The researchers from the Stowers Institute for Medical Research have come up with a breakthrough discovery of two proteins in the living cells. These proteins work to facilitate communication between the nucleus and cytoplasm (its exterior compartment) of the cell. This scientific research provides new clues into the crucial working of proteins, found in organisms from yeast to humans. The study was published in the Journal of Cell Biology.
The study basically focused on a protein called Ndc1. It controls when and where a cell inserts holes into the double-walled membrane, surrounding its nucleus. In the yeast cells, these holes become the sites for two essential structures,
- Passageways called nuclear pore complexes
- Spindle pole bodies, anchoring the cytoskeletal filaments
The latter pulls the chromosomes to opposite sides of a dividing cell.
According to the researchers, too many or too few insertion sites will have disastrous consequences. It explains that new nuclear pore complexes and spindle pole bodies must be created each time a cell prepares to divide. This ensures the proper distribution of the genetic material. Thus, the daughter cells are equipped for gene activation and protein production.
According to the study, the interaction of Ndc1with a protein called Mps3 appears to govern Ndc1’s distribution on the nuclear envelope. Scientists chose to study Ndc1 because it is absolutely crucial for the survival of a cell.
It was already known that Ndc1 is embedded in the nuclear envelope of the yeast cells. Moreover, it is needed for the insertion of both nuclear pore complexes and spindle pole bodies. However, these cells are so sensitive to the changes in Ndc1 that the scientists had been unable to learn much about how the protein functions.
What do the researchers say?
The researchers mention that traditional genetic strategies of eliminating, altering, or increasing Ndc1, to test its function, typically killed cells. It is very critical to have the exact right amount of Ndc1. This is what makes working with the gene very challenging, technically.
For the study, the scientists created yeast with mutations in the ndc1 gene. These changes disrupted the interaction of Ndc1 with its known partners in the nuclear core complex or the spindle pole body. However, there was one mutation that puzzled scientists.
The altered Ndc1 protein bound to the expected components of both the nuclear pore complex and the spindle pole body, just like the normal protein. According to the previous studies, if Ndc1 can functionally bind to these components, both the spindle pole body and the nuclear pore complex should be fine but when the yeast cells produced this changed version, they died.
This led the scientists to suggest that there may be another very critical interaction partner present. Thus, the team turned to a method called “a yeast two-hybrid assay.” In this technique, the researchers linked the proteins under test to two different parts of a gene activator. If the proteins associate with one another inside the cell, they bring along the gene activator components. This allows them to work together to switch on an easily detectable gene.
The membrane-based yeast two-hybrid assay could detect interactions at the nuclear envelope. As a result, the researchers found that Ndc1 bound to a protein called Mps3. The yeast cells needed the protein to duplicate their spindle pole bodies prior to cell division. However, Mps3’s interaction with Ndc1 was a surprise.
The team was proposed to use a sophisticated imaging technique called “fluorescence cross-correlation microscopy.” It was expected to provide a direct look at the Mps3 protein inside living cells and its interactions with Ndc1.
What were the results?
The results of the study revealed that Ndc1 and Mps3 moved either independently within the cell, or physically linked to each other.
This study allowed the experts to look inside living yeast cells. They were able to study the complexes in real time, in the context of the nuclear envelope. They didn’t have to touch or break open the cells and rather sat there and watched.
The imaging confirmed the interaction between Mps3 and Ndc1. Moreover, it allowed the team to track exactly where the interaction occurred. Both the proteins, Mps3 and Ndc1, came together in the nuclear envelope but away from the two structures the team had set out to study. Thus, the scientists are wondering if the Mps3 might help shuttle Ndc1 to the sites where it is needed. This is the possible mechanism predicted regarding the control and the distribution of nuclear pore complexes and spindle pole bodies.
The research team is now planning further experiments to test how Ndc1 and Mps3 maintain the appropriate balance of these critical structures.