Figure 2. Asymmetric Rac activity localizes Apc2 to control centrosome position in interphase GSCs.

(A) Bar graphs of GSC centrosome phenotypes in wild-type, Rac mutant, or Rac^V12 GSCs. One, one cortical centrosome located at the CpC-GSC interface; Migrating, two cortical centrosomes one at the interface and one migrating; At interface, two cortical centrosomes separated by 180° one of which is at the interface; Not at interface, two cortical centrosome separated by 180° neither of which is at the interface; Non-cortical, one or both centrosomes detached from cortex. (B–E) Cortical migration of one centrosome after centrosome duplication in wild-type GSCs (arrowhead, centrosome). (F–I) Defects in centrosome position in Rac mutant (Rac1^J10 Rac2^Δ Mtl^Δ/Rac1^J10 Rac2^Δ+) (F,H) or in Rac1^V12 GSCs (G,I). (F,G) At least one centrosome is not at cortex (arrowheads). (H,I) In GSCs with two cortical centrosomes separated by 180° (double-headed arrows), neither centrosome is at CpC-GSC interface. (J–K) Graphs with angles, in individual control GSCs (J) or GSCs with perturbations in Rac activity (K), between the CpC-GSC interface and the line connecting two cortical centrosomes separated by 180°. Black dot, one of two centrosomes at the CpC-GSC interface. Red dot, neither centrosome at the CpC-GSC interface. Percentage (%) of GSCs with each phenotype compared to total in the graph. (L–N) Apc2 concentration at the CpC-GSC interface (L) is disrupted in GSCs with altered Rac activity (M,N). Inset: white, anti-Apc2 staining; asterisk, control GSC (L,M); dot, Rac mutant GSC (M) or Rac1^V12 GSC (N). (O–P) Apc2^d40 GSC with non-cortical centrosomes (O, arrowheads); Apc2^d40 GSC with two cortical centrosomes, neither of which is at the CpC-GSC interface (P, double-headed arrow). (Q) Uniform cytoplasmic localization of Apc2-GFP expressed in the germ line. Inset: white, anti-GFP; asterisk, Apc2-GFP-expressing GSC. (R) Expression of Apc2-GFP causes the same classes of centrosome defects as are observed in Apc2^d40 mutants: a GSC with two cortically located centrosomes, neither of which is present at the CpC-GSC interface (double-headed arrow), and a GSC with centrosomes detached from the cortex (arrowheads). (S) In wild-type interphase GSCs, microtubules are organized as a bundled network near the CpC-GSC interface. (T) In Rac1^V12-expressing GSCs, a microtubule network is present uniformly around the GSC cell cortex. (U) In Apc2^d40 GSCs, the microtubule staining is reduced and uniformly localized. (S–U) Inset: white, anti-α-tubulin staining. (V) A Rac-RNAi GSC with a mitotic spindle (double-headed arrow) perpendicular to the CpC-GSC interface and with one pole at the interface. All mitotic Rac-RNAi GSCs have the same orientation of the mitotic spindle. (B–I,O–P,R) Anti-γ-tubulin, centrosomes. (B–E,G,I,L,N–R) Anti-Vasa, germline cytoplasm. (F,H,M) Absence of anti-β-galactosidase, Rac mutant GSC. (S–V) Anti-α-tubulin, cytoplasmic microtubules (S–U), and mitotic spindle (V). (B–I,L–V) Arrow, CpC niche; solid line, CpC-GSC interface; dashed outline, individual GSC. (F–I,O–P,V) Percentage (%) of total (N) GSCs with the specific phenotype displayed in the panel.