SRC64 Regulates the Localization of a Tec-Family Kinase Required for Drosophila Ring Canal Growth  Douglas J Guarnieri, G.Steven Dodson, Michael A Simon  Molecular Cell  Volume 1, Issue 6, Pages 831-840 (May 1998) DOI: 10.1016/S1097-2765(00)80082-9

Figure 1 Screen for Dominant Enhancers of Src64 Mutagenized autosomes were followed in this scheme using the P[w+, F]4–2 and ebony phenotypes. These mutagenized chromosomes are indicated by an asterisk. Five females heterozygous for each mutagenized autosome and homozygous for Src64Δ17 were allowed to mate with their siblings. Female fertility was evaluated by counting the number of progeny, and candidate enhancers were chosen from vials yielding few progeny. Molecular Cell 1998 1, 831-840DOI: (10.1016/S1097-2765(00)80082-9)

Figure 2 E(Src64)2Ae482 Enhances the Src64Δ17 Small Ring Canal Phenotype Early stage 10A egg chambers were immunostained for HTS–RC. The images are composed of a series of optical sections that were merged. Anterior is to the left. (A) Wild-type egg chamber; (B) Src64Δ17 egg chamber; (C) E(Src64)2Ae482/+; Src64Δ17 egg chamber. Scale bar, 10 μm. Molecular Cell 1998 1, 831-840DOI: (10.1016/S1097-2765(00)80082-9)

Figure 3 The Outer Diameter of E(Src64)2Ae482 Ring Canals Is Reduced throughout Oogenesis (A) Ring canal size distribution of wild-type, Src64Δ17, and E(Src64)2Ae482/+; Src64Δ17 stage 5 egg chambers. The ring canal outer diameter of wild-type is 2.5 ± 0.12 μm, Src64Δ17 is 2.2 ± 0.2 μm, and E(Src64)2Ae482/+; Src64Δ17 is 2.0 ± 0.17 μm. (B) Ring canal size distribution of wild-type, Src64Δ17, and E(Src64)2Ae482/+; Src64Δ17 early stage 10A egg chambers. The ring canal outer diameter of wild-type is 7.5 ± 0.58 μm, Src64Δ17 is 5.6 ± 0.51 μm, and E(Src64)2Ae482/+; Src64Δ17 is 4.2 ± 0.55 μm. The sizes reported are the average ± the standard deviation of the mean. The sizes determined for each genotype are significantly different from each other (p value is less than 0.05 using a Student's t test) at both stages. Molecular Cell 1998 1, 831-840DOI: (10.1016/S1097-2765(00)80082-9)

Figure 4 E(Src64)2Ae482 and l(2)k05610 Contain Mutations in Tec29 The genomic interval containing the Tec29 gene is depicted. R indicates an EcoR1 site and a double backslash represents 5 kb. The heavy black bar represents genomic DNA sequenced in this study (four new introns were identified), and the rest of the map is derived from previously published work (Gregory et al. 1987). The Tec29 transcription unit is shown below the genomic map with the protein coding regions in heavy black bars, and the untranslated regions are indicated by thin black bars. The location of the first exon has not been determined precisely but lies 7–12 kb from the second exon. The l(2)k05610 P element is inserted between the first and second exons of the Tec29 gene and is indicated by an open triangle. The hatched box represents the approximate location of the insertion. We sequenced the Tec29 gene from the E(Src64)2Ae482 chromosome and discovered a single nucleotide transition that changes a tryptophan to a stop codon in the catalytic domain. Molecular Cell 1998 1, 831-840DOI: (10.1016/S1097-2765(00)80082-9)

Figure 5 Tec29 Mutant Egg Chambers Have Smaller Ring Canals and Significantly Reduced Ring Canal Phosphotyrosine Content These early stage 10A images were derived from serial optical sections that were then merged. (A) Egg chamber, derived from a germline clone of a wild-type Tec29 allele, immunostained for HTS–RC. (B) Tec29k05610 egg chamber immunostained for HTS–RC. (C) Egg chamber, derived from a germline clone of a wild-type Tec29 allele, stained with anti-phosphotyrosine antibodies. (D) Tec29k05610 egg chamber stained with anti-phosphotyrosine antibodies. Note the reduction in ring canal phosphotyrosine content while the cortical staining is still visible. Residual phosphotyrosine content is visible on the mutant ring canals after extensive amplification of the signal. Scale bar, 10 μm. Molecular Cell 1998 1, 831-840DOI: (10.1016/S1097-2765(00)80082-9)

Figure 6 The Outer Diameter of Tec29 Mutant Ring Canals Is Reduced throughout Oogenesis (A) Ring canal size distribution of wild-type and Tec29k05610 stage 5 egg chambers. The ring canal outer diameter of wild-type is 2.5 ± 0.16 μm and Tec29k05610 is 1.8 ± 0.082 μm. (B) Ring canal size distribution of wild-type and Tec29k05610 early stage 10A egg chambers. The ring canal outer diameter of wild-type is 7.1 ± 0.44 μm and Tec29k05610 is 4.5 ± 0.63 μm. The sizes are reported as the average ± the standard deviation of the mean. The sizes determined for wild-type and Tec29k05610 are significantly different from each other (p value is less than 0.05 using a Student's t test) at both stages. Molecular Cell 1998 1, 831-840DOI: (10.1016/S1097-2765(00)80082-9)

Figure 7 TEC29 Requires Src64 for Localization to the Ring Canal (A–F) Stage 10A egg chamber images are composed of serial optical sections that were merged. (A) and (C) only show a portion of the entire series of optical sections. (A) Egg chamber, derived from a germline clone of a wild-type Tec29 allele, immunostained for SRC64. SRC64 is seen at the cortical regions of the nurse cells and occasionally a ring canal can be seen (arrow). (B) Src64Δ17 egg chamber immunostained for SRC64. The cortical actin and ring canal staining is absent in these egg chambers. (C) Tec29k05610 egg chamber immunostained for SRC64. This staining pattern is the same as seen in (A). (D) Wild-type egg chamber immunostained for TEC29. (E) Src64Δ17 egg chamber immunostained for TEC29. The intense ring canal staining observed in (D) is greatly reduced. Src64Δ17 does not affect the somatic follicle cell staining seen in (D). (F) Tec29k05610 egg chamber immunostained for TEC29. The TEC29 localization seen in (D) is absent, except for the somatic follicle cell staining. Scale bar for (A)–(F), 20 μm. (G) A wild-type stage 10 ring canal stained for F-actin. (H) The same ring canal as in (G) stained for TEC29. When (G) and (H) are merged, the two images completely overlap at the ring canal. Scale bar for (G) and (H), 5 μm. Molecular Cell 1998 1, 831-840DOI: (10.1016/S1097-2765(00)80082-9)

Figure 8 Protein Levels of TEC29 Are Not Altered in Src64Δ17 Ovaries An immunoblot using TEC29 antibody on wild-type and Src64Δ17 ovary extracts shows that they have relatively equivalent TEC29 levels. The migration of known molecular weight markers is shown in kilodaltons. The doublet (arrowheads) represents one of the two reported forms of TEC29 (Vincent et al. 1989). The other reported form, p55, was not detected and is presumably not expressed in ovaries. The identity of the p95 band is not known and is unaltered in the Src64 mutant ovaries. Amido black staining showed that the total amount of protein loaded in each lane was equivalent. Molecular Cell 1998 1, 831-840DOI: (10.1016/S1097-2765(00)80082-9)