Isolation of ciliary ectosomes and analysis of amidated peptide-mediated chemotaxis in Chlamydomonas

Abstract

Ciliary ectosomes are vesicles that bud from the ciliary membrane. Isolation and analysis of these structures can shed light on their bioactive cargoes and identify proteins and biomolecules involved in intercellular communication and various physiological processes. Most published methods to isolate ciliary ectosomes are based on their size (100nm to 1μm) to separate cilia-derived vesicles from isolated cilia and/or intact cells. However, it is often difficult to determine the origin of extracellular vesicles and to distinguish ciliary ectosomes from ectosomes budded from the plasma membrane or from exosomes that derive from multivesicular bodies. Here, we describe procedures to isolate and purify ciliary ectosomes from the unicellular green alga, Chlamydomonas reinhardtii, through differential and iodixanol density gradient ultracentrifugation; in this organism, the ciliary membrane is the only membrane directly exposed to the environment and thus ectosomes are of known origin. Ciliary ectosomes contain enzymes and α-amidated peptide products required to mediate peptidergic-signaling cascades; one identified amidated peptide acts as a chemotactic modulator for C. reinhardtii gametes. Classical methods used to assess chemotaxis do not provide quantitative measurements of the chemotactic gradient or the real-time effects on the migration of fast moving cells. Consequently, we developed a chemotaxis assay protocol using microfluidic channel slides that provides quantitative and qualitative measurements of the chemotactic gradient and cell migration. Here, we describe how to establish a stable gradient of a bioactive substance in microfluidic channel slides and perform quantitative assays to assess chemotaxis of both individual cells and populations of C. reinhardtii.

Keywords: Chemotaxis; Chlamydomonas; Cilia; Ectosome; Extracellular vesicle; Peptide amidation; Peptidergic signaling.