RUI: Tubulin Folding and Assembly in Chlamydomonas

Haverford College, Haverford PA

Investigators

Abstract

Eukaryotic cells contain elaborate internal structures called microtubules, which are hollow, pipe-like polymers of tubulin proteins involved in the determination of the cell's shape, organization, mitotitc and meiotic divisions and movement. The production of new tubulin subunits begins with the translation of alpha and beta tubulin-encoding mRNAs, followed by folding of the nascent polypeptide chains and formation of alpha-beta dimers. The dimers then become part of the pool of free subunits available for microtubule assembly. This translation and dimerization pathway involves special proteins called molecular chaperones that assist in the maturation of tubulin subunits. A key participant is a large multimeric complex called CCT (for Chaperonin Containing TCP- 1). CCT has been studied biochemically in a variety of cell types and genetically in yeast; however, relatively little is known about how the folding pathway is spatially organized within the complex environment of the cytoplasm, or how it may play a role in regulating microtubule assembly through dimer availability. In this project, Dr. Johnson will perform experiments directed at localizing CCT and other components of the tubulin folding pathway in the single-celled green alga Chlamydomonas. Chlamydomonas is an important model organism for the study of a variety of significant cellular features, including flagellar motility. Its pair of long, whip-like flagella extend from the apical cytoplasm of each cell and beat in wave-form, oar-like strokes to propel the cell though its aqueous environment. Each flagellum is a complex miniature machine containing a bundle of specialized microtubules that are animated by also-specialized dynein molecular motors; this complex longitudinal array of microtubules and motors is termed an axoneme and is characteristic of flagella in general. These flagella can be experimentally removed from the Chlamydomonas cells, and such removal results in the triggering of a flagellar regeneration process in which replacement structures, each as long as the cell body itself, are produced within 90 minutes. This process, which can be conveniently induced in the laboratory, involves massive translation of new tubulin proteins within the cell body, movement of tubulin dimers into and through the flagellar space and polymerization of the dimers into nascent microtubules at the distal (far) end of the elongating structure. It is within this context of spatially separated synthesis, transport and assembly that Dr. Johnson will investigate the organization of the tubulin folding pathway. Using a combination of molecular and biochemical approaches, Dr. Johnson will characterize genes encoding Chlamydomonas CCT subunits and study their expression during flagellar regeneration. Specific antibody probes to the subunits will be used to localize CCT within the cell using immunofluorescence and immunoEM techniques. These experiments will be carried out in a predominantly undergraduate institution and will involve extensive participation by undergraduates in the research process. This investigation offers significant opportunities for novel insight into the spatial organization of tubulin biogenesis in the living cell. Because microtubule architectures, and the processes that cells use to create them, are highly conserved in all eukaryotes, such studies will lead to a better understanding of microtubule biology in general.