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Global defects in collagen secretion in a Mia3/TANGO1 knockout mouse.

Wilson DG, Phamluong K, Li L, Sun M, Cao TC, Liu PS, Modrusan Z, Sandoval WN, Rangell L, Carano RA, Peterson AS, Solloway MJ - J. Cell Biol. (2011)

Bottom Line: These changes are associated with intracellular accumulation of collagen and the induction of a strong unfolded protein response, primarily within the developing skeleton.Chondrocyte maturation and bone mineralization are severely compromised in Mia3- embryos, leading to dwarfism and neonatal lethality.Thus, Mia3's role in protein secretion is much broader than previously realized, and it may, in fact, be required for the efficient secretion of all collagen molecules in higher organisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, Genentech, South San Francisco, CA 94080, USA.

ABSTRACT
Melanoma inhibitory activity member 3 (MIA3/TANGO1) [corrected] is an evolutionarily conserved endoplasmic reticulum resident transmembrane protein. Recent in vitro studies have shown that it is required for the loading of collagen VII, but not collagen I, into COPII-coated transport vesicles. In this paper, we show that mice lacking Mia3 are defective for the secretion of numerous collagens, including collagens I, II, III, IV, VII, and IX, from chondrocytes, fibroblasts, endothelial cells, and mural cells. Collagen deposition by these cell types is abnormal, and extracellular matrix composition is compromised. These changes are associated with intracellular accumulation of collagen and the induction of a strong unfolded protein response, primarily within the developing skeleton. Chondrocyte maturation and bone mineralization are severely compromised in Mia3- embryos, leading to dwarfism and neonatal lethality. Thus, Mia3's role in protein secretion is much broader than previously realized, and it may, in fact, be required for the efficient secretion of all collagen molecules in higher organisms.

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Abnormal collagen glycosylation, secretion, maturation, and ECM deposition in Mia3- cells. (A–C) Immunofluorescent staining of wt and Mia3−/− primary MEFs (A; Col7a1) and chondrocytes (B and C; Col2a1 and Col3a1) reveals abnormal punctate ECM deposition (asterisks) and an increase in ER-retained collagen upon cell permeabilization. Bars, 10 µm. (D and E) Comparison and quantitation of ECM and cell-associated collagens isolated from wt and Mia3- chondrocytes (left) and MEFs (right). Values are ± SEM. n = 3 per genotype. KO, knockout. (F) Western blot analyses of cell-associated collagen 1 (Col1) from wt and Mia3−/− MEFs show substantial changes in collagen processing in  cells. Distribution plots summarize data (n = 4 per genotype). Arrows indicate processed collagen types. (G) Timed protease digestion of embryonic lysates blotted for Col2a1 further highlights the presence of alternately modified collagen (arrows and boxed region) in Mia3- tissues. Molecular masses are given in kilodaltons.
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fig5: Abnormal collagen glycosylation, secretion, maturation, and ECM deposition in Mia3- cells. (A–C) Immunofluorescent staining of wt and Mia3−/− primary MEFs (A; Col7a1) and chondrocytes (B and C; Col2a1 and Col3a1) reveals abnormal punctate ECM deposition (asterisks) and an increase in ER-retained collagen upon cell permeabilization. Bars, 10 µm. (D and E) Comparison and quantitation of ECM and cell-associated collagens isolated from wt and Mia3- chondrocytes (left) and MEFs (right). Values are ± SEM. n = 3 per genotype. KO, knockout. (F) Western blot analyses of cell-associated collagen 1 (Col1) from wt and Mia3−/− MEFs show substantial changes in collagen processing in cells. Distribution plots summarize data (n = 4 per genotype). Arrows indicate processed collagen types. (G) Timed protease digestion of embryonic lysates blotted for Col2a1 further highlights the presence of alternately modified collagen (arrows and boxed region) in Mia3- tissues. Molecular masses are given in kilodaltons.

Mentions: Consistent with the observation that Col7a1 binds MIA3 and accumulates in cells in which MIA3 transcripts are reduced by RNAi knockdown (Saito et al., 2009b), comparisons of Col7a1 deposition between wt and Mia3−/− MEFs with and without membrane permeabilization reveal a slight reduction in the overall amount of extracellular collagen secreted by the cells and a dramatic intracellular accumulation of Col7a1 adjacent to the nucleus (Fig. 5 A). The profound and cell-autonomous defects in chondrocyte differentiation, however, suggest a broader role for Mia3 than secretion of Col7a1. Indeed, chondrocyte-enriched primary cell cultures derived from 14.5-dpc Mia3−/− ribcages show a similar accumulation of Col2a1 and, to a lesser extent, Col3a1 within the cells (Fig. 5, B and C). Although some collagen is deposited outside of the knockout cells, it appears to be abnormally aggregated and unevenly dispersed throughout the extracellular fibrils. Quantitation of total cell-associated collagen using SDS-PAGE and Western blot analysis confirmed these observations, with 2.2–4.7-fold increases in the amount of Col1, Col3a1, and Col9a1 in Mia3- MEFs relative to controls (Fig. S3, G–I) despite normal levels of transcription (Fig. S3 C). To confirm that these changes were driven by increased retention, we compared ECM-resident versus cell-associated levels of Col1 in chondrocytes and MEFs (Fig. 5 D). A 50% reduction is observed in the amount of ECM-deposited Col1 in Mia3- primary chondrocytes (Fig. 5 E, P = 0.006, n = 3 per genotype). In MEFs, cell-associated Col1 is increased twofold in Mia3- cells (P = 0.05), whereas the level of ECM-associated collagen is barely detectable and not significantly different (Fig. 5 E). We conclude that the transit of several collagens is hampered by the loss of Mia3, and reduced levels are deposited within the ECM.


Global defects in collagen secretion in a Mia3/TANGO1 knockout mouse.

Wilson DG, Phamluong K, Li L, Sun M, Cao TC, Liu PS, Modrusan Z, Sandoval WN, Rangell L, Carano RA, Peterson AS, Solloway MJ - J. Cell Biol. (2011)

Abnormal collagen glycosylation, secretion, maturation, and ECM deposition in Mia3- cells. (A–C) Immunofluorescent staining of wt and Mia3−/− primary MEFs (A; Col7a1) and chondrocytes (B and C; Col2a1 and Col3a1) reveals abnormal punctate ECM deposition (asterisks) and an increase in ER-retained collagen upon cell permeabilization. Bars, 10 µm. (D and E) Comparison and quantitation of ECM and cell-associated collagens isolated from wt and Mia3- chondrocytes (left) and MEFs (right). Values are ± SEM. n = 3 per genotype. KO, knockout. (F) Western blot analyses of cell-associated collagen 1 (Col1) from wt and Mia3−/− MEFs show substantial changes in collagen processing in  cells. Distribution plots summarize data (n = 4 per genotype). Arrows indicate processed collagen types. (G) Timed protease digestion of embryonic lysates blotted for Col2a1 further highlights the presence of alternately modified collagen (arrows and boxed region) in Mia3- tissues. Molecular masses are given in kilodaltons.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3105544&req=5

fig5: Abnormal collagen glycosylation, secretion, maturation, and ECM deposition in Mia3- cells. (A–C) Immunofluorescent staining of wt and Mia3−/− primary MEFs (A; Col7a1) and chondrocytes (B and C; Col2a1 and Col3a1) reveals abnormal punctate ECM deposition (asterisks) and an increase in ER-retained collagen upon cell permeabilization. Bars, 10 µm. (D and E) Comparison and quantitation of ECM and cell-associated collagens isolated from wt and Mia3- chondrocytes (left) and MEFs (right). Values are ± SEM. n = 3 per genotype. KO, knockout. (F) Western blot analyses of cell-associated collagen 1 (Col1) from wt and Mia3−/− MEFs show substantial changes in collagen processing in cells. Distribution plots summarize data (n = 4 per genotype). Arrows indicate processed collagen types. (G) Timed protease digestion of embryonic lysates blotted for Col2a1 further highlights the presence of alternately modified collagen (arrows and boxed region) in Mia3- tissues. Molecular masses are given in kilodaltons.
Mentions: Consistent with the observation that Col7a1 binds MIA3 and accumulates in cells in which MIA3 transcripts are reduced by RNAi knockdown (Saito et al., 2009b), comparisons of Col7a1 deposition between wt and Mia3−/− MEFs with and without membrane permeabilization reveal a slight reduction in the overall amount of extracellular collagen secreted by the cells and a dramatic intracellular accumulation of Col7a1 adjacent to the nucleus (Fig. 5 A). The profound and cell-autonomous defects in chondrocyte differentiation, however, suggest a broader role for Mia3 than secretion of Col7a1. Indeed, chondrocyte-enriched primary cell cultures derived from 14.5-dpc Mia3−/− ribcages show a similar accumulation of Col2a1 and, to a lesser extent, Col3a1 within the cells (Fig. 5, B and C). Although some collagen is deposited outside of the knockout cells, it appears to be abnormally aggregated and unevenly dispersed throughout the extracellular fibrils. Quantitation of total cell-associated collagen using SDS-PAGE and Western blot analysis confirmed these observations, with 2.2–4.7-fold increases in the amount of Col1, Col3a1, and Col9a1 in Mia3- MEFs relative to controls (Fig. S3, G–I) despite normal levels of transcription (Fig. S3 C). To confirm that these changes were driven by increased retention, we compared ECM-resident versus cell-associated levels of Col1 in chondrocytes and MEFs (Fig. 5 D). A 50% reduction is observed in the amount of ECM-deposited Col1 in Mia3- primary chondrocytes (Fig. 5 E, P = 0.006, n = 3 per genotype). In MEFs, cell-associated Col1 is increased twofold in Mia3- cells (P = 0.05), whereas the level of ECM-associated collagen is barely detectable and not significantly different (Fig. 5 E). We conclude that the transit of several collagens is hampered by the loss of Mia3, and reduced levels are deposited within the ECM.

Bottom Line: These changes are associated with intracellular accumulation of collagen and the induction of a strong unfolded protein response, primarily within the developing skeleton.Chondrocyte maturation and bone mineralization are severely compromised in Mia3- embryos, leading to dwarfism and neonatal lethality.Thus, Mia3's role in protein secretion is much broader than previously realized, and it may, in fact, be required for the efficient secretion of all collagen molecules in higher organisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, Genentech, South San Francisco, CA 94080, USA.

ABSTRACT
Melanoma inhibitory activity member 3 (MIA3/TANGO1) [corrected] is an evolutionarily conserved endoplasmic reticulum resident transmembrane protein. Recent in vitro studies have shown that it is required for the loading of collagen VII, but not collagen I, into COPII-coated transport vesicles. In this paper, we show that mice lacking Mia3 are defective for the secretion of numerous collagens, including collagens I, II, III, IV, VII, and IX, from chondrocytes, fibroblasts, endothelial cells, and mural cells. Collagen deposition by these cell types is abnormal, and extracellular matrix composition is compromised. These changes are associated with intracellular accumulation of collagen and the induction of a strong unfolded protein response, primarily within the developing skeleton. Chondrocyte maturation and bone mineralization are severely compromised in Mia3- embryos, leading to dwarfism and neonatal lethality. Thus, Mia3's role in protein secretion is much broader than previously realized, and it may, in fact, be required for the efficient secretion of all collagen molecules in higher organisms.

Show MeSH
Related in: MedlinePlus