TY - JOUR
T1 - Continuous translocation of Rac2 and the NADPH oxidase component p67(phox) during phagocytosis
AU - van Bruggen, Robin
AU - Anthony, Eloise
AU - Fernandez-Borja, Mar
AU - Roos, Dirk
PY - 2004
Y1 - 2004
N2 - In this study, the translocation of the NADPH oxidase components p67(phox) and Rac2 was studied during phagocytosis in living cells. For this purpose, green fluorescent protein (GFP)-tagged versions of these proteins were expressed in the myeloid cell line PLB-985. First, the correct localization of p67GFP and GFP-Rac2 was shown during phagocytosis of serum-treated zymosan by wild-type PLB-985 cells and PLB-985 X-CGD ( chronic granulomatous disease) cells, which lack expression of flavocytochrome b(558). Subsequently, these constructs were used for fluorescence recovery after photobleaching studies to elucidate the turnover of these proteins on the phagosomal membrane. The turnover of p67GFP and GFP-Rac2 proved to be very high, indicating a continuous exchange of flavocytochrome b(558)-bound p67GFP and GFP-Rac2 for cytosolic, free p67GFP and GFP-Rac2. Furthermore, the importance of an intact actin cytoskeleton for correct localization of these proteins was investigated by disrupting the actin cytoskeleton with cytochalasin B. However, cytochalasin B treatment of PLB-985 cells did not alter the localization of p67GFP and GFP-Rac2 once phagocytosis was initiated. In addition, the continuous exchange of flavocytochrome b(558)-bound p67GFP and GFP-Rac2 for cytosolic p67GFP and GFP-Rac2 was still intact in cytochalasin B-treated cells, indicating that the translocation of these proteins does not depend on a rearrangement of the actin cytoskeleton
AB - In this study, the translocation of the NADPH oxidase components p67(phox) and Rac2 was studied during phagocytosis in living cells. For this purpose, green fluorescent protein (GFP)-tagged versions of these proteins were expressed in the myeloid cell line PLB-985. First, the correct localization of p67GFP and GFP-Rac2 was shown during phagocytosis of serum-treated zymosan by wild-type PLB-985 cells and PLB-985 X-CGD ( chronic granulomatous disease) cells, which lack expression of flavocytochrome b(558). Subsequently, these constructs were used for fluorescence recovery after photobleaching studies to elucidate the turnover of these proteins on the phagosomal membrane. The turnover of p67GFP and GFP-Rac2 proved to be very high, indicating a continuous exchange of flavocytochrome b(558)-bound p67GFP and GFP-Rac2 for cytosolic, free p67GFP and GFP-Rac2. Furthermore, the importance of an intact actin cytoskeleton for correct localization of these proteins was investigated by disrupting the actin cytoskeleton with cytochalasin B. However, cytochalasin B treatment of PLB-985 cells did not alter the localization of p67GFP and GFP-Rac2 once phagocytosis was initiated. In addition, the continuous exchange of flavocytochrome b(558)-bound p67GFP and GFP-Rac2 for cytosolic p67GFP and GFP-Rac2 was still intact in cytochalasin B-treated cells, indicating that the translocation of these proteins does not depend on a rearrangement of the actin cytoskeleton
U2 - https://doi.org/10.1074/jbc.M309284200
DO - https://doi.org/10.1074/jbc.M309284200
M3 - Article
C2 - 14623873
SN - 0021-9258
VL - 279
SP - 9097
EP - 9102
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 10
ER -