Measuring the number of protein molecules inside cells

Jul 18, 2014
These images show the different planes of a cell (black) expressing CENP-A (yellow dots). Credit: Dani Bodor (IGC).

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 this week 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 , a process essential for . 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 .

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."

Explore further: Faithful cell division requires tightly controlled protein placement at the centromeres

add to favorites email to friend print save as pdf

Related Stories

Biologists identify proteins vital to chromosome segregation

Dec 24, 2012

New York University biologists have identified how a vital protein is loaded by others into the centromere, the part of the chromosome that plays a significant role in cell division. Their findings shed new light on genome ...

New light shed on cell division

Jun 14, 2011

Genes control everything from eye color to disease susceptibility, and inheritance - the passing of the genes from generation to generation after they have been duplicated - depends on centromeres. Located in the little pinched ...

Portuguese scientists working on chromosome segregation

Jul 01, 2009

Lars Jansen's work on the formation of the centromere, a key cellular structure in powering and controlling chromosome segregation and accurate cell division, has just earned him a paper in Nature Cell Bi ...

Chromosome centromeres are inherited epigenetically

Nov 03, 2011

Centromeres are specialised regions of the genome, which can be identified under the microscope as the primary constriction in X-shaped chromosomes. The cell skeleton, which distributes the chromosomes to ...

Recommended for you

How calcium regulates mitochondrial carrier proteins

10 hours ago

Mitochondrial carriers are a family of proteins that play the key role of transporting a chemically diverse range of molecules across the inner mitochondrial membrane. Mitochondrial aspartate/glutamate carriers are part of ...

Precise measurements of microbial ecosystems

11 hours ago

The Luxembourg Centre for Systems Biomedicine (LCSB) has succeeded for the first time in describing the complex relationships within an ecosystem in unprecedented detail. For their work, carried out in collaboration ...

Students create microbe to weaken superbug

22 hours ago

A team of undergraduate students from the University of Waterloo have designed a synthetic organism that may one day help doctors treat MRSA, an antibiotic-resistant superbug.

User comments : 0

Please sign in to add a comment. Registration is free, and takes less than a minute. Read more

Click here to reset your password.
Sign in to get notified via email when new comments are made.