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Plasma membrane overgrowth causes fibrotic collagen accumulation and immune activation in Drosophila adipocytes.

Zang Y, Wan M, Liu M, Ke H, Ma S, Liu LP, Ni JQ, Pastor-Pareja JC - Elife (2015)

Bottom Line: Deposits also form in the absence of negative Toll immune regulator Cactus, excess PM being caused in this case by increased secretion.Finally, we show that trimeric Collagen accumulation, downstream of Toll or endocytic defects, activates a tissue damage response.It also places fibrotic deposits both downstream and upstream of immune signaling, consistent with the chronic character of fibrotic diseases.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Tsinghua University, Beijing, China.

ABSTRACT
Many chronic diseases are associated with fibrotic deposition of Collagen and other matrix proteins. Little is known about the factors that determine preferential onset of fibrosis in particular tissues. Here we show that plasma membrane (PM) overgrowth causes pericellular Collagen accumulation in Drosophila adipocytes. We found that loss of Dynamin and other endocytic components causes pericellular trapping of outgoing Collagen IV due to dramatic cortex expansion when endocytic removal of PM is prevented. Deposits also form in the absence of negative Toll immune regulator Cactus, excess PM being caused in this case by increased secretion. Finally, we show that trimeric Collagen accumulation, downstream of Toll or endocytic defects, activates a tissue damage response. Our work indicates that traffic imbalances and PM topology may contribute to fibrosis. It also places fibrotic deposits both downstream and upstream of immune signaling, consistent with the chronic character of fibrotic diseases.

No MeSH data available.


Related in: MedlinePlus

(A) Confocal images of adipocytes from shi1and shi2 thermosensitive mutants.Shifting larvae to restrictive temperature for 3 hr causes mild pericellular accumulation of Collagen IV (Vkg-GFP in green). myr-RFP membrane marker in red. (B) Western blots of hemolymph probed with an anti-Cg25C antibody (1:5000). Hemolymph was collected by turning 10 larvae inside-out inside 100 μl of PBS. 10 μl of 2-Mercaptoethanol-reduced sample (equivalent to the blood of 1 larva) were loaded per genotype. We bled wild type larvae (w1118) and larvae where vkg or Cg25C were knocked down in adipocytes (Cg>vkgi+tub-GAL80ts and Cg>Cg25Ci+tub-GAL80ts respectively). For vkg and Cg25C knock-down, and in order to circumvent embryonic/L1 lethality, temporary inhibition of GAL4-driven knock-down was achieved with thermosensitive GAL4 inhibitor tub-GAL80ts (larvae were grown at 18°C to prevent knock-down, transferred to 30°C to initiate knock-down in L1/L2 stage and bled 3 days later in L3 stage). Note that knock-down of Vkg increases Cg25C signal, expected as monomeric Cg25C cannot be incorporated into BMs in the absence of Viking (Pastor-Pareja and Xu, 2011). (C) Pericellular Vkg accumulation in adipocytes from BM-40-SPARC>Hrsi, >RN-trei, >AP-2αi and >AP-2μi larvae. (D) Western blots of hemolymph extracted from wild type (w1118), BM-40-SPARC>shii, >cacti and >Tl10B larvae probed with an anti-GFP antibody (1:5000). The amount of blood loaded in each well is equivalent to 1 larva.DOI:http://dx.doi.org/10.7554/eLife.07187.004
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fig1s1: (A) Confocal images of adipocytes from shi1and shi2 thermosensitive mutants.Shifting larvae to restrictive temperature for 3 hr causes mild pericellular accumulation of Collagen IV (Vkg-GFP in green). myr-RFP membrane marker in red. (B) Western blots of hemolymph probed with an anti-Cg25C antibody (1:5000). Hemolymph was collected by turning 10 larvae inside-out inside 100 μl of PBS. 10 μl of 2-Mercaptoethanol-reduced sample (equivalent to the blood of 1 larva) were loaded per genotype. We bled wild type larvae (w1118) and larvae where vkg or Cg25C were knocked down in adipocytes (Cg>vkgi+tub-GAL80ts and Cg>Cg25Ci+tub-GAL80ts respectively). For vkg and Cg25C knock-down, and in order to circumvent embryonic/L1 lethality, temporary inhibition of GAL4-driven knock-down was achieved with thermosensitive GAL4 inhibitor tub-GAL80ts (larvae were grown at 18°C to prevent knock-down, transferred to 30°C to initiate knock-down in L1/L2 stage and bled 3 days later in L3 stage). Note that knock-down of Vkg increases Cg25C signal, expected as monomeric Cg25C cannot be incorporated into BMs in the absence of Viking (Pastor-Pareja and Xu, 2011). (C) Pericellular Vkg accumulation in adipocytes from BM-40-SPARC>Hrsi, >RN-trei, >AP-2αi and >AP-2μi larvae. (D) Western blots of hemolymph extracted from wild type (w1118), BM-40-SPARC>shii, >cacti and >Tl10B larvae probed with an anti-GFP antibody (1:5000). The amount of blood loaded in each well is equivalent to 1 larva.DOI:http://dx.doi.org/10.7554/eLife.07187.004

Mentions: To gain new insights into Collagen biogenesis, we conducted a screening for genes affecting production of Collagen IV by fat body adipocytes, its main source in the Drosophila larva (Figure 1A). We used BM-40-SPARC-GAL4 (Venken et al., 2011) to drive expression in adipocytes of the RNAi transgenes in the TRiP collection (8459 lines targeting 6200 genes) (Ni et al., 2008, 2011) and analyzed the localization of Vkg-GFP, a functional GFP-trap fusion to the Collagen IV chain Vkg (Morin et al., 2001). While a majority of hits produced intracellular Collagen IV accumulation (full results to be published later), a distinct phenotypical category consisted of 60 genes causing accumulation at or near the PM (Supplementary file 1). Among the strongest hits in this category were two different RNAi transgenes targeting shibire (shi), encoding fly Dynamin, a GTPase involved in excision of endocytic vesicles (Ferguson and De Camilli, 2012). shibire knock-down (shii), as well as expression of dominant negative Dynamin (ShiK44A) (Moline et al., 1999), caused Vkg accumulation in adipocytes (Figure 1B). In validation of the phenotype, antibody staining confirmed reduced Dynamin expression in shii cells, whereas the staining increased after Shi.K44A overexpression (Figure 1C), attesting to the sensitivity of the antibody. Since Collagen IV is a heterotrimer combining the α2 chain Vkg with Cg25C α1 chains, we performed a staining with an anti-Cg25C antibody we generated for this study (see Figure 1—figure supplement 1). This staining revealed that Cg25C, same as Vkg, accumulates in shii adipocytes (Figure 1D), thus confirming Collagen IV accumulation in these cells. The accumulation of Collagen IV occurs at the cell periphery, under a basement membrane surrounding the tissue in the wild type and which still forms in shii adipocytes (Figure 1D; see also Figure 3B later). Further validating the requirement of shibire in normal Collagen IV distribution, shi1 and shi2 thermosensitive mutations (Kim and Wu, 1990) also caused Vkg accumulation in adipocytes when larvae grew at restrictive temperature (Figure 1—figure supplement 1).10.7554/eLife.07187.003Figure 1.Endocytic defects cause Collagen accumulation in Drosophila adipocytes.


Plasma membrane overgrowth causes fibrotic collagen accumulation and immune activation in Drosophila adipocytes.

Zang Y, Wan M, Liu M, Ke H, Ma S, Liu LP, Ni JQ, Pastor-Pareja JC - Elife (2015)

(A) Confocal images of adipocytes from shi1and shi2 thermosensitive mutants.Shifting larvae to restrictive temperature for 3 hr causes mild pericellular accumulation of Collagen IV (Vkg-GFP in green). myr-RFP membrane marker in red. (B) Western blots of hemolymph probed with an anti-Cg25C antibody (1:5000). Hemolymph was collected by turning 10 larvae inside-out inside 100 μl of PBS. 10 μl of 2-Mercaptoethanol-reduced sample (equivalent to the blood of 1 larva) were loaded per genotype. We bled wild type larvae (w1118) and larvae where vkg or Cg25C were knocked down in adipocytes (Cg>vkgi+tub-GAL80ts and Cg>Cg25Ci+tub-GAL80ts respectively). For vkg and Cg25C knock-down, and in order to circumvent embryonic/L1 lethality, temporary inhibition of GAL4-driven knock-down was achieved with thermosensitive GAL4 inhibitor tub-GAL80ts (larvae were grown at 18°C to prevent knock-down, transferred to 30°C to initiate knock-down in L1/L2 stage and bled 3 days later in L3 stage). Note that knock-down of Vkg increases Cg25C signal, expected as monomeric Cg25C cannot be incorporated into BMs in the absence of Viking (Pastor-Pareja and Xu, 2011). (C) Pericellular Vkg accumulation in adipocytes from BM-40-SPARC>Hrsi, >RN-trei, >AP-2αi and >AP-2μi larvae. (D) Western blots of hemolymph extracted from wild type (w1118), BM-40-SPARC>shii, >cacti and >Tl10B larvae probed with an anti-GFP antibody (1:5000). The amount of blood loaded in each well is equivalent to 1 larva.DOI:http://dx.doi.org/10.7554/eLife.07187.004
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Related In: Results  -  Collection

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fig1s1: (A) Confocal images of adipocytes from shi1and shi2 thermosensitive mutants.Shifting larvae to restrictive temperature for 3 hr causes mild pericellular accumulation of Collagen IV (Vkg-GFP in green). myr-RFP membrane marker in red. (B) Western blots of hemolymph probed with an anti-Cg25C antibody (1:5000). Hemolymph was collected by turning 10 larvae inside-out inside 100 μl of PBS. 10 μl of 2-Mercaptoethanol-reduced sample (equivalent to the blood of 1 larva) were loaded per genotype. We bled wild type larvae (w1118) and larvae where vkg or Cg25C were knocked down in adipocytes (Cg>vkgi+tub-GAL80ts and Cg>Cg25Ci+tub-GAL80ts respectively). For vkg and Cg25C knock-down, and in order to circumvent embryonic/L1 lethality, temporary inhibition of GAL4-driven knock-down was achieved with thermosensitive GAL4 inhibitor tub-GAL80ts (larvae were grown at 18°C to prevent knock-down, transferred to 30°C to initiate knock-down in L1/L2 stage and bled 3 days later in L3 stage). Note that knock-down of Vkg increases Cg25C signal, expected as monomeric Cg25C cannot be incorporated into BMs in the absence of Viking (Pastor-Pareja and Xu, 2011). (C) Pericellular Vkg accumulation in adipocytes from BM-40-SPARC>Hrsi, >RN-trei, >AP-2αi and >AP-2μi larvae. (D) Western blots of hemolymph extracted from wild type (w1118), BM-40-SPARC>shii, >cacti and >Tl10B larvae probed with an anti-GFP antibody (1:5000). The amount of blood loaded in each well is equivalent to 1 larva.DOI:http://dx.doi.org/10.7554/eLife.07187.004
Mentions: To gain new insights into Collagen biogenesis, we conducted a screening for genes affecting production of Collagen IV by fat body adipocytes, its main source in the Drosophila larva (Figure 1A). We used BM-40-SPARC-GAL4 (Venken et al., 2011) to drive expression in adipocytes of the RNAi transgenes in the TRiP collection (8459 lines targeting 6200 genes) (Ni et al., 2008, 2011) and analyzed the localization of Vkg-GFP, a functional GFP-trap fusion to the Collagen IV chain Vkg (Morin et al., 2001). While a majority of hits produced intracellular Collagen IV accumulation (full results to be published later), a distinct phenotypical category consisted of 60 genes causing accumulation at or near the PM (Supplementary file 1). Among the strongest hits in this category were two different RNAi transgenes targeting shibire (shi), encoding fly Dynamin, a GTPase involved in excision of endocytic vesicles (Ferguson and De Camilli, 2012). shibire knock-down (shii), as well as expression of dominant negative Dynamin (ShiK44A) (Moline et al., 1999), caused Vkg accumulation in adipocytes (Figure 1B). In validation of the phenotype, antibody staining confirmed reduced Dynamin expression in shii cells, whereas the staining increased after Shi.K44A overexpression (Figure 1C), attesting to the sensitivity of the antibody. Since Collagen IV is a heterotrimer combining the α2 chain Vkg with Cg25C α1 chains, we performed a staining with an anti-Cg25C antibody we generated for this study (see Figure 1—figure supplement 1). This staining revealed that Cg25C, same as Vkg, accumulates in shii adipocytes (Figure 1D), thus confirming Collagen IV accumulation in these cells. The accumulation of Collagen IV occurs at the cell periphery, under a basement membrane surrounding the tissue in the wild type and which still forms in shii adipocytes (Figure 1D; see also Figure 3B later). Further validating the requirement of shibire in normal Collagen IV distribution, shi1 and shi2 thermosensitive mutations (Kim and Wu, 1990) also caused Vkg accumulation in adipocytes when larvae grew at restrictive temperature (Figure 1—figure supplement 1).10.7554/eLife.07187.003Figure 1.Endocytic defects cause Collagen accumulation in Drosophila adipocytes.

Bottom Line: Deposits also form in the absence of negative Toll immune regulator Cactus, excess PM being caused in this case by increased secretion.Finally, we show that trimeric Collagen accumulation, downstream of Toll or endocytic defects, activates a tissue damage response.It also places fibrotic deposits both downstream and upstream of immune signaling, consistent with the chronic character of fibrotic diseases.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Tsinghua University, Beijing, China.

ABSTRACT
Many chronic diseases are associated with fibrotic deposition of Collagen and other matrix proteins. Little is known about the factors that determine preferential onset of fibrosis in particular tissues. Here we show that plasma membrane (PM) overgrowth causes pericellular Collagen accumulation in Drosophila adipocytes. We found that loss of Dynamin and other endocytic components causes pericellular trapping of outgoing Collagen IV due to dramatic cortex expansion when endocytic removal of PM is prevented. Deposits also form in the absence of negative Toll immune regulator Cactus, excess PM being caused in this case by increased secretion. Finally, we show that trimeric Collagen accumulation, downstream of Toll or endocytic defects, activates a tissue damage response. Our work indicates that traffic imbalances and PM topology may contribute to fibrosis. It also places fibrotic deposits both downstream and upstream of immune signaling, consistent with the chronic character of fibrotic diseases.

No MeSH data available.


Related in: MedlinePlus