Figure 6.

Caveolin-1 tubule frequency is enhanced by energy depletion and inhibited by both overexpression and inhibition of dynamin-2. Caveolin-1-GFP (Cav1; 1 μg of plasmid) was expressed in SK-BR-3 cells alone, or together with 3 μg of one of the following plasmids, as indicated: PLAP, HA-dynamin-2 (WT dynamin), HA-dynamin-2-K44A (DN dynamin), HA-ATGL, HA-ITSN1, or HA-spartin. (A) Where indicated, cells were energy-depleted or treated with dynasore as described under Materials and Methods or treated with LatA for 10 min (bottom half of graph). Cells were processed for immunofluorescence microscopy, detecting PLAP with anti-PLAP antibodies and the HA-tagged proteins with anti-HA antibodies. The percentage of cells expressing both caveolin-1-GFP and any cotransfected construct in which caveolin-1-GFP was in long tubules is shown. (B) Cells transfected with the indicated constructs were incubated with 35 μg/ml Texas Red-Tf for 5 min before fixation, and the percentage of caveolin-1-GFP–expressing cells containing internalized Tf (≥3 internal puncta) is shown. Expression of PLAP or the dynamin proteins in parallel dishes not incubated with Tf was verified by immunofluorescence microscopy; ≥85% of cells expressing caveolin-1-GFP also expressed the cotransfected construct. The mean ± SEM of at least three experiments (counting at least 150 cells on each slide) is shown.