Department of Cell and Developmental Biology

PACSIN2-dependent apical endocytosis regulates the morphology of epithelial microvilli

AUTHORS

ABSTRACT

Apical microvilli are critical for the homeostasis of transporting epithelia, yet mechanisms that control the assembly and morphology of these protrusions remain poorly understood. Previous studies in intestinal epithelial cell lines suggested a role for F-BAR domain protein PACSIN2 in normal microvillar assembly. Here we report the phenotype of PACSIN2 KO mice and provide evidence that through its role in promoting apical endocytosis, this molecule functions in controlling microvillar morphology. PACSIN2 KO enterocytes exhibit reduced numbers of microvilli and defects in microvillar ultrastructure, with membranes lifting away from rootlets of core bundles. Dynamin2, a PACSIN2 binding partner, and other endocytic factors were also lost from their normal localization near microvillar rootlets. To determine if loss of endocytic machinery could explain defects in microvillar morphology, we examined the impact of PACSIN2 KD and endocytosis inhibition on live intestinal epithelial cells. These assays revealed that when endocytic vesicle scission fails, tubules are pulled into the cytoplasm and this, in turn, leads to a membrane lifting phenomenon reminiscent of that observed in PACSIN2 KO brush borders. These findings lead to a new model where inward forces generated by endocytic machinery on the plasma membrane control the membrane wrapping of cell surface protrusions. Video S1 Video S1 . Spinning Disk confocal imaging of an induced, scramble control Ls174T-W4 cell expressing EGFP-CAAX (green, membrane) and mCherry-UtrCH (magenta, F-actin). Movie was acquired every 30 seconds for 60 minutes and is played at 12.5 FPS. Scale bar, 5 μm. Video S2 Video S2 . Spinning Disk confocal imaging of an induced, IRTKS KD Ls174T-W4 cell expressing EGFP-CAAX (green, membrane) and mCherry-UtrCH (magenta, F-actin). Movie was acquired every 30 seconds for 90 minutes and is played at 12.5 FPS. Scale bar, 5 μm. Video S3 Video S3 . Spinning disk confocal imaging of an induced Ls174T-W4 cell expressing EGFP-CAAX (green, membrane) and mCherry-UtrCH (magenta, F-actin). Cell was treated with 80 μM DMSO 10 min prior to acquisition. Movie was acquired every 30 seconds for 68 minutes and is played at 12.5 FPS. Scale bar, 5 μm. Video S4 Video S4 . Spinning disk confocal imaging of an induced Ls174T-W4 cell expressing EGFP-CAAX (green, membrane) and mCherry-UtrCH (magenta, F-actin). Cell was treated with 80 μM Dynasore 10 min prior to acquisition. Movie was acquired every 30 seconds for 90 minutes and is played at 12.5 FPS. Scale bar, 5 μm. Video S5 Video S5 . Spinning disk confocal imaging of an induced Ls174T-W4 cell expressing EGFP-CAAX (green, membrane) and mCherry-UtrCH (magenta, F-actin). Cell was treated with 30 μM Pitstop 2 10 min prior to acquisition Movie was acquired every 30 seconds for 90 minutes and is played at 12.5 FPS. Scale bar, 5 μm.

Tags: Publications, Student Publications