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Files in this Data Supplement:
Fig. S1. Molecular cloning of tbc-2. (A) Cloning of tbc-2. The top bar indicates the genetic map of the tbc-2 region and the lower panels show the rescue of tbc-2(qx20). Non-transgenic and transgenic embryos at the 2-fold stage were scored for each construct. At least 15 animals (transgenic or non-transgenic) from each independent transgenic line were scored for all lines obtained, as indicated in parentheses. The tbc-2 gene structure is shown, with filled boxes representing the exons and the interspersed lines indicating the introns. The arrow pointing away from the filled boxes shows the direction of tbc-2 transcription. The mutation identified in the qx20 mutant is marked and the gray bar below tbc-2 transcript delineates the actual region of the tbc-2 gene removed by the deletion mutant tm2241, with the sizes of the deletion and insertion shown. (B) TBC-2 is expressed in the known engulfing cells. The expression of TBC-2::GFP driven by the tbc-2 promoter was examined in wild-type animals. TBC-2::GFP was seen in several known engulfing cell types, such as hypodermal cells (a,b), pharyngeal muscle cells (c,d) and intestinal cells (e,f). TBC-2::GFP was also found to cluster around the cell corpse as indicated by the arrows in g and h. (C) GDP-locked RAB-5 failed to associate with phagosomes. DIC and fluorescence images of a wild-type embryo expressing GFP::RAB-5(S33N), a GDP-locked RAB-5, are shown. GFP::RAB-5(S33N) failed to cluster around cell corpses (indicated by arrows) and displayed a diffused cytoplasmic pattern. Scale bars: 5 µm.
Fig. S2. C. elegans TBC-2 is homologous to TBC1D2B in other organisms. Sequence alignment of C. elegans TBC-2 (c.eTBC-2), mouse TBC1D2B (mTBC1D2B) and Xenopus TBC1D2B (x.tTBC1D2B) is shown. Identical residues are shaded in black, and similar residues are in gray. The three conserved domains (PH, SMC and TBC) are indicated and boxed. The deletion site of the tm2241 mutant and the mutation identified in the qx20 mutant are marked by blue arrowheads. The conserved Arginine residue that is critical for the GAP activity is indicated by the red arrowhead.
Movie 1. Phagosomal release of GFP::RAB-5 in a wild-type embryo. The phagosomal release of GFP::RAB-5 in a wild-type embryo is shown. The phagosome followed is indicated with an arrow. Frames were collected every 3 minutes and displayed every 1 second. Selected images are shown in Fig. 5A.
Movie 2. Phagosomal release of GFP::RAB-5 is disturbed in the tbc-2(qx20) mutant. The phagosomal release of GFP::RAB-5 in a tbc-2(qx20) embryo is shown. The phagosome followed is indicated with an arrow. Frames were collected every 3 minutes and displayed every 1 second. Selected images are shown in Fig. 5A.
Movie 3. PtdIns(3)P transiently accumulates on phagosomes in wild-type animals. The dynamic accumulation of PtdIns(3)P on phagosomes in a wild-type embryo, as indicated by YFP::2×FYVE, is shown. The phagosome followed is indicated with an arrow. Frames were collected every 3 minutes and displayed every 1 second. Selected images are shown in Fig. 5B.
Movie 4. PtdIns(3)P persists longer on phagosomes in the tbc-2(qx20) mutant. The dynamic accumulation of PtdIns(3)P on phagosomes in a tbc-2(qx20) embryo, as indicated by YFP::2×FYVE, is shown. Phagosomes followed are indicated with arrows. Frames were collected every 3 minutes and displayed every 1 second. Selected images are shown in Fig. 5B.
Movie 5. GTP-locked RAB-5 persists longer on phagosomes. The phagosomal release of GFP::RAB-5(Q78L) in a wild-type embryo is shown. Frames were collected every 3 minutes and displayed every 1 second. The phagosome followed is indicated with an arrow. Selected images are shown in Fig. 5A.
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