Separation of mother and daughter cells

doi:10.1016/S0076-6879(02)51865-6

Methods in Enzymology

Volume 351, 2002, Pages 468-477

https://doi.org/10.1016/S0076-6879(02)51865-6 Get rights and content

Publisher Summary

The budding yeast Saccharomyces cerevisiae (S. cerevisiae) divides asymmetrically. In vegetative growth, yeast cells reproduce by budding, and the position where the bud forms ultimately determines the plane of cell division. This chapter describes the detailed procedures for the separation and isolation of mothers and daughters. These protocols have been used by investigators studying aging, bud site selection, and other aspects of asymmetric cell division. The chapter describes the procedures for performing life span analysis by micromanipulation and the steps for the large-scale collection of old cells. At the beginning and the end of a life span, it can be difficult to distinguish mothers from daughters. At most points in the life span, daughter cells are smaller than the mothers that produced them. In addition, mother cells will generally bud a second time before their daughter cells form their first bud. One method for effective isolation of virgin daughter cells from mother cells, but not for recovery of old mothers, is called a “baby machine.” Mother cells are attached to a membrane and allowed to divide. Daughter cells from these attached cells are eluted continuously by washing the membrane.

References (24)

P.-A. Defossez et al.
Curr Opin. Microbiol.
(1998)
N.K. Egilmez et al.
J. Biol. Chem.
(1989)
T. Smeal et al.
Cell
(1996)
B.K. Kennedy et al.
Cell
(1997)
D.A. Sinclair et al.
Cell
(1997)
A. Grzelak et al.
FEBS Lett.
(2001)
J. Chant
Annu. Rev. Cell Dev. Biol.
(1999)
A.A. Hyman et al.
J. Cell Biol.
(1987)
R. Kraut et al.
Nature
(1996)
J.E. Haber
Anna. Rev. Genet.
(1998)

R.K. Mortimer et al.

Nature

(1959)

B. Gompertz

Philos. Trans. R. Soc.

(1825)

Cited by (46)

Loss of Smi1, a protein involved in cell wall synthesis, extends replicative life span by enhancing rDNA stability in Saccharomyces cerevisiae
2021, Journal of Biological Chemistry
In Saccharomyces cerevisiae, replicative life span (RLS) is primarily affected by the stability of ribosomal DNA (rDNA). The stability of the highly repetitive rDNA array is maintained through transcriptional silencing by the NAD⁺-dependent histone deacetylase Sir2. Recently, the loss of Smi1, a protein of unknown molecular function that has been proposed to be involved in cell wall synthesis, has been demonstrated to extend RLS in S. cerevisiae, but the mechanism by which Smi1 regulates RLS has not been elucidated. In this study, we determined that the loss of Smi1 extends RLS in a Sir2-dependent manner. We observed that the smi1Δ mutation enhances transcriptional silencing at the rDNA locus and promotes rDNA stability. In the absence of Smi1, the stress-responsive transcription factor Msn2 translocates from the cytoplasm to the nucleus, and nuclear-accumulated Msn2 stimulates the expression of nicotinamidase Pnc1, which serves as an activator of Sir2. In addition, we observed that the MAP kinase Hog1 is activated in smi1Δ cells and that the activation of Hog1 induces the translocation of Msn2 into the nucleus. Taken together, our findings suggest that the loss of Smi1 leads to the nuclear accumulation of Msn2 and stimulates the expression of Pnc1, thereby enhancing Sir2-mediated rDNA stability and extending RLS in S. cerevisiae.
Advances in quantitative biology methods for studying replicative aging in Saccharomyces cerevisiae
2020, Translational Medicine of Aging
Citation Excerpt :
The procedures for both of these new technologies begin by employing previously published techniques [49,50] that allow mother cells in liquid culture to be sequestered from their progeny and toward the surface of a magnet. This is accomplished by biotinylating the cell walls of a starter culture and using magnetic streptavidin beads to bind the biotin on the surface of the cells and draw them to the magnetic surface [49,50]. Daughter cells do not inherit the biotin-coated surface from their mothers and are therefore invisible to the magnetic field [49].
Aging is a complex, yet pervasive phenomenon in biology. As human cells steadily succumb to the deteriorating effects of aging, so too comes a host of age-related ailments such as neurodegenerative disorders, cardiovascular disease and cancer. Therefore, elucidation of the molecular networks that drive aging is of paramount importance to human health. Progress toward this goal has been aided by studies from simple model organisms such as Saccharomyces cerevisiae. While work in budding yeast has already revealed much about the basic biology of aging as well as a number of evolutionarily conserved pathways involved in this process, recent technological advances are poised to greatly expand our knowledge of aging in this simple eukaryote. Here, we review the latest developments in microfluidics, single-cell analysis and high-throughput technologies for studying single-cell replicative aging in S. cerevisiae. We detail the challenges each of these methods addresses as well as the unique insights into aging that each has provided. We conclude with a discussion of potential future applications of these techniques as well as the importance of single-cell dynamics and quantitative biology approaches for understanding cell aging.
Changes in transcription and metabolism during the early stage of replicative cellular senescence in budding yeast
2014, Journal of Biological Chemistry
Citation Excerpt :
Daughter cells were removed by gentle agitation with a dissecting needle and scored every 2 h. For each of the 48 cell lines, buds from each mother cell were counted until division of living cells ceased, or cells were stained with phloxine B. Isolation of old cells was performed as described previously (19). Cells were grown in YPD medium to A600 of 1.0.
Age-related damage accumulates and a variety of biological activities and functions deteriorate in senescent cells. However, little is known about when cellular aging behaviors begin and what cellular aging processes change. Previous research demonstrated age-related mRNA changes in budding yeast by the 18th to 20th generation, which is the average replicative lifespan of yeast (i.e. about half of the population is dead by this time point). Here, we performed transcriptional and metabolic profiling for yeast at early stages of senescence (4th, 7th, and 11th generation), that is, for populations in which most cells are still alive. Transcriptional profiles showed up- and down-regulation for ∼20% of the genes profiled after the first four generations, few further changes by the 7th generation, and an additional 12% of the genes were up- and down-regulated after 11 generations. Pathway analysis revealed that these 11th generation cells had accumulated transcripts coding for enzymes involved in sugar metabolism, the TCA cycle, and amino acid degradation and showed decreased levels of mRNAs coding for enzymes involved in amino acid biosynthetic pathways. These observations were consistent with the metabolomic profiles of aging cells: an accumulation of pyruvic acid and TCA cycle intermediates and depletion of most amino acids, especially branched-chain amino acids. Stationary phase-induced genes were highly expressed after 11 generations even though the growth medium contained adequate levels of nutrients, indicating deterioration of the nutrient sensing and/or signaling pathways by the 11th generation. These changes are presumably early indications of replicative senescence.
Cellular aging is associated with increased ubiquitylation of histone H2B in yeast telomeric heterochromatin
2013, Biochemical and Biophysical Research Communications
Epigenetic changes in chromatin state are associated with aging. Notably, two histone modifications have recently been implicated in lifespan regulation, namely acetylation at H4 lysine 16 in yeast and methylation at H3 lysine 4 (H3K4) in nematodes. However, less is known about other histone modifications. Here, we report that cellular aging is associated with increased ubiquitylation of histone H2B in yeast telomeric heterochromatin. An increase in ubiquitylation at histone H2B lysine 123 and methylations at both H3K4 and H3 lysine 79 (H3K79) was observed at the telomere-proximal regions of replicatively aged cells, coincident with decreased Sir2 abundance. Moreover, deficiencies in the H2B ubiquitylase complex Rad6/Bre1 as well as the deubiquitylase Ubp10 reduced the lifespan by altering both H3K4 and H3K79 methylation and Sir2 recruitment. Thus, these results show that low levels of H2B ubiquitylation are a prerequisite for a normal lifespan and the trans-tail regulation of histone modifications regulates age-associated Sir2 recruitment through telomeric silencing.
Changes in reactive oxygen species begin early during replicative aging of Saccharomyces cerevisiae cells
2011, Free Radical Biology and Medicine
Increased reactive oxygen species (ROS) are a feature of aging cells, but little is known about when ROS generation begins as cells age. Here we show how ROS change in Saccharomyces cerevisiae cells throughout their early replicative life span using the fluorescent ROS indicator dihydroethidium (DHE), which has some specificity for the superoxide anion. Cells in a particular age range were heterogeneous with respect to their ROS burden. Surprisingly, some cells as young as 5–7 generations acquired a greatly increased level of ROS detected by DHE relative to virgin cells. By 12 generations 50% of cells had a substantial ROS burden despite being only halfway through their life span. In contrast to the wild type, cells of a sir2 mutant had lower levels of ROS reacting with DHE. Daughters from older mothers had low ROS levels, and this asymmetric distribution of ROS was SIR2-independent. Mitochondrial fragmentation also began to occur in cells after 4 generations and increased markedly as cells aged. Daughter cells regenerated normal tubular mitochondria despite the fragmentation of mitochondria in the mother cells, whereas daughters of the sir2 mutant had fragmented mitochondria at all ages.
The absence of a mitochondrial genome in rho<sup>0</sup> yeast cells extends lifespan independently of retrograde regulation
2009, Experimental Gerontology
The absence of mtDNA in rho⁰ yeast cells affects both respiration and mitochondrial–nuclear communication (e.g., retrograde regulation, intergenomic signaling, or pleiotropic drug resistance). Previously, it has been reported that some rho⁰ strains have increased replicative lifespans, attributable to the lack of respiration and retrograde regulation. Here, we have been able to confirm that rho⁰ cells exhibit increased replicative lifespans but have found that this is not associated with the lack of respiration or reduced oxidative stress but instead, is related to the lack of mtDNA per se in rho⁰ cells. Also, we find no correlation between the strength of retrograde regulation and lifespan. Furthermore, we find that pdr3⁻ or rtg2⁻ mutations are not responsible for lifespan extension in rho⁰ cells, ruling out a specific role for PDR3-pleiotropic drug resistance or RGT2-retrograde regulation pathways in the extended lifespans of rho⁰ cells. Surprisingly, Rtg3p, which acts downstream of Rtg2p, is required for lifespan increase in rho⁰ cells. Together, these findings indicate that the loss of mtDNA per se and not the lack of respiration lead to extended longevity in rho⁰ cells. They also suggest that Rtg3p, acting independently of retrograde regulation, mediates this effect, possibly via intergenomic signaling.

View all citing articles on Scopus

View full text

Separation of mother and daughter cells

Publisher Summary

Curr Opin. Microbiol.

J. Biol. Chem.

Cell

Cell

Cell

FEBS Lett.

Annu. Rev. Cell Dev. Biol.

J. Cell Biol.

Nature

Anna. Rev. Genet.

Nature

Philos. Trans. R. Soc.