title: Building cells for quantitative, live-cell analyses of collective motor protein functions.
abstract: Examining the collective mechanical behaviors of interacting cytoskeletal motors has become increasingly important to dissecting the complex and multifaceted mechanisms that regulate the transport and trafficking of materials in cells. Although studying these processes in living cells has been challenging, the development of new Synthetic Biology techniques has opened unique opportunities to both manipulate and probe how these motors function in groups as they navigate the native cytoskeleton. Here, we describe an approach to engineer mammalian cells for a new class of inducible cargo motility assays that utilize drug-dependent protein dimerization switches to regulate motor-cargo coupling and transport. Our adaptations provide genetic-level control over the densities of motor proteins coupled to, as well as the sizes of endogenous vesicular cargos in these assays. By allowing the examination of transport responses to changes in motor density and cargo size-dependent viscous drag force, such control can enable quantitative comparisons of mechanistic distinctions between the collective behaviors of different types of processive cytoskeletal motors.
authors.affiliation: Department of Chemistry, Rice University, Houston, TX, USA; Department of Bioengineering, Rice University, Houston, TX, USA.
authors.forename: Eric A
authors.initials: EA
authors.lastname: Kumar
authors.affiliation: Department of Chemistry, Rice University, Houston, TX, USA; Department of Bioengineering, Rice University, Houston, TX, USA.
authors.forename: David
authors.initials: D
authors.lastname: Tsao
authors.affiliation: Department of Chemistry, Rice University, Houston, TX, USA; Department of Bioengineering, Rice University, Houston, TX, USA.
authors.forename: Anand
authors.initials: A
authors.lastname: Radhakrishnan
authors.affiliation: Department of Chemistry, Rice University, Houston, TX, USA; Department of Bioengineering, Rice University, Houston, TX, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Diehl
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001692:Biological Transport / Q000502:physiology*; D019556:COS Cells; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology*; D002522:Chlorocebus aethiops; D004398:Dyneins / Q000378:metabolism; D016547:Kinesins / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism; D055503:Protein Multimerization / Q000187:drug effects; D020123:Sirolimus / Q000378:metabolism; D022021:Tacrolimus Binding Proteins / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; C054562:yellow fluorescent protein, Bacteria; D009218:Myosins; D004398:Dyneins; D016547:Kinesins; D022021:Tacrolimus Binding Proteins; D020123:Sirolimus
keywords: Cellular engineering; Intracellular transport; Motor cooperativity; Synthetic biology