(21): 2111-22, 2006 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-17084695

RESUMO

BACKGROUND: Asymmetric division of the C. elegans zygote is due to the posterior-directed movement of the mitotic spindle during metaphase and anaphase. During this movement along the anterior-posterior axis, the spindle oscillates transversely. These motions are thought to be driven by a force-generating complex-possibly containing the motor protein cytoplasmic dynein-that is located at the cell cortex and pulls on microtubules growing out from the spindle poles. A theoretical analysis indicates that the oscillations might arise from mechanical coordination of the force-generating motors, and this coordination is mediated by the load dependence of the motors' detachment from the microtubules. The model predicts that the motor activity must exceed a threshold for oscillations to occur. RESULTS: We have tested the existence of a threshold by using RNA interference to gradually reduce the levels of dynein light intermediate chain as well as GPR-1 and GPR-2 that are involved in the G protein-mediated regulation of the force generators. We found an abrupt cessation of oscillations as expected if the motor activity dropped below a threshold. Furthermore, we can account for the complex choreography of the mitotic spindle-the precise temporal coordination of the buildup and die-down of the transverse oscillations with the posterior displacement-by a gradual increase in the processivity of a single type of motor machinery during metaphase and anaphase. CONCLUSIONS: The agreement between our results and modeling suggests that the force generators themselves have the intrinsic capability of generating oscillations when opposing forces exceed a threshold.


Assuntos
Caenorhabditis elegans/citologia , Divisão Celular/fisiologia , Fuso Acromático/fisiologia , Anáfase/fisiologia , Animais , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Dineínas/genética , Dineínas/metabolismo , Metáfase/fisiologia , Modelos Biológicos , Interferência de RNA
13.
Mol Biol Cell ; 29(26): 3093-3104, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30332325

RESUMO

During asymmetric cell division, the molecular motor dynein generates cortical pulling forces that position the spindle to reflect polarity and adequately distribute cell fate determinants. In Caenorhabditis elegans embryos, despite a measured anteroposterior force imbalance, antibody staining failed to reveal dynein enrichment at the posterior cortex, suggesting a transient localization there. Dynein accumulates at the microtubule plus ends, in an EBP-2EB-dependent manner. This accumulation, although not transporting dynein, contributes modestly to cortical forces. Most dyneins may instead diffuse to the cortex. Tracking of cortical dynein revealed two motions: one directed and the other diffusive-like, corresponding to force-generating events. Surprisingly, while dynein is not polarized at the plus ends or in the cytoplasm, diffusive-like tracks were more frequently found at the embryo posterior tip, where the forces are higher. This asymmetry depends on GPR-1/2LGN and LIN-5NuMA, which are enriched there. In csnk-1(RNAi) embryos, the inverse distribution of these proteins coincides with an increased frequency of diffusive-like tracks anteriorly. Importantly, dynein cortical residence time is always symmetric. We propose that the dynein-binding rate at the posterior cortex is increased, causing the polarity-reflecting force imbalance. This mechanism of control supplements the regulation of mitotic progression through the nonpolarized dynein detachment rate.


Assuntos
Divisão Celular Assimétrica , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Dineínas/genética , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular , Dineínas/metabolismo , Embrião não Mamífero , Regulação da Expressão Gênica , Genes Reporter , Proteínas Luminescentes , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Mitose , Fuso Acromático/metabolismo , Fuso Acromático/ultraestrutura , Proteína Vermelha Fluorescente
14.
J Cell Biol ; 201(5): 653-62, 2013 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-23690175

RESUMO

During the first embryonic division in Caenorhabditis elegans, the mitotic spindle is pulled toward the posterior pole of the cell and undergoes vigorous transverse oscillations. We identified variations in spindle trajectories by analyzing the outwardly similar one-cell stage embryo of its close relative Caenorhabditis briggsae. Compared with C. elegans, C. briggsae embryos exhibit an anterior shifting of nuclei in prophase and reduced anaphase spindle oscillations. By combining physical perturbations and mutant analysis in both species, we show that differences can be explained by interspecies changes in the regulation of the cortical Gα-GPR-LIN-5 complex. However, we found that in both species (1) a conserved positional switch controls the onset of spindle oscillations, (2) GPR posterior localization may set this positional switch, and (3) the maximum amplitude of spindle oscillations is determined by the time spent in the oscillating phase. By investigating microevolution of a subcellular process, we identify new mechanisms that are instrumental to decipher spindle positioning.


Assuntos
Caenorhabditis elegans/embriologia , Polaridade Celular , Embrião não Mamífero/citologia , Fuso Acromático/fisiologia , Animais , Evolução Biológica , Caenorhabditis/embriologia , Caenorhabditis/metabolismo , Caenorhabditis/ultraestrutura , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/ultraestrutura , Proteínas de Caenorhabditis elegans/análise , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiologia , Proteínas de Ciclo Celular/análise , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia
15.
PLoS One ; 5(8): e12301, 2010 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-20808841

RESUMO

Asymmetric positioning of the mitotic spindle in C. elegans embryos is mediated by force-generating complexes that are anchored at the plasma membrane and that pull on microtubules growing out from the spindle poles. Although asymmetric distribution of the force generators is thought to underlie asymmetric positioning of the spindle, the number and location of the force generators has not been well defined. In particular, it has not been possible to visualize individual force generating events at the cortex. We discovered that perturbation of the acto-myosin cortex leads to the formation of long membrane invaginations that are pulled from the plasma membrane toward the spindle poles. Several lines of evidence show that the invaginations, which also occur in unperturbed embryos though at lower frequency, are pulled by the same force generators responsible for spindle positioning. Thus, the invaginations serve as a tool to localize the sites of force generation at the cortex and allow us to estimate a lower limit on the number of cortical force generators within the cell.


Assuntos
Caenorhabditis elegans/citologia , Caenorhabditis elegans/embriologia , Membrana Celular/metabolismo , Mitose , Actomiosina/metabolismo , Animais , Fenômenos Biomecânicos , Microscopia , Microtúbulos/metabolismo , Imagem Molecular , Fatores de Tempo
16.
Phys Rev Lett ; 97(9): 098103, 2006 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-17026406

RESUMO

Membrane fusion is an important process in cell biology. While the molecular mechanisms of fusion are actively studied at a very local scale, the consequences of fusion at a larger scale on the shape and stability of the membrane are still not explored. In this Letter, the evolution of the membrane tension during the fusion of positive small unilamellar vesicles with a negative giant unilamellar vesicle has been experimentally investigated and compared to an existing theoretical model. The tension has been deduced using videomicroscopy from the measurement of the fluctuation spectrum and of the time correlation function of the fluctuations. We show that fusion induces a strong decrease in the effective tension of the membrane which eventually reaches negative values. Under these conditions, we show that localized instabilities appear on the vesicle. The membrane finally collapses, forming dense lipid structures.


Assuntos
Lipídeos/química , Algoritmos , Vesículas Revestidas , Bicamadas Lipídicas , Microscopia de Vídeo , Ultrassom
17.
Biophys J ; 87(1): 419-29, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15240476

RESUMO

In this work, we have investigated a new and general method for the reconstitution of membrane proteins into giant unilamellar vesicles (GUVs). We have analyzed systematically the reconstitution of two radically different membrane proteins, the sarcoplasmic reticulum Ca(2+)-ATPase and the H(+) pump bacteriorhodopsin. In a first step, our method involved a detergent-mediated reconstitution of solubilized membrane proteins into proteoliposomes of 0.1-0.2 microm in size. In a second step, these preformed proteoliposomes were partially dried under controlled humidity followed, in a third step, by electroswelling of the partially dried film to give GUVs. The physical characteristics of GUVs were analyzed in terms of morphology, size, and lamellarity using phase-contrast and differential interference contrast microscopy. The reconstitution process was further characterized by analyzing protein incorporation and biological activity. Both membrane proteins could be homogeneously incorporated into GUVs at lipid/protein ratios ranging from 5 to 40 (w/w). After reconstitution, both proteins retained their biological activity as demonstrated by H(+) or Ca(2+) pumping driven by bacteriorhodopsin or Ca(2+)-ATPase, respectively. This constitutes an efficient new method of reconstitution, leading to the production of large unilamellar membrane protein-containing vesicles of more than 20 microm in diameter, which should prove useful for functional and structural studies through the use of optical microscopy, optical tweezers, microelectrodes, or atomic force microscopy.


Assuntos
Bacteriorodopsinas/química , ATPases Transportadoras de Cálcio/química , Proteínas de Membrana/química , Proteolipídeos/química , Retículo Sarcoplasmático/química , Animais , Corantes Fluorescentes/química , Microscopia de Força Atômica
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