ABSTRACT
Many cellular constituents travel along microtubules in association with multiple copies of motor proteins. How the activity of these motors is regulated during cargo sorting is poorly understood. In this study, we address this issue using a novel in vitro assay for the motility of localising Drosophila mRNAs bound to native dynein-dynactin complexes. High precision tracking reveals that individual RNPs within a population undergo either diffusive, or highly processive, minus end-directed movements along microtubules. RNA localisation signals stimulate the processive movements, with regulation of dynein-dynactin's activity rather than its total copy number per RNP, responsible for this effect. Our data support a novel mechanism for multi-motor translocation based on the regulation of dynein processivity by discrete cargo-associated features. Studying the in vitro responses of RNPs to microtubule-associated proteins (MAPs) and microtubule ends provides insights into how an RNA population could navigate the cytoskeletal network and become anchored at its destination in cells. DOI: http://dx.doi.org/10.7554/eLife.01596.001.
Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Dyneins/metabolism , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , RNA, Messenger/metabolism , Ribonucleoproteins/metabolism , Animals , Diffusion , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Dynactin Complex , Dyneins/genetics , Microtubule-Associated Proteins/genetics , Microtubules/genetics , Protein Transport , RNA, Messenger/genetics , Ribonucleoproteins/genetics , Time FactorsABSTRACT
Microtubule-based transport mediates the sorting and dispersal of many cellular components and pathogens. However, the mechanisms by which motor complexes are recruited to and regulated on different cargos remain poorly understood. Here we describe a large-scale biochemical screen for novel factors associated with RNA localization signals mediating minus end-directed mRNA transport during Drosophila development. We identified the protein Lissencephaly-1 (Lis1) and found that minus-end travel distances of localizing transcripts are dramatically reduced in lis1 mutant embryos. Surprisingly, given its well-documented role in regulating dynein mechanochemistry, we uncovered an important requirement for Lis1 in promoting the recruitment of dynein and its accessory complex dynactin to RNA localization complexes. Furthermore, we provide evidence that Lis1 levels regulate the overall association of dynein with dynactin. Our data therefore reveal a critical role for Lis1 within the mRNA localization machinery and suggest a model in which Lis1 facilitates motor complex association with cargos by promoting the interaction of dynein with dynactin.
Subject(s)
Drosophila Proteins/metabolism , Drosophila/metabolism , Dyneins/metabolism , Microtubule-Associated Proteins/metabolism , RNA, Messenger/metabolism , Animals , Dynactin Complex , Protein Transport , RNA TransportABSTRACT
Microstructures that generate shear-protected regions in microchannels can rapidly immobilize cells for cell-based biosensing and drug screening. Here, a two-step fabrication method is used to generate double microgrooves with various depth ratios to achieve controlled double-level cell patterning while still providing shear protection. Six microgroove geometries are fabricated with different groove widths and depth ratios. Two modes of cell docking are observed: cells docked upstream in sufficiently deep and narrow grooves, and downstream in shallow, wide grooves. Computational flow simulations link the groove geometry and bottom shear stress to the experimental cell docking patterns. Analysis of the experimental cell retention in the double grooves demonstrates its linear dependence on inlet flow speed, with slope inversely proportional to the sheltering provided by the groove geometry. Thus, double-grooved microstructures in microfluidic channels provide shear-protected regions for cell docking and immobilization and appear promising for cell-based biosensing and drug discovery.