title: Eisosomes are dynamic plasma membrane domains showing pil1-lsp1 heteroligomer binding equilibrium.
abstract: Eisosomes are plasma membrane domains concentrating lipids, transporters, and signaling molecules. In the budding yeast Saccharomyces cerevisiae, these domains are structured by scaffolds composed mainly by two cytoplasmic proteins Pil1 and Lsp1. Eisosomes are immobile domains, have relatively uniform size, and encompass thousands of units of the core proteins Pil1 and Lsp1. In this work we used fluorescence fluctuation analytical methods to determine the dynamics of eisosome core proteins at different subcellular locations. Using a combination of scanning techniques with autocorrelation analysis, we show that Pil1 and Lsp1 cytoplasmic pools freely diffuse whereas an eisosome-associated fraction of these proteins exhibits slow dynamics that fit with a binding-unbinding equilibrium. Number and brightness analysis shows that the eisosome-associated fraction is oligomeric, while cytoplasmic pools have lower aggregation states. Fluorescence lifetime imaging results indicate that Pil1 and Lsp1 directly interact in the cytoplasm and within the eisosomes. These results support a model where Pil1-Lsp1 heterodimers are the minimal eisosomes building blocks. Moreover, individual-eisosome fluorescence fluctuation analysis shows that eisosomes in the same cell are not equal domains: while roughly half of them are mostly static, the other half is actively exchanging core protein subunits.
authors: Laboratorio de Biología Celular de Membranas, Institut Pasteur de Montevideo, Montevideo, Uruguay.
authors: Agustina
authors: A
authors: Olivera-Couto
authors: Laboratorio de Biología Celular de Membranas, Institut Pasteur de Montevideo, Montevideo, Uruguay.
authors: Valentina
authors: V
authors: Salzman
authors: Laboratorio de Biología Celular de Membranas, Institut Pasteur de Montevideo, Montevideo, Uruguay.
authors: Milagros
authors: M
authors: Mailhos
authors: Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California-Irvine, Irvine, California; Centre for Bioactive Discovery in Health and Ageing, School of Science and Technology, University of New England, Armidale, Australia.
authors: Michelle A
authors: MA
authors: Digman
authors: Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California-Irvine, Irvine, California. Electronic address: egratton22@gmail.com.
authors: Enrico
authors: E
authors: Gratton
authors: Laboratorio de Biología Celular de Membranas, Institut Pasteur de Montevideo, Montevideo, Uruguay. Electronic address: pablo.aguilar@pasteur.edu.uy.
authors: Pablo S
authors: PS
authors: Aguilar
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
chemical_list: C487328:LSP1 protein, S cerevisiae; C487327:PIL1 protein, S cerevisiae; D010750:Phosphoproteins; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins