1. Membrane cholesterol removal changes mechanical properties of cells and induces secretion of a specific pool of lysosomes.
- Author
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Hissa B, Pontes B, Roma PM, Alves AP, Rocha CD, Valverde TM, Aguiar PH, Almeida FP, Guimarães AJ, Guatimosim C, Silva AM, Fernandes MC, Andrews NW, Viana NB, Mesquita ON, Agero U, and Andrade LO
- Subjects
- Actins genetics, Actins metabolism, Animals, Bridged Bicyclo Compounds, Heterocyclic pharmacology, Cell Line, Cell Membrane ultrastructure, Cholesterol deficiency, Cytoskeleton drug effects, Cytoskeleton ultrastructure, Exocytosis drug effects, Fibroblasts cytology, Fibroblasts metabolism, Gene Expression, Lysosomes classification, Membrane Fluidity drug effects, Mice, RNA, Small Interfering genetics, RNA, Small Interfering metabolism, Synaptotagmins antagonists & inhibitors, Synaptotagmins genetics, Synaptotagmins metabolism, Thiazolidines pharmacology, rho GTP-Binding Proteins genetics, rho GTP-Binding Proteins metabolism, Cell Membrane drug effects, Cholesterol chemistry, Fibroblasts drug effects, Lysosomes metabolism, beta-Cyclodextrins pharmacology
- Abstract
In a previous study we had shown that membrane cholesterol removal induced unregulated lysosomal exocytosis events leading to the depletion of lysosomes located at cell periphery. However, the mechanism by which cholesterol triggered these exocytic events had not been uncovered. In this study we investigated the importance of cholesterol in controlling mechanical properties of cells and its connection with lysosomal exocytosis. Tether extraction with optical tweezers and defocusing microscopy were used to assess cell dynamics in mouse fibroblasts. These assays showed that bending modulus and surface tension increased when cholesterol was extracted from fibroblasts plasma membrane upon incubation with MβCD, and that the membrane-cytoskeleton relaxation time increased at the beginning of MβCD treatment and decreased at the end. We also showed for the first time that the amplitude of membrane-cytoskeleton fluctuation decreased during cholesterol sequestration, showing that these cells become stiffer. These changes in membrane dynamics involved not only rearrangement of the actin cytoskeleton, but also de novo actin polymerization and stress fiber formation through Rho activation. We found that these mechanical changes observed after cholesterol sequestration were involved in triggering lysosomal exocytosis. Exocytosis occurred even in the absence of the lysosomal calcium sensor synaptotagmin VII, and was associated with actin polymerization induced by MβCD. Notably, exocytosis triggered by cholesterol removal led to the secretion of a unique population of lysosomes, different from the pool mobilized by actin depolymerizing drugs such as Latrunculin-A. These data support the existence of at least two different pools of lysosomes with different exocytosis dynamics, one of which is directly mobilized for plasma membrane fusion after cholesterol removal.
- Published
- 2013
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