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. 2015 Feb 2;25(3):379-384.
doi: 10.1016/j.cub.2014.11.066. Epub 2015 Jan 8.

Proteomic Analysis of Isolated Ciliary Transition Zones Reveals the Presence of ESCRT Proteins

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Proteomic Analysis of Isolated Ciliary Transition Zones Reveals the Presence of ESCRT Proteins

Dennis R Diener et al. Curr Biol. .
Free PMC article

Abstract

The transition zone (TZ) is a specialized region of the cilium characterized by Y-shaped connectors between the microtubules of the ciliary axoneme and the ciliary membrane [1]. Located near the base of the cilium, the TZ is in the prime location to act as a gate for proteins into and out of the ciliary compartment, a role supported by experimental evidence [2-6]. The importance of the TZ has been underscored by studies showing that mutations affecting proteins located in the TZ result in cilia-related diseases, or ciliopathies, presenting symptoms including renal cysts, retinal degeneration, and situs inversus [7-9]. Some TZ proteins have been identified and shown to interact with each other through coprecipitation studies in vertebrate cells [4, 10, 11] and genetics studies in C. elegans [3]. As a distinct approach to identify TZ proteins, we have taken advantage of the biology of Chlamydomonas to isolate TZs. Proteomic analysis identified 115 proteins, ten of which were known TZ proteins related to ciliopathies, indicating that the preparation was highly enriched for TZs. Interestingly, six proteins of the endosomal sorting complexes required for transport (ESCRT) were also associated with the TZs. Identification of these and other proteins in the TZ will provide new insights into functions of the TZ, as well as candidate ciliopathy genes.

Figures

Figure 1
Figure 1
A. Electron micrograph of the transition zone. The transition zone (TZ) of Chlamydomonas is characterized by a bipartite cylinder bisected by a transverse plate, which together look like an “H” in midsagittal section. Electron dense wedge connectors (W) extend from the axonemal microtubules to the membrane. The flagella pass through a flagellar collar (C) to exit the cell wall (CW). B. Diagram of the formation of the TZ vesicle. 1-3. Prior to cell division the flagellar microtubules depolymerize and the flagella shorten. 4. The TZ detaches from the basal body (BB) forming a vesicle inside the flagellar collar (C). The transitional fibers (TF), which attach the basal body to the membrane, are not a part of the TZ. C-E. Transition zones are trapped in flagellar collars of isolated cell walls. C. Isolated cell walls retain pairs of flagellar collars (left panel, arrows) many of which contain a punctum of acetylated α-tubulin (center panel, arrows). D. In an end-on view, a TZ is seen in the center of each flagellar collar. E. CEP290, a protein known to be associated with the TZ in situ, is also present in many of the shed TZs.
Figure 2
Figure 2
Electron microscopy of isolated TZs. A. Negative stain electron microscopy of a band from a cesium chloride gradient of isolated cell walls reveals flagellar collars containing an electron dense vesicle. B. At higher magnification fibers of the flagellar collar can be seen entrapping the vesicle. C. After detergent treatment of the grid, the central cylinder characteristic of the TZ is seen inside the vesicles. The lower vesicle has lysed releasing the central cylinder, which separated into its proximal and distal components (arrows). D. Purified TZs, identified by the presence of the central cylinder in medial sections, are seen by electron microscopy of a section through a pellet of material fractionated on an iodixanol gradient. Tangential sections of TZs reveal a crosshatching of electron dense material on the inner surface of the membrane (arrows). E. In longitudinal section the distal and proximal sections of the central cylinder are visible although the transverse plate is missing (arrow). F. In cross section, although the microtubules had depolymerized, attachments can be seen between the central cylinder and the membrane. G and H: Virtual 6 nm sections of tomographic reconstructions of a TZ after isolation (G) or in situ (H). Y-shaped connectors can be seen between the outer doublet microtubules and the membrane in the TZs in situ (H, arrows). The Y-shape can sometimes be seen in the membrane attachments in the isolated TZs (G, arrows). See also Figs. S1 and S2 and Movie S1.
Figure 3
Figure 3
POC2 and VPS4 are localized at the TZ. Isolated flagella/basal body complexes (upper panels) stained for acetylated α-tubulin (green) show a pair of POC2 (left, red) and VPS4 (right, red) puncta at their base. Immunostaining of TZs in isolated cell walls (lower panels) confirms the presence of POC2 and VPS4 in the TZ.

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