Bouquet formation,
homologous pairing and crossing over in early meiosis are all processes that
are strongly dependent on the centromere.
When meiosis is initiated one of the first structures that is
seen to form in almost all organisms is the chromosome bouquet. The chromosome
bouquet is an arrangement where telomeres bunch together in a confined area of
the nuclear periphery with centromeres at a polar position to them, which as
its name suggests resembles a bouquet of flowers. This structure
allows for the the pairing of homolgous chromosomes within the cell.
Tetrahymena thermophilia is a unicellular ciliated protist
who’s micronuclei elongate and stretch dramatically during its meiotic
prophase, which is the point at which Tetrahymenas
exaggerated bouquet forms. Loidl, Lukaszewicz, Howard-Till and Koestler at the
University of Vienna have released a paper that may help to explain what
mechanisms are taking place during this process in the unicellular protist.
This paper investigates the importance of Double stranded
breaks (DSB’s) and centromere function in Tetrahymena’s
bouquet formation, suggesting that centromeres have essential functions in
recombination and chromosome pairing.
To begin their investigation Loidl et al attempted to
understand the function of centromeres during Tetrahymenas nuclear elongation. They did this by constructing
strains where the H3 histone Cna1p, was disabled through RNAi depletion. Under
wild type conditions where telomeres and centromeres segregate during the
formation of the bouquet, centromeres cluster at the periphery of the
nuclei. However with this RNAi mutant
immunostaining detected only background staining, with no clear organisation of
centromeres.
This paper also discusses how the bouquet arrangement of
centromeres and telomeres at opposite poles of the nucleus is highly dependent
on in the interaction of microtubules with the kinetochore. It was found
that whilst microtubule interaction is the main contributor to nuclear
elongation and that centromeres play no role in the elongation of the cell. Microtubules
have two known functions in Tetrahymena;
to elongate the nucleus and to hold the centromeres at a fixed position of the
nucleus.
It was already known that DSB’s were needed for the bouquet
to form, as it is an ATR-dependent response. ATR being an enzyme that is
activated in the persistent presence of single stranded DNA, which is a common
intermediate for most DNA damage repair pathways. Loidl et al set out to confirm that the
bouquet was actually necessary for DSB repair by adding nocodazole- a
microtubule inhibitor – to prevent the formation of the bouquet within the Tetrahymenas nuclei.
As an additional measure DSB-formation and repair were
monitored using pulsed-field electrophoresis in both the control and the
nocodazole treated cells. Both of these experiments showed that DSB’s were
repaired independently of bouquet formation. Therefore this proves that whilst
DBS’s are need for the initial formation of the bouquet, the bouquet itself is
not needed for the repair of those DSB’s.
To try and further understand the role of DSB’s and how they
regulate bouquet formation Loidl et al created a scenario where DSBs were
continuously produced. To do this they treated meiotic phase cells with
cisplatin – an inducer of DSBs.
Cells treated with cisplatin were no longer able to exit the
bouquet stage, suggesting that the trigger for the cells release from the
bouquet stage must be an intermediate stage in the DNA repair.
The bouquet structure is highly conserved amongst a vast number
of different species, but it’s function varies
slightly from organism to organism. For example in Arabidopsis, like with Tetrahymena, telomeres are linked with
the nuclear periphery. However, in contrast to Tetrahymena the centromeres do not cluster at a single point at the
opposite pole to the telomeres, but are more dispersed randomly across the
nucleus with no evidence to suggest that they are involved in homologous
chromosome pairing or recombination. Instead chromosome pairing occurs in
zygotene, when a structure loosely similar to the bouquet is formed. In
mammals and budding yeast chromosome pairing has been proved to be led by
telomeres and dependent on the protein SUN1 which anchors the telomere to the
nuclear membrane. A similar protein of which could not be found by Loidl et
al for Tetrahymena.
The next step in the investigation of Tetrahymenas exaggerated bouquet is a more in depth look at the
function of telomeres and telomere associated proteins. By doing this there
will be either confirmation of the views that this paper has put forward or give
us a better insight into the function of telomeres allowing us to appreciate
their involvement in the bouquet forming process.
By understanding Tetrahymenas
bouquet completely and fully we will be able to apply this knowledge to other
organisms to help us determine the processes that govern their bouquet
formation. This would be especially important is we could apply this new
knowledge to ourselves and the processes that go on in our cells during
meiosis. This would give us a much clearer insight into how diseases and
disorders may form, and therefore give us clues on how to prevent these
diseases.
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