Decreased uptake of Vaccinia virus is an indicator of reduced macropinocytosis.

<p>(A) Wild type (Wt) and DGKζ-null (Null) fibroblasts were infected with double-deleted vaccinia virus mature virions engineered to express GFP. GFP expression was assayed by western blotting Tubulin was used as a loading control. (B) Quantification of GFP expression in Wt and Null cells. Values are the mean ± S.E.M. of three independent experiments. The asterisk indicates a statistically significant difference from Wt (p<0.001) by Student’s t-test. (C) Rescue of GFP expression in null cells infected with adenovirus bearing HA-tagged wild type (wt) DGKζ, a kinase-dead mutant (kd), or the MARCKS domain phosphomimetic mutant (M1). GFP expression was assayed by western blotting as in (A) following infection with vaccinia virus. The graph shows the quantification of GFP expression. Statistical analysis was performed by a one-way ANOVA followed by a Holm-Sidak post-hoc multiple comparison test. Asterisks denote a significant difference from uninfected null cells and hashes indicate a significant difference from null cells expressing DGKζ^kd. * or # = p < 0.05, ** or ## = p < 0.01, *** or ### = p < 0.005. (D) Effect of inhibitors on GFP expression. Wild type fibroblasts infected with VV-GFP were treated either with vehicle alone (DMSO), 100 uM amiloride, 200 uM of the Rac inhibitor NSC 23766, or 40 uM of the PAK1 inhibitor IPA-3. The arrow indicates a GFP band present in lysates of infected, but not uninfected, cells. The graph shows the quantification of western blots from two independent experiments. The asterisk indicates a statistically significant difference (p<0.05) from control (VV-GFP + DMSO).</p

Rescue of PDGF-induced macropinocytosis.

<p>DGKζ-null cells were infected with adenoviruses bearing HA-tagged versions of wild type (wt), kinase dead (kd), or phosphomimetic (M1) DGKζ. (A) Representative images of infected cells with (+ PDGF) and without (- PDGF) stimulation. Insets show magnified images of the regions indicated by the white boxes. Scale bars, 20 um. (B—C) Quantification of macropinocytosis in infected DGKζ-null cells. The graphs show the percentage of cells with macropinosomes (B) and the mean macropinosome size (C). Values are the mean ± SEM from three independent experiments. Asterisks denote a significant difference (p<0.05) from kd as determined by a one-way analysis of variance, followed by a Tukey post hoc multiple-comparison test. The western blot shows equivalent levels of HA-DGKζ expression in lysates of infected cells. The lanes correspond to the bars in the graphs above and below.</p

YFP-DGKζ Localization During Macropinosome Biogenesis.

<p>Wild type fibroblast cells were cotransfected with plasmids encoding the N-terminal membrane-targeting domain of neuromodulin fused to the N-terminus of mCherry (NMTD-mCherry) and either YFP-DGKζ or YFP alone. Cells stimulated with 50 ηg/ml PDGF were visualized by time-lapse video microscopy. Shown are representative images of YFP-DGKζ (A-E) and NMTD-mCherry (A’-E’) localization in a single optical plane from a time-lapse sequence during various stages of macropinosome biogenesis including: the formation of an irregular ruffle (A and B, horizontal arrows), transition to a curved ruffle (A, vertical arrow and C, horizontal arrow), closure into a circular ruffle or membrane cup (B and C, vertical arrows), and cup closure (D, vertical arrow and E, arrows). Note the high concentration of YFP-DGKζ surrounding the newly formed macropinosomes (D” and E”, vertical arrows). (F-F”) YFP alone also appears to be concentrated around newly formed macropinosomes. Scale bars = 10 um.</p

Quantitative analysis of macropinosome size in fibroblasts co-expressing Rac1^V12 and DGKζ constructs.

<p>(A) Representative images of wild type MEFs transfected with myc-Rac1^V12 alone or cotransfected with the indicated HA-tagged DGKζ construct. Scale bars = 20 um. Magnified images of the boxed regions are shown at the right. Scale bars = 5 um. (B) Graph showing the average macropinosome size in wild type MEFs expressing Rac1^V12 alone (-) or Rac1^V12 and the indicated DGKζ constructs (wt, kd or M1). Values are the mean ± SEM from three independent experiments. Statistical analysis was performed by a one-way ANOVA followed by a Tukey post-hoc multiple comparison test. Asterisks denote a significant difference (p<0.05) between the indicated conditions. (C) Graph showing the cumulative frequency distribution of macropinosome size (um²). The inset shows the distribution of macropinosomes between 0 and 5 um².</p

Quantitative analysis of macropinocytosis in living wild type and DGKζ-null fibroblasts.

<p>(A) Representative phase-contrast images captured from time-lapse video microscopy of PDGF-stimulated, wild type fibroblasts show macropinosomes (arrowheads) forming from membrane ruffles (white arrows). Two separate sequences (top and bottom) are shown from a 30 min video. Scale bar, 10 μm. (B-D) Graphs showing the percentage of cells with macropinosomes (B), the number of macropinosomes per cell (C) and the mean macropinosome size (D) in wild type and DGKζ-null fibroblasts following 30 min PDGF stimulation. Values are the mean ± SEM from at least four independent experiments. A single asterisk denotes a significant difference (p<0.05) and two asterisks, a highly significant difference (p<0.01), between wild type and null cells by Student’s t test. (E) Cumulative frequency distribution showing the distribution of macropinosome sizes (defined as the two-dimensional area) in PDGF-stimulated wild type and null cells.</p

Quantitative analysis of YFP-DGKζ Localization During Macropinocytosis.

<p>Ratiometric images of wild type and DGKζ-null cells expressing NMTD-mCherry and either YFP-DGKζ, or YFP alone, taken approximately 15–20 seconds after ruffle closure were analyzed as described in Materials and Methods. Individual pixel intensity ratios were calculated for regions immediately surrounding the macropinosomes. The intensity ratios for YFP/NMTD-mCherry (gray bars) and YFP-DGKζ/NMTD-mCherry (black bars) were sorted into bins and plotted as probability distributions. The data were modeled by a three-parameter log-normal distribution (red and blue lines, respectively). Pixel ratios greater than 1 indicate the protein is more concentrated than the mCherry-NMTD membrane marker, while values less than 1 indicate a lower concentration. Asterisks indicate a significant difference in the percentages of pixels in each bin with the indicated intensity ratio.</p

Quantitative analysis of macropinosome size in fibroblasts co-expressing Rac1^V12 and DGKζ constructs.

<p>(A) Representative images of wild type MEFs transfected with myc-Rac1^V12 alone or cotransfected with the indicated HA-tagged DGKζ construct. Scale bars = 20 um. Magnified images of the boxed regions are shown at the right. Scale bars = 5 um. (B) Graph showing the average macropinosome size in wild type MEFs expressing Rac1^V12 alone (-) or Rac1^V12 and the indicated DGKζ constructs (wt, kd or M1). Values are the mean ± SEM from three independent experiments. Statistical analysis was performed by a one-way ANOVA followed by a Tukey post-hoc multiple comparison test. Asterisks denote a significant difference (p<0.05) between the indicated conditions. (C) Graph showing the cumulative frequency distribution of macropinosome size (um²). The inset shows the distribution of macropinosomes between 0 and 5 um².</p