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Mengukur
jumlah molekul protein dalam sel
Measuring the number of protein molecules inside cells
Date:
July 18,
2014
Source:
Instituto Gulbenkian de Ciência
(IGC)
Summary:
The identification of the genes and proteins involved
in a biological process, as well as the way they interact, are essential for
the understanding of that process. However, often little is known about the
dimensions of molecular biological structures. Knowing how many molecules make
up a structure and are required for its function are essential for our
understanding of biological mechanisms, yet poses a difficult challenge. Now,
in a breakthrough study, researchers were able to measure the amount of protein
molecules in living human cells required to form the centromere.
..................
The identification of the genes and proteins involved in a
biological process, as well as the way they interact, are essential for the
understanding of that process. However, often little is known about the
dimensions of molecular biological structures. Knowing how many molecules make
up a structure and are required for its function are essential for our
understanding of biological mechanisms. Yet, quantifying molecules of
infinitesimal size poses a difficult challenge. Now, in a breakthrough study,
Lars Jansen and his team from Instituto Gulbenkian de Ciência (IGC, Portugal)
were able to measure the amount of protein molecules in living human cells
required to form an important structure of the chromosome, the centromere.
This study,
published in the open access scientific journal eLife, presents new
methodologies that may also be used to unveil other biological problems.
Centromeres
are protein structures present at chromosomes. These structures recruit the
necessary molecular machinery that drives the segregation of chromosomes into
the daughter cells, a process essential for cell division. If the location of
centromeres is changed or if the proteins that compose these structures are
impaired, abnormal cell divisions may arise. Lars Jansen's laboratory, together
with other research groups, have identified the components of centromeres and
found one protein, called CENP-A to be central to centromere function. What has
been lacking thus far is a measure of how many of these molecules are present
which is important to understand how centromeres are built and maintained.
Dani Bodor,
PhD candidate at Jansen's laboratory and first author of this study, explains
the context of this study: "We knew the CENP-A protein was playing a
crucial role in the formation of centromeres. Previous studies showed that
without this protein, cells failed to divide properly, with consequences in the
number of chromosomes transmitted to the daughter cells. But exactly how much
CENP-A was required to form a centromere? We needed to find a way to count
CENP-A molecules, that have a size in the order of nanometers (1.000.000 times
smaller than 1 millimeter)."
The research
team set to develop tools that allow for such a measurement. Using modern
genetic engineering they fused a gene that codes for a fluorescent protein to
the CENP-A gene. By using this genetic trick, all CENP-A proteins produced by
cells became fluorescent. Next, the researchers observed these cells under the
microscope, and were able to quantify the total amount of fluorescence present
in the cell and the fraction of fluorescence at centromeres. Ultimately, these
measurements allowed them to determine that approximately 400 molecules of
CENP-A are present on the centromeres of human cells.
Dani Bodor
says: "We were inspired by a methodology used in yeast. But until now, no
one had used it to measure molecules in more 'complex' cells. Yeast cells have
more or less the same shape and volume, but human cells differ in shapes and
volumes which increases the degree of complexity when this kind of techniques
are used."
To confirm
their calculations were accurate, the researchers used two other techniques.
Their results showed that independently of the technique used they would always
reach a number around 400.
Lars Jansen
says: "Centromeres need to be very stable structures to ensure the
faithful transmission of chromosomes to the daughter cells during cell
division. When cells divide, the CENP-A proteins are distributed to the
daughter cells, and the number of molecules that each cell receives may vary.
By having 400 molecules the cell can assure that a sufficient number of CENP-A
is passed to form the centromeres. The calculation of the number of CENP-A
molecules allows us to propose a mechanistic framework that can explain the
formation and inheritance of centromeres."
When asked
about the technical difficulties faced during this study Lars says: "We
took 5 years to conduct this work, and for sure we would not be able to have
done it 10 years ago. We need to develop new techniques continuously to be able
to go further and answer novel questions, even to old biological problems. We
have arrived at a time in biology where more and more laboratories will start
looking at the quantitative aspects of the biological problem they are
studying. The techniques we have employed can be quite helpful for that."
Lars Jansen
received a Consolidator Grant from the European Research Council (ERC) in the
beginning of this year to continue his studies on centromeres and on the
mechanisms that control the faithful transmission of non-genetic information
from mother to daughter cells.
Story
Source:
The above
story is based on materials provided by Instituto Gulbenkian de Ciência (IGC). Note: Materials may be edited
for content and length.
Journal
Reference:
- D. L. Bodor, J. F. Mata, M. Sergeev, A. F. David, K. J. Salimian, T. Panchenko, D. W. Cleveland, B. E. Black, J. V. Shah, L. E. Jansen. The quantitative architecture of centromeric chromatin. eLife, 2014; 3 (0): e02137 DOI: 10.7554/eLife.02137