Engineering Approaches to Biomolecular Motors

Engineering Approaches to Biomolecular Motors: From in vitro to in vivo Friday Speaker Abstracts

Manipulating Kinesin-Tubulin-MTOC Interactions for Engineering Polarized Networks Timothy D. Riehlman, Zachary T. Olmsted, Janet L. Paluh . SUNY Polytechnic Institute CNSE, Albany, NY, USA. In the semiconductor industry, photolithography through a reticle generates mesoscale and larger fixed patterns in contrast to the dynamic self-assembling and adaptable systems in Nature. To advance novel nanomanufacturing strategies we are interested in generating dynamic mesoscale self-assembling patterns by the ability to control polarized networks in vitro utilizing Kinesin- Tubulin and Microtubule organizing center (MTOC) linkages. Here we define the MTOC as the g-tubulin ring complex (g-TuRC) that nucleates microtubules and in vivo attaches to the mammalian centrosome or fission yeast spindle pole body. To fulfill our goal we biochemically purified MTOC g-TuRC from fission yeast and mammalian cells, demonstrated cross- compatibility in vivo of g-TuRC and Kinesin-14 and Kinesin-5 proteins from human to yeast, established in vitro nucleation assays from g-TuRC, and identified and minimally isolated Kinesin control elements for regulation of g-TuRC allowing us to separate MTOC control from Kinesin-tubulin roles. We are experimenting with nanoprinting of the g-TuRC through antibody tethers and designing photoactivatable microtubule connectors based on known sequences from Kinesin-14’s and non-motor microtubule associated proteins, MAPs. Printing capture molecules- antibodies, peptides, proteins or g-TuRC- is accomplished using PDMS stamps with 3um pillar features at 3um spacing. The substrate on which they are printed is silanized to facilitate surface bonding. The printing of g-TuRC into desired patterns is a critical step towards setting polarity points from which to anchor self-assembling networks. We are validating printing by immunofluorescence probing and have patterned antibodies, peptides, and WCE and FPLC purified g-TuRC, and antibody orienting protein A-Fc tethers. The next step is to optimize nucleation control from tethered g-TuRC. The introduction of g-TuRC to in vitro kinesin-tubulin assays represents an important next generation step that extends previous elegant studies by numerous groups in an effort to more fully exploit self-assembling systems.

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