Abstract
Macropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the three-dimensional structure and dynamics of macropinosome formation relative to PI3K activity. We show that multiple ruffling morphologies produce macropinosomes and that the majority form through collisions of adjacent PI3K-rich ruffles. By combining multiple volumetric representations of the plasma membrane structure and PI3K products, we show that PI3K activity begins early throughout the entire ruffle volume and continues to increase until peak activity concentrates at the base of the ruffle after the macropinosome closes. Additionally, areas of the plasma membrane rich in ruffling had increased PI3K activity and produced many macropinosomes of various sizes. Pharmacologic inhibition of PI3K activity had little effect on the rate and morphology of membrane ruffling, demonstrating that early production of 3′-phosphoinositides within ruffles plays a minor role in regulating their morphology. However, 3′-phosphoinositides are critical for the fusogenic activity that seals ruffles into macropinosomes. Taken together, these data indicate that local PI3K activity is amplified in ruffles and serves as a priming mechanism for closure and sealing of ruffles into macropinosomes.
Highlights
Macropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner
The images and movies we present advance our understanding of the spatial dynamics of membrane ruffling, the morphologies that lead to macropinosomes, the spatial distribution of PI3K activity during macropinocytosis, and acknowledge the structural variability among different cell lines during macropinocytosis in fetal liver macrophages (FLMs), bone marrow-derived macrophages (BMDMs), and RAW 264.7 macrophages
lattice light sheet microscopy25 (LLSM) imaging was performed on FLMs stably expressing the fluorescent proteins mNeonGreen localized to the plasma membrane via the lipidation signal sequence from Lck (Mem-mNG) and the pleckstrin homology domain of Akt fused to mScarlet-I (AktPH-mSc)
Summary
Macropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. The production of 3′ phosphoinositides (PIs) by PI 3-kinase (PI3K) is required to generate isolated patches of phosphatidylinositol 3,4,5,triphosphate (PIP3) on the plasma membrane[18,19], and the sequential breakdown of PIP3 into PI(3,4)P2 and PI is necessary for successful macropinosome formation[20]. It is only in the cellular slime mold Dictyostelium that the signal coordination throughout the three-dimensional (3D) ruffle volume during macropinocytosis has been well described[18], and there is still more to learn about how these events are spatially coordinated in metazoan cells[21]. LY294002, have demonstrated that PI3K activity is only required for macropinosome closure but does not inhibit ruffling[17]
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