MSU researchers develop tool to observe complex molecular choreography in real time

A lot of things need to go right on a molecular level for immune cells to launch an adaptive response to an infection.

The B cells can produce different classes of antibodies tailored for specific infections by controlled DNA damage and repair to alter the genetic information needed to encode the antibodies. When the process goes awry, it can produce mutations or genomic rearrangements that promote formation of tumors.

Now for the first time, MSU researchers developed a tool to observe this complex molecular choreography in real time. Their work, published June 23 in the journal Molecular Cell, is shedding light on one of the enduring mysteries of the process: how a key protein locates the correct regions of the immune cell genome to initiate the adaptive response.

The study was led by Mariia Mikhova, PhD, formerly a graduate research assistant in the Michigan State University College of Natural Science's Department of Biochemistry and Molecular Biology and now conducting post-doctoral work at Boston Children's Hospital. Mikhova was supervised by Jens Schmidt, PhD, associate professor in MSU's Institute for Quantitative Health Science and Engineering and the MSU College of Human Medicine's Department of Obstetrics, Gynecology and Reproductive Biology, and Kefei Yu, PhD, professor in the Colleges of Human Medicine and Natural Sciences, Department of Microbiology, Genetics, & Immunology.

The team examined how a DNA mutator protein - activation-induced cytidine deaminase, or AID - gets recruited to the specific sites of the B cell genome to initiate a DNA recombination event called class switch recombination, which enables B cells to change the class of antibodies that they produce.

The question that remained, Yu said, was: "How does this protein know when and where it's supposed to go?"

To answer that, the team leveraged a microscopy technique previously developed by Schmidt to observe individual molecules in real time. Using live mouse cells edited by Yu, the innovative approach allowed them to track the movement of AID in living cells.

The malleable and mobile nature of live B cells complicated the imaging process. Schmidt credited lead author Mikhova with devising a technique using a centrifuge to press the cells against glass, allowing a period of imaging before they floated out of view.

"Mariia, who basically ran all the imaging experiments, had to go through and image many, many cells to watch these rare events," Schmidt said. "It was really a heroic effort on her part."

Being able to observe real-time imaging is "like watching a movie," Yu said.

"And because you are looking at individual molecules in a live cell, it's very different from conventional assays where you are looking at the summary of thousands or even tens of thousands of events," he said.

The study proposes that transcription - creation of an RNA copy of a gene's DNA sequence - produces a "dynamic RNA hub" at specific switch regions of the genome.

You have this cloud of RNA because of the active transcription. We knew that AID likes to bind to RNA. It's super, super specific. It's only in these switch regions. And so it's like a homing beacon."

Jens Schmidt, PhD, associate professor in MSU's Institute for Quantitative Health Science and Engineering and MSU College of Human Medicine's Department of Obstetrics, Gynecology and Reproductive Biology

Once that binding happens, AID can then target the nearby DNA so the genome breaks at the right location to permit class switch recombination.

"I think this idea that the RNA hub recruits the AID would be a very elegant way for the cell to make sure that AID only goes where it's supposed to go and doesn't target other things where it could cause harm," Schmidt said.

The research was supported by two National Institute of Health grants: a New Innovator Award to Schmidt and a Research Project grant to Yu. They also were recently notified of an additional $2,863,560 in NIH funding to continue this work over the next four years.

A lot remains to explore, and the initial findings have multiple implications for further research. This includes examining why some human patients fail to produce certain classes of antibodies, why compromised antibody class switching contributes to allergies or autoimmunity issues, and how off-target effects of AID can lead to B-cell tumors.

"There are many things we still don't understand, but our study established a platform to further analyze the targeting mechanism of this important protein in immunology and also cancer development," Yu said. "So we now have a new set of tools that nobody has had."