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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 showing the PM of shii and Tl10B adipocytes stained with fixable Texas-Red-coupled Dextrans (3000 and 70,000 MW), labelling PM around Collagen IV (Vkg-GFP) accumulations.(B) Electron micrographs of the PM in BM-40-SPARC>shii adipocytes showing instances of connection (arrows) between the pericellular accumulations (asterisks) and the extracellular space showing that these pockets are not isolated cisternae, but part of a very intricate PM. (C) Confocal images of wing discs (posterior ventral hinge) showing localization of Cg25C-GFP and Cg25C-RFP in the basement membrane after expression in the fat body controlled by Cg-GAL4 (Cg>Cg25C-GFP and Cg>Cg25C-RFP respectively). Images of wild type (w1118) discs are shown as controls to exclude auto-fluorescence. Nuclei stained with DAPI (blue). (D) Images of live larvae expressing Cg25C-RFP and Cg25C-GFP in the fat body (Cg>Cg25C-GFP and Cg>Cg25C-RFP). Knock-down of PH4αEFB, required for Collagen IV trimerization, causes tagged Cg25C to accumulate in the blood (note strong fluorescent signal filling the body cavity).DOI:http://dx.doi.org/10.7554/eLife.07187.007
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fig2s1: (A) Confocal images showing the PM of shii and Tl10B adipocytes stained with fixable Texas-Red-coupled Dextrans (3000 and 70,000 MW), labelling PM around Collagen IV (Vkg-GFP) accumulations.(B) Electron micrographs of the PM in BM-40-SPARC>shii adipocytes showing instances of connection (arrows) between the pericellular accumulations (asterisks) and the extracellular space showing that these pockets are not isolated cisternae, but part of a very intricate PM. (C) Confocal images of wing discs (posterior ventral hinge) showing localization of Cg25C-GFP and Cg25C-RFP in the basement membrane after expression in the fat body controlled by Cg-GAL4 (Cg>Cg25C-GFP and Cg>Cg25C-RFP respectively). Images of wild type (w1118) discs are shown as controls to exclude auto-fluorescence. Nuclei stained with DAPI (blue). (D) Images of live larvae expressing Cg25C-RFP and Cg25C-GFP in the fat body (Cg>Cg25C-GFP and Cg>Cg25C-RFP). Knock-down of PH4αEFB, required for Collagen IV trimerization, causes tagged Cg25C to accumulate in the blood (note strong fluorescent signal filling the body cavity).DOI:http://dx.doi.org/10.7554/eLife.07187.007

Mentions: Accumulations of Collagen IV in shii adipocytes were found in close proximity to the PM, as revealed by the membrane marker myr-mRFP (myristoylation domain of Src fused to mRFP; Figure 2A) and by phalloidin staining of F-actin, normally enriched in the cell cortex (Figure 2B). To determine whether Collagen IV accumulations were intracellular or extracellular we stained shii adipocytes with the lipophilic, cell-impermeable dye FM4-64 (Bolte et al., 2004) to label membrane directly in contact with the extracellular space (PM). Short incubation with FM4-64 showed that the accumulations were surrounded by PM (Figure 2C) and, thus, likely extracellular. Labeling of membrane around the accumulations was equally observed when shii adipocytes were fixed and stained with fluorescently-labeled fixable dextrans of molecular weights 70,000 (Figure 2D) and 3000 (Figure 2—figure supplement 1). To confirm that the Collagen IV accumulations in shii adipocytes were extracellular, we performed antibody stainings without permeabilizing the cells (no detergent in washing or blocking solutions). As a control, we stained Tango1i adipocytes, known to retain Collagen IV intracellularly (Pastor-Pareja and Xu, 2011), and found that intracellularly retained Collagen IV could not be stained without permeabilization (Figure 2E). In contrast to this lack of staining, accumulations of Collagen IV in shii adipocytes were still labeled (Figure 2E), indicating that they were indeed extracellular. Altogether, these data show that Collagen IV accumulations in shii adipocytes are pericellular accumulations located outside of the PM (see also electron micrographs in Figure 2—figure supplement 1).10.7554/eLife.07187.006Figure 2.Collagen accumulation in endocytosis-defective cells is pericellular and autonomous.


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 showing the PM of shii and Tl10B adipocytes stained with fixable Texas-Red-coupled Dextrans (3000 and 70,000 MW), labelling PM around Collagen IV (Vkg-GFP) accumulations.(B) Electron micrographs of the PM in BM-40-SPARC>shii adipocytes showing instances of connection (arrows) between the pericellular accumulations (asterisks) and the extracellular space showing that these pockets are not isolated cisternae, but part of a very intricate PM. (C) Confocal images of wing discs (posterior ventral hinge) showing localization of Cg25C-GFP and Cg25C-RFP in the basement membrane after expression in the fat body controlled by Cg-GAL4 (Cg>Cg25C-GFP and Cg>Cg25C-RFP respectively). Images of wild type (w1118) discs are shown as controls to exclude auto-fluorescence. Nuclei stained with DAPI (blue). (D) Images of live larvae expressing Cg25C-RFP and Cg25C-GFP in the fat body (Cg>Cg25C-GFP and Cg>Cg25C-RFP). Knock-down of PH4αEFB, required for Collagen IV trimerization, causes tagged Cg25C to accumulate in the blood (note strong fluorescent signal filling the body cavity).DOI:http://dx.doi.org/10.7554/eLife.07187.007
© Copyright Policy
Related In: Results  -  Collection

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fig2s1: (A) Confocal images showing the PM of shii and Tl10B adipocytes stained with fixable Texas-Red-coupled Dextrans (3000 and 70,000 MW), labelling PM around Collagen IV (Vkg-GFP) accumulations.(B) Electron micrographs of the PM in BM-40-SPARC>shii adipocytes showing instances of connection (arrows) between the pericellular accumulations (asterisks) and the extracellular space showing that these pockets are not isolated cisternae, but part of a very intricate PM. (C) Confocal images of wing discs (posterior ventral hinge) showing localization of Cg25C-GFP and Cg25C-RFP in the basement membrane after expression in the fat body controlled by Cg-GAL4 (Cg>Cg25C-GFP and Cg>Cg25C-RFP respectively). Images of wild type (w1118) discs are shown as controls to exclude auto-fluorescence. Nuclei stained with DAPI (blue). (D) Images of live larvae expressing Cg25C-RFP and Cg25C-GFP in the fat body (Cg>Cg25C-GFP and Cg>Cg25C-RFP). Knock-down of PH4αEFB, required for Collagen IV trimerization, causes tagged Cg25C to accumulate in the blood (note strong fluorescent signal filling the body cavity).DOI:http://dx.doi.org/10.7554/eLife.07187.007
Mentions: Accumulations of Collagen IV in shii adipocytes were found in close proximity to the PM, as revealed by the membrane marker myr-mRFP (myristoylation domain of Src fused to mRFP; Figure 2A) and by phalloidin staining of F-actin, normally enriched in the cell cortex (Figure 2B). To determine whether Collagen IV accumulations were intracellular or extracellular we stained shii adipocytes with the lipophilic, cell-impermeable dye FM4-64 (Bolte et al., 2004) to label membrane directly in contact with the extracellular space (PM). Short incubation with FM4-64 showed that the accumulations were surrounded by PM (Figure 2C) and, thus, likely extracellular. Labeling of membrane around the accumulations was equally observed when shii adipocytes were fixed and stained with fluorescently-labeled fixable dextrans of molecular weights 70,000 (Figure 2D) and 3000 (Figure 2—figure supplement 1). To confirm that the Collagen IV accumulations in shii adipocytes were extracellular, we performed antibody stainings without permeabilizing the cells (no detergent in washing or blocking solutions). As a control, we stained Tango1i adipocytes, known to retain Collagen IV intracellularly (Pastor-Pareja and Xu, 2011), and found that intracellularly retained Collagen IV could not be stained without permeabilization (Figure 2E). In contrast to this lack of staining, accumulations of Collagen IV in shii adipocytes were still labeled (Figure 2E), indicating that they were indeed extracellular. Altogether, these data show that Collagen IV accumulations in shii adipocytes are pericellular accumulations located outside of the PM (see also electron micrographs in Figure 2—figure supplement 1).10.7554/eLife.07187.006Figure 2.Collagen accumulation in endocytosis-defective cells is pericellular and autonomous.

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