Advanced Light Microscopy Core

Understanding a hijacker

May 13, 2019 By Mariah Cox in Research Tags: Advanced Light Microscopy Core

Figuring out how a virus takes over cells could help with gene therapy

Kinjal Majumder and David Pintel | photo by Roger Meissen, Bond LSC

By Mariah Cox | Bond LSC

When we catch a cold or contract the flu, we usually attribute it to picking up a virus from a friend or someone we know. Our bodies’ built-up immune systems have a way of attacking viruses to help us stay healthy, but sometimes viruses can hide.

A study published in eLife by lead researcher Kinjal Majumder, a postdoctoral fellow in the Bond LSC lab of David Pintel, sought to understand where a virus goes when it hijacks a host cell. This basic science may one day help researchers use viruses to better treat human diseases.

In general, when cancer-causing viruses infect a host cell, they translocate their genetic material into the DNA of healthy cells, taking over and causing disease in the host organism.

Majumder and his collaborators developed an experimental system to identify the ‘zip codes’ within the nucleus where the virus localizes.

“When looking at the mouse DNA in infected cells we noticed that the virus seems to associate with particular sites,” Majumder said. “When we mapped where those sites were, we found that the virus localizes to regions of the genome that are prone to DNA breaks. If you unravel all the DNA, which comes out to be about six meters in length, there are very distinct sites where the virus seems to associate.”

Through this research, Majumder was able to figure out where the virus goes when it infects a host cell and why it is attracted to those certain locations. These DNA sequence sites are prone to having breaks which is indicated by the presence of DNA repair machinery, making them more vulnerable to viruses. His current research focuses on how viruses hijack these break sites and take over the repair machinery to replicate.

“Viruses are very intelligent,” Majumder said. “What we’re now trying to do is to understand how they move about in the nucleus. We found out the where, we think we know the why, now we want to know how they know to attach to these sites.”

In order to conduct his research, Majumder is using a simple ‘test-tube’ model virus called minute virus of mice (MVM), which is a rodent parvovirus, to look at host-virus interactions. This model is particularly easy to work with in a lab setting and results are easily transferable to other viruses in animals and humans.

Other cancer-causing viruses that function similarly to parvovirus, such as human papillomavirus (HPV) and hepatitis B, have also been found to integrate into breaks in DNA chains and drive cancer progression.

“There is a close relationship between a replicating virus and the cell’s DNA repair machinery which normally protects us from cancer. Now we are starting to see that MVM localizes to the sites in the nucleus which contain a lot of these repair proteins,” Majumder said. “It’s an interesting phenomenon and we are starting to study the mechanism of how this might be happening.”

Majumder adapted an experimental system called chromosome conformation capture assay by chemically cross-linking virus DNA and the cellular DNA that are close together and generating hybrid DNA fragments in a test tube. After sequencing these fragments, Majumder and his team could determine the exact sites that the viruses associated with.

In order to confirm the location of these sites, they used powerful confocal microscopes in MU’s Advanced Light Microscopy Core. By using a laser to make a DNA break in a cell, Majumder could also show that virus localized near the induced breaks.

Kinjal Majumder uses a laser to make breaks in a DNA sequence using the confocal microscopes in MU’s Advanced Light Microscopy Core. | photo by Roger Meissen, Bond LSC

“If we can figure out how the virus is going to those regions, it opens up the possibility of finding whether other viruses, such as HPV or hepatitis B, utilize similar mechanisms to go to those sites,” Majumder said. “Knowing that may help study how oncogenic viruses cause cancer.”

Understanding how viruses hijack host cells and utilize cellular DNA breaks may allow future researchers to design improved cancer therapies.

Parvoviruses, in a broader sense, are used in oncolytic therapies to treat cancers using viruses. Cancer cells can arise due to dysregulation of DNA repair machinery in cells. They can also occur due to mutations in DNA breaks that can cause translocations. Parvoviruses have the ability to replicate in cancer cells, which is why they are being used as gene therapy vectors in clinical trials to target cancer.

“Knowing where gene therapy vectors stay long-term and how they interact with DNA repair proteins is important because they can express throughout their lifetime,” Majumder said. “For example, if you have cystic fibrosis, you have a mutation in one particular gene, so you don’t make specific proteins and as a result, you get a lot of health problems. Gene therapy adds a wild-type piece of DNA to express the correct version of the protein.”

For his Ph.D., Majumder looked at how the genome folds upon itself, gets packaged into the nucleus, and how this packaging regulates immune system development. His previous research on the genome is currently helping him advance his work with viruses.

“When I started working with viruses, I realized I could easily utilize those techniques to map how viral DNA interacts with the host’s DNA,” Majumder said.

Majumder hopes a better understanding of how viruses interact with DNA damage responses will move forward gene therapy.

“If you know how these viruses interact with the host’s proteins in the nucleus, you can design more effective gene therapy vectors.”

This research was published in the Journal “eLife” in July 2018 and was funded by the National Institute of Allergy and Infectious Disease of the National Institutes of Health.

#IAmScience Frank Baker

February 15, 2019 By Roger Meissen in #IAmScience Tags: Advanced Light Microscopy Core

By Danielle Pycior | Bond Life Sciences Center

With eyes wide open, glued to metal tubes, researchers from across the University of Missouri’s campus are searching for answers that are unseeable to the unequipped human eye.

The Advanced Light Microscopy Core is a resource for anyone to come and further understand their research. The Core’s Imaging Specialist, Frank Baker, has been an intermediary between the facilities equipment and researchers for four years.

Molecular cytology is the localization of molecules within the cell. In other words, researchers look at molecular methods occurring to understand cellular functions. The core has a number of resources such as confocal, super-resolution and digital light-sheet microscopes.

Though microscopes are normally thought of as a scope to look directly into, new technology is available that can create 3D images on a monitor of the cells.

Pointing — Baker points at the 3D image of an individual cell. In this case, the colors are stained to show the cell wall. Researchers can manipulate the image to analyze certain aspects of a cell. | photo by Danielle Pycior, Bond LSC

“One thing I like about working here is it gives me a perspective about the diversity of work being done at the university,” Baker said.

For Baker, one of the greatest parts about MU is its interdisciplinary nature. Any researcher from any department can bring their goals and their problems to the MCC and search for answers, and Baker gets to be a part of that journey.

“And sometimes when the problem is especially confounding, and you have all of these pieces that don’t make sense, you have that mental flash when everything integrates together into an answer. It’s a really beautiful moment,” Baker said.

Baker began his science journey here at MU where he received his undergraduate degree. He then moved on to Berkley to study plant developmental genetics and then did his postdoc at Pennsylvania State.

“I’ve always liked microscopy quite a bit, even in my undergrad days,” Baker said. “I thought, I really do like doing this, and so I decided to make a transfer.”

CloseupBaker — Baker looks through a microscope at a cell culture that is used to train researchers who come in to use the equipment. | photo by Danielle Pycior, Bond LSC

Baker has been in the MCC for four years now and said he is content for the moment.

“It’s always interesting because people come with new problems and new types of experiments, and so with the job, there is always a goal to keep learning,” Baker said.

On the horizon, Baker can see a future where he directs his own core. He said it’s important to him that it stays fairly broad to serve multiple purposes, similar to the MCC. Baker believes in setting goals and collaborating with others to reach them.

“I enjoy this job for a number of reasons. I like the diversity of work being done here… it does provide a sense of mission. Because the work that is being done here is critical to a lot of work being done at the university,” Baker said. “I enjoy that sense of working to achieve both the universities goal to support researchers and also to see work happen that has a positive effect.”

While goals can vary is size and scope, Baker makes it a priority to see both the small and big picture. When he can’t understand something about his research, he finds it useful to step back and take a look at the larger community, or to look at previous work being done on the topic. He values the complexity of science and how a change in perspective can be a shift in understanding.

“What I love about science is looking into the unknown, not knowing what the answer is, devising a hypothesis based on what you do know, and then doing the experiments to find out what the answer is,” Baker said.

More than meets the eye

June 23, 2016 By Roger Meissen in Events and speakers, Research Tags: Advanced Light Microscopy Core, Anand Chandrasekhar, Microscopes

Advanced Light Microscopy magnifies scope of research

A sample is shown in the foreground that can be used in the digital light sheet microscope at MU's Molecular Cytology Core as Anand Chandrasekhar explains how he uses it to study neuronal development in zebrafish. | photo by Roger Meissen, Bond LSC — A sample is shown in the foreground that can be used in the digital light sheet microscope at MU’s Advanced Light Microscopy Core as Anand Chandrasekhar explains how he uses it to study neuronal development in zebrafish. | photo by Roger Meissen, Bond LSC

By Phillip Sitter | MU Bond Life Sciences Center

Microscopes have come a long way since Anton van Leeuwenhoek first looked at single-cell organisms in the 1600s.

Now, cutting-edge microscopes allow scientists a better look at how cells interact and work.

The results were easy to see Tuesday morning when a new digital light sheet illuminated all the cells in a zebrafish embryo of Anand Chandrasekhar, a Bond Life Sciences Center scientist and professor of biological sciences. The fish, with its two eyes, brain and spinal cord lit up like a green-colored digital ghost floating in invisible black waters of the monitor screen.

This new microscope joins an array in the Advanced Light Microscopy Core, or MCC, located at Bond LSC. The MCC is one of nine core facilities at MU that provide vital services across campus, from DNA sequencing to imaging.

Researchers and staff at the MCC showed on Tuesday how the new capabilities of the technology give them and MU a competitive edge in their research through better visualizations of their experiments.

The digital reconstruction produced by the light sheet combines “a whole bunch of images over an extended period of time,” Chandrasekhar said. Thousands of images of a cell or organism can be generated by the new equipment, at speeds of hundreds of frames per second, creating a picture that can easily take up a terabyte of hard drive space.

At those speeds, Chandrasekhar said he can “literally watch neurons in the brain light up” in real time. He observes how neuron changes occur in the fish’s brain as the animal goes about its different routine behaviors like avoiding possible predators and searching for food. With this capability, researchers like him can study how neuronal networks develop.

The new digital light sheet uses less damaging light than traditional microscopes and allows for clearer pictures, often with a 3-D look at their structure. More advanced imaging equipment also allow for faster, larger volume experiments that are gentler on the biological samples used in them.

Thomas Phillips, MCC Director and professor of biological sciences, explained how his center now can better serve scientists across campus.

In addition to the digital light sheet imaging system, the MCC also has two new super-resolution microscopy systems, what Phillips said researchers across campus expressed they needed most. The presence of these super-resolution systems in particular puts MU at the forefront of microscopic research.

“Whereas every school has a confocal, less than 10 percent have super-resolution capabilities,” Phillips said, and now MU has two super-resolution systems. “The new equipment adds totally new capabilities without interfering with the traditional confocal activities.”

Traditional confocal microscopy systems, while still useful, have limitations in their resolution which super-resolution systems overcome by how the specimens are illuminated.

In addition to having technology other institutions do not, MU has a higher quality version of super-resolution. Phillips explained that while systems at other institutions use two different lasers in the internal mechanisms of their imaging equipment, MU is one of less than 10 or 12 schools that has access to a super-resolution system that has a unique three laser combination that increases the resolution of the system.

“Super-resolution microscopy allows us to see how individual proteins are interacting inside cells in ways we haven’t been able to before,” he said.

Sergiy Sukhanov uses one of the super-resolution microscopes in cardiology research that looks at failure of organ systems. Sukhanov studies heart failure and atherosclerosis — the chronic, dangerous build-up of plaque in arteries that can create blockages that lead to heart attacks, strokes and death.

Sergiy Sukhanov explains how he uses a confocal microscope in MU's Molecular Cytology Core to study atherosclerosis and heart disease. | photo by Roger Meissen, Bond LSC — Sergiy Sukhanov explains how he uses a confocal microscope in MU’s Advanced Light Microscopy Core to study atherosclerosis and heart disease. | photo by Roger Meissen, Bond LSC

The associate research professor at MU’s School of Medicine showed on Tuesday images of protein interactions in the smooth muscle cells that line arteries. The goal of understanding these interactions is to help keep these smooth muscles in the healthy condition they need to be in to prevent catastrophic blockages.

With its capability of a resolution of up to 30 nanometers, Sukhanov said that the new equipment’s advantage for him is that he can actually see the cell he’s working on. Soon, he hopes to be able to work with live cells so he can observe changes in their protein structures in response to changes in their environment in real time.

Availability of the new equipment lured Sukhanov and his research team to MU over other institutions in 2014. He explained that he struggled to find the equipment he needed for his experiments at Tulane University in New Orleans or elsewhere in Louisiana. Other systems at other institutions usually only have a resolution of up to 50 nanometers.

Sukhanov’s decision is an example of the growth that the new imaging capabilities at the MCC can promote. As director Phillips explained, “you don’t plan experiments ahead of time if you don’t have the apparatus for it.”

MCC not only provides services across campus, but can give scientists insight into how to better look at their specimens.

First-year graduate student Jennifer Wolf displayed a super-resolution image of cancerous liver cells infected with Hepatitis C that been treated with a drug thought to prevent the virus from spreading. The drug aggregates viral capsid molecules – the outer part of a virus – within the infected cells to effectively contain them.

Jennifer Wolf, a first year grad student working in Stefan Sarafianos' lab, explains an image of hepatitis C infected liver cancer cells captured by a super-resolution 3-D microscope housed at MU's Molecular Cytology Core. | photo by Roger Meissen, Bond LSC — Jennifer Wolf, a first year grad student working in Stefan Sarafianos’ lab, explains an image of hepatitis C infected liver cancer cells captured by a super-resolution 3-D microscope housed at MU’s Advanced Light Microscopy Core. | photo by Roger Meissen, Bond LSC

Wolf said with the new equipment she is now able to see an individual molecule, and using that see the overlap of proteins, RNA and DNA fragments, which can help determine the effectiveness of drugs in treatment.

Associate director of the MCC Alexander Jurkevich explained that the super-resolution equipment also allows for the compilation of separate images into an even more detailed 3-D projection.

Wolf pulled up an image of green-colored mitochondria surrounded by red micro-tubules, green hubs of cellular activity connected by red highways that looked almost like a city from space at night. Wolf used the 3-D capability and rotated the image. She turned the biological intricacy on its side until it looked something like a galaxy on a cosmic horizon, only this view that maybe even fewer people have witnessed is microscopic.

“It’s important to use new technology like this to help the University of Missouri to stay on top,” Wolf said.