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1.
Philos Trans A Math Phys Eng Sci ; 379(2205): 20200317, 2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34275365

RESUMO

In this paper, we formulate the space-dependent variable-order fractional master equation to model clustering of particles, organelles, inside living cells. We find its solution in the long-time limit describing non-uniform distribution due to a space-dependent fractional exponent. In the continuous space limit, the solution of this fractional master equation is found to be exactly the same as the space-dependent variable-order fractional diffusion equation. In addition, we show that the clustering of lysosomes, an essential organelle for healthy functioning of mammalian cells, exhibit space-dependent fractional exponents. Furthermore, we demonstrate that the non-uniform distribution of lysosomes in living cells is accurately described by the asymptotic solution of the space-dependent variable-order fractional master equation. Finally, Monte Carlo simulations of the fractional master equation validate our analytical solution. This article is part of the theme issue 'Transport phenomena in complex systems (part 1)'.


Assuntos
Lisossomos , Animais , Análise por Conglomerados , Difusão
2.
J Cell Sci ; 128(4): 755-67, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25588841

RESUMO

Endosomal sorting complexes required for transport (ESCRT)-0 sorts ubiquitylated EGFR within the early endosome so that the receptor can be incorporated into intralumenal vesicles. An important question is whether ESCRT-0 acts solely upon EGFR that has already entered the vacuolar early endosome (characterised by the presence of EEA1) or engages EGFR within earlier compartments. Here, we employ a suite of software to determine the localisation of ESCRT-0 at subpixel resolution and to perform particle-based colocalisation analysis with other endocytic markers. We demonstrate that although some of the ESCRT-0 subunit Hrs (also known as HGS) colocalises with the vacuolar early endosome marker EEA1, most localises to a population of peripheral EEA1-negative endosomes that act as intermediates in transporting EGFR from the cell surface to more central early endosomes. The peripheral Hrs-labelled endosomes are distinct from APPL1-containing endosomes, but co-label with the novel endocytic adaptor SNX15. In contrast to ESCRT-0, ESCRT-I is recruited to EGF-containing endosomes at later times as they move to more a central position, whereas ESCRT-III is also recruited more gradually. RNA silencing experiments show that both ESCRT-0 and ESCRT-I are important for the transit of EGF to EEA1 endosomes.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/fisiologia , Receptores ErbB/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Ativação Enzimática , Fator de Crescimento Epidérmico/metabolismo , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transporte Proteico , Interferência de RNA , RNA Interferente Pequeno , Nexinas de Classificação/metabolismo , Vesículas Transportadoras/metabolismo , Ubiquitinação , Proteínas de Transporte Vesicular/genética
3.
EMBO J ; 35(11): 1155-7, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27099300
4.
Nat Cell Biol ; 9(1): 113-20, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17173037

RESUMO

The early endosome is organised into domains to ensure the separation of cargo. Activated mitogenic receptors, such as epidermal growth factor (EGF) receptor, are concentrated into vacuoles enriched for the small GTPase Rab5, which progressively exclude nutrient receptors, such as transferrin receptor, into neighbouring tubules. These vacuoles become enlarged, increase their content of intralumenal vesicles as EGF receptor is sorted from the limiting membrane, and eventually mature to late endosomes. Maturation is governed by the loss of Rab5 and is accompanied by the movement of endosomes along microtubules towards the cell centre. Here, we show that EGF relocates to the cell centre in a dynein-dependent fashion, concomitant with the sorting away of transferrin receptor, although it remains in Rab5-positive early endosomes. When dynein function is acutely disrupted, efficient recycling of transferrin from EGF-containing endosomes is retarded, loss of Rab5 is slowed and endosome enlargement is reduced.


Assuntos
Dineínas/metabolismo , Dineínas/fisiologia , Endossomos/metabolismo , Morfogênese , Endossomos/fisiologia , Fator de Crescimento Epidérmico/farmacocinética , Células HeLa , Humanos , Microinjeções , Microscopia de Fluorescência/métodos , Microscopia de Vídeo/métodos , Transporte Proteico , Receptores da Transferrina/metabolismo , Transdução de Sinais , Transfecção , Transferrina/farmacocinética , Proteínas de Transporte Vesicular , Proteínas rab5 de Ligação ao GTP/metabolismo
5.
Phys Biol ; 10(3): 036002, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23574726

RESUMO

Particle tracking experiments with high speed digital microscopy yield the positions and trajectories of lipid droplets inside living cells. Angular correlation analysis shows that the lipid droplets have uncorrelated motion at short time scales (τ < 1 ms) followed by anti-persistent motion for lag times in the range of 1 ⩽ τ ⩽ 10 ms. The angular correlation at longer time scales, τ > 10 ms, becomes persistent, indicating directed movement. The motion at all time scales is associated with the lipid droplets being tethered to and driven along the microtubule network. The point at which the angular correlation changes from anti-persistent to persistent motion corresponds to the cross over between sub-diffusive and super diffusive motion, as observed by mean square displacement analysis. Correct analysis of the angular correlations of the detector noise is found to be crucial in modelling the observed phenomena.


Assuntos
Lipídeos/análise , Microtúbulos/metabolismo , Modelos Biológicos , Movimento (Física) , Algoritmos , Linhagem Celular , Difusão , Humanos , Metabolismo dos Lipídeos , Microscopia , Probabilidade
6.
J Cell Biol ; 222(5)2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-36946995

RESUMO

Cytoplasmic dynein-driven movement of chromosomes during prophase I of mammalian meiosis is essential for synapsis and genetic exchange. Dynein connects to chromosome telomeres via KASH5 and SUN1 or SUN2, which together span the nuclear envelope. Here, we show that KASH5 promotes dynein motility in vitro, and cytosolic KASH5 inhibits dynein's interphase functions. KASH5 interacts with a dynein light intermediate chain (DYNC1LI1 or DYNC1LI2) via a conserved helix in the LIC C-terminal, and this region is also needed for dynein's recruitment to other cellular membranes. KASH5's N-terminal EF-hands are essential as the interaction with dynein is disrupted by mutation of key calcium-binding residues, although it is not regulated by cellular calcium levels. Dynein can be recruited to KASH5 at the nuclear envelope independently of dynactin, while LIS1 is essential for dynactin incorporation into the KASH5-dynein complex. Altogether, we show that the transmembrane protein KASH5 is an activating adaptor for dynein and shed light on the hierarchy of assembly of KASH5-dynein-dynactin complexes.


Assuntos
Proteínas de Ciclo Celular , Dineínas do Citoplasma , Complexo Dinactina , Proteínas Associadas aos Microtúbulos , Animais , Cálcio/metabolismo , Dineínas do Citoplasma/genética , Dineínas do Citoplasma/metabolismo , Complexo Dinactina/genética , Complexo Dinactina/metabolismo , Mamíferos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Telômero/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo
7.
J Cell Sci ; 123(Pt 2): 202-12, 2010 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-20048338

RESUMO

LIS1, NDE1 and NDEL1 modulate cytoplasmic dynein function in several cellular contexts. However, evidence that they regulate dynein-dependent organelle positioning is limited. Here, we show that depletion of NDE1 or NDEL1 alone profoundly affected the organisation of the Golgi complex but did not cause it to disperse, and slightly affected the position of endocytic compartments. However, striking dispersal of organelles was observed when both NDE1 and NDEL1 were depleted. A substantial portion of NDE1 and NDEL1 is membrane associated, and depletion of these proteins led to complete loss of dynein from membranes. Knockdown of LIS1 also caused the Golgi complex to fragment and disperse throughout the cell, and caused endocytic compartments to relocalise to the periphery. Depletion of LIS1, which is primarily cytosolic, led to partial loss of membrane-associated dynein, without affecting NDE1 and NDEL1. These data suggest that NDE1 and NDEL1 act upstream of LIS1 in dynein recruitment, and/or activation, on the membrane. Consistent with this hypothesis, expression of exogenous NDE1 or NDEL1 rescued the effects of LIS1 depletion on Golgi organisation, whereas LIS1 was only partially effective at rescuing the loss of NDE1 and NDEL1.


Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Proteínas de Transporte/metabolismo , Dineínas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Organelas/metabolismo , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Mitose , Fenótipo , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo
8.
Biochem Soc Trans ; 39(5): 1169-78, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21936784

RESUMO

The organization and function of eukaryotic cells rely on the action of many different molecular motor proteins. Cytoplasmic dynein drives the movement of a wide range of cargoes towards the minus ends of microtubules, and these events are needed, not just at the single-cell level, but are vital for correct development. In the present paper, I review recent progress on understanding dynein's mechanochemistry, how it is regulated and how it binds to such a plethora of cargoes. The importance of a number of accessory factors in these processes is discussed.


Assuntos
Dineínas do Citoplasma/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Transporte Biológico/fisiologia , Dineínas do Citoplasma/química , Dineínas do Citoplasma/genética , Microtúbulos/metabolismo , Modelos Biológicos , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo
9.
Nat Cell Biol ; 4(10): E236-42, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12360306

RESUMO

The Golgi apparatus is a dynamic organelle through which nascent secretory and transmembrane proteins are transported, post-translationally modified and finally packaged into carrier vesicles for transport along the cytoskeleton to a variety of destinations. In the past decade, studies have shown that a number of 'molecular motors' are involved in maintaining the proper structure and function of the Golgi apparatus. Here, we review just some of the many functions performed by these mechanochemical enzymes - dyneins, kinesins, myosins and dynamin - in relation to the Golgi apparatus.


Assuntos
Complexo de Golgi/metabolismo , Proteínas Motores Moleculares/metabolismo , Biossíntese de Proteínas , Transporte Proteico/fisiologia , Animais , Dinaminas/metabolismo , Dineínas/metabolismo , Complexo de Golgi/ultraestrutura , Humanos , Cinesinas/metabolismo , Miosinas/metabolismo , Proteínas/metabolismo , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/ultraestrutura
10.
Sci Rep ; 11(1): 16230, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376706

RESUMO

The endoplasmic reticulum (ER) is a eukaryotic subcellular organelle composed of tubules and sheet-like areas of membrane connected at junctions. The tubule network is highly dynamic and undergoes rapid and continual rearrangement. There are currently few tools to evaluate network organisation and dynamics. We quantified ER network organisation in Vero and MRC5 cells, and developed an analysis workflow for dynamics of established tubules in live cells. The persistence length, tubule length, junction coordination number and angles of the network were quantified. Hallmarks of imbalances in ER tension, indications of interactions with microtubules and other subcellular organelles, and active dynamics were observed. Clear differences in dynamic behaviour were observed for established tubules at different positions within the cell using itemset mining. We found that tubules with activity-driven fluctuations were more likely to be located away from the cell periphery and a population of peripheral tubules with no signs of active motion was found.


Assuntos
Retículo Endoplasmático/fisiologia , Fibroblastos/fisiologia , Pulmão/fisiologia , Microtúbulos/fisiologia , Animais , Chlorocebus aethiops , Fibroblastos/citologia , Humanos , Pulmão/citologia , Células Vero
11.
Phys Chem Chem Phys ; 12(15): 3753-61, 2010 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-20358070

RESUMO

The first passage probability (FPP), of trafficked intracellular particles reaching a displacement L, in a given time t or inverse velocity S = t/L, can be calculated robustly from measured particle tracks. The FPP gives a measure of particle movement in which different types of motion, e.g. diffusion, ballistic motion, and transient run-rest motion, can readily be distinguished in a single graph, and compared with mathematical models. The FPP is attractive in that it offers a means of reducing the data in the measured tracks, without making assumptions about the mechanism of motion. For example, it does not employ smoothing, segmentation or arbitrary thresholds to discriminate between different types of motion in a particle track. In contrast to conventional mean square displacement analysis, FPP is sensitive to a small population of trafficked particles that move long distances (> or = 5 microm), which are thought to be crucial for efficient long range signaling in theories of network dynamics. Taking experimental data from tracked endocytic vesicles, and calculating the FPP, we see how molecular treatments affect the trafficking. We show the FPP can quantify complicated movement which is neither completely random nor completely deterministic, making it highly applicable to trafficked particles in cell biology.


Assuntos
Modelos Teóricos , Algoritmos , Difusão , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Probabilidade , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
12.
Elife ; 92020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32207687

RESUMO

Intracellular transport is predominantly heterogeneous in both time and space, exhibiting varying non-Brownian behavior. Characterization of this movement through averaging methods over an ensemble of trajectories or over the course of a single trajectory often fails to capture this heterogeneity. Here, we developed a deep learning feedforward neural network trained on fractional Brownian motion, providing a novel, accurate and efficient method for resolving heterogeneous behavior of intracellular transport in space and time. The neural network requires significantly fewer data points compared to established methods. This enables robust estimation of Hurst exponents for very short time series data, making possible direct, dynamic segmentation and analysis of experimental tracks of rapidly moving cellular structures such as endosomes and lysosomes. By using this analysis, fractional Brownian motion with a stochastic Hurst exponent was used to interpret, for the first time, anomalous intracellular dynamics, revealing unexpected differences in behavior between closely related endocytic organelles.


Assuntos
Fenômenos Bioquímicos/fisiologia , Transporte Biológico/fisiologia , Movimento/fisiologia , Redes Neurais de Computação , Vesículas Transportadoras/metabolismo , Humanos , Modelos Biológicos , Movimento (Física)
13.
J Cell Biol ; 156(3): 495-509, 2002 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-11815631

RESUMO

The mammalian Golgi complex is comprised of a ribbon of stacked cisternal membranes often located in the pericentriolar region of the cell. Here, we report that during apoptosis the Golgi ribbon is fragmented into dispersed clusters of tubulo-vesicular membranes. We have found that fragmentation is caspase dependent and identified GRASP65 (Golgi reassembly and stacking protein of 65 kD) as a novel caspase substrate. GRASP65 is cleaved specifically by caspase-3 at conserved sites in its membrane distal COOH terminus at an early stage of the execution phase. Expression of a caspase-resistant form of GRASP65 partially preserved cisternal stacking and inhibited breakdown of the Golgi ribbon in apoptotic cells. Our results suggest that GRASP65 is an important structural component required for maintenance of Golgi apparatus integrity.


Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Glicoproteínas , Complexo de Golgi/enzimologia , Complexo de Golgi/ultraestrutura , Proteínas de Membrana/metabolismo , Peptídeo Hidrolases/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Autoantígenos , Caspase 3 , Inibidores Enzimáticos/farmacologia , Proteínas da Matriz do Complexo de Golgi , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Proteínas Luminescentes/genética , Glicoproteínas de Membrana/metabolismo , Microscopia Eletrônica , Microscopia de Fluorescência , Microscopia de Vídeo , N-Acetilgalactosaminiltransferases/metabolismo , Oligopeptídeos/farmacologia , Proteínas/metabolismo , Polipeptídeo N-Acetilgalactosaminiltransferase
14.
Elife ; 82019 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-31718774

RESUMO

Cortical collapse factors affect microtubule (MT) dynamics at the plasma membrane. They play important roles in neurons, as suggested by inhibition of axon growth and regeneration through the ARF activator Efa6 in C. elegans, and by neurodevelopmental disorders linked to the mammalian kinesin Kif21A. How cortical collapse factors influence axon growth is little understood. Here we studied them, focussing on the function of Drosophila Efa6 in experimentally and genetically amenable fly neurons. First, we show that Drosophila Efa6 can inhibit MTs directly without interacting molecules via an N-terminal 18 amino acid motif (MT elimination domain/MTED) that binds tubulin and inhibits microtubule growth in vitro and cells. If N-terminal MTED-containing fragments are in the cytoplasm they abolish entire microtubule networks of mouse fibroblasts and whole axons of fly neurons. Full-length Efa6 is membrane-attached, hence primarily blocks MTs in the periphery of fibroblasts, and explorative MTs that have left axonal bundles in neurons. Accordingly, loss of Efa6 causes an increase of explorative MTs: in growth cones they enhance axon growth, in axon shafts they cause excessive branching, as well as atrophy through perturbations of MT bundles. Efa6 over-expression causes the opposite phenotypes. Taken together, our work conceptually links molecular and sub-cellular functions of cortical collapse factors to axon growth regulation and reveals new roles in axon branching and in the prevention of axonal atrophy. Furthermore, the MTED delivers a promising tool that can be used to inhibit MTs in a compartmentalised fashion when fusing it to specifically localising protein domains.


Assuntos
Axônios/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Membrana/metabolismo , Microtúbulos/metabolismo , Polimerização , Motivos de Aminoácidos , Animais , Membrana Celular/metabolismo , Células Cultivadas , Proteínas de Drosophila/química , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Cones de Crescimento/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Proteínas de Membrana/química , Camundongos , Células NIH 3T3 , Peptídeos/metabolismo , Domínios Proteicos , Pseudópodes/metabolismo
15.
Curr Biol ; 15(7): 678-83, 2005 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-15823542

RESUMO

Mitochondria display a variety of shapes, ranging from small and spherical or the classical tubular shape to extended networks. Shape transitions occur frequently and include fusion, fission, and branching. It was reported that some mitochondrial shape transitions are developmentally regulated, whereas others were linked to disease or apoptosis. However, if and how mitochondrial function controls mitochondrial shape through regulation of mitochondrial fission and fusion is unclear. Here, we show that inhibitors of electron transport, ATP synthase, or the permeability transition pore (mtPTP) induced reversible mitochondrial fission. Mitochondrial fission depended on dynamin-related protein 1 (DRP1) and F-actin: Disruption of F-actin attenuated fission and recruitment of DRP1 to mitochondria. In contrast, uncoupling of electron transport and oxidative phosphorylation caused mitochondria to adopt a distinct disk shape. This shape change was independent of the cytoskeleton and DRP1 and was most likely caused by swelling. Thus, disruption of mitochondrial function rapidly and reversibly altered mitochondrial shape either by activation of DRP1-dependent fission or by swelling, indicating a close relationship between mitochondrial fission, shape, and function. Furthermore, our results suggest that the actin cytoskeleton is involved in mitochondrial fission by facilitating mitochondrial recruitment of DRP1.


Assuntos
Actinas/metabolismo , Dinaminas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Animais , Western Blotting , Células Cultivadas , Chlorocebus aethiops , Imunofluorescência , Proteínas Luminescentes , Camundongos , Microscopia Eletrônica de Transmissão , Mitocôndrias/ultraestrutura , Dilatação Mitocondrial/fisiologia , Proteína Vermelha Fluorescente
16.
Sci Rep ; 7(1): 16474, 2017 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-29184084

RESUMO

The endoplasmic reticulum (ER) is a single organelle in eukaryotic cells that extends throughout the cell and is involved in a large number of cellular functions. Using a combination of fixed and live cells (human MRC5 lung cells) in diffraction limited and super-resolved fluorescence microscopy (STORM) experiments, we determined that the average persistence length of the ER tubules was 3.03 ± 0.24 µm. Removing the branched network junctions from the analysis caused a slight increase in the average persistence length to 4.71 ± 0.14 µm, and provides the tubule's persistence length with a moderate length scale dependence. The average radius of the tubules was 44.1 ± 3.2 nm. The bending rigidity of the ER tubule membranes was found to be 10.9 ± 1.2 kT (17.0 ± 1.3 kT without branch points). We investigated the dynamic behaviour of ER tubules in live cells, and found that the ER tubules behaved like semi-flexible fibres under tension. The majority of the ER tubules experienced equilibrium transverse fluctuations under tension, whereas a minority number of them had active super-diffusive motions driven by motor proteins. Cells thus actively modulate the dynamics of the ER in a well-defined manner, which is expected in turn to impact on its many functions.


Assuntos
Retículo Endoplasmático/metabolismo , Imagem Molecular , Biomarcadores , Linhagem Celular Tumoral , Imunofluorescência , Humanos , Microscopia de Fluorescência , Imagem Molecular/métodos
17.
Sci Rep ; 6: 27456, 2016 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-27272132

RESUMO

Microtubules and their associated proteins (MAPs) underpin the polarity of specialised cells. Adenomatous polyposis coli (APC) is one such MAP with a multifunctional agenda that requires precise intracellular localisations. Although APC has been found to associate with kinesin-2 subfamily members, the exact mechanism for the peripheral localization of APC remains unclear. Here we show that the heavy chain of kinesin-1 directly interacts with the APC C-terminus, contributing to the peripheral localisation of APC in fibroblasts. In rat hippocampal neurons the kinesin-1 binding domain of APC is required for its axon tip enrichment. Moreover, we demonstrate that APC requires interactions with both kinesin-2 and kinesin-1 for this localisation. Underlining the importance of the kinesin-1 association, neurons expressing APC lacking kinesin-1-binding domain have shorter axons. The identification of this novel kinesin-1-APC interaction highlights the complexity and significance of APC localisation in neurons.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/genética , Genes Supressores de Tumor , Cinesinas/fisiologia , Células HeLa , Humanos
18.
J Cell Biol ; 207(4): 499-516, 2014 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-25422374

RESUMO

Cytoplasmic dynein 1 (dynein) is a minus end-directed microtubule motor protein with many cellular functions, including during cell division. The role of the light intermediate chains (LICs; DYNC1LI1 and 2) within the complex is poorly understood. In this paper, we have used small interfering RNAs or morpholino oligonucleotides to deplete the LICs in human cell lines and Xenopus laevis early embryos to dissect the LICs' role in cell division. We show that although dynein lacking LICs drives microtubule gliding at normal rates, the LICs are required for the formation and maintenance of a bipolar spindle. Multipolar spindles with poles that contain single centrioles were formed in cells lacking LICs, indicating that they are needed for maintaining centrosome integrity. The formation of multipolar spindles via centrosome splitting after LIC depletion could be rescued by inhibiting Eg5. This suggests a novel role for the dynein complex, counteracted by Eg5, in the maintenance of centriole cohesion during mitosis.


Assuntos
Dineínas do Citoplasma/metabolismo , Cinesinas/antagonistas & inibidores , Mitose/fisiologia , Fuso Acromático/patologia , Animais , Linhagem Celular Tumoral , Movimento Celular , Centríolos/fisiologia , Dineínas do Citoplasma/genética , Complexo Dinactina , Feminino , Células HEK293 , Células HeLa , Humanos , Cinetocoros , Proteínas dos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Dados de Sequência Molecular , Interferência de RNA , RNA Interferente Pequeno , Fuso Acromático/genética , Xenopus laevis
19.
Phys Rev E Stat Nonlin Soft Matter Phys ; 86(3 Pt 1): 031910, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23030947

RESUMO

The first-passage-probability can be used as an unbiased method for determining the phases of motion of individual organelles within live cells. Using high speed microscopy, we observe individual lipid droplet tracks and analyze the motor protein driven motion. At short passage lengths (<10(-2)µm), a log-normal distribution in the first-passage-probability as a function of time is observed, which switches to a Gaussian distribution at longer passages due to the running motion of the motor proteins. The mean first-passage times () as a function of the passage length (L), averaged over a number of runs for a single lipid droplet, follow a power law distribution ~L(α), α>2, at short times due to a passive subdiffusive process. This changes to another power law at long times where 1<α<2, corresponding to sub-ballistic superdiffusive motion, an active process. Subdiffusive passive mean square displacements are observed as a function of time, ~t(ß), where 0<ß<1 at short times again crossing over to an active sub-ballistic superdiffusive result 1<ß<2 at longer times. Consecutive runs of the lipid droplets add additional independent Gaussian peaks to a cumulative first-passage-probability distribution indicating that the speeds of sequential phases of motion are independent and biochemically well regulated. As a result we propose a model for motor driven lipid droplets that exhibits a sequential run behavior with occasional pauses.


Assuntos
Modelos Biológicos , Transporte Biológico Ativo , Sobrevivência Celular , Probabilidade
20.
PLoS One ; 6(9): e24479, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21915335

RESUMO

Microtubule-dependent movement is crucial for the spatial organization of endosomes in most eukaryotes, but as yet there has been no systematic analysis of how a particular microtubule motor contributes to early endosome dynamics. Here we tracked early endosomes labeled with GFP-Rab5 on the nanometer scale, and combined this with global, first passage probability (FPP) analysis to provide an unbiased description of how the minus-end microtubule motor, cytoplasmic dynein, supports endosome motility. Dynein contributes to short-range endosome movement, but in particular drives 85-98% of long, inward translocations. For these, it requires an intact dynactin complex to allow membrane-bound p150(Glued) to activate dynein, since p50 over-expression, which disrupts the dynactin complex, inhibits inward movement even though dynein and p150(Glued) remain membrane-bound. Long dynein-dependent movements occur via bursts at up to ∼8 µms(-1) that are linked by changes in rate or pauses. These peak speeds during rapid inward endosome movement are still seen when cellular dynein levels are 50-fold reduced by RNAi knock-down of dynein heavy chain, while the number of movements is reduced 5-fold. Altogether, these findings identify how dynein helps define the dynamics of early endosomes.


Assuntos
Dineínas/metabolismo , Endossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Complexo Dinactina , Dineínas/genética , Humanos , Proteínas Associadas aos Microtúbulos/genética , Reação em Cadeia da Polimerase , Interferência de RNA , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo
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