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Phosphoinositide regulation of integrin trafficking required for muscle attachment and maintenance.

Ribeiro I, Yuan L, Tanentzapf G, Dowling JJ, Kiger A - PLoS Genet. (2011)

Bottom Line: Depletion of mtm leads to increased integrin turnover at the sarcolemma and an accumulation of integrin with PI(3)P on endosomal-related membrane inclusions, indicating a role for Mtm phosphatase activity in endocytic trafficking.The depletion of Class II, but not Class III, PI3-kinase rescued mtm-dependent defects, identifying an important pathway that regulates integrin recycling.Importantly, similar integrin localization defects found in human XLMTM myofibers signify conserved MTM1 function in muscle membrane trafficking.

View Article: PubMed Central - PubMed

Affiliation: Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California, United States of America.

ABSTRACT
Muscles must maintain cell compartmentalization when remodeled during development and use. How spatially restricted adhesions are regulated with muscle remodeling is largely unexplored. We show that the myotubularin (mtm) phosphoinositide phosphatase is required for integrin-mediated myofiber attachments in Drosophila melanogaster, and that mtm-depleted myofibers exhibit hallmarks of human XLMTM myopathy. Depletion of mtm leads to increased integrin turnover at the sarcolemma and an accumulation of integrin with PI(3)P on endosomal-related membrane inclusions, indicating a role for Mtm phosphatase activity in endocytic trafficking. The depletion of Class II, but not Class III, PI3-kinase rescued mtm-dependent defects, identifying an important pathway that regulates integrin recycling. Importantly, similar integrin localization defects found in human XLMTM myofibers signify conserved MTM1 function in muscle membrane trafficking. Our results indicate that regulation of distinct phosphoinositide pools plays a central role in maintaining cell compartmentalization and attachments during muscle remodeling, and they suggest involvement of Class II PI3-kinase in MTM-related disease.

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Mtm depletion leads to myofiber detachment and morphological defects common with human myotubular myopathy.(A) Timeline of fly and muscle development, indicating stages of mtm requirements; days after puparium formation (APF). (B–B′) Normal body wall muscles in OreR and mtmΔ77/mtmz2-4747 third instar larvae. F-actin. (C) Percent viable and lethal progeny 13 days after egg lays with 24B-GAL4 and DMef2-GAL4 muscle-targeted mtm RNAi. (D–D′) Pharate dorsal abdominal muscles F-actin. Large IOMs (arrows) and smaller adult muscles (open arrowheads) span tergites (numerals). (D′) Detached IOMs (arrowheads) seen with mtm RNAi. (E–F) IOMs in filleted abdomens with 24B-GAL4 or DMef2-GAL4 expression of 1 or 2 copies of mtm RNAi hairpins. (E) Number IOMs present in tergites 3 and 4, including detached but present IOMs. (F) Number of present, visibly detached IOMs. (G–G′) Timelapse microscopy of GFP in IOMs imaged in same animals 1, 2, 3 and 4 days APF. (G′) With mtm RNAi, normal IOM formation (1d APF), survival upon histolysis of non-persistent muscles (2d APF), myofiber thinning (3d APF) and rethickening (4d APF) preceded detachment (4d APF, arrowheads). (H–H′) Individual IOMs. F-actin, red; DNA, blue. Projections, merged and nuclei images; central z-sections, F-actin. (H) Contractile myofibrils are normally tightly packed (double arrow) around linear aligned nuclei (arrowheads). (H′) With mtm RNAi, intact peripheral myofibrils surround expanded central area (double arrow) with unaligned nuclei (arrowheads). (I) Distance (µm) of nuclei from IOM midline. (J) Number nuclei per IOM. Scale bar 200 µm, except H–H′ 20 µm.
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pgen-1001295-g001: Mtm depletion leads to myofiber detachment and morphological defects common with human myotubular myopathy.(A) Timeline of fly and muscle development, indicating stages of mtm requirements; days after puparium formation (APF). (B–B′) Normal body wall muscles in OreR and mtmΔ77/mtmz2-4747 third instar larvae. F-actin. (C) Percent viable and lethal progeny 13 days after egg lays with 24B-GAL4 and DMef2-GAL4 muscle-targeted mtm RNAi. (D–D′) Pharate dorsal abdominal muscles F-actin. Large IOMs (arrows) and smaller adult muscles (open arrowheads) span tergites (numerals). (D′) Detached IOMs (arrowheads) seen with mtm RNAi. (E–F) IOMs in filleted abdomens with 24B-GAL4 or DMef2-GAL4 expression of 1 or 2 copies of mtm RNAi hairpins. (E) Number IOMs present in tergites 3 and 4, including detached but present IOMs. (F) Number of present, visibly detached IOMs. (G–G′) Timelapse microscopy of GFP in IOMs imaged in same animals 1, 2, 3 and 4 days APF. (G′) With mtm RNAi, normal IOM formation (1d APF), survival upon histolysis of non-persistent muscles (2d APF), myofiber thinning (3d APF) and rethickening (4d APF) preceded detachment (4d APF, arrowheads). (H–H′) Individual IOMs. F-actin, red; DNA, blue. Projections, merged and nuclei images; central z-sections, F-actin. (H) Contractile myofibrils are normally tightly packed (double arrow) around linear aligned nuclei (arrowheads). (H′) With mtm RNAi, intact peripheral myofibrils surround expanded central area (double arrow) with unaligned nuclei (arrowheads). (I) Distance (µm) of nuclei from IOM midline. (J) Number nuclei per IOM. Scale bar 200 µm, except H–H′ 20 µm.

Mentions: Given the role for myotubularins in human myopathy, and our discovery of an mtm requirement in muscle essential for fly viability [18], we investigated the contribution of mtm-dependent phosphoinositide regulation to muscle cell function and compartmentalization. Loss of mtm function using either alleles or muscle-directed RNAi had no visible effects on muscle in larvae, which remained mobile and exhibited normal body wall muscle formation, attachments and growth (Figure 1A and 1B–1B′, Figure S1A–S1C′). However, targeted RNAi depletion revealed muscle requirements for mtm at later developmental stages. Muscle-specific mtm knockdown, as indicated by protein depletion, showed either animal lethality (24B-GAL4) or developmental delay (DMef2-GAL4) around the stage of adult eclosion that was rescued by co-expression of either wildtype mtm or human MTMR2 (Figure 1A and 1C, Figure S1B).


Phosphoinositide regulation of integrin trafficking required for muscle attachment and maintenance.

Ribeiro I, Yuan L, Tanentzapf G, Dowling JJ, Kiger A - PLoS Genet. (2011)

Mtm depletion leads to myofiber detachment and morphological defects common with human myotubular myopathy.(A) Timeline of fly and muscle development, indicating stages of mtm requirements; days after puparium formation (APF). (B–B′) Normal body wall muscles in OreR and mtmΔ77/mtmz2-4747 third instar larvae. F-actin. (C) Percent viable and lethal progeny 13 days after egg lays with 24B-GAL4 and DMef2-GAL4 muscle-targeted mtm RNAi. (D–D′) Pharate dorsal abdominal muscles F-actin. Large IOMs (arrows) and smaller adult muscles (open arrowheads) span tergites (numerals). (D′) Detached IOMs (arrowheads) seen with mtm RNAi. (E–F) IOMs in filleted abdomens with 24B-GAL4 or DMef2-GAL4 expression of 1 or 2 copies of mtm RNAi hairpins. (E) Number IOMs present in tergites 3 and 4, including detached but present IOMs. (F) Number of present, visibly detached IOMs. (G–G′) Timelapse microscopy of GFP in IOMs imaged in same animals 1, 2, 3 and 4 days APF. (G′) With mtm RNAi, normal IOM formation (1d APF), survival upon histolysis of non-persistent muscles (2d APF), myofiber thinning (3d APF) and rethickening (4d APF) preceded detachment (4d APF, arrowheads). (H–H′) Individual IOMs. F-actin, red; DNA, blue. Projections, merged and nuclei images; central z-sections, F-actin. (H) Contractile myofibrils are normally tightly packed (double arrow) around linear aligned nuclei (arrowheads). (H′) With mtm RNAi, intact peripheral myofibrils surround expanded central area (double arrow) with unaligned nuclei (arrowheads). (I) Distance (µm) of nuclei from IOM midline. (J) Number nuclei per IOM. Scale bar 200 µm, except H–H′ 20 µm.
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Related In: Results  -  Collection

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pgen-1001295-g001: Mtm depletion leads to myofiber detachment and morphological defects common with human myotubular myopathy.(A) Timeline of fly and muscle development, indicating stages of mtm requirements; days after puparium formation (APF). (B–B′) Normal body wall muscles in OreR and mtmΔ77/mtmz2-4747 third instar larvae. F-actin. (C) Percent viable and lethal progeny 13 days after egg lays with 24B-GAL4 and DMef2-GAL4 muscle-targeted mtm RNAi. (D–D′) Pharate dorsal abdominal muscles F-actin. Large IOMs (arrows) and smaller adult muscles (open arrowheads) span tergites (numerals). (D′) Detached IOMs (arrowheads) seen with mtm RNAi. (E–F) IOMs in filleted abdomens with 24B-GAL4 or DMef2-GAL4 expression of 1 or 2 copies of mtm RNAi hairpins. (E) Number IOMs present in tergites 3 and 4, including detached but present IOMs. (F) Number of present, visibly detached IOMs. (G–G′) Timelapse microscopy of GFP in IOMs imaged in same animals 1, 2, 3 and 4 days APF. (G′) With mtm RNAi, normal IOM formation (1d APF), survival upon histolysis of non-persistent muscles (2d APF), myofiber thinning (3d APF) and rethickening (4d APF) preceded detachment (4d APF, arrowheads). (H–H′) Individual IOMs. F-actin, red; DNA, blue. Projections, merged and nuclei images; central z-sections, F-actin. (H) Contractile myofibrils are normally tightly packed (double arrow) around linear aligned nuclei (arrowheads). (H′) With mtm RNAi, intact peripheral myofibrils surround expanded central area (double arrow) with unaligned nuclei (arrowheads). (I) Distance (µm) of nuclei from IOM midline. (J) Number nuclei per IOM. Scale bar 200 µm, except H–H′ 20 µm.
Mentions: Given the role for myotubularins in human myopathy, and our discovery of an mtm requirement in muscle essential for fly viability [18], we investigated the contribution of mtm-dependent phosphoinositide regulation to muscle cell function and compartmentalization. Loss of mtm function using either alleles or muscle-directed RNAi had no visible effects on muscle in larvae, which remained mobile and exhibited normal body wall muscle formation, attachments and growth (Figure 1A and 1B–1B′, Figure S1A–S1C′). However, targeted RNAi depletion revealed muscle requirements for mtm at later developmental stages. Muscle-specific mtm knockdown, as indicated by protein depletion, showed either animal lethality (24B-GAL4) or developmental delay (DMef2-GAL4) around the stage of adult eclosion that was rescued by co-expression of either wildtype mtm or human MTMR2 (Figure 1A and 1C, Figure S1B).

Bottom Line: Depletion of mtm leads to increased integrin turnover at the sarcolemma and an accumulation of integrin with PI(3)P on endosomal-related membrane inclusions, indicating a role for Mtm phosphatase activity in endocytic trafficking.The depletion of Class II, but not Class III, PI3-kinase rescued mtm-dependent defects, identifying an important pathway that regulates integrin recycling.Importantly, similar integrin localization defects found in human XLMTM myofibers signify conserved MTM1 function in muscle membrane trafficking.

View Article: PubMed Central - PubMed

Affiliation: Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California, United States of America.

ABSTRACT
Muscles must maintain cell compartmentalization when remodeled during development and use. How spatially restricted adhesions are regulated with muscle remodeling is largely unexplored. We show that the myotubularin (mtm) phosphoinositide phosphatase is required for integrin-mediated myofiber attachments in Drosophila melanogaster, and that mtm-depleted myofibers exhibit hallmarks of human XLMTM myopathy. Depletion of mtm leads to increased integrin turnover at the sarcolemma and an accumulation of integrin with PI(3)P on endosomal-related membrane inclusions, indicating a role for Mtm phosphatase activity in endocytic trafficking. The depletion of Class II, but not Class III, PI3-kinase rescued mtm-dependent defects, identifying an important pathway that regulates integrin recycling. Importantly, similar integrin localization defects found in human XLMTM myofibers signify conserved MTM1 function in muscle membrane trafficking. Our results indicate that regulation of distinct phosphoinositide pools plays a central role in maintaining cell compartmentalization and attachments during muscle remodeling, and they suggest involvement of Class II PI3-kinase in MTM-related disease.

Show MeSH
Related in: MedlinePlus