Sequential activities of phosphoinositide 3-kinase, PKB/Aakt, and Rab7 during macropinosome formation in Dictyostelium.
Macropinocytosis plays an important role in the internalization of antigens by dendritic cells and is the route of entry for many bacterial pathogens; however, little is known about the molecular mechanisms that regulate the formation or maturation of macropinosomes. Like dendritic cells, Dictyostelium amoebae are active in macropinocytosis, and various proteins have been identified that contribute to this process. As described here, microscopic analysis of null mutants have revealed that the class I phosphoinositide 3-kinases, PIK1 and PIK2, and the downstream effector protein kinase B (PKB/Akt) are important in regulating completion of macropinocytosis. Although actin-rich membrane protrusions form in these cell lines, they recede without forming macropinosomes. Imaging of cells expressing green fluorescent protein (GFP) fused to the pleckstrin homology domain (PH) of PKB (GFP-PHPKB) indicates that D3 phosphoinositides are enriched in the forming macropinocytic cup and remain associated with newly formed macropinosomes for <1 minute. A fusion protein, consisting of GFP fused to an F-actin binding domain, overlaps with GFP-PHPKB in the timing of association with forming macropinosomes. Although macropinocytosis is reduced in cells expressing dominant negative Rab7, microscopic imaging studies reveal that GFP-Rab7 associates only with formed macropinosomes at approximately the time that F-actin and D3 phosphoinositide levels decrease. These results support a model in which F-actin modulating proteins and vesicle trafficking proteins coordinately regulate the formation and maturation of macropinosomes.
Rupper
A
A
Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, Shreveport, LA 71130, USA.
Lee
K
K
Knecht
D
D
Cardelli
J
J
D019870:1-Phosphatidylinositol 4-Kinase / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000818:Animals; D004023:Dictyostelium / Q000166:cytology* / Q000201:enzymology*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008247:Lysosomes / Q000201:enzymology / Q000378:metabolism; D018613:Microscopy, Confocal; D008954:Models, Biological; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010873:Pinocytosis; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D029701:Saccharomyces cerevisiae Proteins; D013997:Time Factors; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D000090802:rab7 GTP-Binding Proteins
D019870:1-Phosphatidylinositol 4-Kinase; D000199:Actins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D019869:Phosphatidylinositol 3-Kinases; D010716:Phosphatidylinositols; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D029701:Saccharomyces cerevisiae Proteins; D020691:rab GTP-Binding Proteins; D000090802:rab7 GTP-Binding Proteins; C441760:PIK1 protein, S cerevisiae