Dataset Information

Mechanical coupling of microtubule-dependent motor teams during peroxisome transport in Drosophila S2 cells.

ABSTRACT: Intracellular transport requires molecular motors that step along cytoskeletal filaments actively dragging cargoes through the crowded cytoplasm. Here, we explore the interplay of the opposed polarity motors kinesin-1 and cytoplasmic dynein during peroxisome transport along microtubules in Drosophila S2 cells.We used single particle tracking with nanometer accuracy and millisecond time resolution to extract quantitative information on the bidirectional motion of organelles. The transport performance was studied in cells expressing a slow chimeric plus-end directed motor or the kinesin heavy chain. We also analyzed the influence of peroxisomes membrane fluidity in methyl-?-ciclodextrin treated cells. The experimental data was also confronted with numerical simulations of two well-established tug of war scenarios.The velocity distributions of retrograde and anterograde peroxisomes showed a multimodal pattern suggesting that multiple motor teams drive transport in either direction. The chimeric motors interfered with the performance of anterograde transport and also reduced the speed of the slowest retrograde team. In addition, increasing the fluidity of peroxisomes membrane decreased the speed of the slowest anterograde and retrograde teams.Our results support the existence of a crosstalk between opposed-polarity motor teams. Moreover, the slowest teams seem to mechanically communicate with each other through the membrane to trigger transport.

SUBMITTER: De Rossi MC

PROVIDER: S-EPMC5796658 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Publications

Mechanical coupling of microtubule-dependent motor teams during peroxisome transport in Drosophila S2 cells.

De Rossi María Cecilia MC Wetzler Diana E DE Benseñor Lorena L De Rossi María Emilia ME Sued Mariela M Rodríguez Daniela D Gelfand Vladimir V Bruno Luciana L Levi Valeria V

Biochimica et biophysica acta. General subjects 20170919 12

<h4>Background</h4>Intracellular transport requires molecular motors that step along cytoskeletal filaments actively dragging cargoes through the crowded cytoplasm. Here, we explore the interplay of the opposed polarity motors kinesin-1 and cytoplasmic dynein during peroxisome transport along microtubules in Drosophila S2 cells.<h4>Methods</h4>We used single particle tracking with nanometer accuracy and millisecond time resolution to extract quantitative information on the bidirectional motion o ...[more]

PMID: 28935608

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Project description:BACKGROUND:S2 cells are one of the most widely used Drosophila melanogaster cell lines. A series of studies has shown that they are particularly suitable for RNAi-based screens aimed at the dissection of cellular pathways, including those controlling cell shape and motility, cell metabolism, and host-pathogen interactions. In addition, RNAi in S2 cells has been successfully used to identify many new mitotic genes that are conserved in the higher eukaryotes, and for the analysis of several aspects of the mitotic process. However, no detailed and complete description of S2 cell mitosis at the ultrastructural level has been done. Here, we provide a detailed characterization of all phases of S2 cell mitosis visualized by transmission electron microscopy (TEM). RESULTS:We analyzed by TEM a random sample of 144 cells undergoing mitosis, focusing on intracellular membrane and microtubule (MT) behaviors. This unbiased approach provided a comprehensive ultrastructural view of the dividing cells, and allowed us to discover that S2 cells exhibit a previously uncharacterized behavior of intracellular membranes, involving the formation of a quadruple nuclear membrane in early prometaphase and its disassembly during late prometaphase. After nuclear envelope disassembly, the mitotic apparatus becomes encased by a discontinuous network of endoplasmic reticulum membranes, which associate with mitochondria, presumably to prevent their diffusion into the spindle area. We also observed a peculiar metaphase spindle organization. We found that kinetochores with attached k-fibers are almost invariably associated with lateral MT bundles that can be either interpolar bundles or k-fibers connected to a different kinetochore. This spindle organization is likely to favor chromosome alignment at metaphase and subsequent segregation during anaphase. CONCLUSIONS:We discovered several previously unknown features of membrane and MT organization during S2 cell mitosis. The genetic determinants of these mitotic features can now be investigated, for instance by using an RNAi-based approach, which is particularly easy and efficient in S2 cells.

Dataset Information

Mechanical coupling of microtubule-dependent motor teams during peroxisome transport in Drosophila S2 cells.

Publications

Mechanical coupling of microtubule-dependent motor teams during peroxisome transport in Drosophila S2 cells.

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