![Figure 5. ESCRTs, Rab GTPases, and BMP signaling regulate the exosome biogenesis pathway. (A) The numbers of CD63-GFP–positive puncta secreted into the AG lumen were significantly reduced in SC>Hrs-RNAi, SC>ALiX-RNAi, SC>Rab11DN, SC>Rab11-RNAi, SC>Dad, SC>Rab7-DN, SC>Rab7-RNAi, SC>Rab27-RNAi1, and SC>Rab35-RNAi males compared with control (number of glands above or in the bars) but not SC>Rab27-RNAi2 and SC>Evi-RNAi males. DN, dominant negative. (B–F) SCs from 6-d-old virgin males stained with LysoTracker red. (G–I) Different intracellular compartments were subsequently counted and measured. mMVBLs (asterisks) in SCs expressing CD63-GFP and Hrs-RNAi (C) or ALiX-RNAi (D) contain very few CD63-GFP–positive ILVs (internal puncta), unlike controls (B), and are altered in size (I). Reducing levels of BMP signaling in SCs (Dad overexpression; E) results in significantly smaller mMVBLs (I), typically containing a higher density of internalized fluorescent CD63-GFP than controls. SCs expressing an activated form of the BMP receptor TkvACT (F) contain fewer mMVBLs (G), which are larger (I), and typically surrounded by multiple SVs (e.g., arrow). They also contain more iLEs (H). (G–I) Mean number of mMVBLs (G) and iLEs (H) and mean diameter of largest mMVBL (I) in SCs of different genotypes (I). Quantification in G–I is from a mean of three cells per gland, averaged over n glands (numbers in bars). (J) Western analysis of AG protein extracts from w1118 (wild type [wt]) and flies expressing SC>CD63-GFP with or without (control) different transgenes. (K) Western analysis of protein extracts from reproductive tracts dissected from females mated to males of the same genotypes. CD63-GFP/ANCE is the ratio of signals from Western blots. Full-length CD63-GFP protein is observed in males and females. Error bars indicate ±SE; *, P < 0.05; **, P < 0.01; ***, P < 0.001 relative to control. Data were analyzed using either an unpaired two-tailed Student’s t test or Mann–Whitney U test, for normally and nonnormally distributed datasets, respectively. Approximate outline of SC is marked. Bars, 5 µm.](https://cdn.rupress.org/rup/content_public/journal/jcb/206/5/10.1083_jcb.201401072/6/jcb_201401072r_fig5.jpeg?Expires=1771715749&Signature=gfabG1jdFlcyKbU3NxY~aZ3qsqwk5-GH-BIJKvbIZrr~5JUy6qfxw1RBfWK11AnZig5rTp1Lbo3x4Va1pD3pN-jp5QoW095A55zMxGgz2dtZL2K88iXm159df4Vt0EJdn6Vs-127NKVMQw-Ktmu2XUt3Msnts7ZQOnQin7rUK80OER9B4tBM0oLJn1J3mY0t19kaxAp1W1YPYbNT8~F-LHIbwvzASobAMeDXokFa0P2WjLmMX2RsBdaEjhpww4LmjCYEAKGnKAfDJ9b4NmP-w5x17Kb~UcI5U3kVl7S0ZyG0Js1bFZORzjwCZFu82SgXnktW~ArhsAts5H~P0zru2Q__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
ESCRTs, Rab GTPases, and BMP signaling regulate the exosome biogenesis pathway. (A) The numbers of CD63-GFP–positive puncta secreted into the AG lumen were significantly reduced in SC>Hrs-RNAi, SC>ALiX-RNAi, SC>Rab11DN, SC>Rab11-RNAi, SC>Dad, SC>Rab7-DN, SC>Rab7-RNAi, SC>Rab27-RNAi1, and SC>Rab35-RNAi males compared with control (number of glands above or in the bars) but not SC>Rab27-RNAi2 and SC>Evi-RNAi males. DN, dominant negative. (B–F) SCs from 6-d-old virgin males stained with LysoTracker red. (G–I) Different intracellular compartments were subsequently counted and measured. mMVBLs (asterisks) in SCs expressing CD63-GFP and Hrs-RNAi (C) or ALiX-RNAi (D) contain very few CD63-GFP–positive ILVs (internal puncta), unlike controls (B), and are altered in size (I). Reducing levels of BMP signaling in SCs (Dad overexpression; E) results in significantly smaller mMVBLs (I), typically containing a higher density of internalized fluorescent CD63-GFP than controls. SCs expressing an activated form of the BMP receptor TkvACT (F) contain fewer mMVBLs (G), which are larger (I), and typically surrounded by multiple SVs (e.g., arrow). They also contain more iLEs (H). (G–I) Mean number of mMVBLs (G) and iLEs (H) and mean diameter of largest mMVBL (I) in SCs of different genotypes (I). Quantification in G–I is from a mean of three cells per gland, averaged over n glands (numbers in bars). (J) Western analysis of AG protein extracts from w1118 (wild type [wt]) and flies expressing SC>CD63-GFP with or without (control) different transgenes. (K) Western analysis of protein extracts from reproductive tracts dissected from females mated to males of the same genotypes. CD63-GFP/ANCE is the ratio of signals from Western blots. Full-length CD63-GFP protein is observed in males and females. Error bars indicate ±SE; *, P < 0.05; **, P < 0.01; ***, P < 0.001 relative to control. Data were analyzed using either an unpaired two-tailed Student’s t test or Mann–Whitney U test, for normally and nonnormally distributed datasets, respectively. Approximate outline of SC is marked. Bars, 5 µm.
