Download How did the experiments with cell fusion, oocytes and yeast lead to

How did the experiments with cell fusion, oocytes and yeast lead to our current understanding of
the control of the cell cycle?
Introduction:

The cell cycle is a series of events that take place in a cell, leading to division and duplication
that result in the formation of two daughter cells.

Approximately 10,000 trillion cell divisions occur in the human body over a lifetime.

Experiments involving cell fusion, oocytes and yeast were important in elucidating how
uncontrolled proliferation and genetic replication errors are detected and prevented.

Progression through the cell cycle is tightly regulated, enduring high fidelity – essential for cell
survival and functionality.
Oocyte/Early Embryos:

The early embryos of sea urchins provide an excellent experimental model for analysis of cell
cycle checkpoint mechanisms – allows biochemical features to be readily analysed.

Experimentation by Tim Hunt and Joan Ruderman examined the pattern of gene expression
in oocytes.

During early stages of development, the sea urchin embryonic cell cycles occur
synchronously, allowing all the cells to be studied at the same stage in the cell cycle.

Treating and incubating the oocytes with radiolabelled amino acids causes them to be taken
up into the cell and then incorporated into synthesised proteins.

Using this technique, Hunt and Ruderman were able to monitor the pattern of protein
synthesis by taking samples at regular 10-minute intervals, breaking open the cells,
separating the proteins using SDS-polyacrylamide gel electrophoresis then visualising the
radioactively labelled proteins by autoradiography.

From this they were able to identify ‘cyclins’, which accumulated during interphase and were
lost at mitosis. Further analysis showed that this protein (cyclin B) is synthesised
continuously during the cell cycle and it rapidly degraded when anaphase is reached.
Yeast

The basic organisation of the cell cycle is the same in all eukaryotic cells – the machinery and
control mechanisms have been well-conserved over the course of evolution. It is therefore
possible to study the cell cycle in different organisms.

Experiments involving the budding yeast Saccharomyces cerevisiae and the fission yeast
Saccharomyces pombe allowed us to identify many of the genes encoding cyclins and cyclin
dependent kinases (CDKs). These two classes of regulatory molecules determine a cell’s
progress through the cell cycle.

These yeast species are model organisms as they have: small sizes, short generation times
(doubling time = 1.25-2 hours), high accessibility and are easily cultured. They can also
proliferate in a haploid state – only a single copy of each gene is present in the cell – this makes
it easier to isolate and study mutations.

Temperature-sensitive yeast mutants (conditional mutants) were used to identify mutations
that arrest mutant cells at a discrete step of the cell cycle at non-permissive temperatures, but
function normally at lower temperatures. It was therefore possible to identify genes involved in
cell cycle regulation.

A single cell of S. cerevisiae begins a mitotic cycle with the emergence of a bud on its surface in
G1. This bud grows throughout the cell cycle. Bud size can therefore be used to determine the
stage in the cell cycle that the cell is at, facilitating recognition of mutants.

S. pombe is a rod-shaped “fission yeast”; it elongates before dividing to produce two identical
daughter cells of equal size. Mutants can be recognised by daughter cell length.
Checkpoints:

S. cerevisiae and S. pombe were also used to elucidate ‘check points’ within the cell cycle. Each
checkpoint verifies that the processes in each phase have been carried out correctly, thus
ensuring fidelity of cell division in eukaryotic cells. There are sensor mechanisms at each
checkpoint which assess DNA damage. If damage is found, the checkpoint uses a signal
mechanism to either:
 Stall the cell cycle until DNA is repaired.
 If lesions are unrepairable, target the cell for destruction via apoptosis.
 There are three different checkpoints in the cell cycle – all of which must be regulated.
G1 checkpoint: the cell commits to cell-cycle entry and chromosome duplication.

Cells either proceed to the S phase or exit the cell cycle and enter G0.

This checkpoint is controlled by CDK inhibitor p16. It inhibits cyclin dependent kinase 4/6
(CDK4/6). This prevents CDK4 from interacting with cyclin D1, preventing progression
through the cell cycle.

To pass the checkpoint, growth-induced or oncogenic-induced expression of cyclin D allows
cyclin D to compete with CKI p16 and bind to CDK4/6.

The resulting active CDK4/6-cyclin D complex phosphorylates a protein called Rb. This
relieves inhibition of the transcription factor E2F.

This allows cyclin E to be expressed, which interacts with CDK2, allowing the cell cycle to
progress into the synthesis stage of interphase.
G2/M checkpoint: this control system triggers early mitotic events that lead to chromosome
alignment on the spindle in metaphase.

Cdk1 and cyclin B form a complex (maturation promoting factor MFP) which
phosphorylates a range of proteins regulating nuclear envelope breakdown, chromosome
condensation and spindle reorganisation.

MPF also regulates apoptosis induced by DNA-damage.
Experiments using S. cerevisiae and S. pombe also helped to identify two enzymes controlling CDK1.
Wee1:

Wee1 is a tyrosine kinase that phosphorylates the tyrosine residue at position 15 in CDK1,
inactivating it (by blocking the ATP binding site of cdk1, preventing kinase activity).

It influences cell size by preventing entry into mitosis through inhibition of cdk1. It’s
therefore highly active during interphase. Wee1 activity is decreased by several regulators at
the G2/M checkpoint.
 Decreased Wee1 activity isn’t sufficient alone to enter mitosis however – cyclin
synthesis and phosphorylation by a cyclin activating kinase (CAK) is also needed.
Cdc25:

Cdc25 is a tyrosine phosphatase. It removes the phosphate group, allowing cdk1 to function
as a kinase. Low levels of activity are observed during interphase, ensuring that cdk1 remains
inactive. At the G2/M checkpoint, several regulators increase cdc25 activity.
Wee1 and cdc25 mutants were used to elucidate the role of the enzymes.

Mutants lacking Wee1 produce smaller daughter cells than wild-type cells, since the
cell enters mitosis and divides prematurely.

If Wee1 expression is increased, mitosis is delayed and the daughter cells are
abnormally large.

Cdc25 has been found to be overly expressed in a number of cancers.

In mutants where Cdc25 is lacking, cells may fail to divide (e.g. C. pombe cells will
just become very elongated).
Metaphase checkpoint: ensures segregation of chromosomes is completed with fidelity.
Cell Fusion Experiments:
 Cell fusion experiments conducted by Potu Rao and Robert Johnson demonstrated the
dependence of the S phase on M phase.
 They isolated and fused cells in different phases of the cell cycle to form hybrids.
 When cells in S/G1/G2 are fused with a mitotic cell, the non-mitotic nucleus is driven into
mitosis.
 When G1 cells were fused with S phase cells, the G1 nucleus immediately began to synthesise
DNA. This means that the cytoplasm of cells in S phase contains factors which initiated DNA
synthesis in the G1 nucleus.
 This suggests that positive factors drive entry into S and M phases.
 When G2 cells were fused with S phase cells, the G2 nucleus failed to initiate DNA synthesis.
This showed that DNA synthesis in the G2 nucleus was prevented by a mechanism that
blocked replication of the genome until after mitosis had taken place.
 Further investigation showed that DNA replication is restricted to once per cell cycle by
MCM proteins which bind to origins of replication together with ORC. MCM proteins are
needed for the initiation of DNA replication = positive regulatory factors.
 MCM proteins can only bind to DNA in G1, allowing DNA replication to initiate in S.
 Once initiation has occurred, the MCM proteins are displaced so that replication can’t
initiate again until after mitosis.
Conclusion:

Regulation of the cell cycle involves processes crucial to cell survival, including the detection
and repair of damage to DNA and prevention of uncontrolled cell proliferation.

Experimentation with yeast, oocytes and cells fusion helped to elucidate the regulatory
mechanisms that govern the ordered and directional molecular events in the cell cycle.

Disregulation of the cell cycle can lead to uncontrolled cell division, causing tumour
formation.

Increasing our understanding of the control of the cell cycle regulation will therefore
advance our understanding of cancer and DNA damage amongst other things – it is an
exciting area of research.