(A) High-throughput assay for phagocytosis. GFP-expressing C. albicans (green) were co-incubated with S2 cells to allow phagocytosis. Cells were lightly fixed, and non-phagocytosed C. albicans were secondarily labeled with a rabbit anti–C. albicans antibody and Cy3-labeled anti-rabbit antibody (red). S2 cell DNA (blue) was labeled with Hoechst 33258. Left panels, wild-type S2 cells; right panels: S2 cells treated with RNAi against actin (Act5C).
(B) One hundred eighty-four dsRNAs decreased phagocytosis of C. albicans. The 184 genes were categorized and plotted in a pie graph with the number of genes in each class indicated.
(C–E) Secondary screens used to further test the RNAi-treated S2 cells specificity of phagocytosis of C. albicans (C), E. coli (D), or latex beads (E). (C) Phagocytosis of C. albicans. GFP-expressing C. albicans (green) were co-incubated with S2 cells to allow phagocytosis. Cells were lightly fixed, and non-phagocytosed C. albicans were secondarily labeled with an anti–C. albicans antibody and Cy3-labeled anti-rabbit antibody (red). S2 cell DNA was labeled with Hoechst 33258 to mark the position of the S2 cell. The level of phagocytosis was quantified by counting the percentage of S2 cells that had phagocytosed one or more C. albicans. (D) Drosophila S2 cells phagocytose E. coli. GFP-expressing E. coli (green) were co-incubated with S2 cells to allow phagocytosis in a similar assay as (C). Cells were lightly fixed, and an anti–E. coli antibody was used to label non-phagocytosed E. coli (red). The level of phagocytosis was quantified by counting the percentage of S2 cells that had phagocytosed one or more E. coli. (E) Drosophila S2 cells phagocytose 2-μm latex beads. Yellow-green fluorescent latex beads were co-incubated with S2 cells to allow phagocytosis. The S2 cell filamentous actin cytoskeleton was labeled with rhodamine phalloidin (red) and the DNA with Hoechst 33258 (blue). The level of phagocytosis was quantified by counting the percentage of S2 cells that had phagocytosed one or more latex beads.
(F) One hundred eighty-four dsRNAs disrupt the phagocytosis of C. albicans by S2 cells. The genes required for phagocytosis of C. albicans are listed along with the effect on phagocytosis of E. coli and latex beads. The color-based scale is given below and corresponds to the percentage of S2 cells that phagocytosed one or more C. albicans, E. coli, or latex beads. Genes were categorized based upon function as in (B). Mean values for phagocytosis by wild-type, untreated S2 cells were: C. albicans 52%, E. coli 56%, and latex beads 51%.

(A) S2 cells were treated with dsRNA against SCAR, Mcr, or one of the Drosophila Teps and co-incubated with C. albicans. The percentage of S2 cells phagocytosing one or more C. albicans was quantified and plotted.
(B) The S2 cells treated above were also co-incubated with E. coli, and phagocytosis was quantified.
(C) The RNAi-treated S2 cells above were co-incubated with S. aureus, and phagocytosis was quantified. In all graphs, the 3.5-h timepoints were analyzed using a t-test assuming unequal variance. Those values that differ significantly from untreated cells (p < 0.01) are indicated by asterisks.

(A) Wild-type C. albicans, S. cerevisiae, or Δefg1/Δefg1 mutant C. albicans were co-incubated with conditioned media containing 2% FBS from wild-type S2 cells for 2 h, washed, and analyzed by immunoblotting with anti-Mcr antibody. Lane 1, conditioned media; lane 2, new media; lane 3, Mcr bound to wild-type C. albicans; lane 4, Mcr bound to S. cerevisiae; lane 5, Mcr bound to Δefg1/Δefg1 mutant C. albicans.
(B) Quantification of phagocytosis of C. albicans wild-type and mutant strains and S. cerevisiae by S2 cells. S2 cells and the indicated fungal strain were co-incubated for various times, and the percentage of S2 cells that had phagocytosed one or more C. albicans was quantified by counting 50–100 S2 cells. Results are the average of four experiments, and the error bars indicate the standard deviation. The 3.5-h timepoints were analyzed using a t-test assuming unequal variance. Those values that differ significantly from untreated cells (p < 0.01) are indicated by asterisks.

(A) The Drosophila hemocyte-like S2 cell line phagocytoses C. albicans. S2 cells were co-incubated with GFP expressing C. albicans for the indicated times. Cells were fixed, and the filamentous actin of S2 cells was stained with rhodamine phalloidin and the S2 cell DNA with Hoechst 33258.
(B) Quantification of phagocytosis of C. albicans and S. cerevisiae by S2 cells. S2 cells and the indicated fungal strain were co-incubated for various times, and the percentage of S2 cells that had phagocytosed one or more C. albicans was quantified by counting 50–100 S2 cells. The maximum time shown is 3 h, as the levels of phagocytosis did not significantly increase after this timepoint. Results are the average of four experiments, and the error bars indicate the standard deviation. As described in Materials and Methods, the 3-h results were evaluated for statistical significance using the t-test, assuming unequal variance. As indicated by the asterisks, the values for heat-killed C. albicans and S. cerevisiae were statistically different from that of live C. albicans, with a confidence level p < 0.01.

(A) Schematic representation of α2M-related proteins. Drosophila Mcr is compared with a close homolog in A. gambiae (Ag Mcr [Tep13]), TepI from both Drosophila and Anopheles and the human homologs CD109, α2M, and C3. Various conserved domains are colored as indicated in the gray box. Numbers correspond to amino acid position. The sequences of the conserved thioester domains are given below the schematic. Dm, Drosophila melanogaster; Ag, Anopheles gambiae; Hs, Homo sapiens.
(B) RNAi against SCAR reduces phagocytosis of C. albicans, E. coli, and latex beads (row 2). RNAi against Mcr significantly decreased phagocytosis of only C. albicans (row 3). Cells were stained as in Figure 4C–4E. Column 1, GFP expressing C. albicans—green, S2 cell DNA—blue, non-phagocytosed C. albicans—red; column 2, GFP-expressing E. coli—green, S2 cell DNA—blue, non-phagocytosed E. coli—red; column 3, latex beads—green, S2 cell DNA—blue, S2 cell actin cytoskeleton—red.
(C) dsRNA against both SCAR and Mcr decreases phagocytosis of C. albicans. S2 cells were treated with dsRNA against SCAR and Mcr as described in Materials and Methods and then co-incubated with C. albicans for the indicated times. The percentage of S2 cells phagocytosing one or more C. albicans was quantified and plotted. The 3.5-h timepoints were analyzed using a t-test assuming unequal variance. Those values that differ significantly from untreated cells (p < 0.01) are indicated by asterisks.
(D) Mcr dsRNA does not reduce phagocytosis of E. coli. S2 cells were treated with dsRNA against SCAR and Mcr and then co-incubated with E. coli for the indicated times. The percentage of S2 cells phagocytosing one or more E. coli was quantified and plotted. The 3.5-h timepoints were analyzed using a t-test assuming unequal variance. Those values that differ significantly from untreated cells (p < 0.01) are indicated by asterisks.
(E) Mcr dsRNA does not reduce phagocytosis of latex beads. S2 cells were treated with dsRNA against SCAR and Mcr and then co-incubated with green fluorescent latex beads for the indicated times. The percentage of S2 cells phagocytosing one or more latex beads was quantified and plotted. The 3.5-h timepoints were analyzed using a t-test assuming unequal variance. Those values that differ significantly from untreated cells (p < 0.01) are indicated by asterisks.
(F) An additional dsRNA against the 3′ UTR of Mcr was generated and tested for disruption of C. albicans phagocytosis. S2 cells were treated with RNAi directed against both the coding region and the 3′ UTR of Mcr and then co-incubated with C. albicans for the indicated times. The percentage of S2 cells phagocytosing one or more C. albicans was quantified and plotted. The 3.5-h timepoints were analyzed using a t-test assuming unequal variance. Those values that differ significantly from untreated cells (p < 0.01) are indicated by asterisks.

(A) Mcr is secreted into the culture media. Whole-cell lysates were prepared from S2 cells (lane 1) and compared to Schneider's medium with 2% FBS (lane 2) or Schneider's medium with 2% FBS collected from S2 cells (conditioned media, lane 3) by immunoblotting with an anti-Mcr antibody.
(B) RNAi against Mcr depletes Mcr protein from cell lysates and from the conditioned media. Cell lysates and conditioned media were collected from wild-type S2 cells or cells treated with RNAi against Mcr or SCAR and probed by immunoblotting with an anti-Mcr antibody.
(C) Conditioned media rescues the phagocytosis defect of Mcr RNAi-treated cells. Wild-type S2 cells or cells treated with RNAi against Mcr or SCAR were plated in new Schneider's medium with 10% FBS or conditioned media with 10% FBS from wild-type S2 cells and incubated with C. albicans for various times. The percentage of S2 cells that had phagocytosed one or more C. albicans was quantified and graphed. A t-test was used to test the statistical significance between wild-type cells in new media versus SCAR- or Mcr RNAi–treated cells in new media and wild-type cells in conditioned media versus SCAR- or Mcr RNAi–treated cells in conditioned media (see Materials and Methods). An asterisk indicates comparisons that showed statistically significant differences (p < 0.01). Mcr RNAi–treated cells in wild-type-conditioned media were not significantly different from wild-type cells in conditioned media.
(D) Mcr interacts with C. albicans cells. C. albicans was co-incubated either with new media containing 2% FBS or conditioned media containing 2% FBS from wild-type S2 cells for 2 h, washed, and analyzed by immunoblotting with anti-Mcr. Lane 1, S2 cell lysates; lane 2, new media; lane 3, conditioned media; lane 4, C. albicans incubated in new media; lane 5, C. albicans incubated in conditioned media.

Genes Identified in the Screen as Being Required for Phagocytosis of C. albicans Were Superimposed onto a Drosophila Genomic Yeast Two-Hybrid Interaction Map
Interactions are displayed, and several pathways are outlined. Dark-blue circles indicate genes identified as reduced phagocytosis of C. albicans, with light-blue circles indicating genes present in the two-hybrid map but not identified in the phagocytosis screen. Several functional groups are circled as indicated. This diagram represents only a portion of the complete two-hybrid map [35], indicating that the genes identified in the phagocytosis screen affect a limited number of cellular processes.