GCCRI - USA
Protein interaction with CENP-A
The histone-3 like protein, CENP-A, is a major protein involved in the maintenance of the centromere and the building of the kinetochore. The function of CENP-A is regulated by its ubiquitinylation by its chaperone protein, HJURP, thus allowing the recruitment of other centromeric protein, as well as keeping the centromere position at the same location. A lot of protein involved with the centromere or the DNA interact with the ubiquitinylated form of CENP-A, and if some of them are either obvious or already described, some new proteins candidates retain the attention.
One of them if the DNA repair protein, Rad50, that composes the MRN complex, is a key component for the double-strand break DNA damage repair mechanism. The evidence of interaction has been shown by immuno-precipitation in cancer cells, not only for Rad50, but also for all the protein complex components, Nbs1, and Mre11. Other proteins involved in the DNA repair (ATR and DHX9) also show an interaction with CENP-A.
However, these interactions can be indirect or situational, and more investigations, mainly in vitro, are realized to determine the binding candidate, then the binding domain. And proving the interaction is, of course, not enough! The function of this interaction is also critical: why a constitutive protein like CENP-A interacts with the MRN complex, and in which condition?
RIKEN - JAPAN
Assessing the proteome profile at a single molecule sensitivity
Separating the proteome of a sample, and analyze the trace, is what I mean by proteome profiling. The proteome can be separated by different means, like SDS-PAGE, native-PAGE, capillary electrophoresis or IEF, and can inform about the global evolution of the proteome of a sample. Being able to reach the single-molecule sensitivity will permit to analyze the proteome of a single cell on one part, and on the other part to detect a small change of biomarkers to be able to identify cell type or diagnostic or illness.
This project necessitates the proteome separation and detection of a tiny amount of protein, and then require a single-molecule microscope. To observe the profile, this microscope need to be able to scan a large area of few centimeters long, in case of gels, or to observe inside a capillary. We use in this case a modified light-sheet microscope and need to optimize the condition to enable single-molecule condition
Measurement of labeling homogeneity at the single molecule level
Fluorescence labeling efficiency of a protein is generally bulk measured by spectrophotometry, through the coupling efficiency (CE) or Degree of Labeling (DoL). However, this measure does not permit taking into account the homogeneity of labeling that can appear at the single molecule level. This homogeneity is important to assess the absolute fluorescence quantification, and indispensable for single molecule quantification.
During my time in Japan, at RIKEN, I developed a protocol that allows measuring the proportion of fluorescently labeled protein. This protocol main idea is to immobilize proteins on a microscope coverslip at a given density, then count and compare the maximum density and the density given by the protein, at a single molecule level. To realize this project, I need to develop a strong method for protocol creation and optimization, as well as troubleshooting detection. I also learned how to realize single molecule imaging, and to determine which parameters need to be optimized to improve the imaging system. I also updated my skills in biochemistry as well as chemistry to be able to improve the global labeling efficiency of purified protein and cell lysate. On the analysis side, I developed my programming skills, first with ImageJ for automated image analysis, then with Python (and Scipy) to statistically analyze the generated data.
This work is published in Bioconjugate Chemistry.
Virology laboratory - FRANCE
The Adeno-Associated Virus (AAV) is a parvovirus, known for its nuclear replication, and I studied its interaction with nuclear bodies, like the Cajal or PML bodies, as well as the speckles, and the function of these nuclear bodies on the viral gene expression and replication.
During this time, I developed all the necessary skills for molecular biology (cloning, bacterial transformation, PCR, Wester-Blot) necessary to realize the fluorescent fusion protein needed for this project. I also acquired my first skills in Immuno-fluorescence and then fluorescence microscopy to visualize the nuclear bodies. I also acquired more specific virology skills, like virus production, purification, and utilization, to visualize the effect of the virus on these nuclear bodies, as well on fixed or living cells through IF and time-lapse experiment, and finally observe that the speckles are disrupted around the viral replication center during the infection. This result has then been confirmed by immunoFISH, targeted against the viral DNA or RNA. The disruption of the speckles through siRNA permit to observe a strong diminution in the viral replication, but a more modest diminution in the viral gene expression visualized by RT-qPCR. In a living cell, the viral replication center without speckles lost its concentration and is dispersed in the cell nucleus.
The heavy use of different type of microscopy, confocal, time-lapse and bi-photon during this project made me want to keep in the microscopy field, resulting in my enrolment in the Master IRIV of Strasbourg University about microscopy and imaging.