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Development of a quantitative Correlative Light Electron Microscopy technique to study GLUT4 trafficking.

Hodgson L, Tavaré J, Verkade P - Protoplasma (2014)

Bottom Line: There are many different CLEM techniques, each having its own special advantages but also its technical challenges.It is however the biological question that (should) drive(s) the development and application of a specific CLEM technique in order to provide the answer.Here we describe the development of a CLEM technique that is based on the Tokuyasu cryo immuno-gold labelling technique that has allowed us to quantitatively study GLUT4 trafficking.

View Article: PubMed Central - PubMed

Affiliation: School of Biochemistry, Medical Sciences Building, University of Bristol, University Walk, Bristol, BS8 1TD, UK.

ABSTRACT
Correlative Light Electron Microscopy (CLEM) combines advantages of light microscopy and electron microscopy in one experiment to deliver information above and beyond the capability of either modality alone. There are many different CLEM techniques, each having its own special advantages but also its technical challenges. It is however the biological question that (should) drive(s) the development and application of a specific CLEM technique in order to provide the answer. Here we describe the development of a CLEM technique that is based on the Tokuyasu cryo immuno-gold labelling technique that has allowed us to quantitatively study GLUT4 trafficking.

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Related in: MedlinePlus

Comparison of protein expression, differentiation and GLUT4 localisation in un-transfected and lentivirally transduced 3T3-L1 adipocytes. a 3T3-L1 fibroblasts and adipocytes un-transfected or transduced with pXLG3.HA.GLUT4.GFP were serum starved for 3 h and treated with or without insulin (87 nM) for 20 min prior to lysis in 0.1 % Triton buffer. Samples were separated by SDS-PAGE and analysed by Western blotting using GLUT4, AS160, p-AKT(S473), GFP and Tubulin (loading control) antibodies. b 3T3-L1 adipocytes were serum starved and stimulated in the presence and absence of insulin and fixed in 4 % PFA for 20 min. Un-transfected cells were immunostained with antibodies against endogenous GLUT4 followed by Alexa Fluor 488 secondary antibodies, whilst adipocytes stably expressing pXLG3.HA.GLUT4.GFP were analysed in fixed cells based on their GFP fluorescence. Scale bar = 8 mm
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Fig1: Comparison of protein expression, differentiation and GLUT4 localisation in un-transfected and lentivirally transduced 3T3-L1 adipocytes. a 3T3-L1 fibroblasts and adipocytes un-transfected or transduced with pXLG3.HA.GLUT4.GFP were serum starved for 3 h and treated with or without insulin (87 nM) for 20 min prior to lysis in 0.1 % Triton buffer. Samples were separated by SDS-PAGE and analysed by Western blotting using GLUT4, AS160, p-AKT(S473), GFP and Tubulin (loading control) antibodies. b 3T3-L1 adipocytes were serum starved and stimulated in the presence and absence of insulin and fixed in 4 % PFA for 20 min. Un-transfected cells were immunostained with antibodies against endogenous GLUT4 followed by Alexa Fluor 488 secondary antibodies, whilst adipocytes stably expressing pXLG3.HA.GLUT4.GFP were analysed in fixed cells based on their GFP fluorescence. Scale bar = 8 mm

Mentions: The expression levels of GLUT4, AS160, pAkt and GFP were examined by Western blotting (Fig. 1a). The data reveals that the levels of endogenous GLUT4 and AS160 increased dramatically upon differentiation into mature adipocytes (day 5) and importantly, transduction of cells with lentivirus did not significantly alter this up-regulation. In addition, the levels of pXLG3.HA.GLUT4.GFP, determined by blotting with an anti-GFP antibody, showed a similar increase in expression. The levels of these endogenous and over expressed proteins remained unchanged upon stimulation with insulin. However, stimulation with insulin did increase the amount of phosphorylated Akt in both adipocytes and fibroblasts, with higher levels seen in the latter. No difference was observed between the stable cell line and un-transfected cells, demonstrating effective insulin signalling and activation of Akt within the lentivirally transduced cell line.Fig. 1


Development of a quantitative Correlative Light Electron Microscopy technique to study GLUT4 trafficking.

Hodgson L, Tavaré J, Verkade P - Protoplasma (2014)

Comparison of protein expression, differentiation and GLUT4 localisation in un-transfected and lentivirally transduced 3T3-L1 adipocytes. a 3T3-L1 fibroblasts and adipocytes un-transfected or transduced with pXLG3.HA.GLUT4.GFP were serum starved for 3 h and treated with or without insulin (87 nM) for 20 min prior to lysis in 0.1 % Triton buffer. Samples were separated by SDS-PAGE and analysed by Western blotting using GLUT4, AS160, p-AKT(S473), GFP and Tubulin (loading control) antibodies. b 3T3-L1 adipocytes were serum starved and stimulated in the presence and absence of insulin and fixed in 4 % PFA for 20 min. Un-transfected cells were immunostained with antibodies against endogenous GLUT4 followed by Alexa Fluor 488 secondary antibodies, whilst adipocytes stably expressing pXLG3.HA.GLUT4.GFP were analysed in fixed cells based on their GFP fluorescence. Scale bar = 8 mm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig1: Comparison of protein expression, differentiation and GLUT4 localisation in un-transfected and lentivirally transduced 3T3-L1 adipocytes. a 3T3-L1 fibroblasts and adipocytes un-transfected or transduced with pXLG3.HA.GLUT4.GFP were serum starved for 3 h and treated with or without insulin (87 nM) for 20 min prior to lysis in 0.1 % Triton buffer. Samples were separated by SDS-PAGE and analysed by Western blotting using GLUT4, AS160, p-AKT(S473), GFP and Tubulin (loading control) antibodies. b 3T3-L1 adipocytes were serum starved and stimulated in the presence and absence of insulin and fixed in 4 % PFA for 20 min. Un-transfected cells were immunostained with antibodies against endogenous GLUT4 followed by Alexa Fluor 488 secondary antibodies, whilst adipocytes stably expressing pXLG3.HA.GLUT4.GFP were analysed in fixed cells based on their GFP fluorescence. Scale bar = 8 mm
Mentions: The expression levels of GLUT4, AS160, pAkt and GFP were examined by Western blotting (Fig. 1a). The data reveals that the levels of endogenous GLUT4 and AS160 increased dramatically upon differentiation into mature adipocytes (day 5) and importantly, transduction of cells with lentivirus did not significantly alter this up-regulation. In addition, the levels of pXLG3.HA.GLUT4.GFP, determined by blotting with an anti-GFP antibody, showed a similar increase in expression. The levels of these endogenous and over expressed proteins remained unchanged upon stimulation with insulin. However, stimulation with insulin did increase the amount of phosphorylated Akt in both adipocytes and fibroblasts, with higher levels seen in the latter. No difference was observed between the stable cell line and un-transfected cells, demonstrating effective insulin signalling and activation of Akt within the lentivirally transduced cell line.Fig. 1

Bottom Line: There are many different CLEM techniques, each having its own special advantages but also its technical challenges.It is however the biological question that (should) drive(s) the development and application of a specific CLEM technique in order to provide the answer.Here we describe the development of a CLEM technique that is based on the Tokuyasu cryo immuno-gold labelling technique that has allowed us to quantitatively study GLUT4 trafficking.

View Article: PubMed Central - PubMed

Affiliation: School of Biochemistry, Medical Sciences Building, University of Bristol, University Walk, Bristol, BS8 1TD, UK.

ABSTRACT
Correlative Light Electron Microscopy (CLEM) combines advantages of light microscopy and electron microscopy in one experiment to deliver information above and beyond the capability of either modality alone. There are many different CLEM techniques, each having its own special advantages but also its technical challenges. It is however the biological question that (should) drive(s) the development and application of a specific CLEM technique in order to provide the answer. Here we describe the development of a CLEM technique that is based on the Tokuyasu cryo immuno-gold labelling technique that has allowed us to quantitatively study GLUT4 trafficking.

Show MeSH
Related in: MedlinePlus