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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 is required for βPS-integrin flux from intracellular compartments and localization at sarcolemmal adhesions.(A) Schematic of individual pharate IOM and regions imaged. MTJ, myotendinous junction. (B–B″) βPS-integrin in IOM z-projections. (B) βPS-integrin at MTJs (arrow) and costameres (open arrowheads) in control. (B′–B″) With mtm RNAi, βPS-integrin was absent from detached ends (B″, arrow) and costameres, and detected on abnormal inclusions (arrowheads). (C–C′) IOM sarcolemma highlighting βPS-integrin at costameres in control (C, open arrowheads), absent with mtm RNAi (C′). (D–D′) IOM central z-sections revealing βPS-integrin punctae in control (D), and accumulation on abnormal inclusions with mtm RNAi (D′, arrowheads). DNA, blue. (E–E′) Transmission electron microscopy of IOM cross-sections, showing densely packed central regions in control (E) and large lucent membrane compartments with mtm RNAi (E′, arrowheads). (F) Averaged FRAP recovery curves and mean mobile fraction for larval βPS-integrin:YFP (int/+) in wildtype background (pink) and trans-heterozygous  mtmΔ77/mtmz2-4747 (orange). (G) Little to no βPS-integrin present at the sarcolemma in wildtype pupal IOM, 2.5 days APF. (H) βPS-integrin on central inclusions (arrowheads) detected in wildtype pupal IOM, 2.5 days APF. DNA, blue. (I–I′) βPS-integrin (red, and single channel below) at costameres in z-projections of adult abdominal lateral transversal muscles (I), and sporadically absent from costameres and dispersed in regions of myofibers with mtm RNAi (I′) in 6 day old adult flies. GFP, green; DNA, blue. DMef2-GAL4. (J) Heterozygous mtmΔ77/+ enhanced frequency of adult wing blisters in hemizygous if3/Y flies. Scale bars 10 µm, except E–E′ 1 µm.
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pgen-1001295-g002: Mtm is required for βPS-integrin flux from intracellular compartments and localization at sarcolemmal adhesions.(A) Schematic of individual pharate IOM and regions imaged. MTJ, myotendinous junction. (B–B″) βPS-integrin in IOM z-projections. (B) βPS-integrin at MTJs (arrow) and costameres (open arrowheads) in control. (B′–B″) With mtm RNAi, βPS-integrin was absent from detached ends (B″, arrow) and costameres, and detected on abnormal inclusions (arrowheads). (C–C′) IOM sarcolemma highlighting βPS-integrin at costameres in control (C, open arrowheads), absent with mtm RNAi (C′). (D–D′) IOM central z-sections revealing βPS-integrin punctae in control (D), and accumulation on abnormal inclusions with mtm RNAi (D′, arrowheads). DNA, blue. (E–E′) Transmission electron microscopy of IOM cross-sections, showing densely packed central regions in control (E) and large lucent membrane compartments with mtm RNAi (E′, arrowheads). (F) Averaged FRAP recovery curves and mean mobile fraction for larval βPS-integrin:YFP (int/+) in wildtype background (pink) and trans-heterozygous mtmΔ77/mtmz2-4747 (orange). (G) Little to no βPS-integrin present at the sarcolemma in wildtype pupal IOM, 2.5 days APF. (H) βPS-integrin on central inclusions (arrowheads) detected in wildtype pupal IOM, 2.5 days APF. DNA, blue. (I–I′) βPS-integrin (red, and single channel below) at costameres in z-projections of adult abdominal lateral transversal muscles (I), and sporadically absent from costameres and dispersed in regions of myofibers with mtm RNAi (I′) in 6 day old adult flies. GFP, green; DNA, blue. DMef2-GAL4. (J) Heterozygous mtmΔ77/+ enhanced frequency of adult wing blisters in hemizygous if3/Y flies. Scale bars 10 µm, except E–E′ 1 µm.

Mentions: Given the muscle detachment and myofibril misalignment observed in mtm mutant myofibers, we considered a possible defect in IACs at MTJs and costameres (Figure 2A–2C). We found that βPS-integrin, the single D. melanogaster β-integrin subunit encoded by mys (GenBank NM_080054), was dramatically mislocalized in mtm-depleted muscles (Figure 2B′). In contrast to wildtype muscle, βPS-integrin was absent at the ends of detached myofibers (Figure 2B″) and from costameres (Figure 2C′), consistent with detachment due to disruption of integrin adhesions. Although an intracellular pool of βPS-integrin protein was detected as small punctae within wildtype myofibers (Figure 2D), upon mtm knockdown, βPS-integrin became enriched along abnormal vacuolar inclusions within the myofiber center (Figure 2D′). Ultrastructural analyses revealed large, lucent membrane-bound compartments within the central regions of the mtm-depleted but not control myofibers (Figure 2E–2E″). Other proteins of the integrin adhesion complex, αPS2-integrin and Talin, were both detected at MTJs but not at the inclusions, suggesting βPS-integrin as a primary target of mtm function (Figure S4A–S4B′).


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 is required for βPS-integrin flux from intracellular compartments and localization at sarcolemmal adhesions.(A) Schematic of individual pharate IOM and regions imaged. MTJ, myotendinous junction. (B–B″) βPS-integrin in IOM z-projections. (B) βPS-integrin at MTJs (arrow) and costameres (open arrowheads) in control. (B′–B″) With mtm RNAi, βPS-integrin was absent from detached ends (B″, arrow) and costameres, and detected on abnormal inclusions (arrowheads). (C–C′) IOM sarcolemma highlighting βPS-integrin at costameres in control (C, open arrowheads), absent with mtm RNAi (C′). (D–D′) IOM central z-sections revealing βPS-integrin punctae in control (D), and accumulation on abnormal inclusions with mtm RNAi (D′, arrowheads). DNA, blue. (E–E′) Transmission electron microscopy of IOM cross-sections, showing densely packed central regions in control (E) and large lucent membrane compartments with mtm RNAi (E′, arrowheads). (F) Averaged FRAP recovery curves and mean mobile fraction for larval βPS-integrin:YFP (int/+) in wildtype background (pink) and trans-heterozygous  mtmΔ77/mtmz2-4747 (orange). (G) Little to no βPS-integrin present at the sarcolemma in wildtype pupal IOM, 2.5 days APF. (H) βPS-integrin on central inclusions (arrowheads) detected in wildtype pupal IOM, 2.5 days APF. DNA, blue. (I–I′) βPS-integrin (red, and single channel below) at costameres in z-projections of adult abdominal lateral transversal muscles (I), and sporadically absent from costameres and dispersed in regions of myofibers with mtm RNAi (I′) in 6 day old adult flies. GFP, green; DNA, blue. DMef2-GAL4. (J) Heterozygous mtmΔ77/+ enhanced frequency of adult wing blisters in hemizygous if3/Y flies. Scale bars 10 µm, except E–E′ 1 µm.
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Related In: Results  -  Collection

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pgen-1001295-g002: Mtm is required for βPS-integrin flux from intracellular compartments and localization at sarcolemmal adhesions.(A) Schematic of individual pharate IOM and regions imaged. MTJ, myotendinous junction. (B–B″) βPS-integrin in IOM z-projections. (B) βPS-integrin at MTJs (arrow) and costameres (open arrowheads) in control. (B′–B″) With mtm RNAi, βPS-integrin was absent from detached ends (B″, arrow) and costameres, and detected on abnormal inclusions (arrowheads). (C–C′) IOM sarcolemma highlighting βPS-integrin at costameres in control (C, open arrowheads), absent with mtm RNAi (C′). (D–D′) IOM central z-sections revealing βPS-integrin punctae in control (D), and accumulation on abnormal inclusions with mtm RNAi (D′, arrowheads). DNA, blue. (E–E′) Transmission electron microscopy of IOM cross-sections, showing densely packed central regions in control (E) and large lucent membrane compartments with mtm RNAi (E′, arrowheads). (F) Averaged FRAP recovery curves and mean mobile fraction for larval βPS-integrin:YFP (int/+) in wildtype background (pink) and trans-heterozygous mtmΔ77/mtmz2-4747 (orange). (G) Little to no βPS-integrin present at the sarcolemma in wildtype pupal IOM, 2.5 days APF. (H) βPS-integrin on central inclusions (arrowheads) detected in wildtype pupal IOM, 2.5 days APF. DNA, blue. (I–I′) βPS-integrin (red, and single channel below) at costameres in z-projections of adult abdominal lateral transversal muscles (I), and sporadically absent from costameres and dispersed in regions of myofibers with mtm RNAi (I′) in 6 day old adult flies. GFP, green; DNA, blue. DMef2-GAL4. (J) Heterozygous mtmΔ77/+ enhanced frequency of adult wing blisters in hemizygous if3/Y flies. Scale bars 10 µm, except E–E′ 1 µm.
Mentions: Given the muscle detachment and myofibril misalignment observed in mtm mutant myofibers, we considered a possible defect in IACs at MTJs and costameres (Figure 2A–2C). We found that βPS-integrin, the single D. melanogaster β-integrin subunit encoded by mys (GenBank NM_080054), was dramatically mislocalized in mtm-depleted muscles (Figure 2B′). In contrast to wildtype muscle, βPS-integrin was absent at the ends of detached myofibers (Figure 2B″) and from costameres (Figure 2C′), consistent with detachment due to disruption of integrin adhesions. Although an intracellular pool of βPS-integrin protein was detected as small punctae within wildtype myofibers (Figure 2D), upon mtm knockdown, βPS-integrin became enriched along abnormal vacuolar inclusions within the myofiber center (Figure 2D′). Ultrastructural analyses revealed large, lucent membrane-bound compartments within the central regions of the mtm-depleted but not control myofibers (Figure 2E–2E″). Other proteins of the integrin adhesion complex, αPS2-integrin and Talin, were both detected at MTJs but not at the inclusions, suggesting βPS-integrin as a primary target of mtm function (Figure S4A–S4B′).

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