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. 2013;8(1):e53366.
doi: 10.1371/journal.pone.0053366. Epub 2013 Jan 24.

A role for the membrane in regulating Chlamydomonas flagellar length

William Dentler  1
Affiliations

A role for the membrane in regulating Chlamydomonas flagellar length

William Dentler. PLoS One. 2013.

Abstract

Flagellar assembly requires coordination between the assembly of axonemal proteins and the assembly of the flagellar membrane and membrane proteins. Fully grown steady-state Chlamydomonas flagella release flagellar vesicles from their tips and failure to resupply membrane should affect flagellar length. To study vesicle release, plasma and flagellar membrane surface proteins were vectorially pulse-labeled and flagella and vesicles were analyzed for biotinylated proteins. Based on the quantity of biotinylated proteins in purified vesicles, steady-state flagella appeared to shed a minimum of 16% of their surface membrane per hour, equivalent to a complete flagellar membrane being released every 6 hrs or less. Brefeldin-A destroyed Chlamydomonas Golgi, inhibited the secretory pathway, inhibited flagellar regeneration, and induced full-length flagella to disassemble within 6 hrs, consistent with flagellar disassembly being induced by a failure to resupply membrane. In contrast to membrane lipids, a pool of biotinylatable membrane proteins was identified that was sufficient to resupply flagella as they released vesicles for 6 hrs in the absence of protein synthesis and to support one and nearly two regenerations of flagella following amputation. These studies reveal the importance of the secretory pathway to assemble and maintain full-length flagella.

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Conflict of interest statement

Competing Interests: The author has declared that no competing interests exist.

Figures

Figure 1
Figure 1. BFA reversibly inhibited flagellar regeneration (A–F) and induced fully grown flagella to disassemble (G–N).
A–D: Flagellar lengths on WT cells regenerating with 0 (diamonds), 3.6 µM (triangles), 18 µM (squares), 36 µM (circles), or 54 µM BFA (open diamonds). A: Four hrs after deflagellation, a portion of cells was washed suspended without BFA (B). C–D: percentage of flagellated cells. E, F: Regeneration of deflagellated pf16 without (diamonds) or with (squares) 18 µM BFA. Flagella regrew to control lengths upon BFA removal (triangles). F: Cells regenerated in BFA for 4 hrs (E) were re-deflagellated and incubated without (diamonds) or with (squares) 18 µM BFA. G, H: Flagellar lengths (G) and percentage of flagellated cells (H) when WT cells were treated with 0–36 µM BFA. I: Flagellar lengths on WT cells treated with 0 µM (diamond), 3.6 µM (triangles), 18 µM (squares), 36 µM (dots), 54 µM (open triangles). BFA. J: Flagellar on cells washed free of BFA at 6 hr (I, box). K–N: To determine if BFA induced cells to deflagellate, individual pf16 cells were recorded at 1 min intervals without (K) or with 18 µM (L), 36 µM (M), or 54 µM BFA (N). Each curve represents the length of flagella on an individual cell. Videos of representative cells are shown in Supplementary movies S1, S2, S3, S4.
Figure 2
Figure 2. Analysis of control and biotinylated Chlamydomonas cells, purified flagellar vesicles shed into the medium, and the detergent-insoluble axonemes and detergent-solubilized membrane+matrix from purified flagella.
A. Composition of FV compared with axonemes and membrane+matrix. “+” and “−” refer to the relative quantity of biotinylated proteins in the membrane+matrix (MM) and the flagellar vesicles (FV). B. Identification of endogenously biotinylated proteins in cells but not flagella. Biotinylated (+B) and non-biotinylated (−B) cells were deflagellated. Black dots mark endogenously biotinylated bands. These were not found in flagellar fractions or in FV (compare B with FV in A). C. Biotinylated proteins from flagella and deflagellated cells were affinity-purified by avidin chromatography and separated by SDS-PAGE. Identically loaded blots were stained with AP-streptavidin (Biotin) or antibodies against FMG-1B. D. Flagella isolated from non-biotinylated cells (1st) contained no endogenously biotinylated bands. Deflagellated cells were biotinylated and allowed to regenerate flagella. Regenerated flagella contained the same biotinylated proteins seen when flagellated cells were biotinylated.
Figure 3
Figure 3. Flagellar vesicles continued to be shed and surface proteins replaced as cells were incubated with cycloheximide, to inhibit protein synthesis while maintaining full-length flagella (A, B).
Sodium pyrophosphate induced flagellar shortening and increased the release of flagellar vesicles (C, D). A: Biotinylated cells were incubated without (C) or with (Cx) 10 µg/ml cycloheximide for 6 hrs and then were pelleted, deflagellated, and flagella were extracted with NP-40 to produce axonemes and membrane+matrix. Shed flagellar vesicles (FV) were purified from the medium and cell bodies were sedimented after deflagellation. Identical volumes of axonemes, membrane+matrix, and FV were stained for total protein (S) or biotinylated protein (B). B: Aliquots of cells were fixed during the 6 hr incubation and flagellar lengths were measured on control cells (diamonds) or cycloheximide-treated cells (squares). Cycloheximide did not induce flagellar length changes. C: Cells with full-length flagella (diamonds) maintained full length while 20 mM sodium pyrophosphate induced flagella to shorten (squares). D: Equal volumes of biotinylated cells were incubated without (C) or with (ppi) pyrophosphate for 4 hrs as flagella on pyrophosphate-treated cells resorbed. Shed flagellar vesicles were purified, suspended in equal volumes of buffer, fractionated by SDS-PAGE, and stained for total protein (Coob, Silver) or for biotinylated proteins (Biotin). Densitometric analysis of individual bands, identical in MW to those analyzed in regenerating flagella (Fig. 4) is presented in Table 2.
Figure 4
Figure 4. A pool of flagellar surface proteins on the plasma membrane can supply regenerating flagella.
Cells were biotinylated and flagella were isolated from the freshly biotinylated cells (1st). Cells regenerated flagella, deflagellated (R1), and regenerated flagella a second time (R2). A: Flagellar lengths before the first deflagellation (1st), first (R1) and second (R2) regeneration. B–D: Membrane+Matrix stained with CB or blotted and stained with AP-streptavidin (Biotin). Density of selected bands stained with CB (1C–6C) or AP-streptavidin (6B–13B) presented as a percentage of the density of the bands in flagella isolated immediately after biotinylation (1st). Coomassie blue stained proteins in each fraction were nearly identical (B) but the quantity of biotinylated protein decreased with each regeneration (C), indicating that the pool of biotinylated protein was gradually depleted as flagella grew.
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References

    1. Baldari CT, Rosenbaum JL (2010) Intraflagellar transport: it's not just for cilia anymore. Curr Opinion Cell Biol 22: 75–78. - PMC - PubMed
    1. Emmer BT, Maric D, Engman DM (2010) Molecular mechanisms of protein and lipid targeting to ciliary membranes. J Cell Sci 123: 529–536. - PMC - PubMed
    1. Rohatgi R, Snell WJ (2010) The ciliary membrane. Curr Opin Cell Biol 22: 541–546. - PMC - PubMed
    1. Jekely G, Arendt G (2006) Evolution of intraflagellar transport from coated vesicles and autogenous origin of the eukaryotic cilium. Bioessays 28: 191–198. - PubMed
    1. Dentler WL (2009) Microtubule-membrane interactions in Chlamydomonas flagella. The Chlamydomonas Sourcebook, 2nd edition. G.B. Witman, ed. Academic Press, NY, 283–301.

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