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Title: Septin 7 reduces nonmuscle myosin IIA activity in the SNAP23 complex and hinders GLUT4 storage vesicle docking and fusion

Abstract

Glomerular epithelial cells, podocytes, are insulin responsive and can develop insulin resistance. Here, we demonstrate that the small GTPase septin 7 forms a complex with nonmuscle myosin heavy chain IIA (NMHC-IIA; encoded by MYH9), a component of the nonmuscle myosin IIA (NM-IIA) hexameric complex. We observed that knockdown of NMHC-IIA decreases insulin-stimulated glucose uptake into podocytes. Both septin 7 and NM-IIA associate with SNAP23, a SNARE protein involved in GLUT4 storage vesicle (GSV) docking and fusion with the plasma membrane. We observed that insulin decreases the level of septin 7 and increases the activity of NM-IIA in the SNAP23 complex, as visualized by increased phosphorylation of myosin regulatory light chain. Also knockdown of septin 7 increases the activity of NM-IIA in the complex. The activity of NM-IIA is increased in diabetic rat glomeruli and cultured human podocytes exposed to macroalbuminuric sera from patients with type 1 diabetes. Collectively, the data suggest that the activity of NM-IIA in the SNAP23 complex plays a key role in insulin-stimulated glucose uptake into podocytes. Furthermore, we observed that septin 7 reduces the activity of NM-IIA in the SNAP23 complex and thereby hinders GSV docking and fusion with the plasma membrane. - Highlights: • Septinmore » 7, nonmuscle myosin heavy chain IIA (NMHC-IIA) and SNAP23 form a complex. • Knockdown of septin 7 increases NM-IIA activity in the SNAP23 complex. • Insulin decreases septin 7 level and increases NM-IIA activity in the SNAP23 complex. • Septin 7 hinders GSV docking/fusion by reducing NM-IIA activity in the SNAP23 complex.« less

Authors:
;  [1];  [2];  [3]; ; ;  [4];  [5];  [5];  [1];  [5];  [4];  [5];  [5];  [6];  [1]
  1. Department of Pathology, University of Helsinki, 00014 Helsinki (Finland)
  2. Department of Pathology, University of Helsinki and Helsinki University Hospital, 00290 Helsinki, 05850 Hyvinkää (Finland)
  3. Institute of Biotechnology, University of Helsinki, 00014 Helsinki (Finland)
  4. Folkhälsan Institute of Genetics, Folkhälsan Research Center, 00290 Helsinki (Finland)
  5. (Finland)
  6. (Australia)
Publication Date:
OSTI Identifier:
22649814
Resource Type:
Journal Article
Resource Relation:
Journal Name: Experimental Cell Research; Journal Volume: 350; Journal Issue: 2; Other Information: Copyright (c) 2016 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; CELL MEMBRANES; COMPLEXES; GLOMERULI; GLUCOSE; INSULIN; MYOSIN; PATIENTS; PHOSPHORYLATION; RATS; RECEPTORS; UPTAKE

Citation Formats

Wasik, Anita A., Dumont, Vincent, Tienari, Jukka, Nyman, Tuula A., Fogarty, Christopher L., Forsblom, Carol, Lehto, Markku, Abdominal Center Nephrology, University of Helsinki and Helsinki University Hospital, 000290 Helsinki, Diabetes&Obesity Research Program, Research Program´s Unit, 00014 University of Helsinki, Lehtonen, Eero, Laboratory Animal Centre, University of Helsinki, 00014 Helsinki, Groop, Per-Henrik, Abdominal Center Nephrology, University of Helsinki and Helsinki University Hospital, 000290 Helsinki, Diabetes&Obesity Research Program, Research Program´s Unit, 00014 University of Helsinki, Baker IDI Heart & Diabetes Institute, 3004 Melbourne, and Lehtonen, Sanna, E-mail: sanna.h.lehtonen@helsinki.fi. Septin 7 reduces nonmuscle myosin IIA activity in the SNAP23 complex and hinders GLUT4 storage vesicle docking and fusion. United States: N. p., 2017. Web. doi:10.1016/J.YEXCR.2016.12.010.
Wasik, Anita A., Dumont, Vincent, Tienari, Jukka, Nyman, Tuula A., Fogarty, Christopher L., Forsblom, Carol, Lehto, Markku, Abdominal Center Nephrology, University of Helsinki and Helsinki University Hospital, 000290 Helsinki, Diabetes&Obesity Research Program, Research Program´s Unit, 00014 University of Helsinki, Lehtonen, Eero, Laboratory Animal Centre, University of Helsinki, 00014 Helsinki, Groop, Per-Henrik, Abdominal Center Nephrology, University of Helsinki and Helsinki University Hospital, 000290 Helsinki, Diabetes&Obesity Research Program, Research Program´s Unit, 00014 University of Helsinki, Baker IDI Heart & Diabetes Institute, 3004 Melbourne, & Lehtonen, Sanna, E-mail: sanna.h.lehtonen@helsinki.fi. Septin 7 reduces nonmuscle myosin IIA activity in the SNAP23 complex and hinders GLUT4 storage vesicle docking and fusion. United States. doi:10.1016/J.YEXCR.2016.12.010.
Wasik, Anita A., Dumont, Vincent, Tienari, Jukka, Nyman, Tuula A., Fogarty, Christopher L., Forsblom, Carol, Lehto, Markku, Abdominal Center Nephrology, University of Helsinki and Helsinki University Hospital, 000290 Helsinki, Diabetes&Obesity Research Program, Research Program´s Unit, 00014 University of Helsinki, Lehtonen, Eero, Laboratory Animal Centre, University of Helsinki, 00014 Helsinki, Groop, Per-Henrik, Abdominal Center Nephrology, University of Helsinki and Helsinki University Hospital, 000290 Helsinki, Diabetes&Obesity Research Program, Research Program´s Unit, 00014 University of Helsinki, Baker IDI Heart & Diabetes Institute, 3004 Melbourne, and Lehtonen, Sanna, E-mail: sanna.h.lehtonen@helsinki.fi. Sun . "Septin 7 reduces nonmuscle myosin IIA activity in the SNAP23 complex and hinders GLUT4 storage vesicle docking and fusion". United States. doi:10.1016/J.YEXCR.2016.12.010.
@article{osti_22649814,
title = {Septin 7 reduces nonmuscle myosin IIA activity in the SNAP23 complex and hinders GLUT4 storage vesicle docking and fusion},
author = {Wasik, Anita A. and Dumont, Vincent and Tienari, Jukka and Nyman, Tuula A. and Fogarty, Christopher L. and Forsblom, Carol and Lehto, Markku and Abdominal Center Nephrology, University of Helsinki and Helsinki University Hospital, 000290 Helsinki and Diabetes&Obesity Research Program, Research Program´s Unit, 00014 University of Helsinki and Lehtonen, Eero and Laboratory Animal Centre, University of Helsinki, 00014 Helsinki and Groop, Per-Henrik and Abdominal Center Nephrology, University of Helsinki and Helsinki University Hospital, 000290 Helsinki and Diabetes&Obesity Research Program, Research Program´s Unit, 00014 University of Helsinki and Baker IDI Heart & Diabetes Institute, 3004 Melbourne and Lehtonen, Sanna, E-mail: sanna.h.lehtonen@helsinki.fi},
abstractNote = {Glomerular epithelial cells, podocytes, are insulin responsive and can develop insulin resistance. Here, we demonstrate that the small GTPase septin 7 forms a complex with nonmuscle myosin heavy chain IIA (NMHC-IIA; encoded by MYH9), a component of the nonmuscle myosin IIA (NM-IIA) hexameric complex. We observed that knockdown of NMHC-IIA decreases insulin-stimulated glucose uptake into podocytes. Both septin 7 and NM-IIA associate with SNAP23, a SNARE protein involved in GLUT4 storage vesicle (GSV) docking and fusion with the plasma membrane. We observed that insulin decreases the level of septin 7 and increases the activity of NM-IIA in the SNAP23 complex, as visualized by increased phosphorylation of myosin regulatory light chain. Also knockdown of septin 7 increases the activity of NM-IIA in the complex. The activity of NM-IIA is increased in diabetic rat glomeruli and cultured human podocytes exposed to macroalbuminuric sera from patients with type 1 diabetes. Collectively, the data suggest that the activity of NM-IIA in the SNAP23 complex plays a key role in insulin-stimulated glucose uptake into podocytes. Furthermore, we observed that septin 7 reduces the activity of NM-IIA in the SNAP23 complex and thereby hinders GSV docking and fusion with the plasma membrane. - Highlights: • Septin 7, nonmuscle myosin heavy chain IIA (NMHC-IIA) and SNAP23 form a complex. • Knockdown of septin 7 increases NM-IIA activity in the SNAP23 complex. • Insulin decreases septin 7 level and increases NM-IIA activity in the SNAP23 complex. • Septin 7 hinders GSV docking/fusion by reducing NM-IIA activity in the SNAP23 complex.},
doi = {10.1016/J.YEXCR.2016.12.010},
journal = {Experimental Cell Research},
number = 2,
volume = 350,
place = {United States},
year = {Sun Jan 15 00:00:00 EST 2017},
month = {Sun Jan 15 00:00:00 EST 2017}
}
  • In adipocytes and myocytes, insulin stimulation translocates glucose transporter 4 (Glut4) storage vesicles (GSVs) from their intracellular storage sites to the plasma membrane (PM) where they dock with the PM. Then, Glut4 is inserted into the PM and initiates glucose uptake into these cells. Previous studies using chemical inhibitors demonstrated that myosin II participates in fusion of GSVs and the PM and increase in the intrinsic activity of Glut4. In this study, the effect of myosin IIA on GSV trafficking was examined by knocking down myosin IIA expression. Myosin IIA knockdown decreased both glucose uptake and exposures of myc-tagged Glut4more » to the cell surface in insulin-stimulated cells, but did not affect insulin signal transduction. Interestingly, myosin IIA knockdown failed to decrease insulin-dependent trafficking of Glut4 to the PM. Moreover, in myosin IIA knockdown cells, insulin-stimulated binding of GSV SNARE protein, vesicle-associated membrane protein 2 (VAMP2) to PM SNARE protein, syntaxin 4 was inhibited. These data suggest that myosin IIA plays a role in insulin-stimulated docking of GSVs to the PM in 3T3-L1 adipocytes through SNARE complex formation.« less
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  • In this study, we investigated the relative participation of N-ethylmaleimide-sensitive factor (NSF) in vivo in a complex multistep vesicle trafficking system, the translocation response of GLUT4 to insulin in rat adipose cells. Transfections of rat adipose cells demonstrate that over-expression of wild-type NSF has no effect on total, or basal and insulin-stimulated cell-surface expression of HA-tagged GLUT4. In contrast, a dominant-negative NSF (NSF-D1EQ) can be expressed at a low enough level that it has little effect on total HA-GLUT4, but does reduce both basal and insulin-stimulated cell-surface HA-GLUT4 by {approx}50% without affecting the GLUT4 fold-translocation response to insulin. However, highmore » expression levels of NSF-D1EQ decrease total HA-GLUT4. The inhibitory effect of NSF-D1EQ on cell-surface HA-GLUT4 is reversed when endocytosis is inhibited by co-expression of a dominant-negative dynamin (dynamin-K44A). Moreover, NSF-D1EQ does not affect cell-surface levels of constitutively recycling GLUT1 and TfR, suggesting a predominant effect of low-level NSF-D1EQ on the trafficking of GLUT4 from the endocytic recycling compared to the intracellular GLUT4-specific compartment. Thus, our data demonstrate that the multiple fusion steps in GLUT4 trafficking have differential quantitative requirements for NSF activity. This indicates that the rates of plasma and intracellular membrane fusion reactions vary, leading to differential needs for the turnover of the SNARE proteins.« less
  • Research highlights: {yields} Vimentin is shown to bind to the N-terminus of insulin-responsive aminopeptidase (IRAP), a major cargo protein of GLUT4 vesicles in 3T3-L1 adipocytes. {yields} GLUT4 translocation to the plasma membrane by insulin is decreased in vimentin-depleted adipocytes. {yields} An interaction between vimentin and IRAP functions to sequester GLUT4 vesicles to the peri-nuclear region of the cell. -- Abstract: Insulin-responsive aminopeptidase (IRAP) and GLUT4 are two major cargo proteins of GLUT4 storage vesicles (GSVs) that are translocated from a postendosomal storage compartment to the plasma membrane (PM) in response to insulin. The cytoplasmic region of IRAP is reportedly involvedmore » in retention of GSVs. In this study, vimentin was identified using the cytoplasmic domain of IRAP as bait. The validity of this interaction was confirmed by pull-down assays and immunoprecipitation in 3T3-L1 adipocytes. In addition, it was shown that GLUT4 translocation to the PM by insulin was decreased in vimentin-depleted adipocytes, presumably due to dispersing GSVs away from the cytoskeleton. These findings suggest that the IRAP binding protein, vimentin, plays an important role in retention of GSVs.« less
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