Sequential breakdown of 3-phosphorylated phosphoinositides is essential for the completion of macropinocytosis

Abstract

Macropinocytosis is a highly conserved endocytic process by which extracellular fluid and solutes are internalized into cells. Macropinocytosis starts with the formation of membrane ruffles at the plasma membrane and ends with their closure. The transient and sequential emergence of phosphoinositides PI(3,4,5)P 3 and PI(3,4) P 2 in the membrane ruffles is essential for macropinocytosis. By making use of information in the Caenorhabditis elegans mutants defective in fluid-phase endocytosis, we found that mammalian phosphoinositide phosphatase MTMR6 that dephosphorylates PI(3)P to PI, and its binding partner MTMR9, are required for macropinocytosis. INPP4B, which dephosphorylates PI(3,4)P 2 to PI(3)P, was also found to be essential for macropinocytosis. These phosphatases operate after the formation of membrane ruffles to complete macropinocytosis. Finally, we showed that KCa3.1, a Ca 2+ -activated K + channel that is activated by PI(3)P, is required for macropinocytosis. We propose that the sequential breakdown of PI(3,4,5)P 3 → PI(3,4)P 2 → PI(3)P → PI controls macropinocytosis through specific effectors of the intermediate phosphoinositides.

Delivery of the CSF-1R to the lumen of macropinosomes promotes its destruction in macrophages

“…Furthermore, inhibition of the actin cytoskeleton blocked macropinocytosis but had little effect on CSF-1R internalization. Thus, the CSF-1R must stimulate macropinocytosis through signaling by, for example, PI3K, Ras and PLC (Amyere et al, 2000;Maekawa et al, 2014;Veltman et al, 2014), which can diffuse away from the CSF-1R during its accumulation at sites of small vesicle endocytosis (Fig. 6).…”

Uses and abuses of macropinocytosis

“…When PIP3 production is attenuated either with drugs or genetically, macropinocytosis is inhibited to striking extents (Araki et al, 2007(Araki et al, , 1996Buczynski et al, 1997;Hoeller et al, 2013;Zhou et al, 1998). In both mammalian cells and Dictyostelium, PIP3 is lost from macropinocytic vesicles shortly after they close, giving rise first to phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P 2 ], which (in mammalian cells at least) is then converted to phosphatidylinositol 3-phosphate as the vesicle progresses through the endocytic pathway (Araki et al, 2007;Dormann et al, 2004;Maekawa et al, 2014;Swanson, 2014;Welliver and Swanson, 2012;Yoshida et al, 2009;Egami et al, 2014). The evolutionary conservation between mammals and Dictyostelium of these Ras and phosphoinositides functions shows that they can be regarded as core features of macropinocytosis, presumably dating back to the last common ancestor of eukaryotes.…”

Phosphoinositides in endocytosis

“…The formation of circular dorsal ruffles, a specialized form of macropinocytosis that occurs at the dorsal surface rather than the leading edge of cells, depends on SHIP2-mediated formation of PI(3,4)P 2 from PI(3,4,5)P 3 [141]. Furthermore, a recent detailed analysis of PIs during macropinocytosis showed that in addition to SHIP2 the PI(3,4)P 2 -specific 4-phosphatase INPP4B as well as myotubularinrelated 3-phosphatases MTMR6 and 9 are essential for completion of macropinosome formation, but not for membrane ruffling [142]. Consistently, it has been found that both PI(3)P and Rab5 associate with macropinosomes prior to cup closure [140].…”

“…PI(3)P produced by INPP4B-mediated hydrolysis of PI(3,4)P 2 was shown to increase conductance of the Ca 2+ -activated K + -channel KCa3.1. Inhibition of KCa3.1 or expression of a PI(3)P-activation deficient mutant of KCa3.1 both interfere with macropinocytosis [142], suggesting that MTMR6/9 may be required to restrict PI(3)P levels and KCa3.1 opening on the macropinocytic cup before macropinosome formation. In summary, while we have obtained detailed insight into the PI conversions during macropinocytosis the effectors that bind to these PIs and their role in macropinosome formation remain largely elusive.…”