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First published online 30 August 2006
doi: 10.1242/dev.02544

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Depletion of the co-chaperone CDC-37 reveals two modes of PAR-6 cortical association in C. elegans embryos

Department of Molecular Biology and Genetics, 101 Biotechnology Building, Cornell University, Ithaca, NY 14853, USA.

View larger version (84K): [in a new window]   Fig. 1. Depletion of CDC-37 causes polarity defects. Anterior is to the left for all embryos in all figures. (A-J) Timed sequences of differential interference contrast photomicrographs of a wild-type embryo (A-E) and a cdc-37(RNAi) embryo (F-J) dissected 20 hours after dsRNA injection. The arrows in B,G mark the pseudocleavage furrow. Note the central meeting of the two pronuclei (H). Note the nearly equal size of the two blastomeres (I) and the abnormal spindle orientation (bars, J). Scale bar: 10 µM.

View larger version (67K): [in a new window]   Fig. 2. MEX-5 protein and P-granule distributions are disrupted in cdc-37(RNAi) embryos. (A) GFP::MEX-5 in control embryos. (B) MEX-5::GFP in cdc-37(RNAi) embryos. (C) GFP::PGL-1 in control embryos. (D) GFP::PGL-1 in cdc-37(RNAi) embryos. All images are from live embryos. Scale bar: 10 µm.

View larger version (57K): [in a new window]   Fig. 3. PAR proteins are mislocalized in cdc-37(RNAi) embryos. (A-F) Confocal immunofluorescence images of late-prophase stage one-cell embryos showing distributions of PAR-3 (red) and PAR-2 (green) in wild-type (A-C) and cdc-37(RNAi) (D-F) embryos. (G-I) Wide-field immunofluorescence images of a cdc-37(RNAi) metaphase one-cell embryo showing the complete overlap of the PAR-2 (green) and PKC-3 (red) domains.

View larger version (122K): [in a new window]   Fig. 4. Depletion of CDC-37 affects the distribution of PAR-3 and PAR-6 differently. Each column shows multiple images from a wide-field microscopy time-lapse movie of a single embryo through the first cell cycle. Note that, in control embryos, cortical PAR-3::GFP (column 1) and GFP::PAR-6 (column 3) are co-localized and asymmetric, whereas the two proteins behave differently in cdc-37(RNAi) embryos (columns 2 and 4; see Results). The arrows point to the pericentriolar accumulation of GFP::PAR-6 in the cdc-37(RNAi) embryos. Scale bar: 10 µM.

View larger version (72K): [in a new window]   Fig. 5. Distributions of PAR-6 and PKC-3 after single and double depletions of CDC-37 and anterior PAR proteins. All embryos in the immunofluorescence images are at late prophase or metaphase of the first cell cycle. (A-D) PAR-6 distribution (A,B) and PKC-3 distribution (C,D) in par-3(it71) and par-3(it71); cdc-37(RNAi) treated embryos. (E,F) PAR-6 localization in the pkc-3(RNAi) or pkc-3(RNAi); cdc-37(RNAi) background. (G,H) PKC-3 localization in par-6(zu222) or par-6(zu222); cdc-37(RNAi) treated embryos. The cytoplasmic particles in A, B and E are P granules that are detected with some preparations of affinitypurified anti-PAR-6 antibody.

View larger version (80K): [in a new window]   Fig. 6. PAR-6 protein does not accumulate at the cortex in cdc-37(RNAi); cdc-42(RNAi) embryos. (A,C) GFP::PAR-6 embryo treated with cdc-42(RNAi). (A) Punctate GFP::PAR-6 becomes somewhat asymmetric early in the cell cycle. (C) At metaphase, GFP::PAR-6 becomes undetectable at the cortex. (B,D) GFP::PAR-6 embryos treated with cdc-37(RNAi); cdc-42(RNAi). GFP::PAR-6 does not become cortical at any stage in the cell cycle. Control uninjected or cdc-37(RNAi) embryos treated on the same day looked the same as embryos in Fig. 4, columns 3 and 4, respectively.

View larger version (59K): [in a new window]   Fig. 7. PAR-6 puncta that do not contain PAR-3 are rare in cdc-42(RNAi) embryos. (A-F) Immunofluorescence images of anterior cortical patches of control embryos (A-C) or cdc-42(RNAi) embryos (D-F), showing the distributions of PAR-3 (red) and GFP::PAR-6 (green) puncta. In A, examples of puncta positive for only anti-PAR-3 are marked with red asterisks. In B, examples of puncta positive for only anti-GFP are marked with green asterisks. Note the larger puncta and the much more extensive overlap of anti-PAR-3 and anti-GFP positive puncta in D-F. Embryos examined were at late prophase of the first mitotic cell cycle.

View larger version (104K): [in a new window]   Fig. 8. GFP::CDC-37 is cytoplasmic with accumulation in the nucleoplasm and central spindle (marked by the arrow) during mitosis. Images are taken from a time-lapse movie of an embryo expressing GFP::CDC-37.

View larger version (48K): [in a new window]   Fig. 9. PKC-3 and PAR-4 are affected by the depletion of CDC-37 by RNAi. (A,B) Western blots of protein extracts from embryos probed with either anti-PKC-3 antibody (A) or anti-PAR-4 antibody (B). Tubulin was used as a loading control. The upper arrow in B points to a phosphorylated form of PAR-4 that is also reduced in cdc-37(RNAi) embryos.

View larger version (16K): [in a new window]   Fig. 10. Two possible modes of cortical association for PAR-6.Mode 1: (gray circle and oval) one or more CDC-37 clients (indicated by `?') are required for PAR-6 to bind to a PAR-3-containing complex, perhaps through direct binding to PAR-3, in a CDC-42-independent manner. Mode 2: (open shapes) one or more CDC-37 clients act to prevent CDC-42-dependent binding of PAR-6 at the cortex. PAR-3 or a protein regulated by PAR-3 acts locally to inhibit the action of the client protein, allowing CDC-42-dependent binding of PAR-6 only in the anterior.