![Figure 5. pVHL reduces intrinsic GTPase activity of MTs, resulting in higher frequencies of GTP caps and GTP remnants along the MT lattice. (A) MAP-purified tubulin was incubated with GST-VHL30, GST-VHL30(Δ95–123), or GST. Release of free 32P from γ-[32P]GTP was measured at the indicated time points. Error bars represent SD of n = 9 or n = 3 independent measurements for GST and GST-VHL30 or GST-VHL30(Δ95–123), respectively (GST vs. GST-VHL30(Δ95–123), NS; *, P = 0.04 and 0.01 [60 min]; *, P = 0.003 and 0.0007 [90 min]; *, P = 7 × 10−5 and 0.01 [180 min]; Student’s t test [GST vs. GST-VHL30 and GST-VHL30(Δ95–123) vs. GST-VHL30]). (B) Frequency of GTP-capped MTs in RCC-4 cells expressing no VHL (VHL−/−), wild-type long form (VHL30), or wild-type short form (VHL19). Mean ± SD is shown (***, P = 3.3 × 10−7). (C) Scatter plot of shrinkage probability (see legend for Fig. 4 C) to GTP cap frequency (percentage of GTP-capped MTs) in RCC-4 cells expressing various naturally occurring VHL point mutants (Fig. 4 A; mean of n = 12 cells from three independent stainings, a total of >250 measurements per condition; Spearman correlation). (D) Distance between GTP-tubulin remnants in RCC-4 cells expressing no VHL (VHL−/−), wild-type long form (VHL30), or wild-type short form (VHL19; mean ± SD of n = 12 cells from three independent stainings; *, P = 0.03; ***, P = 3.4 × 10−6). (E) Scatter plot of relative time in shrinkage (see legend for Fig. 4 C) to GTP remnant distance in RCC-4 cells expressing various naturally occurring VHL point mutants (Fig. 4 A; mean of n = 12 cells from three independent stainings, a total of >500 measurements per condition; Spearman correlation). (F) Immunofluorescence costaining with anti–α-tubulin (green) and anti–GTP-tubulin (red) of RCC-4 cells reconstituted with VHL30 or the empty vector as control (VHL−/−). Zoomed areas (right) are indicated by a dashed square in the panels to the left. Yellow arrows show GTP-capped MTs. Red lines indicate perinuclear area where the GTP remnant signal was measured. Bars: (left) 10 µm; (right) 2 µm. (G) GTP remnant signal intensity relative to tubulin intensity of RCC-4 cells reconstituted with VHL30 or the empty vector as control (VHL−/−; mean ± SD of n = 9 cells from three independent stainings; *, P < 0.05; Student’s t test).](https://cdn.rupress.org/rup/content_public/journal/jcb/190/6/10.1083_jcb.201006059/9/jcb_201006059_rgb_fig5.jpeg?Expires=1771348628&Signature=ZE--fJeQ1MtM9hM4t2tNTACeqYJbpNnN6Gnr-eGigyA8GUAuTffztitmXevEwc3U~~5oO-HZTy1L3NF29NZ5u~z8e3tlkZMJEZLeBmhKqPtsBA0-XVHk~s2wlTTEzzMVwHDcqbcBVR~LwX2yf8q1MkzB0PH2YZ~VTpPwvgSNXv-zm9xvUO8JV5jRLXl1KzLapNOXAINInV3pzqbkd41v6ILnui-eYP4plEntgzTrleJPrgnz~vHDjNOWX2mAaCljPcfN66NPt-J1RE60PfZjUInHb6QyKldoLxs2jgYLoJDlgTp~9LZCxzBE6jxWUGnk3R-wY-vsYMqK52s~3Soklg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
pVHL reduces intrinsic GTPase activity of MTs, resulting in higher frequencies of GTP caps and GTP remnants along the MT lattice. (A) MAP-purified tubulin was incubated with GST-VHL30, GST-VHL30(Δ95–123), or GST. Release of free 32P from γ-[32P]GTP was measured at the indicated time points. Error bars represent SD of n = 9 or n = 3 independent measurements for GST and GST-VHL30 or GST-VHL30(Δ95–123), respectively (GST vs. GST-VHL30(Δ95–123), NS; *, P = 0.04 and 0.01 [60 min]; *, P = 0.003 and 0.0007 [90 min]; *, P = 7 × 10−5 and 0.01 [180 min]; Student’s t test [GST vs. GST-VHL30 and GST-VHL30(Δ95–123) vs. GST-VHL30]). (B) Frequency of GTP-capped MTs in RCC-4 cells expressing no VHL (VHL−/−), wild-type long form (VHL30), or wild-type short form (VHL19). Mean ± SD is shown (***, P = 3.3 × 10−7). (C) Scatter plot of shrinkage probability (see legend for Fig. 4 C) to GTP cap frequency (percentage of GTP-capped MTs) in RCC-4 cells expressing various naturally occurring VHL point mutants (Fig. 4 A; mean of n = 12 cells from three independent stainings, a total of >250 measurements per condition; Spearman correlation). (D) Distance between GTP-tubulin remnants in RCC-4 cells expressing no VHL (VHL−/−), wild-type long form (VHL30), or wild-type short form (VHL19; mean ± SD of n = 12 cells from three independent stainings; *, P = 0.03; ***, P = 3.4 × 10−6). (E) Scatter plot of relative time in shrinkage (see legend for Fig. 4 C) to GTP remnant distance in RCC-4 cells expressing various naturally occurring VHL point mutants (Fig. 4 A; mean of n = 12 cells from three independent stainings, a total of >500 measurements per condition; Spearman correlation). (F) Immunofluorescence costaining with anti–α-tubulin (green) and anti–GTP-tubulin (red) of RCC-4 cells reconstituted with VHL30 or the empty vector as control (VHL−/−). Zoomed areas (right) are indicated by a dashed square in the panels to the left. Yellow arrows show GTP-capped MTs. Red lines indicate perinuclear area where the GTP remnant signal was measured. Bars: (left) 10 µm; (right) 2 µm. (G) GTP remnant signal intensity relative to tubulin intensity of RCC-4 cells reconstituted with VHL30 or the empty vector as control (VHL−/−; mean ± SD of n = 9 cells from three independent stainings; *, P < 0.05; Student’s t test).
