Humphey - Cell Cycle Control Mechanisms and Protocols.pdf - Biologia - adam001d

METHODS IN MOLECULAR BIOLOGY TM

Volume 296

Cell Cycle

Control

Mechanisms and Protocols

Edited by

Tim Humphrey

Gavin Brooks

Cell Cycle

Control

Mechanisms and Protocols

Edited by

Tim Humphrey

Gavin Brooks

The Budding and Fission Yeasts

Cell Cycle Molecules and Mechanisms of the Budding

and Fission Yeasts

Tim Humphrey and Amanda Pearce

Summary

The cell cycles of the budding yeast Saccharomyces cerevisiae and the fission yeast,

Schizosaccharomyces pombe are currently the best understood of all eukaryotes. Studies in

these two evolutionarily divergent organisms have identified common control mechanisms,

which have provided paradigms for our understanding of the eukaryotic cell cycle. This

chapter provides an overview of our current knowledge of the molecules and mechanisms

that regulate the mitotic cell cycle in these two yeasts.

Key Words

Cell cycle; Saccharomyces cerevisiae ; Schizosaccharomyces pombe ; fission yeast; bud-

ding yeast; review.

1. Introduction

The eukaryotic cell cycle can be considered as two distinct events, DNA replication

(S-phase) and mitosis (M-phase), separated temporally by gaps known as G 1 and G 2 .

These events must be regulated to ensure that they occur in the correct order with

respect to each other and that they occur only once per cell cycle. Moreover, these

discontinuous events must be coordinated with continuous events such as cell growth,

in order to maintain normal cell size (reviewed in ref. 1 ). Significant advances in

understanding such cell cycle controls have arisen from the study of these yeasts. The

use of yeast as a model system for studying the cell cycle provides a number of advan-

tages: yeasts are single-celled, rapidly dividing eukaryotes that can exist in the haploid

form. Thus yeast are readily amenable to powerful genetic analyses, and molecular

tools are available (reviewed in refs. 2 and 3 ). Although both yeasts are evolutionarily

divergent (4) , common mechanisms control their cell cycles that are conserved

throughout eukaryotes (reviewed in refs. 5 and 6 ). Moreover, following the sequenc-

ing of both yeast genomes (7 , 8) , systematic genetic analyses together with reverse

From: Methods in Molecular Biology, vol. 296, Cell Cycle Control: Mechanisms and Protocols

Edited by: T. Humphrey and G. Brooks © Humana Press Inc., Totowa, NJ

Humphrey and Pearce

genetics are beginning to provide global insights into the cell cycle control of these

model organisms, and hence all eukaryotes.

2. Yeast Life Cycles

S. cerevisiae proliferates by budding, during which organelles, and ultimately a

copy of the genome, are deposited into a daughter bud, which grows out of the mother

cell. The bud grows to a minimal size and after receiving a full complement of chro-

mosomes pinches off from the mother cell in a process called cytokinesis. Budding

yeast can exist in a haploid (16 chromosomes) or diploid (32 chromosomes) state (re-

viewed in ref. 9 ).

In contrast, S. pombe grows by medial fission, whereby newly born daughter cells

grow from the tips of their cylindrical rod shape by a process known as new-end take-

off. Once a mature length is reached, the cell ceases growth and produces a septum

that bisects the mother cell into two daughter cells. Fission yeasts exist naturally in a

haploid form (one set of three chromosomes), limiting the diploid phase to the zygotic

nucleus, which enters meiosis immediately (reviewed in ref. 10 ).

Conditions of nitrogen starvation have the same consequences for both yeasts and

may result in several developmental fates. If the culture contains cells of a single mat-

ing type, then the cell cycle will arrest in stationary phase in G 1 and enter G 0 . How-

ever, if the opposite mating type is also available, pheromone production will result in

conjugation to form diploid cells, which will undergo meiosis and form spores. Bud-

ding yeasts are distinct from fission yeasts in that they can arrest in G 1 in the absence

of nitrogen starvation and may exist as diploids in the mitotic cell cycle (reviewed in

refs. 9 and 10 ).

3. The Mitotic Cell Cycle of Yeasts

3.1. Budding Yeast

In budding yeast, a point exists in mid-G1 after which the cell becomes committed

to the mitotic cell cycle. This point is commonly referred to as Start (11) . Start plays

an important role in coordinating division with growth. Growth is rate-limiting for the

cell cycle, and if a critical size requirement is not reached, cells cannot progress

through Start. Prior to Start (in early G 1 ), cells can respond to the environment. If

nutrients are plentiful, they can proceed into the next cell cycle; however, if nutrients

are limiting, they can make the decision to enter stationary phase or meiosis. In addi-

tion, passage through Start may be inhibited by mating factors from other yeasts; hence

if two haploid yeast of the opposite mating types detect each other’s pheromones, then

they will “schmoo” toward one another, mate and form a diploid. Having passed Start,

cells are programmed to complete the cell cycle irrespective of the nutrient state or

exposure to pheromones.

Entry into mitosis is classically defined by three physiological events in eukary-

otes: the formation of the mitotic spindle, breakdown of the nuclear membrane and

chromosomal condensation. Both yeasts undergo what is termed a closed mitosis, in

which the mitotic nuclear membrane, remains intact. In addition, S. cerevisiae is dis-

tinct from other eukaryotic cells in that the mitotic spindle begins to form during early

The Budding and Fission Yeasts

S-phase. Thus S. cerevisiae does not have a clear landmark event distinguishing the G 2

and M-phase, and thus the G 2 /M transition is difficult to define in this organism (re-

viewed in ref. 12 ).

3.2. Fission Yeast

In fission yeast the G 1 and S-phases are relatively short (each accounting for 10%

of the time it takes to complete the cell cycle), whereas G 2 is considerably longer (70%

of the time is spent in this phase, in which most growth occurs; reviewed in ref. 10 ).

Again, a critical Start point exists, and passage through this point is dependent on the

prior completion of mitosis in the previous cell cycle and on the cell reaching a critical

minimal size (13) . Following spore germination or nutrient starvation, when cells are

unusually small, a period of growth before Start is required such that a critical size is

obtained. However, under nonlimiting conditions, cells have already achieved a mini-

mal size requirement for passage through G 1 . Consequently, G 1 is usually cryptic in

logarithmically dividing cultures of S. pombe , and S-phase directly follows comple-

tion of nuclear division, resulting in cells that are already in G 2 at the time of cell

separation (14) .

The G 2 /M transition is the major control point in the cell cycle of fission yeast and

determines the timing of entry into mitosis (as opposed to S. cerevisiae , in which Start

in G 1 is the major control point). Entry into mitosis is dependent on the cell having

previously completed S-phase; on repairing any DNA damage; and on reaching a criti-

cal size. Cells coordinate size such that if G 2 is shortened, G 1 will be lengthened and

vice versa (reviewed in ref. 10 ).

4. Cell Cycle Molecules

4.1. cdc Mutants

Much of what we know about the cell cycle was discovered through the isolation of

temperature sensitive ( ts ), cell division cycle ( cdc ) mutants. In 1970 Hartwell et al.

(15) discovered that a number of these ts mutants, upon shifting to the restrictive tem-

perature, arrested the cell population with the same morphology, suggesting that the

mutant product was required only at a specific point in the cell cycle. Approximately

60 different cdc mutants have been isolated in budding yeast, and approx 30 have been

isolated in fission yeasts. In addition to cdc genes, a large number of new cell cycle

genes have been identified on the basis of interactions with preexisting cell cycle genes

(reviewed in refs. 10 and 12 ).

4.2. Cyclin-Dependent Kinases

A highly conserved class of molecules termed the cyclin-dependent kinases (CDKs)

plays a central role in coordinating the cell cycles of all eukaryotes. In both fission and

budding yeasts, the cell cycle is controlled both at the G 1 /S transition and the G 2 /M

transition by a single highly conserved CDK, encoded by the CDC28 and cdc2 + genes

of S. cerevisiae and S. pombe , respectively. In budding yeast, ts mutations in CDC28

allowed the definition of Start. The cdc28ts mutant blocked budding and cell cycle

progression at a point in the G 1 -phase at which cells could still enter the sexual cycle

Humphrey and Pearce

instead of proceeding with the mitotic cycle. From this work, Start could be defined

genetically as the point in the cell cycle at which budding, DNA replication, and spindle

pole body (SPB) duplication become insensitive to loss of Cdc28 function (11) .

In fission yeasts, different mutations in cdc2 + result in the cells either elongating

(16) or conversely becoming smaller (17) , a phenotype suggesting that Cdc2 might

function in the timing of division. CDC28 and cdc2 + share 63% identity, and both are

required for passage through Start as well as mitosis. Indeed, these genes are con-

served, with the human CDC2 gene displaying the same properties, demonstrating

conservation of essential features of the cell cycle in all eukaryotes (6) .

Active CDKs generally phosphorylate serine or threonine residues that are followed

by a proline and a consensus sequence of K/R, S/T, P, X, K/R (reviewed in ref. 12 ).

Although many CDK targets have been identified, a comprehensive analysis of CDK

targets remains an important goal.

4.3. Cyclins

All CDKs require positive regulatory partners for activity, known as cyclins (1) ,

which additionally impart CDK substrate specificity. Cyclins were identified as pro-

teins that oscillated in abundance through the cell cycle in rapidly cleaving early

embryonic cells (18) . Not all cyclins show this cell cycle-dependent pattern of synthe-

sis and degradation. However, all cyclins share homology over a domain called the

cyclin box , a region required for binding and activation of CDKs. In S. cerevisiae , a

number of cyclins have been identified that associate with Cdc28: G1 cyclins (Cln1,

Cln2, and Cln3), S-phase cyclins (Clb5 and Clb6), and G 2 cyclins (Clb1–4. Clb1–6)

are all B-type cyclins (19) . S. pombe cyclins include Puc1 (a G 1 cyclin), three B-type

cyclins (Cig1 and Cig2; S-phase cyclins), and Cdc13 (a G 2 cyclin) (reviewed in ref.

20 ). Cyclins bind to Cdc28/Cdc2, forming an active complex, which is associated with

histone H1 kinase activity. In order to bind, cyclins recognize a binding motif present

on CDKs known as the PSTAIR motif (corresponding to the conserved amino acids

within this domain). Cyclins accumulate at specific times during the cell cycle, lead-

ing to overlapping activation of different CDK/cyclin complexes, which in turn regu-

late the cell cycle (reviewed in refs. 10 and 12 ).

5. Regulation of the Yeast CDK/Cyclin Complex

The activity of the CDK/cyclin complex is key to cell cycle progression and can be

considered the cell cycle “engine” (1) . Thus CDK/cyclin complexes are subject to a

high degree of regulation through a number of posttranslational mechanisms includ-

ing phosphorylation, inhibition by cyclin-kinase inhibitors, destruction of cyclins, and

destruction of the inhibitors at the appropriate time in the cell cycle. These mecha-

nisms ensure that the cell cycle progresses in an orderly fashion. In addition, the peri-

odic activity of particular CDK/cyclin complexes is achieved through feedback loops

within the cell cycle: In G 1 /S, G 1 cyclins activate the Clb cyclins, which then turn off

the G 1 cyclins. Similarly, in mitosis, the mitotic cyclins promote spindle formation

and turn on the anaphase-promoting complex (APC), or cyclosome, which then de-

grades the mitotic cyclins needed for the first step. The molecular basis of these regu-

latory events in yeast is described below in Subheadings 5.1.–5.3. ( see also Fig. 1 ).

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