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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 disrupts integrin trafficking at endosomal compartments.(A–A″) GFP:LAMP (green, and single channels) found as punctae throughout IOM controls (A) and localized to inclusions with mtm RNAi (A′, arrowheads; A″). F-actin, red; DNA, blue. (B) GFP:Rab5 (green, and single channels) found as punctae with normally little overlap with βPS-integrin (red) throughout IOM controls. (B′–B″) Rab5 partially co-localized with βPS-integrin on inclusions and accumulated at the plasma membrane and perinuclear with mtm RNAi. DNA, blue. (C–C″) PI(3)P detected by GFP:2xFYVE (green, and single channels) and βPS-integrin (red) exhibited little overlap in control (C), but co-localized on inclusions with mtm RNAi (C′–C″, arrowheads). (D–D″) PI(3)P detected by GFP:2xFYVE (green) and Dlg (red) exhibited little overlap in control (D), but co-localized on inclusions with mtm RNAi (D′–D″, arrowheads). Scale bars 10 µm, except zooms A″, C″, D″ 2.5 µm.
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pgen-1001295-g004: Mtm depletion disrupts integrin trafficking at endosomal compartments.(A–A″) GFP:LAMP (green, and single channels) found as punctae throughout IOM controls (A) and localized to inclusions with mtm RNAi (A′, arrowheads; A″). F-actin, red; DNA, blue. (B) GFP:Rab5 (green, and single channels) found as punctae with normally little overlap with βPS-integrin (red) throughout IOM controls. (B′–B″) Rab5 partially co-localized with βPS-integrin on inclusions and accumulated at the plasma membrane and perinuclear with mtm RNAi. DNA, blue. (C–C″) PI(3)P detected by GFP:2xFYVE (green, and single channels) and βPS-integrin (red) exhibited little overlap in control (C), but co-localized on inclusions with mtm RNAi (C′–C″, arrowheads). (D–D″) PI(3)P detected by GFP:2xFYVE (green) and Dlg (red) exhibited little overlap in control (D), but co-localized on inclusions with mtm RNAi (D′–D″, arrowheads). Scale bars 10 µm, except zooms A″, C″, D″ 2.5 µm.

Mentions: The disrupted βPS-integrin localization together with the enlarged membrane inclusions suggested defective membrane trafficking in mtm mutant myofibers. Characterization of the central inclusions could point to a specific compartment or trafficking step that normally requires Mtm phosphatase activity in muscle remodeling. The inclusions did not noticeably contain markers of endoplasmic reticulum, the trans-Golgi network or autophagosomes (Figure S5E–S5E′ KDEL; Figure S5F–S5F′ PH-FAPP1; Figure S5G–S5G′ Atg8). In contrast, the majority of inclusions were decorated by the endosome-lysosomal marker, GFP:LAMP (Figure 4A–4A″). The inclusions were frequently colocalized with an indicator of early endosomes, GFP:Rab5 (Figure 4B–4B″), and infrequently by the Rab5 effector, Rbsn5 (Figure S5H–S5H′), but not an indicator of late endosome identity, GFP:Rab7 (Figure S5I–S5I′). Together, these results suggest a relationship between the inclusions and early endocytic traffic, and that mtm depletion disrupts endocytic traffic.


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 disrupts integrin trafficking at endosomal compartments.(A–A″) GFP:LAMP (green, and single channels) found as punctae throughout IOM controls (A) and localized to inclusions with mtm RNAi (A′, arrowheads; A″). F-actin, red; DNA, blue. (B) GFP:Rab5 (green, and single channels) found as punctae with normally little overlap with βPS-integrin (red) throughout IOM controls. (B′–B″) Rab5 partially co-localized with βPS-integrin on inclusions and accumulated at the plasma membrane and perinuclear with mtm RNAi. DNA, blue. (C–C″) PI(3)P detected by GFP:2xFYVE (green, and single channels) and βPS-integrin (red) exhibited little overlap in control (C), but co-localized on inclusions with mtm RNAi (C′–C″, arrowheads). (D–D″) PI(3)P detected by GFP:2xFYVE (green) and Dlg (red) exhibited little overlap in control (D), but co-localized on inclusions with mtm RNAi (D′–D″, arrowheads). Scale bars 10 µm, except zooms A″, C″, D″ 2.5 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3037412&req=5

pgen-1001295-g004: Mtm depletion disrupts integrin trafficking at endosomal compartments.(A–A″) GFP:LAMP (green, and single channels) found as punctae throughout IOM controls (A) and localized to inclusions with mtm RNAi (A′, arrowheads; A″). F-actin, red; DNA, blue. (B) GFP:Rab5 (green, and single channels) found as punctae with normally little overlap with βPS-integrin (red) throughout IOM controls. (B′–B″) Rab5 partially co-localized with βPS-integrin on inclusions and accumulated at the plasma membrane and perinuclear with mtm RNAi. DNA, blue. (C–C″) PI(3)P detected by GFP:2xFYVE (green, and single channels) and βPS-integrin (red) exhibited little overlap in control (C), but co-localized on inclusions with mtm RNAi (C′–C″, arrowheads). (D–D″) PI(3)P detected by GFP:2xFYVE (green) and Dlg (red) exhibited little overlap in control (D), but co-localized on inclusions with mtm RNAi (D′–D″, arrowheads). Scale bars 10 µm, except zooms A″, C″, D″ 2.5 µm.
Mentions: The disrupted βPS-integrin localization together with the enlarged membrane inclusions suggested defective membrane trafficking in mtm mutant myofibers. Characterization of the central inclusions could point to a specific compartment or trafficking step that normally requires Mtm phosphatase activity in muscle remodeling. The inclusions did not noticeably contain markers of endoplasmic reticulum, the trans-Golgi network or autophagosomes (Figure S5E–S5E′ KDEL; Figure S5F–S5F′ PH-FAPP1; Figure S5G–S5G′ Atg8). In contrast, the majority of inclusions were decorated by the endosome-lysosomal marker, GFP:LAMP (Figure 4A–4A″). The inclusions were frequently colocalized with an indicator of early endosomes, GFP:Rab5 (Figure 4B–4B″), and infrequently by the Rab5 effector, Rbsn5 (Figure S5H–S5H′), but not an indicator of late endosome identity, GFP:Rab7 (Figure S5I–S5I′). Together, these results suggest a relationship between the inclusions and early endocytic traffic, and that mtm depletion disrupts endocytic traffic.

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