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.