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First published online 7 April 2009
doi: 10.1242/jcs.045096

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Identification of dyneins that localize exclusively to the proximal portion of Chlamydomonas flagella

¹ Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, 113-0033, Japan
² Department of Biological Science, Graduate School of Science, Kyoto University, Kyoto, 606-8502, Japan

View larger version (38K): [in this window] [in a new window]   Fig. 1. Fractionation of inner-arm dyneins. (A) Elution pattern of inner-arm dyneins. Inner-arm dyneins were extracted with high salt from outer-armless axonemes of oda1 cells, and fractionated by ion-exchange chromatography on a MonoQ column. The elution positions of dynein a-g are indicated by arrowheads. (B) SDS-PAGE of peak fractions showing the DHC bands. Only the high molecular mass region of the gel is shown. Peak fractions were subjected to SDS-PAGE with a 3-5% acrylamide gradient and a 3-8 M urea gradient. For identification of the DHCs in each dynein species (arrowheads), bands were cut out from the gel and analyzed by mass-spectroscopy. CE, crude dynein extract.

View larger version (34K): [in this window] [in a new window]   Fig. 2. Detection of novel DHC bands in SDS-PAGE gels. (A) SDS-PAGE of mutant axonemes. SDS-PAGE was carried out with large amounts of oda1, oda1ida4 and oda1ida5 axonemes. Only the high molecular mass region of the gel, corresponding to the sizes of the DHCs, is shown. In the oda1 lane, two faint bands (filled and open arrows) were detected above the f band of dynein f/I1 (arrowhead). In oda1ida4 lane, the upper band of this pair is absent. In oda1ida5 lane, the upper band is absent and the lower band is reduced in intensity. Mass-spectroscopic analyses showed that the upper and lower bands are encoded by DHC11 and DHC4, respectively. The strong intensity bands (bracket) above the DHC bands are from contaminating membrane proteins. (B) Quantification of relative DHC4 and DHC11 contents in axonemes. (Left) SDS-PAGE pattern of oda1 axonemes stained with Sypro Ruby, a fluorescent dye with a wide range of staining linearity. DHC11 and DHC4 band intensities are weaker than the DHC1 band intensity (f DHC of dynein f/I1). (Right) Relative band intensities of DHC4, DHC11 and DHC1 quantified from the Sypro-Ruby-stained gel. The DHC11 and DHC4 band intensities are 11.7±4.6% and 9.4±4.8%, respectively, of the DHC1 band intensity (n=5).

View larger version (66K): [in this window] [in a new window]   Fig. 3. Immunoblot analysis using specific DHC antibodies. (A) Analysis of oda1 axonemes before and after photocleavage of DHCs. SDS-PAGE was carried out on 4% acrylamide and 6 M urea gel. The gel was stained with silver (far left two lanes), or analyzed by immunoblot using DHC3, DHC4, and DHC11 antibodies. UV+ and - denote axoneme samples with or without UV irradiation in the presence of ATP and vanadate, a condition that induces photocleavage of DHCs. With each antibody, a band in the DHC region (filled arrowheads) was detected in the sample without UV irradiation. After UV irradiation, the band disappeared or weakened, and a new, 150-200 kDa band (open arrowheads) was detected. The band appearing after UV irradiation is most probably the N-terminal DHC fragment produced by photocleavage. In DHC3 and DHC11, weak bands migrating below the DHC bands (filled circles) also appeared to undergo photocleavage. These weak bands might be DHC degradation products or splice variants. Note that the DHC3 band appears at a significantly higher position than DHC4 and DHC11 bands. (B) Immunoblot analyses of oda1, oda1ida4 and oda1ida5 axonemes using DHC3, DHC4, DHC11 and DHC5 antibodies. DHC5 antibody was used as a positive control. Only the high molecular mass region of the blot containing the area around DHC bands is shown. DHC3 and DHC4 antibodies detected their respective bands in all three axoneme samples, but the band intensities were significantly reduced in oda1ida5 (filled and open arrowheads). By contrast, the DHC11 antibody detected a band only in oda1 axomemes. DHC5 antibody detected a band in all three samples.

View larger version (48K): [in this window] [in a new window]   Fig. 4. Immunolocalization of DHC11, DHC9 and DHC5 in the axoneme. (A) (Upper panels) DHC11 localization. Nucleoflagellar apparatuses (see Materials and Methods) were stained with (left) DHC11 antibody and (middle) an -tubulin antibody. The far right panel shows the merged images. The DHC11 antibody preferentially stained a portion near the proximal end of each axoneme, and the -tubulin antibody uniformly stained the entire length of the axoneme. (Middle and lower panels) DHC9 and DHC5 localization. DHC9 antibody showed staining of axonemes along their entire length. This antibody also stains the nucleus. DHC5 antibody also stained axonemes along their entire length; but in 60% of axonemes, DHC5 antibody signal was weaker in a short region near the proximal end of the axoneme (white arrowheads) than in the rest. In the remaining 40% of axonemes, the axonemes appeared to be uniformly stained along their entire length. Scale bar: 10 µm. (B) Examples of DHC distribution. (Upper and middle panels) Higher magnification immunofluorescence images with DHC and -tubulin antibodies, respectively. (Lower panels) DHC (green) and -tubulin (magenta) signal distributions along the length of the axonemes. The regions analyzed are indicated by double arrows in upper panels. Strong DHC11 signals were present in the proximal 2 µm region. The signal in the more distal region was 10% that of the proximal region. By contrast, DHC9 signals were uniformly intense along the entire length of the flagellum. DHC5 signals appear to be present along the entire flagellar length; however, the signal intensity was weaker in the proximal 2 µm region of the axoneme than in the distal region. The signal intensity in the proximal region was 50% that of the distal region. Because of experimental limitations such as non-specific binding of antibodies, we cannot conclude whether DHC11 and DHC5 are completely absent from the distal and proximal regions, respectively, of the axoneme. However, the variability of DHC5 distribution as mentioned above appears to suggest that the change in localization of these DHCs does not occur in an all-or-none manner. Scale bar: 10 µm.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Change in the relative contents of minor and major DHCs during flagellar growth. (A) Flagellar regeneration in oda1 after pH-shock deflagellation. The pH of the medium was neutralized immediately after deflagellation, and the cells were allowed to regenerate flagella. Flagella grew to the original length by 120 minutes after amputation (not shown). (B) Immunoblot analyses of equal amounts of axonemes (10 µg) prepared at the indicated time points after flagellar amputation. Only the high molecular mass region of the blot, around DHC bands, is shown. The immunoblot shows that DHC3, DHC4 and DHC11 bands are strongest 30 minutes after flagellar amputation (filled arrowheads). By contrast, the DHC5 band (the DHC of dynein b) is very weak at 30 minutes (open arrowhead). The DHC9 band intensity (the DHC of dynein c) was constant throughout the entire period of flagellar regeneration. (C) Immunoblot analysis of changes in relative DHC content during flagellar growth.

View larger version (36K): [in this window] [in a new window]   Fig. 6. Localization of DHC11, DHC9 and DHC5 in axonemes of various lengths. (A) Immunofluorescence localization of DHC11, DHC9 (DHC of dynein c), and DHC5 (DHC of dynein b), in detached flagella of various lengths. Images of flagella stained with DHC or -tubulin antibodies and merged images (right panels) are shown. Short flagella were obtained from cells that had grown flagella for 30 minutes or 60 minutes following amputation, and mixed with normal-length flagella obtained from untreated cells. DHC11 signals were detected in a short region at one end of each flagellum, and DHC9 signals were detected along the entire length of each flagellum. DHC5 staining was detected along the entire length of flagella with lengths >3 µm, frequently with a region of weaker signal at one end (arrows). In contrast to DHC9 and DHC11, only negligible DHC5 signals were present in <3 µm length flagella (arrowheads). In addition, a flagellum 6 µm long displayed weaker DHC signals than those observed in fully grown flagella (double arrowhead). Scale bar: 10 µm. (B) (Upper panels) Magnified images of DHCs and -tubulin localization in short and long axonemes. Scale bar: 5 µm. (Lower panels) DHC11, DHC9, and DHC5 signal distribution along the lengths of short and long axonemes (red and blue, respectively) assessed by densitometry. Note that an extremely low DHC5 signal is detected in the short axoneme. (C) Distinct distribution of the three dynein along the axoneme length. Diagram of the localization of DHC5 (DHC of dynein b, green), DHC9 (DHC of dynein c, blue), and DHC11 (red).

View larger version (22K): [in this window] [in a new window]   Fig. 7. Phylogenetic tree of Chlamydomonas dynein heavy chains. A phylogenetic tree was constructed for cytoplasmic and flagellar DHCs in Chlamydomonas using full-length sequences. For dyneins with cDNA sequences not yet determined, the predicted sequences from the Chlamydomonas genome database were used (see Materials and Methods). The single-headed DHC type can be classified into three subgroups, IAD3, IAD4, and IAD5 types, as reported previously (Morris et al., 2006; Wickstead and Gull, 2007). Minor DHCs identified in this study are indicated by arrowheads. The protein ID 206178, which was putatively assigned to conventional cytoplasmic dynein (Porter et al., 1999), might belong to the axonemal type (Wickstead and Gull, 2007).

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