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Tagging strategies strongly affect the fate of overexpressed caveolin-1.

Han B, Tiwari A, Kenworthy AK - Traffic (2015)

Bottom Line: A significant amount of our current knowledge about caveolins and caveolae is derived from studies of transiently overexpressed, C-terminally tagged caveolin proteins.These findings suggest that differences in tagging strategies may be a source of variation in previously published studies of Cav1 and that overexpressed Cav1 may exert functional effects outside of caveolae.They also highlight the need for a critical re-evaluation of current knowledge based on transient overexpression of tagged Cav1.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN, USA.

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The aggregates of tagged wild-type Cav1 are mainly composed of 8S-like complexes. COS-7 cells expressing (A, B) Cav1-myc, (C, D) Cav1-GFP, (E, F) Cav1-mCherry, (G, H) P132L-myc or (I, J) P132L-GFP were lysed in 0.2% Triton-X-100 and 0.4% SDS at room temperature. Extracts were run through 10–40% sucrose velocity gradients and fractions were analyzed by SDS–PAGE/western blot (A, C, E, G, I) and the levels of overexpressed Cav1 and endogenous Cav1 in each fraction were quantified by densitometry (B, D, F, H, J). The position of the 8S complex containing endogenous Cav1 is indicated by black arrows. Bars represent mean ± SD for two independent experiments.
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fig09: The aggregates of tagged wild-type Cav1 are mainly composed of 8S-like complexes. COS-7 cells expressing (A, B) Cav1-myc, (C, D) Cav1-GFP, (E, F) Cav1-mCherry, (G, H) P132L-myc or (I, J) P132L-GFP were lysed in 0.2% Triton-X-100 and 0.4% SDS at room temperature. Extracts were run through 10–40% sucrose velocity gradients and fractions were analyzed by SDS–PAGE/western blot (A, C, E, G, I) and the levels of overexpressed Cav1 and endogenous Cav1 in each fraction were quantified by densitometry (B, D, F, H, J). The position of the 8S complex containing endogenous Cav1 is indicated by black arrows. Bars represent mean ± SD for two independent experiments.

Mentions: A large fraction of the Cav1-FPs and Cav1-myc was found in high molecular weight aggregates in the velocity gradient centrifugation experiments (Figure 8). To further investigate the nature of these aggregates, prior to the velocity gradient centrifugation, we lysed the cells with a combined detergent solution containing 0.2% Triton-X-100 and 0.4% SDS previously shown to disassemble the 70S complexes (60). Under these conditions, Cav1-myc and Cav1-mCherry disassembled into 8S-like complexes (Figure 9A, B, E, F), whereas for Cav1-GFP, a combination of monomers or small oligomers and 8S-like complexes was observed (Figure 9C,D). The GFP tag may, thus, partially interfere with the formation of 8S-like oligomers. Consistent with previous findings that P132L tends to oligomerize poorly (60,84), the P132L constructs dissociated into low molecular weight oligomers (Figures 9G–J and S4B). These findings suggest that although both wild-type and P132L form irregular aggregates and high molecular weight oligomers, the aggregates are formed from different building blocks.


Tagging strategies strongly affect the fate of overexpressed caveolin-1.

Han B, Tiwari A, Kenworthy AK - Traffic (2015)

The aggregates of tagged wild-type Cav1 are mainly composed of 8S-like complexes. COS-7 cells expressing (A, B) Cav1-myc, (C, D) Cav1-GFP, (E, F) Cav1-mCherry, (G, H) P132L-myc or (I, J) P132L-GFP were lysed in 0.2% Triton-X-100 and 0.4% SDS at room temperature. Extracts were run through 10–40% sucrose velocity gradients and fractions were analyzed by SDS–PAGE/western blot (A, C, E, G, I) and the levels of overexpressed Cav1 and endogenous Cav1 in each fraction were quantified by densitometry (B, D, F, H, J). The position of the 8S complex containing endogenous Cav1 is indicated by black arrows. Bars represent mean ± SD for two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440517&req=5

fig09: The aggregates of tagged wild-type Cav1 are mainly composed of 8S-like complexes. COS-7 cells expressing (A, B) Cav1-myc, (C, D) Cav1-GFP, (E, F) Cav1-mCherry, (G, H) P132L-myc or (I, J) P132L-GFP were lysed in 0.2% Triton-X-100 and 0.4% SDS at room temperature. Extracts were run through 10–40% sucrose velocity gradients and fractions were analyzed by SDS–PAGE/western blot (A, C, E, G, I) and the levels of overexpressed Cav1 and endogenous Cav1 in each fraction were quantified by densitometry (B, D, F, H, J). The position of the 8S complex containing endogenous Cav1 is indicated by black arrows. Bars represent mean ± SD for two independent experiments.
Mentions: A large fraction of the Cav1-FPs and Cav1-myc was found in high molecular weight aggregates in the velocity gradient centrifugation experiments (Figure 8). To further investigate the nature of these aggregates, prior to the velocity gradient centrifugation, we lysed the cells with a combined detergent solution containing 0.2% Triton-X-100 and 0.4% SDS previously shown to disassemble the 70S complexes (60). Under these conditions, Cav1-myc and Cav1-mCherry disassembled into 8S-like complexes (Figure 9A, B, E, F), whereas for Cav1-GFP, a combination of monomers or small oligomers and 8S-like complexes was observed (Figure 9C,D). The GFP tag may, thus, partially interfere with the formation of 8S-like oligomers. Consistent with previous findings that P132L tends to oligomerize poorly (60,84), the P132L constructs dissociated into low molecular weight oligomers (Figures 9G–J and S4B). These findings suggest that although both wild-type and P132L form irregular aggregates and high molecular weight oligomers, the aggregates are formed from different building blocks.

Bottom Line: A significant amount of our current knowledge about caveolins and caveolae is derived from studies of transiently overexpressed, C-terminally tagged caveolin proteins.These findings suggest that differences in tagging strategies may be a source of variation in previously published studies of Cav1 and that overexpressed Cav1 may exert functional effects outside of caveolae.They also highlight the need for a critical re-evaluation of current knowledge based on transient overexpression of tagged Cav1.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN, USA.

Show MeSH
Related in: MedlinePlus