Sex and longevity

Or: why mating of eukaryotic single cell organisms may be less

boring than you think

If it can find a suitable partner, yeast prefers to mate to become diploid rather than haploid

Processes required for mating to occur successfully

Processes required for dating to occur successfully

• Sensing of the partner• Signaling to the partner• Orientation towards the partner• Synchronization of behavior

Processes required for mating to occur successfully cont’d

• Synchronization of Cell cycle• Fusion (cell/nuclear)• Repression of Haploid specific genes after

mating

Signalling to/Sensing of the partner

Haploid yeast cells produce pheromones:

a cells produce a- factor (12 aa peptide)

a “ “ a- factor (13 aa peptide)

Haploid yeast cells produce pheromone receptors that localized to the cell membrane:

a cells produce a- factor-receptor Ste2p

a “ “ a- factor receptor Ste3p

Intercellular signalling! Analogous/Homologous to cell-cell signalling in higher eukaryotes

- Environmental signalling pathway

Selection of mutants unable to mate

• Lee Hartwell, 1980• Seminal work on cell cycle regulation (Nobel

Prize 2001 for Medicine/Physiology)• Interest in factors that regulate the cell cycle• S. cerevisiae cells arrest their cell cycle in

G1 phase upon exposure to mating factor

Selection scheme• Mutants were selected for their ability to grow on

media containing mating factor (unresponsive/

resistant to mating factor) at high temperature (34oC) but not at low temperature (22oC) (temperature sensitive “ts” mutants)

• These mutants can be tested for their ability to mate by mixing with cells of opposite mating type and selecting for diploids via unique markers present in the haploids

• Mutants deficient for mating (sterile) at high temperature can be sorted into complementation groups

• STE genes

Cell-to-cell signal recognition and transduction

Ste2p/Ste3p

• 7-transmembrane receptor molecules(highly conserved cell membrane signal transduction

molecules; e.g. opiate receptors in humans)

- C-terminus involved in regulation of degradation of Ste2/3 (ubiquitination pathway), phosphorylation sites

• Interact with the Heterotrimeric G-protein

Gpa1p = a subunit, Ste18p = b-subunit, Ste4p = g-subunit

Upon binding of pheromone release of b, g-subunits of heterotrimeric G-protein, initiation of MAP-kinase cascade

The mating signalling pathway is a MAP – kinase pathway

MAPK = mitogen activated protein kinase (FUS3)

MAPKK= mitogen activated protein kinase-kinase (STE7)

MAPKKK= ….. (STE11)

MAPKKKKK= …..(STE20)

“MAP kinase cascade”

Activation of expression of genes required for changes in cells necessary for mating

Dependent on recruitment of “Scaffold protein” Ste5p to the plasma membrane

This slide was nicked from internet lecture notes of a

course held at the Universität München

http://biochemie.web.med.uni-muenchen.de/Yeast_Biol/

Conservation of Signal Transduction Mechanisms in Eukaryotes

Fig. 1. Scheme of distinct MAPK signalling pathways in mammals, yeast and plants. Note the general similarity in the organization of MAPK pathways in all three eukaryotic systems. MAPKKK, mitogen activated protein kinase kinase kinase; MAPKK, mitogen activated protein kinase kinase; MAPK, mitogen activated protein kinase. Scaffolding proteins (depicted in dark blue) are integrating signalling pathways.

Samaj J, Baluska F, Hirt H. From signal to cell polarity: mitogen-activated protein kinases as sensors and effectors of cytoskeleton dynamicity.J Exp Bot. 2004 Jan;55(395):189-98. Epub 2003 Dec 12

Multiple MAP kinase (MAPK) cascades using shared components regulate growth and differentiation in S. cerevisiae. Mating, invasive growth, pseudohyphal development, high-osmolarity/glycerol response and maintenance of cell wall integrity are each regulated by structurally similar but functionally distinct MAPK cascades that are activated by different upstream signals but have in common at least three kinds of kinases: a MAPKKK, a MAPKK (or MEK) and a MAPK. Yellow highlighting indicates the kinases that are shared by the different pathways. Note that for simplicity Ste50p is not shown in this figure, although it associates with Ste11p and is required for optimal signaling through all of the pathways shown. Details can be found elsewhere (Gustin et al., 1998; Elion, 2000; Pan et al., 2000). (Elion EA.The Ste5p scaffold.J Cell Sci. 2001 Nov;114(Pt 22):3967-78. Review).

Far1 Ste12 Ste12Tec1Transcription

Factors/Effectors ?

What confers specificity to kinase cascades?

βγαS

te5

Ste11

Ste7

Fus3

Ste20

Kss1Tec1Ste12

GTP

GDP

Distinct “scaffolds” for distinct signaling pathways?

Cartoon of Ste5p, Pbs2p and Far1p scaffolds. Ste5p is required for activation of the mating MAPK cascade in response to mating pheromone and does not have an intrinsic kinase activity, whereas Pbs2p encodes the MAPKK of the high osmolarity/glycerol pathway that is activated by increased osmolarity. Far1p is required for oriented polarized growth in response to mating pheromone. Pbs2p and Far1p are postulated to be analogs of Ste5p on the basis of their ability to associate with multiple components of an individual signal transduction pathway (Posas and Saito, 1997; Butty et al., 1998; Nern and Arkowitz, 1998; Nern and Arkowitz, 1999; Rait et al., 2000), but it is not known whether they simultaneously bind to associated signaling components. Similarities between Ste5p, Pbs2p and Far1p include the ability to associate with an uppermost component of a pathway that is membrane associated and senses the external signal, as well as to downstream components that regulate the activity of effectors within a pathway. In addition, all three scaffolds link signaling components that also associate with a Rho-type G protein (Cdc42p). Ste5p and Far1p share two domains of homology (Leberer et al., 1992), one of which overlaps with the RING-H2 domain that is thought to associate with the Gß subunit Ste4p of the same heterotrimeric G protein. It is not known whether the RING-H2 domains have a function in ubiquitin-mediated proteolysis (Borden, 2001). Elion EA.The Ste5p scaffold. J Cell Sci. 2001 Nov;114(Pt 22):3967-78. Review

Mechanisms of cell polarization

Yeast cells undergo polarized cell growth during the mating reponse

- In response to mating factor, yeast cells “grow” towards the highest concentration of mating factor forming a “shmoo”

- identification of factors involved in shmooing allowed dissection of cell polarization mechanisms

Isolation of shmooing-deficient mutants• Ethyl-methane sulfonate mutagenesis of yeast cells (ade2

deficient, red)• Plating of mutagenized cells at low cell titer on YPD plates• Replica plating of mutagenized cells onto a lawn of

inefficiently mating yeast strains and incubated for 6 hours to allow for mating

• Mating plates were replica plated on selective media to select for diploids

• Mutants unable to mate were identified by inability to grow on selective plate (but leaving a red “shadow” of dying cells due to ade2 mutation) while cells that were able to mate produced white colonies

• (Also other screens to identify mutants e.g. in bud site selection etc)

• mutations isolated in FAR1, CDC24, CDC42, BEM1 and other factors investigation of mutant phenotypes

Cell polarization steps• Cell surface site determination using

intrinsic (e.g.for cell budding) or extrinsic (e.g. for mating) cues

• Marking of cell surface by ”Landmark protein” (Polarity cue)

• Establishment of polarity by activation of small GTPase proteins in vicinity of the landmark

• Cytoskeletal re-orientation/polarisation (actin and other polarized components)

Cell polarizationModel for the localization of Cdc24 during budding and mating. a, In the G1 phase of the cell cycle, Far1 localizes Cdc24 in the nucleus. b, Activation of the Cdc28–Cln kinase triggers degradation of Far1 in the nucleus, allowing rapid export of Cdc24 to the cytoplasm. Cytoplasmic Cdc24 is then recruited to the incipient bud site, possibly marked by Rsr1/Bud1-GTP. c, In the presence of pheromones, the Far1–Cdc24 complex is exported by Msn5 into the cytoplasm, where Far1 targets Cdc24 to Gβγ at the site of activated receptor. As a result, the actin cytoskeleton polarizes towards the position of the mating partner, signalled by the morphogenetic pheromone gradient (Shimada Y, Gulli MP, Peter M. Nuclear sequestration of the exchange factor Cdc24 by Far1 regulates cell polarity during yeast mating.Nat Cell Biol. 2000 Feb;2(2):117-24.)

Bud1

Cdc42

GDP

Far1p

Cdc24

βγ

α

G1 phase

Bud1

Cdc42

GDP

Far1p

Cdc24

βγ

α

Budding

GTP

Cdc42

Msn5Bud1

βγ

αMap kinase

cascade

Fus3

Activates?

Far1p

Cdc24

GDP

GTP

Bem1

Reorientation of Actin networks during

shmooing

Polarity Pathways in Yeast

 Schematic representation of the molecular pathways leading to a polarized organization of the cytoskeleton in response to internal cues during budding and external signals during mating. Common to both pathways is the asymmetric activation of Cdc42p at the site determined by the landmarks. In turn, activated Cdc42p triggers the polarized assembly of the actin cytoskeleton by binding to various effectors. The mediators Bud1p and Far1p are thought to recruit and activate the GEF Cdc24p downstream of the specific landmark proteins. Far1p interacts directly with activated Gβγ at activated receptors, whereas Bud1p is regulated at landmarks by the GEF Bud5p and the GAP Bud2p. The mechanisms required to localize the landmark proteins Bud10p, Bud8p and Bud9p are not fully understood, but in the case of Bud9p and Bud10p require an intact septin structure. Genetic analysis has identified a number of novel proteins, which alter the budding pattern of diploid cells, and may thus be involved in localizing these landmark proteins. Chang F, Peter M.Yeasts make their mark. Nat Cell Biol. 2003 Apr;5(4):294-9. Review.

A model for cell specialization

Mating type regulation

• Differ in expression from MAT (mating type) locus and subsequent gene activation/repression

• Haploid cells are unable to sporulate but can mate to form diploid a/a cells

• Main players: a1 activator, a2 repressor, a1 co-repressor, Mcm1p co-activator/repressor

• a2-mediated repression is dependent on the yeast general repression complex Ssn6p/Tup1p

Three yeast cell types:

haploid diploid

a cells a cells a/a cells

Mating type regulation by a1, a1, a2 and Mcm1 proteins

Herskowitz I. A regulatory hierarchy for cell specialization in yeast.Nature. 1989 Dec 14;342(6251):749-57. Review.

Mcm1p can act as an activator or as a repressor depending on interacting factors

Ssn6/Tup1

Herskowitz I. A regulatory hierarchy for cell specialization in yeast.Nature. 1989 Dec 14;342(6251):749-57. Review.

Homothallic life cycle of S. cerevisiae

 Homothallic life cycle of Saccharomyces. A MAT a haploid cell that has divided can switch to the opposite mating-type and the original cell and its switched partner can conjugate to form a MAT a/MAT diploid cell. Meiosis and sporulation will regenerate haploid cells. Homothallic switching is confined to cells that have previously divided (mother cells) by the action of the Ash1 repressor protein that is localized to the newly formed bud that gives rise to a daughter cell. Ash1p represses a positive regulator of HO endonuclease expression, Swi5p. In germinating spores, it is most likely the absence of Swi5p itself that prevents MAT switching until the cells have budded and divided Haber JE.Mating-type gene switching in Saccharomyces cerevisiae. Annu Rev Genet. 1998;32:561-99. Review.

1

2

3

Independent of mating type, yeast cells contain the information for both a and a- specific regulators

• All genes for determination of the expression of mating type are physically present in a and a cells

• These genes are contained in so called mating type loci HMRa and HML a which are silenced (no gene expression)

• A third locus (MAT) carries the expressed information

• In HO+ cells, mating type can be switched by homologous recombination of one of the silent cassettes into the active MAT locus

• The switch is initiated by a double-strand cut of the HO endonuclease in the MAT locus

Fig. 1. Mating-type switching: switching from (a) MATa or (b) MATa. An HO endonuclease-induced double-strand break at MAT initiates gene conversion/replacement of the Ya region with Ya sequences copied from HMLa. MATa cells express Mata1p and Mata2p regulatory proteins, while MATa encodes Mata1p. HMLa and HMRa contain complete copies of mating-type genes but are not expressed because of the silencing imposed through the adjacent E and I silencer sequences that organize a repressed chromatin structure (indicated by hatched lines). HML shares more sequences (regions W, X, Z1 and Z2) with MAT than does HMR (regions X and Z1). MATa strains preferentially recombine with HMLa, even if HMR also contains Y a instead of Ya. Donor preference is dependent on a 244 bp cis-acting recombination enhancer (RE).

Trends in Genetics Volume 14, Issue 8, 1 December 1998, Pages 317-321A locus control region regulates yeast recombination James E. Haber*

W X Z1 Z2

W X Z1 Z2

W X Z1 Z2

W X Z1 Z2

X Z1

X Z1

Gene silencing

Silencing of mating type loci• Expression from HMRa and HMLα needs to be

turned off tightly to ensure proper regulation of mating type genes

• Turning off these loci ( as well as other regions, e.g. telomeres) is achieved by a process called silencing, which results in heterochromatin formation over the silcenced region

• Heterochromatin is “a specialized chromatin structure that blocks expression of most genes within the silenced domain, irrespective of which activator or RNA polymerase is used”

Silencing is a process that is mechanistically

different from repression • Silencing turns off the expression of existing genes

permanently in an organism

• Repression turns of genes temporarily, but can be relieved due to changing environment (nutrients, growth factors)

• Silencing in yeast is present at the HML/HMR loci and at telomeres

• Epigenetic phenomenon (change in expression without change in DNA sequence)

• Can you think of a silencing event in humans?

Main components involved in silencing

• Rap1p, Orc1p, Abf1p: DNA binding proteins with affinity to Sir proteins

• Sir (silent information regulator) proteins:- Sir1p: required for establishment of silencing- Sir2p: histone deacetylase- Sir3p: structural protein, interacts with deacetylated histones- Sir4p:structural protein, interacts with Sir2p in a 1:1 complex; interacts with deacetylated histones

Cis-acting elements required for silencing in yeast

First: “Nucleation event”Sir1 (silent information regulator 1) protein required for establishment of silencing but not for maintenance

(Sir1p temperature sensitive mutant:- Permissive temperature -> loci silent- Restrictive temperature-> most loci will stay

silent, but no new establishment)- Sir1p binds to ORC (origin recognition

complex)- targeting of Sir1p to ORC is sufficient for

establishment of silencing- Passing through S-phase in cell cycle is

required for establishment of silencing of mating type loci

Sir1p is only required in Mating type loci silencing!

Hierarchy of establishment of silencing in yeast

• Rap1p, Orc1p, Abf1p binding of silencer region• Recruitment of Sir1p• Sir4p recruited to silencer by binding to Sir1p and

Rap1p• Sir2p joins the complex by physical interaction with

Sir4p• Sir3p interacts via Sir4p, Rap1p and Abf1p• Deacetylation of Histones by Sir2p results in further

recruitment of Sir3p and Sir4p, leading to chain reaction-like chromatin spreading

Orc1p

Abf1p

Sir1p

Rap1p

Sir4pSir2pSir4p

Sir2pSir4pSir2p

Sir4p

TATATATA TATATATA

AcAcAc AcAcAcAc Ac

Grunstein M. Yeast heterochromatin: regulation of its assembly and inheritance by histones.Cell. 1998 May 1;93(3):325-8. Review

Limiting of Chromating spreading• Uncontrolled spreading of chromatin would

be detrimental to the cell• Spreading is limited by several machanisms:

– boundary elements (e.g pol III transcription complex on tRNA promoter proximal to HMR)

– anti-silencing modifications on nucleosomes (acetylation of specific histone residues)

– Intrinsic limitations of the spreading mechanism due to rapid turnover and limited availability of Sir proteins

Epigenetic Inheritance of Silenced chromatin

• Epigenetic Inheritance: “Heritability of two different states in otherwise identical cells”

• Ability of a specialized chromatin structure (or protein structure) to template its own reformation

• Model for epigenetic inheritance:- during DNA replication, epigenetically

marked (acetylated or methylated) histones (H3 and H4) are distributed randomly between the sister DNA strands

- presence of epigenetically marked histones/ nucleosomes on DNA strands causes propagation of silenced chromatin

Yeast as a model for aging processes

Sir2p and longevity• Unlike E. coli cells, S. cerevisiae

cells are not immortal – the replicative life span (i.e. how often a cell can make buds) of yeast is finite = yeast cells die of old age!

• Replicative life span is assessed by counting bud scars on yeast cells

• Life span is strain dependent (18 – 100 budding events)

• Model for research on human longevity?

Bud scar

Screen for genes involved in aging • Guarente group (Harvard)• Prolonged life span is a complex trait (several

genes involved)• Life span for individual cells not easy to score• Higher stress resistance (e.g resistance

starvation) is associated with longevity • Mutagenesis of a short-lived, starvation

sensitive yeast strains and selection for starvation resistant clones led to the isolation of yeast mutants with an increased life span

Silent information regulator genes affect life span

• One long-lived mutant (uth2-42) had additional phenotypes that co-segregated with longevity: poor mating and bipolar budding pattern (like diploid cells)

• Cloning of the mutation by library complementation, selecting for mating-competent transformants

Grow mutant cells

High frequency transformation with

genomic library

Grow cells of opposite

mating type

Spread on Petri dish to make a

lawn of cells

Replica plate to allow for mating

Select diploids

Mating-competent clones carried the complementing plasmid

The uth2-42 mutant was complemented by SIR4

SIR genes affect longevity• The uth2-42 mutation was a gain of function

mutation that redirected the Sir2p/Sir3p/Sir4p complex away from silenced loci (telomeres and mating type loci) to the nucleolus

• Deletion mutations of SIR2, SIR3 and SIR4 caused shortened life span

• Overexpression of yeast SIR2 increases life span

• SIR2 mediates the beneficial effects of calorie restriction (CR) on longevity in yeast (at least in some strain backgrounds)

Sirtuins and aging• Role in silencing and suppression of recombination at the ribosomal

DNA locus (which can give rise to toxic extrachromosomal rDNA circles - ERCs)– The yeast rDNA locus on chromosome XII is known to give rise to 3 μm circular

forms of rDNA, composed of a single rDNA repeat and containing an ARS– The accumulation of ERCs is such that after 15 divisions, they can reach levels

equal to the DNA content of the yeast genome (Sinclair et al., TIGS 23, Vol4, 1998)

• As a result of yeast senescence, ERCs accumulate in yeast cells, and the Sir protein complex disassociates from silenced loci and moves to the nucleolus to inactivate ERCs

• “A redistribution of chromatin modifying factors results in epigenetic changes that promote aging phenotypes”

• Sir proteins are also required for the repair of DNA double strand breaks• DNA - damage induced relocalization of SIR complex results in aging• Some aspects of these mechanisms are conserved in humans

Fig. 2. Characteristics of yeast aging. The replicative life span of yeast is defined as the number of daughter cells produced by a mother cell before cessation of cell division and senescence. Yeast cells undergo many age-related changes, including an increase in cell size, a slowing of the cell cycle, enlargement and fragmentation of the nucleolus (red), and the redistribution of the Sir3 silencing protein (green) from telomeres and HM loci to the nucleolus (Sinclair et al., TIGS 23, Vol 4, 1998).

Sirtuins in higher eukaryotes • SIR2 is conserved in eukaryotes

• Overexpression of SIR2.1 in worms increases life span*• Mammals with 7 Sirtuin variants• Beneficial effects of calorie restriction in mammals mediated in part by

SIRT1

Chen D and Guarente L. TIMM Vol. 13 No.2

*This may not be true!!

http://www.nature.com/news/2011/110921/full/

news.2011.549.html

Mammalian Sir homologues (Sirtuins) are major regulators of aging in mammals