ABSTRACT
In yeast, actin cables are F-actin bundles that are essential for cell division through their function as tracks for cargo movement from mother to daughter cell. Actin cables also affect yeast lifespan by promoting transport and inheritance of higher-functioning mitochondria to daughter cells. Here, we report that actin cable stability declines with age. Our genome-wide screen for genes that affect actin cable stability identified the open reading frame YKL075C. Deletion of YKL075C results in increases in actin cable stability and abundance, mitochondrial fitness, and replicative lifespan. Transcriptome analysis revealed a role for YKL075C in regulating branched-chain amino acid (BCAA) metabolism. Consistent with this, modulation of BCAA metabolism or decreasing leucine levels promotes actin cable stability and function in mitochondrial quality control. Our studies support a role for actin stability in yeast lifespan, and demonstrate that this process is controlled by BCAA and a previously uncharacterized ORF YKL075C, which we refer to as actin, aging and nutrient modulator protein 1 (AAN1).
Subject(s)
Actin Cytoskeleton , Longevity , Mitochondria , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Actin Cytoskeleton/metabolism , Actins/metabolism , Longevity/genetics , Mitochondria/metabolism , Nutrients/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
This chapter describes labeling methods and optical approaches for live-cell imaging of the cytoskeleton and of a specific organelle-cytoskeleton interaction in budding yeast.
Subject(s)
Cytoskeleton/metabolism , Microtubules/metabolism , Mitochondria/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Actins/metabolism , Carbocyanines/metabolism , Coloring Agents/metabolism , Cytoskeleton/genetics , Cytoskeleton/ultrastructure , Fluorescent Antibody Technique , Fluorescent Dyes/metabolism , Green Fluorescent Proteins/metabolism , Indoles/metabolism , Membrane Glycoproteins/metabolism , Microfilament Proteins/metabolism , Microtubules/genetics , Microtubules/ultrastructure , Mitochondria/genetics , Mitochondria/ultrastructure , Pyridinium Compounds/metabolism , Rhodamine 123/metabolism , Saccharomyces cerevisiae/ultrastructure , Staining and Labeling/methods , Tropomyosin/metabolismABSTRACT
MCAK belongs to the Kinesin-13 family, whose members depolymerize microtubules rather than translocate along them. We defined the minimal functional unit of MCAK as the catalytic domain plus the class specific neck (MD-MCAK), which is consistent with previous reports. We used steady-state ATPase kinetics, microtubule depolymerization assays, and microtubule.MCAK cosedimentation assays to compare the activity of full-length MCAK, which is a dimer, with MD-MCAK, which is a monomer. Full-length MCAK exhibits higher ATPase activity, more efficient microtubule end binding, and reduced affinity for the tubulin heterodimer. Our studies suggest that MCAK dimerization is important for its catalytic cycle by promoting MCAK binding to microtubule ends, enhancing the ability of MCAK to recycle for multiple rounds of microtubule depolymerization, and preventing MCAK from being sequestered by tubulin heterodimers.
