Studying the role of membrane domains in cellular aging

SALZMAN, VALENTINA; CORREA, FRANCISCO; BUSTAMANTE-TORRES MOISES; ESTRADA,LAURA; AGUILAR, PABLO S.

Mendoza

Congreso; LVIII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; 2022

SAIB

The plasma membrane (PM) of eukaryotic cells is compartmentalized into domains enriched in specific lipids and proteins. Saccharomyces cerevisiae contains different nanodomains that exhibit different morphologies and dynamic behaviors. Particularly,eisosomes are nanoscale PM Invaginations structured by scaffolds composed mainly by two cytoplasmic proteins Pil1 and Lsp1.More than 25 proteins including transporters, signaling molecules and proteins of unknown function have been localized in eisosomes. We are interested in understanding eisosomes´ role in cell lifespan using S. cerevisiae as a model. Replicative lifespan (RLS) is the number of daughter cells a mother yeast cell can produce before senescing, traditionally measured by manuallydissecting mother cells from daughter cells. Using this method we found that knockout strains for the PIL1 gene (eisosomes´dissasembled) have significantly enhanced longevity. No effect was observed when LSP1 was deleted (eisosomes assembled), suggesting that eisosomes´ structure plays a key role in yeast aging. Microfluidic systems have been developed to automate the dissection process, accelerating RLS determination. We were able to measure RLS of wt and eisosome´s mutants yeast strains analyzing microscope bright field images of mother cells trapped in microfluidic devices during 90 h. A decrease in the concentration of glucose or certain amino acids in the culture medium extends RLS in S. cerevisiae. In order to understand the mechanism underlying pil1 mutant extended lifespan we studied if the absence of eisosomal organization leads to a nutrient imbalance state and/or alters nutrient signaling extending RLS. We found that Extension of longevity in pil1 mutant is not given by a difference in glucose consumption. Eisosomes are storage compartments of many amino acids transporters including arginine and tryptophan permeases Can1 and Tat2. Deletion of CAN1 and TAT2 genes decreases amino acid cytosolic contents and correlates with an extension in RLS. While the importance of proper domain association for protein functionality has been demonstrated for Can1, the role of eisosomes in Tat2 functionality and/or availability was unknown. Measuring 3H-Trp import in vivo we found thatPIL1 deletion does not generate a decrease in Trp incorporation. Therefore, a deficiency in Tat2 activity does not seem to be underling the RLS extension mechanism in study. General Amino acid Control pathway activity was determined performing reporter gene assays in eisosomal mutants. Together with cytosolic amino acid quantifications by NMR these experiments will enable us to determine whether an amino acids imbalance state is underlying eisosome disassembly-dependent RLS extension. Our work on microfluidic devices in conjunction with mutants´s physiology analysis will be key in order to under eisosomes´ role in aging, likely contributing to further describing the complex aging process and nanoscale PM domains function.