Abstract
Lysosome function is essential in cellular homeostasis. In addition to its recycling role, the lysosome has recently been recognized as a cellular signaling hub. We have shown in mammary epithelial cells, both in vivo and in vitro, that signal transducer and activator of transcription 3 (Stat3) modulates lysosome biogenesis and can promote the release of lysosomal proteases that culminates in cell death. To further investigate the impact of Stat3 on lysosomal function, we conducted a proteomic screen of changes in lysosomal membrane protein components induced by Stat3 using an iron nanoparticle enrichment strategy. Our results show that Stat3 activation not only elevates the levels of known membrane proteins but results in the appearance of unexpected factors, including cell surface proteins such as annexins and flotillins. These data suggest that Stat3 may coordinately regulate endocytosis, intracellular trafficking, and lysosome biogenesis to drive lysosome-mediated cell death in mammary epithelial cells. The methodologies described in this study also provide significant improvements to current techniques used for the purification and analysis of the lysosomal proteome.
Highlights
Lysosome function is essential in cellular homeostasis
We have shown in mammary epithelial cells, both in vivo and in vitro, that signal transducer and activator of transcription 3 (Stat3) modulates lysosome biogenesis and can promote the release of lysosomal proteases that culminates in cell death
By transmission electron microscopy (TEM) we observed that fluid phase uptake of nanoparticles by EpH4 cells led to the specific loading of degradative lysosomal vacuoles (Fig. 1A)
Summary
Lysosome function is essential in cellular homeostasis. In addition to its recycling role, the lysosome has recently been recognized as a cellular signaling hub. Our results show that Stat activation elevates the levels of known membrane proteins but results in the appearance of unexpected factors, including cell surface proteins such as annexins and flotillins These data suggest that Stat may coordinately regulate endocytosis, intracellular trafficking, and lysosome biogenesis to drive lysosome-mediated cell death in mammary epithelial cells. Lysosomes can be highly heterogeneous in nature, and in biological contexts where their function is impaired or altered (e.g. in lysosomal storage disorders) changes in lysosomal buoyant density result in their redistribution across a density gradient, hampering their purification by differential centrifugation [13, 18, 19] To overcome these difficulties, we and others have utilized magnetic iron nanoparticles to isolate lysosomes from different cell types [13, 18, 20, 21]. The utility of this method for LC-MS/MS analysis of cel-
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