![Figure 7. The MIA complex affects I1 dynein stability in the axoneme. (A) I1 dynein is missing along the entire length of DMT1 in the mia mutants. Tomographic slices of axonemal average of individual outer DMTs from pWT (top row), mia2 (middle row), and mia1 (bottom row). The first two columns show the axonemal repeat of DMT1 in the proximal (left) and distal (middle) regions; the right column depicts the combined average of the axonemal repeats from DMTs 2–9 that do not show obvious structural differences between the proximal and distal regions (for individual DMT averages, see Fig. S3). In wild type, I1 dynein is missing from the proximal region of DMT1 (white outlines) but is present in the distal region (red outlines). In contrast, in both mia mutants, I1 dynein is missing along the entire length of DMT1 (white outlines). The pWT data were refined from data originally reported by Heuser et al. (2009) and Lin et al. (2012b). Other structures labeled are single-headed inner dynein arms (IDAs 2–6 and X), the N-DRC, and the inner SUB5-6 bridge. Bars, 25 nm. (B) Representative ion-exchange chromatography (Mono Q; GE Healthcare) elution profiles of axonemal dyneins show that I1 dynein levels (black arrows) appear relatively normal in mia1-2 extracts compared with that of wild-type extracts (left graph), consistent with the immunoblotting results using I1 dynein subunit antibodies (Fig. 4 B). In contrast, I1 dynein is drastically reduced in the mia1-2 × oda6 double mutant (red arrow) compared with the oda1 single mutant (right graph), suggesting that I1 dynein becomes unstable without both ODAs and the MIA complex. Typical Mono Q elution patterns are shown (A280 protein estimate in arbitrary units is indicated to the right of each scan [y axis]; elution time in minutes is shown on the x axis), and at least two experiments were performed for each strain. Elution of dynein complexes began at ∼12 min. (C) Immunoblot analysis of isolated axonemes using the anti-DHC1 (I1 dynein HC-α) and IC97 antibodies confirms the reduction of I1 dynein in mia × oda double mutants. I1 dynein appears normal to slightly reduced in the single mia mutants relative to wild type (wt).](https://cdn.rupress.org/rup/content_public/journal/jcb/201/2/10.1083_jcb.201211048/5/jcb_201211048_fig7.jpeg?Expires=1771560126&Signature=DDS0mjMcn-i0lnbJyjKWz82qbqZ2SLCtyRsSwCUZvludskhXJt60L2zSx84pD5Vkk4Q3XWGVVQaw7ZuXh7vOYYUmDbrlwSJrruecmzp6PgM1d0gchLsn-8b~5vigzQbQCZwncJoB6kq-vtPyfoH5iMTW17~yClVTGIGlhZ-yxTbebNsoUGaLV6hTVtqkwyDnGncPAADTz5i7brqEE7RF1F9t3hxkOg91rCHyw3QnzCs0-QUsOptN7sIojxZL5q~3fcFbWEnVN0tGYw7j4Ud0cW1meOnuRmIiqe40T6KezxlRyPKRnOYLTbImmwHWJrGyVcEHknN57sGW9WTJsJ8CmQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The MIA complex affects I1 dynein stability in the axoneme. (A) I1 dynein is missing along the entire length of DMT1 in the mia mutants. Tomographic slices of axonemal average of individual outer DMTs from pWT (top row), mia2 (middle row), and mia1 (bottom row). The first two columns show the axonemal repeat of DMT1 in the proximal (left) and distal (middle) regions; the right column depicts the combined average of the axonemal repeats from DMTs 2–9 that do not show obvious structural differences between the proximal and distal regions (for individual DMT averages, see Fig. S3). In wild type, I1 dynein is missing from the proximal region of DMT1 (white outlines) but is present in the distal region (red outlines). In contrast, in both mia mutants, I1 dynein is missing along the entire length of DMT1 (white outlines). The pWT data were refined from data originally reported by Heuser et al. (2009) and Lin et al. (2012b). Other structures labeled are single-headed inner dynein arms (IDAs 2–6 and X), the N-DRC, and the inner SUB5-6 bridge. Bars, 25 nm. (B) Representative ion-exchange chromatography (Mono Q; GE Healthcare) elution profiles of axonemal dyneins show that I1 dynein levels (black arrows) appear relatively normal in mia1-2 extracts compared with that of wild-type extracts (left graph), consistent with the immunoblotting results using I1 dynein subunit antibodies (Fig. 4 B). In contrast, I1 dynein is drastically reduced in the mia1-2 × oda6 double mutant (red arrow) compared with the oda1 single mutant (right graph), suggesting that I1 dynein becomes unstable without both ODAs and the MIA complex. Typical Mono Q elution patterns are shown (A280 protein estimate in arbitrary units is indicated to the right of each scan [y axis]; elution time in minutes is shown on the x axis), and at least two experiments were performed for each strain. Elution of dynein complexes began at ∼12 min. (C) Immunoblot analysis of isolated axonemes using the anti-DHC1 (I1 dynein HC-α) and IC97 antibodies confirms the reduction of I1 dynein in mia × oda double mutants. I1 dynein appears normal to slightly reduced in the single mia mutants relative to wild type (wt).
