In RAW 264. their PKC–expressing counterparts, blocked at the pseudopod-extension phase.

In RAW 264. their PKC–expressing counterparts, blocked at the pseudopod-extension phase. In summary, we have shown that PS and C1B are necessary and sufficient for targeting PKC- to phagosomes, where its catalytic activity is required for membrane delivery and pseudopod extension. 0.05 was considered significant. RESULTS Primary mouse macrophages require PKC- for efficient phagocytosis With the use of the RAW 264.7 cell line, we reported that PKC- concentrates at phagocytic cups and formed phagosomes and that it is necessary for efficient phagocytosis [4, 6]. The availability of PKC-?/? mice enabled us to validate the RAW cell results in primary BMDM. Like RAW cells, PKC–GFP concentrated at phagocytic cups and internalized phagosomes in WT BMDM (Fig. 2 and Supplemental Video 1). Calculated from live imaging, PKC-?/? BMDM (KO) internalized 2 m IgG-opsonized beads at a significantly slower rate than their WT counterparts (KO: 78.14.9 s/target; WT: 53.73.9 s/target; P<0.001, n=40 from three independent experiments). The phagocytosis rate was restored upon expression of PKC–GFP in KO; the average time for internalization of a single bead was 54.3 3.3 s for reconstituted KO cells and 87.1 6.5 s for KO-expressing GFP (n=77C79, three experiments; P<0.001). Flow cytometry established that FcR expression is 5-hydroxymethyl tolterodine equivalent in WT and PKC-?/? BMDM (Supplemental Fig. 1), eliminating this trivial explanation for the observed 5-hydroxymethyl tolterodine decrease in phagocytosis in KO cells. Thus, RAW cells and BMDM require PKC- for efficient phagocytosis. Figure 2. PKC- concentrates at phagosomes during IgG-mediated phagocytosis in primary mouse macrophages. PKC- is necessary for membrane mobilization in response to FcR ligation As phagocytosis involves pseudopod extension, and PKC- is involved in cell spreading [31] and neurite extension [12], we asked whether PKC- contributes to membrane mobilization in response to FcR ligation. 5-hydroxymethyl tolterodine To maximize membrane recruitment, BMDM were subjected to frustrated phagocytosis on IgG surfaces [17]. As a result of the tight binding of cells to the IgG surface, the area of 5-hydroxymethyl tolterodine attached cells can be calculated from the black holes produced by labeling the exposed IgG with Alexa 488 secondary antibodies (Fig. 3, inset). When measured with time, WT were significantly larger than their KO counterparts after 5 min (Fig. 3). The slopes of the time-course plots provided a measure of spreading rate. A comparison of the rates confirmed that WT cells spread significantly faster than KO (603 m2/min vs. 332 m2/min; P<0.05). Finally, the difference between WT and KO RYBP cells was Ca2+-independent and was seen for primary BMDM (Fig. 3) and elicited peritoneal macrophages (not shown), suggesting that the spreading defect is intrinsic and not a function of the in vitro differentiation of BM precursors. Flow cytometry indicates that WT and KO cells are similar in size and 5-hydroxymethyl tolterodine granularity (forward- and side-scatter, respectively; Supplemental Fig. 1). Thus, the smaller spread area in KO cells could be a result of less overall plasma membrane or an inability of PKC-?/? cells to access their plasma membrane (as a result of defects in cortical actin structure, etc.). Whole-cell patch-clamping, the gold standard for quantitation of membrane area, was used to measure the capacitance of the plasma membrane. WT and KO macrophages were patched 7.5C10 min into frustrated phagocytosis. On IgG surfaces, WT had a significantly higher capacitance than KO (25.50.76 pF vs. 21.90.53 pF; P<0.001), consistent with having more plasma membrane (Fig. 4). To determine whether membrane delivery was specific to receptor ligation or a response to cell attachment, we repeated the experiment on PLL surfaces, to which cells attach via electrostatic interactions [32]. On PLL, the capacitance of WT and KO cells was equivalent (WT: 19.70.37 pF vs. KO: 20.30.49 pF) and not different from KO cells on IgG (Fig. 4). This validates the flow data and demonstrates that WT, but not KO, cells add membrane to their surface upon FcR ligation. It is notable that the 5-pF capacitance increase of WT on IgG translates to a 30% increase in surface membrane. To probe the requirement for PKC- in spreading, PKC--GFP was expressed in KO cells. Reconstituted cells were significantly more.