Summary The release of membrane vesicles from the surface of cells into their surrounding environment is now recognized as an important pathway for the delivery of proteins to extracellular sites of biological function. Membrane vesicles of this kind, termed exosomes and ectosomes, are the result of active processes and have been shown to carry a wide array of biological effector molecules that can play roles in cell-to-cell communication and remodeling of the extracellular space [1–7]. Degradation of the extracellular matrix (ECM) through the regulated release of proteolytic enzymes is a key process for development, morphogenesis and cell migration in animal and plant cells. Here we show that the unicellular alga, Chlamydomonas, achieves the timely degradation of its mother cell wall, a type of ECM, through the budding of ectosomes directly from the membranes of their flagella. Using a combination of immunoelectron microscopy, immunofluorescence microscopy, and functional analysis, we demonstrate that these vesicles, which we term ciliary ectosomes, act as carriers of the proteolytic enzyme necessary for the liberation of daughter cells following mitosis [8, 9]. Chlamydomonas has proven to be the key unicellular model for the highly conserved mechanisms of mammalian cilia, and our results suggest that cilia may be an under-appreciated source of bioactive, extracellular membrane vesicles.

To analyze the function of ciliary polycystic kidney disease 2 (PKD2) and its relationship to intraflagellar transport (IFT), we cloned the gene encoding Chlamydomonas reinhardtii PKD2 (CrPKD2), a protein with the characteristics of PKD2 family members. Three forms of this protein (210, 120, and 90 kD) were detected in whole cells; the two smaller forms are cleavage products of the 210-kD protein and were the predominant forms in flagella. In cells expressing CrPKD2–GFP, about 10% of flagellar CrPKD2–GFP was observed moving in the flagellar membrane. When IFT was blocked, fluorescence recovery after photobleaching of flagellar CrPKD2–GFP was attenuated and CrPKD2 accumulated in the flagella. Flagellar CrPKD2 increased fourfold during gametogenesis, and several CrPKD2 RNA interference strains showed defects in flagella-dependent mating. These results suggest that the CrPKD2 cation channel is involved in coupling flagellar adhesion at the beginning of mating to the increase in flagellar calcium required for subsequent steps in mating.

Blue light as an environmental cue plays a pivotal role in controlling the progression of the sexual life cycle in the green alga Chlamydomonas reinhardtii. Phototropin was considered a prime candidate for the blue-light receptor involved. By using the RNA interference method, knockdown strains with reduced phototropin levels were isolated. Those with severely reduced levels of this photoreceptor were partially impaired in three steps of the life cycle: in gametogenesis, the maintenance of mating ability, and the germination of zygotes. These observations suggest that phototropin is the principal sensory molecule used by this alga for the control of its life cycle by light.

Summary Primary and motile cilia/flagella function as cellular antennae, receiving signals from the environment, and subsequently activating signaling pathways that are critical for cellular homeostasis and differentiation [1-3]. Recent work with the green alga Chlamydomonas and the nematode C. elegans demonstrated that ectosomes can be released from the cilium and can mediate the intercellular communication [4-9]. To better understand the function of flagellar ectosomes, we have compared their protein composition to that of the flagellar membrane from which they are derived. Ectosomes released from flagella have a unique protein composition, being enriched in a subset of flagellar membrane proteins, proteases, proteins from the endosomal sorting complex required for transport (ESCRT) [10-12], small GTPases, and ubiquitinated proteins. Live imaging showed that an ESCRT-related protein (PDCD6) was enriched in ectosomes released from flagella during gamete activation. We devised a sensitive and rapid assay to monitor ectosome release using luciferase fused to PDCD6 and a mutated ubiquitin. Ectosome release increased when cells underwent flagellar resorption. Knockdown of two ESCRT-related proteins, PDCD6 and VPS4, attenuated ectosome release during flagellar shortening and shortening was slowed. These data suggest that the ESCRT proteins mediate ectosome release and thereby influence flagellar shortening in Chlamydomonas. In addition, the prevalence of receptors such as agglutinin and ubiquitinated proteins in ciliary ectosomes suggests that they are involved in cell signaling and turnover of ciliary proteins.