Recruitment of Arp2/3 complex at the site of phagocytosis

<p><b>(A)</b> Purified recombinant Kinase domain (KD) of EhAK1 or K85A (1μg) was incubated in the presence of γ-32P-ATP, MgCl<sup>2</sup> and substrate EhARPC1-GST ((2μg) at 30°C for 1 h. KD showed phosphorylation of EhARPC1-GSTwhereas K85A mutant of EhAK1 exhibits no autophosphorylation and substrate phosphorylation activities. The products were analysed on SDS-PAGE and visualized in a phosphorimager. Red color star marks autophosphorylation band of EhAK1-KD and yellow color star marks substrate phosphorylation band. <b>(B)</b> Total cell- lysate of <i>E</i>. <i>histolytica</i> was passed through agarose conjugated with either anti-EhARPC1/ purified anti–EhARPC2 antibody or pre-immune serum. Co-Immunoprecipitation of EhARPC1 and EhARPC2 was checked using respective antibodies. <b>(C)</b> Imaging of EhARPC2 with respect to EhARPC1 and Actin was done during erythrophagocytosis where <i>E</i>. <i>histolytica</i> cells were incubated with RBCs for indicated times at 37°C. The cells were then fixed and immunostained with anti-EhARPC1 and anti EhARPC2 antibody followed by Pacific blue-410 and Alexa 488-labelled secondary antibodies respectively. F-actin was stained with TRITC phalloidin. Images with Pacific blue-410 labelled anti-EhARPC1 antibody were given pseudo-color to gray for efficient visualization. Arrowhead indicate phagocytic cups, asterisk closed cups before scission, star marks phagosome and yellow arrowheads indicate RBC to be phagocytosed. Scale Bar represents 10 μm. <b>(D)</b> Colocalization analysis and PCC (r) from 25 cells was done by using Olympus Fluoview FV1000 software. The values obtained by a pairwise analysis of EhARPC2 with EhARPC1, actin and EhAK1 from phagocytic cups are indicated. <b>(E)</b> Immunostaining was performed for amoebic cells containing EhAK1 antisense construct grown in presence or absence of 30μg/ml tet and were incubated with RBCs for 5min at 37°C. The cells were then fixed and immunostained with anti-EhAK1 and anti-EhARPC2 antibody as indicated and double stained with Pacific blue-410 and Alexa 488-labelled secondary antibodies respectively. F-actin was stained with TRITC-phalloidin. Images with Pacific blue-410 labelled anti-EhAK1 (in presence of tetracycline) is pseudo-color to gray for efficient visualization. Yellow color arrowheads show the site of RBC attachment. Scale Bar represents 10 μm.</p