Proteins of the ciliary axoneme are found on cytoplasmic membrane vesicles during growth of cilia

Abstract

The cilium is a specialized extension of the cell in which many specific proteins are admitted and retained, while many others are excluded or expelled. In order to maintain the organelle, the cell must possess mechanisms for the selective gating of protein entry, as well as for the targeted transport of proteins to the cilium from their sites of synthesis within the cell [1-4]. We hypothesized that the cell employs cytoplasmic vesicles as vehicles not only for the transport of proteins destined for the ciliary membrane but also for the transport of axonemal proteins to the cilium by means of peripheral association with vesicles. To test this hypothesis, we employed two different experimental strategies: (1) isolation and biochemical characterization of cytoplasmic vesicles that carry ciliary proteins, and (2) in situ localization of ciliary proteins on cytoplasmic vesicle surfaces using gold labeling and electron microscopy. Our findings indicate that structural proteins destined for the ciliary axoneme are attached to the outer surfaces of cytoplasmic vesicles that carry integral ciliary membrane proteins during the process of ciliary growth.

Figure 1. Isolation of cytoplasmic vesicles with associated ciliary proteins

A. Cytoplasmic extract, obtained by mechanical disruption of cells, was placed at the bottom of a seven-step, discontinuous OptiPrep (iodixanol) density gradient. During high-speed centrifugation, various classes of membrane vesicles in the extract float up to different interfaces of the gradient according to their density (bands indicated by white arrow heads). B. Isolated gradient fractions were subjected to SDSPAGE and analyzed on immunoblots probed with antibodies specific for ciliary membrane and axonemal proteins. The class of membrane vesicles that floats to the 20% gradient interface carries with it PKD2, IFT and radial spoke proteins (RSPs). A fraction from the bottom of the gradient (30%) was loaded to the lane labeled B. C. The fraction at the 20% gradient interface was determined by negative staining and TEM to be composed of membrane vesicles ranging in diameter from ~60 – 120 nm. The scale bar in panel C1 represents 100 nm. D. Silver-enhanced, immunogold labeling of intact, negatively-stained vesicles from the 20% gradient interface fraction showed the presence of PKD2, IFT46, RSPs, and α-tubulin on their outside surfaces (1 – 5). Double labeling performed sequentially with differential silver enhancement revealed that RSP3 (larger silver-enhanced gold particles) could be found associated with the same vesicles as PKD2 and IFT46 (smaller silver-enhanced gold particles) (6 – 9). The scale bar in panel D1 represents 50 nm.

Figure 2. Proteins of the ciliary membrane and axoneme associate with cytoplasmic vesicles in increased amounts during flagellar regeneration

The 20% interface vesicle fraction was obtained from control (full length flagella) and cells undergoing flagellar growth (regenerating) and compared by SDSPAGE and immunoblotting. Shown to the left are silver-stained SDSPAGE gels resulting from equal protein loads of control and regenerating vesicle fractions. Prominent band differences are given asterisks. Shown to the right are immunoblots probed with antibodies specific for the indicated ciliary proteins.

Figure 3. In situ immunogold labeling of ciliary membrane and axonemal proteins during flagellar regeneration

A. White arrows indicate PKD2 specific gold particles clustering on the membrane of a newly forming Chlamydomonas flagellum (emphasized by dotted outline), and on membrane vesicles nearby in the cytoplasm. B – D. In addition to labeling the growing axoneme (C, D), gold particles specific for radial spoke proteins are observed on the surface of membrane vesicles in the cytoplasm beneath newly forming flagella (RSP1, RSP2 and RSP3; white arrows). E – G. Cross sections proximal to the base of newly forming flagella show transitional fibers radiating from basal bodies. RSP specific gold particles are found clustered at the cell membrane where transitional fibers terminate. H, I. An rsp3 null mutant cell line was rescued with an HA-tagged version of RSP3. Immunogold labeling with HA-specific antibodies showed labeling of newly forming flagellar axonemes (H, C1 and C2) and large clusters of RSP3-HA-specific gold particles were found at the region where transitional fibers attach to the cell membrane at the base of flagella (H, C1; I). J – M. Immunofluorescence localization with antibodies specific for RSP3-HA (J, K) and IFT46 (L, M) show similar patters of localization at the base of flagella, consistent with the immunogold localization results. N, O. Representative images of RSP3-HA-specific gold particles observed on membrane vesicles in the cytoplasm beneath growing flagella. The scale bars represent 200 nm.

Figure 4. A model for the movement of ciliary proteins from the cytoplasm to the base of the cilium by association with cytoplasmic vesicles

A. 1. IFT proteins (green) and axonemal proteins (red) are synthesized on free polysomes (blue) in the cytoplasm. 2. IFT proteins associate with the outer surface of cytoplasmic vesicles. 3. Axonemal proteins associate with IFT complexes on the surface of cytoplasmic vesicles. 4. IFT and axonemal protein complexes are delivered to the inner surface of the cell membrane following fusion (exocytosis) of cytoplasmic vesicles. 5. IFT trains carrying axonemal proteins emerge from a membrane-associated pool of complexes around the transitional fibers (yellow) at the ciliary base. B. A cartoon representation of a cross sectional view through the region where the transitional fibers radiate from the basal body to their points of contact with the cell membrane at the base of the cilium. A pool of IFT and axonemal proteins accumulates at the place where transitional fibers meet the membrane. C. A cartoon representation of a longitudinal sectional view through the base of the cilium. Transitional fibers (yellow) extend from the basal body to the cell membrane. IFT trains are shown originating from the membrane-associated pool of IFT and axonemal proteins observed where the transitional fibers meet the cell membrane. The locations of membrane-spanning ciliary proteins within this model are shown in purple.