Moving Towards Self-Assembling Machines: Harnessing Spindle Components for Biosynthetic Devices

Abstract

The remarkably dynamic and complex manufacturing environment of the eukaryotic cell is currently unrivaled by manmade systems. Through dissection and harnessing of biological machines and underlying processes, and merger with synthetic materials, we hope to scale manufacturing into realms currently restricted from physical manipulation. Spindle assembly is the first critical step in chromosome segregation and a primary target for anti-cancer therapeutics. An assemblage of nanoscale components and communication networks must be integrated for spindle formation and function. We are applying nanotechnology in parallel with traditional methods of yeast genetics, timelapse microscopy, AFM and biochemistry. Dramatic reorganization of interphase microtubules into a bipolar organization for chromosome segregation requires focused nucleation from spindle pole body microtubule organizing centers (MTOCs). The γ-tubulin small complex (γ-TuSC) MTOC associates with growing microtubules and is part of a larger ring complex (γ-TuRC). The complex controls microtubule nucleation, organization and dynamics. Three conserved kinesin-like protein (Klp) families in fission yeast contribute in critical roles in spindle assembly. Kinesin-14 Pkl1 and Kinesin-5 associate with γ-TuSC at poles and provide counterbalanced roles in microtubule nucleation and organization, while Kinesin-6 acts on overlapping anti-parallel microtubules. The coupled action of the MTOC and Klps enables multiple levels of control. By purifying the γ-TuSC and γ-TuRC complexes from human and fission yeast cells using superose 6 FPLC chromatography, co-immunoprecipitation with magnetic beads and western analysis and attachment of these complexes to functionalized surfaces we are analyzing minimal components and frameworks for generating anti-parallel, bipolar and more complex microtubule array structures in the presence of multiple Klps. Our goal is to incorporate the MTOC, tubulin and multiple Klp families in different biosynthetic platforms to better understand self-assembling machines and transitional dynamic architectures thereby refining both in vivo models and in vitro advanced applications.