Biology Reference
In-Depth Information
Abstract
Microtubules play essential roles in a wide variety of cellular processes including
cell division, motility, and vesicular transport. Microtubule function depends on
the polymerization dynamics of tubulin and specific interactions between tubulin
and diverse microtubule-associated proteins. To date, investigation of the structural
and functional properties of tubulin and tubulin mutants has been limited by the in-
ability to obtain functional protein from overexpression systems, and by the hetero-
geneous mixture of tubulin isotypes typically isolated from higher eukaryotes. The
budding yeast,
Saccharomyces cerevisiae
, has emerged as a leading system for tu-
bulin structure-function analysis. Yeast cells encode a single beta-tubulin gene
and can be engineered to express just one of two alpha isotypes. Moreover, yeast
allows site-directed modification of tubulin genes at the endogenous loci expressed
under the native promoter and regulatory elements. These advantageous features
provide a homogeneous and controlled environment for analysis of the functional
consequences of specific mutations. Here, we present the techniques to generate
site-specific tubulin mutations in diploid and haploid cells, assess the ability of
the mutated protein to support cell viability, measure overall microtubule stability,
and define changes in the specific parameters of microtubule dynamic instability. We
also outline strategies to determine whether mutations disrupt interactions with
microtubule-associated proteins. Microtubule-based functions in yeast are well de-
fined, which allows the observed changes in microtubule properties to be related to
the role of microtubules in specific cellular processes.
INTRODUCTION
Microtubules are cytoskeletal polymers composed of
-tubulin heterodimers that
exhibit highly dynamic growth behavior, which allows for rapid rearrangement of
microtubule networks to support diverse cellular tasks (
Desai & Mitchison, 1997
).
Specific mutations in tubulin genes result in a spectrum of congenital neurological
disorders in humans (
Cederquist et al., 2012; Jaglin et al., 2009; Tischfield,
Cederquist, Gupta, & Engle, 2011
). Moreover, the pathology of Alzheimer's and
Parkinson's disease has been at least in part attributed to microtubule dysfunction
(
Brandt, Hundelt, & Shahani, 2005; Lei et al., 2010
). The organization and dynamics
of microtubule polymer are also essential for proper function of the mitotic spindle
and fidelity of chromosome segregation. Indeed, small molecules that interfere with
microtubule dynamics are a powerful class of antimitotic agents and represent a
proven strategy in the treatment of various cancers (
Jordan, 2002
). Therefore, under-
standing the structural and functional properties underlying the regulation of micro-
tubule dynamics is of great interest for human health.
Studies directed toward elucidating the structure-function relationship of tubulin
andmicrotubules havebeen challenging. The foldingof tubulinpolypeptides intonative
heterodimers requires interactions with chaperones and protein cofactors (
Lewis, Tian,
ab
