Journal
CYTOSKELETON
Volume 69, Issue 9, Pages 625-643Publisher
WILEY
DOI: 10.1002/cm.21051
Keywords
cortactin; membrane trafficking; actin assembly; endosomal; lysosomal maturation; Golgi
Categories
Funding
- NIH grant [1R01GM075126, 1R21DE018244, CA68485, DK20593, DK58404, HD15052, DK59637, EY08126]
- ACS grant [RSG-118085]
- Vanderbilt International Scholars Award Program
- NCRR/NIH [UL1 RR024975]
- [5T32 CA009592-23]
- [1F31DE021619]
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Cortactin is a branched actin regulator and tumor-overexpressed protein that promotes vesicular trafficking at a variety of cellular sites, including endosomes and the trans-Golgi network. To better understand its role in secretory trafficking, we investigated its function in Golgi homeostasis. Here, we report that knockdown (KD) of cortactin leads to a dramatic change in Golgi morphology by light microscopy, dependent on binding the Arp2/3 actin-nucleating complex. Surprisingly, there was little effect of cortactin-KD on anterograde trafficking of the constitutive cargo vesicular stomatitis virus glycoprotein (VSVG), Golgi assembly from endoplasmic reticulum membranes upon Brefeldin A washout, or Golgi ultrastructure. Instead, electron microscopy studies revealed that cortactin-KD cells contained a large number of immature-appearing late endosomal/lysosomal (LE/Lys) hybrid organelles, similar to those found in lysosomal storage diseases. Consistent with a defect in LE/Lys trafficking, cortactin-KD cells also exhibited accumulation of free cholesterol and retention of the retrograde Golgi cargo mannose-6-phosphate receptor in LE. Inhibition of LE maturation by treatment of control cells with Rab7 siRNA or chloroquine led to a compact Golgi morphology similar to that observed in cortactin-KD cells. Furthermore, the Golgi morphology defects of cortactin-KD cells could be rescued by removal of cholesterol-containing lipids from the media, suggesting that buildup of cholesterol-rich membranes in immature LE/Lys induced disturbances in retrograde trafficking. Taken together, these data reveal that LE/Lys maturation and trafficking are highly sensitive to cortactin-regulated branched actin assembly and suggests that cytoskeletal-induced Golgi morphology changes can be a consequence of altered trafficking at late endosomes. (c) 2012 Wiley Periodicals, Inc.
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