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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 subcellular distribution of transiently overexpressed wild-type Cav1 and P132L Cav1 fusion proteins differ depending on the tag. COS-7 cells were transiently transfected with (A) Cav1-GFP, (B) Cav1-mCherry, (C) Cav1-myc, (D) P132L-GFP, (E) P132L-mCherry or (F) P132L-myc for 24 h, fixed and imaged. Cells expressing myc-tagged Cav1 constructs were immunostained prior to imaging. Bars, 10 µm.
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fig01: The subcellular distribution of transiently overexpressed wild-type Cav1 and P132L Cav1 fusion proteins differ depending on the tag. COS-7 cells were transiently transfected with (A) Cav1-GFP, (B) Cav1-mCherry, (C) Cav1-myc, (D) P132L-GFP, (E) P132L-mCherry or (F) P132L-myc for 24 h, fixed and imaged. Cells expressing myc-tagged Cav1 constructs were immunostained prior to imaging. Bars, 10 µm.

Mentions: In a previous study, we found that overexpressed wild-type Cav1 had a similar subcellular distribution as P132L, but that their distributions varied depending on the nature of the tag (EGFP, mCherry or myc) (81). When transiently expressed in COS-7 cells, Cav1-GFP and P132L-GFP primarily accumulated in the perinuclear region (Figure 1A, D) in the majority of cells (81). The localization of Cav1-mCherry and P132L-mCherry was dramatically different from their GFP counterparts (Figure 1B, E). In about 20% of cells, Cav1-mCherry was diffusely distributed on the plasma membrane and in the ER, while 80% of the cells contained a perinuclear pool of Cav1-mCherry and bright fluorescent puncta within the cytoplasm (81). In most of the P132L-mCherry-transfected cells, P132L-mCherry exists as bright fluorescent puncta with in the cytoplasm (81). Two phenotypes were also observed in Cav1-myc-transfected cells. One phenotype showed perinuclear accumulation, and the other discrete puncta (Figure 1C) (81). Here, we generated a P132L-myc construct for comparison. Consistent with a former report, all of the P132L-myc-transfected cells displayed a classical perinuclear accumulation (Figure 1F) (84). In summary, all of the overexpressed tagged wild-type Cav1 or mutant (P132L) constructs display a perinuclear accumulation phenotype to a different degree as summarized in Table 1.


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

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

The subcellular distribution of transiently overexpressed wild-type Cav1 and P132L Cav1 fusion proteins differ depending on the tag. COS-7 cells were transiently transfected with (A) Cav1-GFP, (B) Cav1-mCherry, (C) Cav1-myc, (D) P132L-GFP, (E) P132L-mCherry or (F) P132L-myc for 24 h, fixed and imaged. Cells expressing myc-tagged Cav1 constructs were immunostained prior to imaging. Bars, 10 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig01: The subcellular distribution of transiently overexpressed wild-type Cav1 and P132L Cav1 fusion proteins differ depending on the tag. COS-7 cells were transiently transfected with (A) Cav1-GFP, (B) Cav1-mCherry, (C) Cav1-myc, (D) P132L-GFP, (E) P132L-mCherry or (F) P132L-myc for 24 h, fixed and imaged. Cells expressing myc-tagged Cav1 constructs were immunostained prior to imaging. Bars, 10 µm.
Mentions: In a previous study, we found that overexpressed wild-type Cav1 had a similar subcellular distribution as P132L, but that their distributions varied depending on the nature of the tag (EGFP, mCherry or myc) (81). When transiently expressed in COS-7 cells, Cav1-GFP and P132L-GFP primarily accumulated in the perinuclear region (Figure 1A, D) in the majority of cells (81). The localization of Cav1-mCherry and P132L-mCherry was dramatically different from their GFP counterparts (Figure 1B, E). In about 20% of cells, Cav1-mCherry was diffusely distributed on the plasma membrane and in the ER, while 80% of the cells contained a perinuclear pool of Cav1-mCherry and bright fluorescent puncta within the cytoplasm (81). In most of the P132L-mCherry-transfected cells, P132L-mCherry exists as bright fluorescent puncta with in the cytoplasm (81). Two phenotypes were also observed in Cav1-myc-transfected cells. One phenotype showed perinuclear accumulation, and the other discrete puncta (Figure 1C) (81). Here, we generated a P132L-myc construct for comparison. Consistent with a former report, all of the P132L-myc-transfected cells displayed a classical perinuclear accumulation (Figure 1F) (84). In summary, all of the overexpressed tagged wild-type Cav1 or mutant (P132L) constructs display a perinuclear accumulation phenotype to a different degree as summarized in Table 1.

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