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1.
Biophys J ; 105(5): 1182-91, 2013 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-24010661

RESUMO

Molecular motor proteins are responsible for long-range transport of vesicles and organelles. Recent works have elucidated the richness of the transport complex, with multiple teams of similar and dissimilar motors and their cofactors attached to individual cargoes. The interaction among these different proteins, and with the microtubules along which they translocate, results in the intricate patterns of cargo transport observed in cells. High-precision and high-bandwidth measurements are required to capture the dynamics of these interactions, yet the crowdedness in the cell necessitates performing such measurements in vitro. Here, we show that endogenous cargoes, lipid droplets purified from Drosophila embryos, can be used to perform high-precision and high-bandwidth optical trapping experiments to study motor regulation in vitro. Purified droplets have constituents of the endogenous transport complex attached to them and exhibit long-range motility. A novel method to determine the quality of the droplets for high-resolution measurements in an optical trap showed that they compare well with plastic beads in terms of roundness, homogeneity, position sensitivity, and trapping stiffness. Using high-resolution and high-bandwidth position measurements, we demonstrate that we can follow the series of binding and unbinding events that lead to the onset of active transport.


Assuntos
Drosophila melanogaster/embriologia , Embrião não Mamífero/metabolismo , Metabolismo dos Lipídeos , Proteínas Motores Moleculares/metabolismo , Animais , Transporte Biológico , Embrião não Mamífero/citologia , Microtúbulos/metabolismo , Pinças Ópticas
2.
PLoS One ; 8(7): e67710, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23840877

RESUMO

To complete meiosis II in animal cells, the male DNA material needs to meet the female DNA material contained in the female pronucleus at the egg center, but it is not known how the male pronucleus, deposited by the sperm at the periphery of the cell, finds the cell center in large eggs. Pronucleus centering is an active process that appears to involve microtubules and molecular motors. For small and medium-sized cells, the force required to move the centrosome can arise from either microtubule pushing on the cortex, or cortically-attached dynein pulling on microtubules. However, in large cells, such as the fertilized Xenopus laevis embryo, where microtubules are too long to support pushing forces or they do not reach all boundaries before centrosome centering begins, a different force generating mechanism must exist. Here, we present a centrosome positioning model in which the cytosolic drag experienced by cargoes hauled by cytoplasmic dynein on the sperm aster microtubules can move the centrosome towards the cell's center. We find that small, fast cargoes (diameter ∼100 nm, cargo velocity ∼2 µm/s) are sufficient to move the centrosome in the geometry of the Xenopus laevis embryo within the experimentally observed length and time scales.


Assuntos
Centrossomo/metabolismo , Dineínas/metabolismo , Xenopus laevis/embriologia , Animais , Transporte Biológico , Citoplasma/metabolismo , Feminino , Masculino , Microtúbulos/metabolismo , Modelos Biológicos
3.
Biophys J ; 103(3): 492-500, 2012 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-22947865

RESUMO

Molecular motor proteins use the energy released from ATP hydrolysis to generate force and haul cargoes along cytoskeletal filaments. Thus, measuring the force motors generate amounts to directly probing their function. We report on optical trapping methodology capable of making precise in vivo stall-force measurements of individual cargoes hauled by molecular motors in their native environment. Despite routine measurement of motor forces in vitro, performing and calibrating such measurements in vivo has been challenging. We describe the methodology recently developed to overcome these difficulties, and used to measure stall forces of both kinesin-1 and cytoplasmic dynein-driven lipid droplets in Drosophila embryos. Critically, by measuring the cargo dynamics in the optical trap, we find that there is memory: it is more likely for a cargo to resume motion in the same direction-rather than reverse direction-after the motors transporting it detach from the microtubule under the force of the optical trap. This suggests that only motors of one polarity are active on the cargo at any instant in time and is not consistent with the tug-of-war models of bidirectional transport where both polarity motors can bind the microtubules at all times. We further use the optical trap to measure in vivo the detachment rates from microtubules of kinesin-1 and dynein-driven lipid droplets. Unlike what is commonly assumed, we find that dynein's but not kinesin's detachment time in vivo increases with opposing load. This suggests that dynein's interaction with microtubules behaves like a catch bond.


Assuntos
Proteínas Motores Moleculares/metabolismo , Pinças Ópticas , Animais , Transporte Biológico , Drosophila melanogaster/embriologia , Embrião não Mamífero/metabolismo , Fatores de Tempo
4.
Fly (Austin) ; 6(4): 303-8, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22992470

RESUMO

Drosophila melanogaster is widely used as a model system for development and disease. Due to the homology between Drosophila and human genes, as well as the tractable genetics of the fly, its use as a model for neurologic disorders, in particular, has been rising. Locomotive impairment is a commonly used diagnostic for screening and characterization of these models, yet a fast, sensitive and model-free method to compare behavior is lacking. Here, we present a high throughput method to quantify the crawling behavior of larvae. We use the mean squared displacement as well as the direction autocorrelation of the crawling larvae as descriptors of their motion. By tracking larvae from wild-type strains and models of the Fragile X mental retardation as well as Alzheimer disease, we show these mutants exhibit impaired crawling. We further show that the magnitude of impairment correlates with the severity of the mutation, demonstrating the sensitivity and the dynamic range of the method. Finally, we study larvae with altered expression of the shaggy gene, a homolog of Glycogen Synthase Kinase-3 (GSK-3), which has been implicated in Alzheimer disease. Surprisingly, we find that both increased and decreased expression of dGSK-3 lead to similar larval crawling impairment. These findings have implications for the use of GSK-3 inhibitors recently proposed for Alzheimer treatment.


Assuntos
Drosophila/genética , Genótipo , Locomoção/genética , Animais , Drosophila/fisiologia , Proteínas de Drosophila/genética , Proteína do X Frágil da Deficiência Intelectual/genética , Estudos de Associação Genética , Quinase 3 da Glicogênio Sintase/genética , Processamento de Imagem Assistida por Computador , Larva/genética , Larva/fisiologia , Mutação , Fenótipo , Imagem com Lapso de Tempo
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