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First published online 3 August 2006
doi: 10.1242/dev.02514

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Milton controls the early acquisition of mitochondria by Drosophila oocytes

Howard Hughes Medical Institute Research Laboratories, Department of Embryology, Carnegie Institution of Washington, 3520 San Martin Drive, Baltimore, MD 21218, USA

View larger version (122K): [in a new window]   Fig. 1. milt mutant oocytes do not form normal Balbiani bodies. (A-D) Prefollicular cysts: (A) schematic; mitochondria (green) in wild-type cysts (broken outlines, B) and milt92 null mutant cysts (broken outlines, C) associate, as expected, with the fusome (magenta). By contrast, mitochondria in class II miltEY01559 cysts (D, broken outlines) accumulate prematurely at the middle of the fusome (arrows). (E-H) Forming follicles: (E) schematic; mitochondria in milt92 null mutants remain at the anterior of the oocyte (G, small broken outline) and fail to form a normal Balbiani body (yellow broken outline) as in wild type (F). In miltEY01559 mutants, excess mitochondria move into the oocyte (H) to form an abnormally large Balbiani body. (I-L) Stage 5 follicles: (I) schematic; in wild type (J), mitochondria are sparse and evenly distributed. Mitochondria remain tightly packed at the anterior of milt92 oocytes (K, arrow). In miltEY01559, mitochondria fill the cytoplasm of the oocyte (L). (M-P) Stage 9 follicles: (M) schematic; the number of mitochondria in wild type (N), milt92 (O) and miltEY01559 (P) is similar. However, in miltEY01559, mitochondria cluster at the anterior of the oocyte (P, arrow), where microtubule minus-ends focus (M). (B-D,F-H,J-L,N-P) ATP synthase, green; (B-D,F,H) 1B1, magenta; (J-L,N-P) phosphotyrosine, magenta; (G) ß-galactosidase marks wild type cells (blue). Scale bars: 10 µm in A-L; 20 µm in M-P.

View larger version (63K): [in a new window]   Fig. 2. Balbiani body-associated components are normal in milt mutants. (A-C) The fusome in milt92 null (B) and miltEY01559 class II mutants (C) is indistinguishable from wild type (A). (D-F) Golgi elements (green) concentrate and move into the oocyte (broken outline) normally; compare wild type (D, arrow) with milt92 (E, arrow) and miltEY01559 (F, arrow). (G-I) Cup protein (green) accumulates preferentially in the oocyte at the time of follicle formation in wild type (G, arrows) as well as in milt92 (H, arrows) and miltEY01559 (I, arrows). (A-C) 1B1; (D-F) mannosidase II, green; (G-I) Cup, green; (D-I) 1B1, magenta. Scale bar: 10 µm.

View larger version (29K): [in a new window]   Fig. 3. milt mutants differentially express milt isoforms. (A) milt encodes two known transcripts, RA and RB. milt92 contains a 2 bp deletion affecting both isoforms. The three class II alleles, k06704, k14514 and EY01559, are all inserted within 100 bp of each other in the first intron of RA. Arrowheads indicate RT-PCR isoform-specific primer sites. (B) Two protein isoforms, Milt PA and PB, differ only at their N termini. Both contain the putative Kinesin-binding site, as uncovered for the mammalian homolog GRIF-1 using yeast two-hybrid analysis. However, some of their predicted HAP-1 domains differ. (C) RT-PCR shows that milt RA and RB are expressed equally in wild type. In miltEY01559, RA is absent and RB is increased. In miltEY01559/k06704, the ratio of RB to RA is still greatly elevated. (D) A Western blot shows that Milt is overexpressed in miltEY01559 and miltEY01559/CyO ovaries. The Milton antibody used recognizes both PA and PB. (E) Model of Milton-mediated bidirectional mitochondrial movement along microtubules. Milt-PA (magenta oval) and possibly Milt-PB (magenta square) link mitochondria to microtubules via the plus-end directed motor Kinesin. Additionally, Milt-PB positively influences the minus-end directed motor Dynein (large arrow).

View larger version (105K): [in a new window]   Fig. 4. Khc mutants resemble milt class II mutants. (A-C) Prefollicular cysts: mutant Khc27 clonal cysts (B, broken outlines) show premature mitochondrial accumulation (green, arrow) at the middle of the fusome (magenta) in contrast to wild-type (A) and Dhc64C6-6/6-12 (C) cysts. (D-F) Forming follicles: the Balbiani body (broken yellow line) is greatly enlarged in Khc27 clones (E) and reduced in Dhc64C6-6/6-12 mutants (F) compared with wild type (D). The Dhc64C6-6/6-12 oocyte (F inset, barbed arrow) can be distinguished from the nurse cells by its selective accumulation of Cup protein (blue). (G-J) Dynein heavy chain (green) localization is indistinguishable between wild type (G,H) and miltEY01559 (I,J), both during follicle formation (G,I) and in older follicles (H,J). By contrast, Dhc accumulates in intense puncta in the posterior region of Khc27 mutant oocytes (arrows, K,L). A wild-type oocyte shows normal diffuse Dhc localization (L, barbed arrow). (A-F) ATP synthase, green; (G-L) Dynein heavy chain, green; (A-J) 1B1, magenta; (B,E,K,L) ß-galactosidase marks wild-type cells (blue). Scale bars: 10 µm in A-F; 10 µm in G,I,K; 20 µm in H,J,L.

View larger version (98K): [in a new window]   Fig. 5. Mitochondrial mislocalization in milt class II mutants reveals microtubule polarity. (A) Known apical polarity of microtubules in follicular epithelial cells. (B,C) Mitochondria (green) are evenly distributed in wild type (B, arrow) and milt92 mutant (C, broken outline) follicle cells. (D,E) Mitochondria cluster at the apical side of miltk06704 mutant follicle cells (D, arrow), as predicted (A). Khc27 mutant follicle cells also accumulate mitochondria at their apical surface (E, arrow) until stage 7, when mitochondrial distribution becomes normal (E, asterisk). (F,G) Mitochondria predominantly localize at the anterior end of wild-type (F, arrows) and milt92 mutant (G, arrow) GSCs (broken outlines) near the spectrosome (magenta). By contrast, in miltk06704 mutant clones (H, arrow) and Khc27 mutant clones (I, arrow) mitochondria aggregate at the posterior of the GSC (broken outlines). This suggests the minus ends of the microtubules are directed towards the posterior of the stem cell, away from the spectrosome (J). (K-M) Mitochondria in dividing cysts are normally spread evenly throughout the cytoplasm (L, broken outline). However, they clump away from the fusome (magenta) in Khc2 7 clones (I, barbed arrow; M, arrows), indicating the location of microtubule minus ends (K). (B-I,L,M) ATP synthase, green; (B,F,G,I,L,M) 1B1, magenta; (C-E,H,I,M) ß-galactosidase marks wild-type cells (blue). Scale bars: 10 µm in B-E; 10 µm in F-M.

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