Membrane trafficking from the cytoplasm, to the flagellum, and into the medium

Kaiyao Huang1, Christopher R. Wood1, Dennis R. Diener1, Stefan Geimer2, and Joel L. Rosenbaum1

1) MCDB, Yale University, New Haven, CT 06520 2) Cell Biology/Electron Microscopy, University of Bayreuth, 95440, Bayreuth, Germany

Cilia and flagella are sensory and motile organelles composed of a microtubule core encased in a specialized domain of the plasma membrane. Although the membranes of cilia and flagella are continuous with the plasma membrane, cells are able to localize proteins and lipids specifically to these domains by mechanisms that are not fully known. Our objective is to characterize the trafficking pathway of membrane vesicles from the cytoplasm, to the flagellar membrane, and into the medium by isolating each vesicle population and analyzing its composition. We have obtained from cytoplasmic extracts a population of vesicles that contains proteins destined for the flagellum, such as polycystin-2 (PKD2), components of IFT and BBS. We have also obtained highly purified membrane preparations from both the flagella and cell bodies of Chlamydomonas and found that several membrane proteins including PKD2 and a voltage-dependent calcium channel (CAV2) are greatly enriched in the flagellar membrane. These data suggest that there is a diffusion barrier or gate between the flagellar and cell body membranes. We have also documented by transmission electron microscopy the pinching-off of these vesicles at the flagellar tip. The vesicles that are secreted from the flagella into the medium contain an enrichment of a subset of flagellar polypeptides such as tubulin, BBS4, PKD2 and high molecular weight ubiquitinated proteins. This secretion probably occurs in almost all cilia and balances the addition of membrane at the base of the cilium. In addition, secreted vesicles from flagella during mating of Chlamydomonas can activate the gametes of opposite mating type, suggesting that cilia/flagella are also secretory organelles for long distance signaling.

e-mail address of presenting author: kaiyao.huang@yale.edu