Chapter 46 Preparation and Reactivation of Spermatozopsis Cytoskeletons

Abstract

Publisher Summary Spermatozopsis similis (Chlorophyceae) is a flagellate green alga, which has crescent and spirally twisted cells. They bear two flagella of subequal length and contain a single chloroplast with an eyespot but lacking a pyrenoid. Cells reproduce asexually in the flagellate state by longitudinal division into two progeny cells. The basal apparatus resembles that of Chlamydomonas reinhardtii in comprising two basal bodies interconnected by distal and proximal connecting fibers and four microtubular flagellar roots. Two types of fibrous flagellar roots occur in S. similis : a single small nucleus-basal body connector (NBBC), which contains the calcium-modulated EF-hand protein centrin (also known as caltractin), and two striated microtubule-associated fibers (SMAFs or system I fibers) that accompany the two-member microtubular flagellar roots over most of their length. The addition of 5 m M MgSO 4 , to the isolation buffer yields nucleoflagellar apparatus complexes in which a stable nuclear remnant (the karyoskeleton) remains attached to the basal bodies by the NBBC. Isolated cytoskeletons can be used as a source for biochemical analysis of cytoskeletal proteins such as tubulin and centrin or to isolate subcomponents of the flagellar apparatus such as basal apparatuses and SMAFs. Some flagellar apparatus functions of S. similis have been reconstituted in vitro —namely, axonemal shedding, axonemal motility, and reorientation of the basal bodies. Calcium concentrations induce centrin-mediated contraction of the distal connecting fiber, resulting in a reorientation of the basal bodies to the parallel orientation. In vivo basal body reorientation in S. similis can be elicited as a phobic shock response by very high photon flux rates or mechanical stimulation. The cell biology of phototaxis in S. similis has been extensively studied in situ and in isolated eyespot apparatuses. This chapter discusses the materials needed, the process of isolation of Spermatozopsis Cytoskeletons and the process of reactivation of isolated cytoskeletons.