Protocol

Visualizing the Subcellular Localization of Actin, β-Tubulin, and DNA in Monosiga brevicollis

¹Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94704, USA
²Department of Integrative Biology, University of California, Berkeley, Berkeley, CA 94704, USA
³Canadian Institute for Advanced Research, Toronto, Ontario M5G 1Z8, Canada
⁴Department of Biology, University of York, York YO10 5YW, United Kingdom
⁵School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom

⁶Corresponding author (b.s.c.leadbeater{at}bham.ac.uk)

This article is also available in Emerging Model Organisms: A Laboratory Manual, Vol. 1. CSHL Press, Cold Spring Harbor, NY, USA, 2009.

INTRODUCTION

Choanoflagellates are heterotrophic nanoflagellates: small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Because they are the closest living relatives of the metazoa, the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. They occur as spherical to ovoid cells exhibiting morphological similarities to the choanocytes (feeding cells) of sponges. Specifically, they are characterized by a single anterior flagellum surrounded by a funnel-shaped collar composed of 30 or more actin-based tentacles or microvilli. These structures can be visualized by staining for actin and β-tubulin. In this protocol, Monosiga brevicollis cells are fixed in dilute formaldehyde, attached to coverslips, and then exposed to antibodies against β-tubulin and an actin-specific stain; a simple DNA stain highlights the nucleus. The central challenge of this protocol is to treat the cells gently so that they retain their ultrastructure and remain attached to the coverslips during processing.