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First published online 22 February 2005
doi: 10.1242/jcs.01644

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Sec6 mutations and the Drosophila exocyst complex

Mala Murthy^1,*, Ravi Ranjan^1,, Natalie Denef², Misao E. L. Higashi¹, Trudi Schupbach² and Thomas L. Schwarz^1,

¹ Division of Neuroscience, Children's Hospital, Harvard Medical School, Boston, MA 02115, USA
² Howard Hughes Medical Institute, Molecular Biology Department, Princeton University, Princeton, NJ 08544, USA

View larger version (8K): [in a new window]   Fig. 1. Molecular characterization of sec6 excisions. The genomic region 55E1-3 is shown, with an enlargement of the immediate vicinity of sec6. Df(2R)PC4 was found to remove the sec6 locus by in situ hybridization to polytene chromosomes (data not shown). The P-element EP2021 (small triangle), located just 3' to sec6 and within the 5' untranslated region of the adjacent gene Eip55E was excised to give rise to three deletions, Ex15, Ex228 and Ex212 (heavy bars). Primers 1-6 were used to amplify DNA from these lines and the extent of the deleted regions was determined by DNA sequencing. Two of the three excisions, Ex15 and Ex228, remove coding sequence from sec6 and 21 base pairs of non-coding sequence from Eip55E. Ex212 removes a 34 kb genomic fragment extending from predicted gene CG30332 to CG30122, as shown.

View larger version (90K): [in a new window]   Fig. 2. Sec5 protein is mislocalized in sec6 excision alleles. (A) Ventral nerve cords from yw larvae at 24 hours and 72 hours AEL, and sec6Ex15/+, sec6Ex212/+, sec6Ex15, sec6Ex212 and sec6Ex15/Ex212 at 72 hours AEL. All nerve cords were stained for Sec5 with monoclonal antibody 22A2. All animals were imaged at the same image settings for comparison. (B) Close ups of the cell body and neuropil regions of the nerve cords from A. (C) Quantitation of mean pixel intensity for a fixed area of the neuropil region of nerve cords from yw 24 hours (n=6), yw 72 hours (n=4), sec6Ex15/+ 72 hours (n=4), sec6Ex212/+ 72 hours (n=5), sec6Ex15 72 hours (n=8), sec6Ex212 72 hours (n=5) and sec6Ex15/Ex212 72 hours (n=8). Statistical significance of the reduction of Sec5 in the mutant neuropil compared with the wild type is: P=0.008 (sec6Ex15 72 hours vs yw 24 hours); P=0.04 (sec6Ex212 72 hours vs yw 24 hours); P=0.07 (sec6Ex15/Ex212 72 hours vs yw 24 hours). (D) Ventral nerve cords from yw 24 hours AEL and sec6Ex15/sec6Ex212 72 hours AEL were stained for cysteine string protein (CSP) and FasII. All scale bars are 20 µm.

View larger version (53K): [in a new window]   Fig. 3. Vesicle trafficking defects in sec5E10. (A) A lateral bd sensory neuron in a wild-type larva that had been fed RU486 at 48 hours AEL and dissected at 59 hours AEL. Anti-HRP immunostaining (gray) was used to find bd sensory neurons in each animal. (B) A lateral bd neuron as in A but from a sec5E10 mutant fed RU486 at 72 hours AEL and dissected at 83 hours AEL. sec5E10 larvae show less cell surface CD8 than the control. (C,D) Additional wild-type and sec5E10 neurons as in A and B. (E) From quantitative fluorescent microscopy of the transport assay, surface-expressed CD8 immunofluorescence, total GFP fluorescence and cell surface area (measured by HRP immunoreactivity) were expressed as fluorescent units after background subtraction. In the sensory neuron soma [n=7 for wild-type larvae fed RU486 (white) and uninduced (blue); n=6 for sec5E10 larvae fed RU486 (gray) and uninduced (black)], there are comparable levels of anti-HRP labeling at the cell surface. Total GFP fluorescence is reduced in the RU486-fed mutants (gray) compared with RU486-fed controls (white), but there is a significant induction of the GFP signal, which is a measure of transgene expression, in the RU486-fed (gray) compared with uninduced (black) mutants. The amount of mCD8 at the cell surface is reduced in the RU486-fed mutants compared to RU486-fed controls, and a 50% reduction is observed when surface mCD8 is normalized to GFP in order to control for the level of transgene expression (P=0.004). (F) To measure the amount of transgene induction by feeding RU486, the uninduced averages (blue and black bars in E) were subtracted from the RU486-induced averages (white and gray bars in E). To control for differences in cell size, induced GFP and induced surface CD8 were then normalized to the anti-HRP signal. The amount of induced mCD8 trafficked to the cell surface was also normalized to the induced GFP signal, revealing the disruption of transport of the newly synthesized protein to the surface.

View larger version (44K): [in a new window]   Fig. 4. Vesicle trafficking defects in sec6Ex15 larvae at 72 hours AEL. (A) From quantitative fluorescent microscopy of the transport assay in neuronal cell bodies, surface-expressed CD8 immunofluorescence, total GFP fluorescence and cell surface area (measured by HRP immunoreactivity) were expressed as fluorescent units after background subtraction. n=10 for wild-type larvae fed RU486 (white); n=8 for wild-type larvae uninduced (blue); n=13 for sec6Ex15 larvae fed RU486 (grey); and n=9 for uninduced sec6Ex15 larvae (black). (B) Representative lateral bd sensory neurons as used in the assay in A. In particular, a wild-type larva that had been fed RU486 at 24 hours AEL and dissected and stained in the absence of Triton X-100 at 36 hours AEL is compared with a sec6Ex15 mutant fed RU486 at 72 hours AEL similarly stained at 84 hours AEL. Anti-HRP immunostaining (gray) was used to find bd sensory neurons in each animal. The sec6Ex15 larvae show less cell surface CD8 than the control, but also showa large decrease in GFP fluorescence. (C) When normalized to the anti-HRP signal, there was a significant induction of the mCD8-GFP transgene in the sec6Ex15 mutant after feeding RU486 (P=0.01), but the amount of mCD8 at the soma surface was not significantly increased. (D) To measure the amount of transgene induction in the cell body by feeding RU486, the uninduced averages (blue and black bars in A) were subtracted from the RU486 averages (white and gray bars in A). To control for differences in cell size, GFP and surface CD8 were also normalized to the anti-HRP signal, as in C. The amount of induced mCD8 trafficked to the cell surface, when normalized to the corrected GFP signal, is decreased in the mutant compared with control.

View larger version (85K): [in a new window]   Fig. 5. The sec5E10 and sec6Ex15 mutants have similar phenotypes in the ovary. (A-G) Egg chambers labeled with phalloidin (red), Hoechst 33342 (blue) and GFP (green in G). Unlike in the wild type (A), egg chambers from sec5E10 germ-line clones (B,C) lack membranes (marked with phalloidin) between nuclei and have ring canals clumped together (arrows). Egg chambers from sec6Ex15 germ-line clones (D-G) exit the germarium and progress through stage 3 (D,E), initially resembling the control. However, after stage 3 (F,G), ring canals clump together (arrows) and phalloidin-marked membranes between nuclei are absent. FRT42D sec6Ex15 homozygous germ lines were generated by mitotic recombination in combination with either FRT42B ovoD (D-F) or FRT42D Ubi-GFP and imaging egg chambers that lacked GFP in the germ line (G). (H-L) Egg chambers labeled with Texas Red-phalloidin (red), Hoechst 33342 (blue) and anti-Gurken antibody (green). Gurken accumulates only in the oocyte, which resides at the posterior end of the egg chamber throughout oogenesis, contacting the posterior follicle cells in the wild type (H). In egg chambers from sec5E10 (I,J) and sec6Ex15 (K,L) germ-line clones, the oocyte is often mispositioned anteriorly (arrows). Anterior (A) and posterior (P) ends of the chamber are marked. All scale bars are 20 µm.

View larger version (108K): [in a new window]   Fig. 6. Sec5 protein is mislocalized in sec6Ex15 germ-line clones. (A) Control egg chamber, stages 3 and 4, labeled with anti-Sec5 antibody (green) and Hoechst 33342 (blue). Sec5 concentrates on membranes and is enriched at the boundary between the oocyte and posterior follicle cells (arrow). (B) In sec6Ex15 germ lines, labeled as in A, Sec5 protein is found in puncta (arrow), many of which are clustered towards the center of the egg chamber. (C,D) Sec5 localization (green) is compared with the submembranous actin cytoskeleton (gray) in sec6Ex15 germ lines. (E1) GFP– sec6Ex15 germ lines (generated as in Fig. 5G) stained for Sec5 (red) and Hoechst 33342 (blue). GFP is shown in green. (E2) GFP– sec6Ex15 germ lines stained for Sec5 (green) and actin (red). (B-E) Sec5 accumulates in intracellular puncta (arrows) and any germ-line membranes separating nuclei have a patchy, discontinuous distribution of Sec5. The Sec5 puncta often accumulate near the clump of ring canals. All scale bars are 20 µm.

View larger version (111K): [in a new window]   Fig. 7. HA-Sec8 localization in late-stage egg chambers. HA-Sec8 was expressed exclusively in the germ line under the control of nanos-Gal4. (A,B) Egg chambers from sec5E13/+ heterozygous controls at stage 7 are labeled with anti-Sec5 antibody (A) or anti-HA antibody and Texas-Red/phalloidin (B). Sec5 and HA-Sec8 both concentrate along the oocyte (*) membrane. Probably owing to overexpression, HA-Sec8 often accumulates in the cytoplasm of some nurse cells. (C-E) Egg chambers from sec5E13/+ heterozygous controls at stage 10 are labeled with Hoechst 33342 and anti-Sec5 antibody (C) or anti-HA antibody and Texas Red-phalloidin (D,E). Sec5 and HA-Sec8 concentrate at anterior corners (arrows) of the oocyte membrane. (F-H) Egg chambers from sec5E13 homozygous germ lines are labeled with Hoechst 33342 and Texas Red-phalloidin (F-H) and with anti-HA antibody (G,H). HA-Sec8 no longer concentrates at the oocyte (*) membrane but rather fills the oocyte cytosol when Sec5 function is compromised by the mutation. In addition, the sec5E13 phenotype (F) is enhanced by the presence of the HA-sec8 transgene (G,H), stunting the oocyte, disrupting nurse cell membranes and causing ring canals to clump together. (I,J) GFP– egg chambers from FRT42D sec6Ex15 homozygous germ lines, generated by mitotic recombination with FRT42D Ubi-GFP. GFP is shown in green (I1,J1). HA-Sec8 (red in I1,J1 and green in I2,J2) accumulates in puncta within the mutant egg chambers and in proximity to actin-rich ring canals (red in I2,J2). All scale bars are 20 µm.

View larger version (21K): [in a new window]   Fig. 8. Organization of the exocyst complex. Interactions are depicted for members of the exocyst complex with each other and with an unknown receptor on the plasma membrane (left), based on a slight modification of the model derived from studies in yeast (Guo et al., 1999) and in accordance with the data from Figs 6, 7. In sec5 mutants, Sec8 is no longer associated with the membrane and other subunits might also become cytosolic (center). In sec6 mutants, however, both Sec5 and Sec8 can remain membrane associated (right).