Abstract
Macropinosomes arise from the closure of plasma membrane ruffles to bring about the non-selective uptake of nutrients and solutes into cells. The morphological changes underlying ruffle formation and macropinosome biogenesis are driven by actin cytoskeleton rearrangements under the control of the Rho GTPase Rac1. We showed previously that Rac1 is activated by diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show DGKζ is required for optimal macropinocytosis induced by growth factor stimulation of mouse embryonic fibroblasts. Time-lapse imaging of live cells and quantitative analysis revealed DGKζ was associated with membrane ruffles and nascent macropinosomes. Macropinocytosis was attenuated in DGKζ-null cells, as determined by live imaging and vaccinia virus uptake experiments. Moreover, macropinosomes that did form in DGKζ-null cells were smaller than those found in wild type cells. Rescue of this defect required DGKζ catalytic activity, consistent with it also being required for Rac1 activation. A constitutively membrane bound DGKζ mutant substantially increased the size of macropinosomes and potentiated the effect of a constitutively active Rac1 mutant on macropinocytosis. Collectively, our results suggest DGKζ functions in concert with Rac1 to regulate macropinocytosis.
Highlights
Macropinocytosis is a form of endocytosis in which extracellular fluid and plasma membrane are internalized in large vesicles [1,2]
To investigate potential roles for DGKz in macropinocytosis, we analyzed phase contrast, time-lapse images of nascent macropinosomes forming from membrane ruffles following stimulation with platelet-derived growth factor (PDGF; S1, S2 and S3 Movies)
To minimize the possibility that potential differences in macropinocytosis between wild type and DGKz-null cells could be due to defects in the process of membrane ruffle formation per se, we only scored macropinosomes that were clearly derived from already-formed membrane ruffles
Summary
Macropinocytosis is a form of endocytosis in which extracellular fluid and plasma membrane are internalized in large vesicles [1,2]. DGKζ Regulates Macropinocytosis peroxidase; PA, phosphatidic acid; PAK1, p21activated kinase 1; PDGF, platelet-derived growth factor; YFP, yellow fluorescent protein. Macropinocytosis contributes to infection since many pathogenic bacteria and viruses exploit it as a pathway to gain entry into cells [5,6,7]. It has been implicated in the modulation of cell-cell adhesion by regulating the internalization of E-cadherin-catenin complexes [8,9]. Recent evidence suggests Ras-transformed cancer cells use macropinocytosis to internalize extracellular protein to supply amino acids for their proliferation and growth [10]. Macropinocytosis is a fundamental cellular process used by a variety of cell types and underpins several different biological functions
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