Abstract
BackgroundMacropinocytosis is an actin-driven endocytic process, whereby membrane ruffles fold back onto the plasma membrane to form large (>0.2 µm in diameter) endocytic organelles called macropinosomes. Relative to other endocytic pathways, little is known about the molecular mechanisms involved in macropinocytosis. Recently, members of the Sorting Nexin (SNX) family have been localized to the cell surface and early macropinosomes, and implicated in macropinosome formation. SNX-PX-BAR proteins form a subset of the SNX family and their lipid-binding (PX) and membrane-curvature sensing (BAR) domain architecture further implicates their functional involvement in macropinosome formation.Methodology/Principal FindingsWe exploited the tractability of macropinosomes through image-based screening and systematic overexpression of SNX-PX-BAR proteins to quantitate their effect on macropinosome formation. SNX1 (40.9+/−3.19 macropinosomes), SNX5 (36.99+/−4.48 macropinosomes), SNX9 (37.55+/−2.4 macropinosomes), SNX18 (88.2+/−8 macropinosomes), SNX33 (65.25+/−6.95 macropinosomes) all exhibited statistically significant (p<0.05) increases in average macropinosome numbers per 100 transfected cells as compared to control cells (24.44+/−1.81 macropinosomes). SNX1, SNX5, SNX9, and SNX18 were also found to associate with early-stage macropinosomes within 5 minutes following organelle formation. The modulation of intracellular PI(3,4,5)P3 levels through overexpression of PTEN or a lipid phosphatase-deficient mutant PTEN(G129E) was also observed to significantly reduce or elevate macropinosome formation respectively; coexpression of PTEN(G129E) with SNX9 or SNX18 synergistically elevated macropinosome formation to 119.4+/−7.13 and 91.4+/−6.37 macropinosomes respectively (p<0.05).Conclusions/SignificanceSNX1, SNX5, SNX9, SNX18, and SNX33 were all found to elevate macropinosome formation and (with the exception of SNX33) associate with early-stage macropinosomes. Moreover the effects of SNX9 and SNX18 overexpression in elevating macropinocytosis is likely to be synergistic with the increase in PI(3,4,5)P3 levels, which is known to accumulate on the cell surface and early-stage macropinocytic cups. Together these findings represent the first systematic functional study into the impact of the SNX-PX-BAR family on macropinocytosis.
Highlights
Macropinocytosis is a high-capacity variant of endocytic uptake first reported by Warren Lewis in 1931 [1], generating large endocytic organelles termed macropinosomes through actindriven evaginations of the plasma membrane
Unlike clathrinmediated endocytosis or phagocytosis, macropinocytosis is not regulated by the binding of cargo to the receptors which recruit effector molecules that aid in vesicle formation [2]; instead the activation of receptor tyrosine-kinases (RTK) in response to growth factor treatment drives the actin-mediated ruffling of the plasma membrane, non-selectively engulfing large volumes of fluid to form phase bright macropinosomes larger than 0.2 mm in diameter [1,3]
Screening Assay Development and Validation To test the hypothesis that the Sorting Nexin (SNX)-Phox homology (PX)-binding (PX) and membrane-curvature sensing (BAR) family is involved in macropinocytosis, a functional assay was developed in order to screen the candidate proteins in a systematic fashion
Summary
Macropinocytosis is a high-capacity variant of endocytic uptake first reported by Warren Lewis in 1931 [1], generating large endocytic organelles termed macropinosomes through actindriven evaginations of the plasma membrane. Unlike clathrinmediated endocytosis or phagocytosis, macropinocytosis is not regulated by the binding of cargo to the receptors which recruit effector molecules that aid in vesicle formation [2]; instead the activation of receptor tyrosine-kinases (RTK) in response to growth factor treatment drives the actin-mediated ruffling of the plasma membrane, non-selectively engulfing large volumes of fluid to form phase bright macropinosomes larger than 0.2 mm in diameter [1,3]. SNX-PX-BAR proteins form a subset of the SNX family and their lipid-binding (PX) and membrane-curvature sensing (BAR) domain architecture further implicates their functional involvement in macropinosome formation
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