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BMP-regulated exosomes from Drosophila male reproductive glands reprogram female behavior.

Corrigan L, Redhai S, Leiblich A, Fan SJ, Perera SM, Patel R, Gandy C, Wainwright SM, Morris JF, Hamdy F, Goberdhan DC, Wilson C - J. Cell Biol. (2014)

Bottom Line: Male reproductive glands secrete signals into seminal fluid to facilitate reproductive success.Exosome release was required to inhibit female remating behavior, suggesting that exosomes are downstream effectors of BMP signaling.These results demonstrate a new function for the MVB-exosome pathway in the reproductive tract that appears to be conserved across evolution.

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Affiliation: Department of Physiology, Anatomy and Genetics and Nuffield Department of Surgical Sciences, University of Oxford, Oxford OX1 3QX, England, UK.

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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.
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fig5: 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.

Mentions: To assess whether CD63-GFP–positive puncta in the AG lumen are exosomes derived from mMVBLs of SCs, we specifically blocked expression or function of a broad range of molecules in SCs, which in other systems are implicated in exosome biogenesis. The ESCRT complex has well-characterized roles in ILV formation, although ESCRT-independent mechanisms have also been reported (Trajkovic et al., 2008). Hrs is a component of the ESCRT-0 complex directly involved in protein sorting into ILVs and ILV formation through subsequent recruitment of downstream ESCRTs (Lloyd et al., 2002; Katzmann et al., 2003). Several recent studies demonstrate a role for Hrs in ESCRT-dependent exosome secretion in mammals and flies (Tamai et al., 2010; Gross et al., 2012; Colombo et al., 2013). ALiX (ALG-2–interacting protein X) interacts with several ESCRT complex proteins (Matsuo et al., 2004; Odorizzi, 2006). Recent knockdown studies in mammalian cells indicate that it is required in exosome biogenesis (Matsuo et al., 2004; Baietti et al., 2012), but this has not been investigated in flies. Fluorescent ILVs inside mMVBLs were almost completely absent in SCs expressing either Hrs-RNAi or ALiX-RNAi constructs, unlike controls (Fig. 5, B–D). The number of CD63-GFP–positive puncta secreted into the AG lumen was also drastically reduced (P < 0.001, n > 9; Fig. 5 A). mMVBLs in ALiX-RNAi– and Hrs-RNAi–expressing SCs were smaller and larger than controls, respectively (Fig. 5 I). These genotypes had normal numbers of iLEs (Fig. 5 H), but Hrs-RNAi–expressing SCs had significantly fewer mMVBLs (Fig. 5 G). Importantly, these ESCRT knockdown SCs still synthesized equivalent levels of the SC-specific CD63-GFP fusion protein by Western blot analysis of whole glands (Fig. 5 J) but transferred much less protein to females upon mating (Fig. 5 K), consistent with the reduced secretion of this exosome marker.


BMP-regulated exosomes from Drosophila male reproductive glands reprogram female behavior.

Corrigan L, Redhai S, Leiblich A, Fan SJ, Perera SM, Patel R, Gandy C, Wainwright SM, Morris JF, Hamdy F, Goberdhan DC, Wilson C - J. Cell Biol. (2014)

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.
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Related In: Results  -  Collection

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fig5: 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.
Mentions: To assess whether CD63-GFP–positive puncta in the AG lumen are exosomes derived from mMVBLs of SCs, we specifically blocked expression or function of a broad range of molecules in SCs, which in other systems are implicated in exosome biogenesis. The ESCRT complex has well-characterized roles in ILV formation, although ESCRT-independent mechanisms have also been reported (Trajkovic et al., 2008). Hrs is a component of the ESCRT-0 complex directly involved in protein sorting into ILVs and ILV formation through subsequent recruitment of downstream ESCRTs (Lloyd et al., 2002; Katzmann et al., 2003). Several recent studies demonstrate a role for Hrs in ESCRT-dependent exosome secretion in mammals and flies (Tamai et al., 2010; Gross et al., 2012; Colombo et al., 2013). ALiX (ALG-2–interacting protein X) interacts with several ESCRT complex proteins (Matsuo et al., 2004; Odorizzi, 2006). Recent knockdown studies in mammalian cells indicate that it is required in exosome biogenesis (Matsuo et al., 2004; Baietti et al., 2012), but this has not been investigated in flies. Fluorescent ILVs inside mMVBLs were almost completely absent in SCs expressing either Hrs-RNAi or ALiX-RNAi constructs, unlike controls (Fig. 5, B–D). The number of CD63-GFP–positive puncta secreted into the AG lumen was also drastically reduced (P < 0.001, n > 9; Fig. 5 A). mMVBLs in ALiX-RNAi– and Hrs-RNAi–expressing SCs were smaller and larger than controls, respectively (Fig. 5 I). These genotypes had normal numbers of iLEs (Fig. 5 H), but Hrs-RNAi–expressing SCs had significantly fewer mMVBLs (Fig. 5 G). Importantly, these ESCRT knockdown SCs still synthesized equivalent levels of the SC-specific CD63-GFP fusion protein by Western blot analysis of whole glands (Fig. 5 J) but transferred much less protein to females upon mating (Fig. 5 K), consistent with the reduced secretion of this exosome marker.

Bottom Line: Male reproductive glands secrete signals into seminal fluid to facilitate reproductive success.Exosome release was required to inhibit female remating behavior, suggesting that exosomes are downstream effectors of BMP signaling.These results demonstrate a new function for the MVB-exosome pathway in the reproductive tract that appears to be conserved across evolution.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Anatomy and Genetics and Nuffield Department of Surgical Sciences, University of Oxford, Oxford OX1 3QX, England, UK.

Show MeSH
Related in: MedlinePlus