Membrane cholesterol removal changes mechanical properties of cells and induces secretion of a specific pool of lysosomes

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 inv...

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Published in:	PloS one Vol. 8; no. 12; p. e82988
Main Authors:	Hissa, Barbara, Pontes, Bruno, Roma, Paula Magda S, Alves, Ana Paula, Rocha, Carolina D, Valverde, Thalita M, Aguiar, Pedro Henrique N, Almeida, Fernando P, Guimarães, Allan J, Guatimosim, Cristina, Silva, Aristóbolo M, Fernandes, Maria C, Andrews, Norma W, Viana, Nathan B, Mesquita, Oscar N, Agero, Ubirajara, Andrade, Luciana O
Format:	Journal Article
Language:	English
Published:	United States Public Library of Science 20-12-2013 Public Library of Science (PLoS)
Subjects:	Actin Actins - genetics Actins - metabolism Animals Bending modulus beta-Cyclodextrins - pharmacology Bridged Bicyclo Compounds, Heterocyclic - pharmacology Calcium Cell Line Cell Membrane - drug effects Cell Membrane - ultrastructure Cholesterol Cholesterol - chemistry Cholesterol - deficiency Cytoskeleton Cytoskeleton - drug effects Cytoskeleton - ultrastructure Defocusing Depolymerization Drugs Dynamic tests Exocytosis Exocytosis - drug effects Fibroblasts Fibroblasts - cytology Fibroblasts - drug effects Fibroblasts - metabolism Gene Expression Laboratory animals Lipids Lysosomes Lysosomes - classification Lysosomes - metabolism Lysosomes - secretion Mechanical properties Membrane Fluidity - drug effects Membrane fusion Mice Microscopy Muscle proteins Polymerization Proteins Relaxation time rho GTP-Binding Proteins - genetics rho GTP-Binding Proteins - metabolism RNA, Small Interfering - genetics RNA, Small Interfering - metabolism Secretion Surface tension Synaptotagmin Synaptotagmins - antagonists & inhibitors Synaptotagmins - genetics Synaptotagmins - metabolism Thiazolidines - pharmacology United States > US Maryland
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Summary:	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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: BH BP NWA NBV ONM UA LOA. Performed the experiments: BH BP PMSR APA CDR TMV PHNA FPA AJG MCF LOA. Analyzed the data: BH BP CDR PHNA AJG CG AMS MCF NBV ONM UA LOA. Contributed reagents/materials/analysis tools: AJG CG AMS NWA NBV ONM UA LOA. Wrote the paper: BH BP LOA.
ISSN:	1932-6203 1932-6203
DOI:	10.1371/journal.pone.0082988