title: Membrane cholesterol removal changes mechanical properties of cells and induces secretion of a specific pool of lysosomes.
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.
authors.affiliation: Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
authors.forename: Barbara
authors.initials: B
authors.lastname: Hissa
authors.affiliation: LPO-COPEA, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
authors.forename: Bruno
authors.initials: B
authors.lastname: Pontes
authors.affiliation: Departamento de Física, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
authors.forename: Paula Magda S
authors.initials: PM
authors.lastname: Roma
authors.affiliation: Departamento de Física, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
authors.forename: Ana Paula
authors.initials: AP
authors.lastname: Alves
authors.affiliation: Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
authors.forename: Carolina D
authors.initials: CD
authors.lastname: Rocha
authors.affiliation: Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
authors.forename: Thalita M
authors.initials: TM
authors.lastname: Valverde
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
authors.forename: Pedro Henrique N
authors.initials: PH
authors.lastname: Aguiar
authors.affiliation: Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
authors.forename: Fernando P
authors.initials: FP
authors.lastname: Almeida
authors.affiliation: Departamento de Microbiologia e Parasitologia, Instituto Biomédico, Universidade Federal Fluminense, Rio de Janeiro, RJ, Brazil.
authors.forename: Allan J
authors.initials: AJ
authors.lastname: Guimarães
authors.affiliation: Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
authors.forename: Cristina
authors.initials: C
authors.lastname: Guatimosim
authors.affiliation: Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
authors.forename: Aristóbolo M
authors.initials: AM
authors.lastname: Silva
authors.affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, United States of America.
authors.forename: Maria C
authors.initials: MC
authors.lastname: Fernandes
authors.affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, United States of America.
authors.forename: Norma W
authors.initials: NW
authors.lastname: Andrews
authors.affiliation: LPO-COPEA, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil ; Instituto de Física, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
authors.forename: Nathan B
authors.initials: NB
authors.lastname: Viana
authors.affiliation: Departamento de Física, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
authors.forename: Oscar N
authors.initials: ON
authors.lastname: Mesquita
authors.affiliation: Departamento de Física, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
authors.forename: Ubirajara
authors.initials: U
authors.lastname: Agero
authors.affiliation: Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
authors.forename: Luciana O
authors.initials: LO
authors.lastname: Andrade
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects* / Q000648:ultrastructure; D002784:Cholesterol / Q000737:chemistry* / Q000172:deficiency; D003599:Cytoskeleton / Q000187:drug effects / Q000648:ultrastructure; D005089:Exocytosis / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D015870:Gene Expression; D008247:Lysosomes / Q000145:classification / Q000378:metabolism*; D008560:Membrane Fluidity / Q000187:drug effects; D051379:Mice; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D050857:Synaptotagmins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D053778:Thiazolidines / Q000494:pharmacology; D047392:beta-Cyclodextrins / Q000494:pharmacology*; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D034741:RNA, Small Interfering; D053778:Thiazolidines; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D050857:Synaptotagmins; D002784:Cholesterol; D020741:rho GTP-Binding Proteins; C037067:latrunculin A