Author: Tyler Tri-Thanh Pham

The following post will describe an update on my project to someone outside of academia. I am currently analyzing a channel located within the mitochondria. The channel is called the Mitochondrial Calcium Uniporter (MCU). MCU is highly selective to Calcium and only allows passage of calcium into the mitochondria.

My lab is studying this specific selectivity for calcium in hopes to better understand the MCU functionality and find better ways to regulate such a channel. Currently, we have identified a subunit located on the MCU called MCUb. It has been proven that over expression of this subunit prohibits the flow of calcium through the channel. However, upon further examination, we discovered that there are few regions between the two that are different. The regions in which we are analyzing is called the motif regions. These regions are the ones lining the inside of the channel and the ones responsible for the high calcium selectivity.

Currently, we are creating mutations of the MCU based upon the differences of the motif regions. These mutations are specifically designed to aid us in narrowing down the specific changes and modifications that result in the changes that we see between MCU and MCUb. To analyze these changes we manually add calcium sensitive fluorescence into the mitochondria. With this fluorescence we are able to track the intake of calcium into the mitochondria through the MCU by measuring the increase of  intensity of fluorescence over time. Through calculations and analysis, we can use our data to directly calculate intake of calcium for different mutations our lab creates.

I have currently examined 4 cell lines each with a different mutation. We are still analyzing the results, but we have seen some positive data indicating that these mutations are causing alterations to how calcium is entering the mitochondria.

With time, I hope to finish up the data analysis and continue to image more cell lines with mutations. One major bottleneck that my project is experiencing is the creation of the mentioned mutated cell lines. Creating stable cell lines with the specific mutations that we desire takes a considerable amount of time and we are currently working on creating as many cell lines as possible so that we can get a wide range of mutations to test. However, we hope to have enough mutations that we can narrow down the specifics of how we can better regulate the Mitochondrial Calcium Uniporter.

Over the past couple of weeks, I have been facing my ultimate nemesis in my project, confocal microscopy. Confocal microscopy is a microscope that is able to measure fluorescence (light) from a fluorescent dye (light inducing). In the beginning, I thought it was going to be an easy task, but I was deeply mistaken. It has taken my a total of 5 weeks to master the microscope and to produce usable data for my project. The main hurdle that I encountered was learning to permeabilized my cells. This was a crucial step because it was going to yield accurate and useful data. If I didn’t master this, my whole project would come falling down.

According to my professor, cell permeabilization is a “dark art”. For the past 5 weeks, I have been perfecting the art of permeabilizing cells. Cell permeabilization is when we use a detergent solution to break down the cellular membrane that surrounds the cells. However, we don’t want to break down the entire membrane, because the cell components would literally spill out everywhere. To have usable cells, we have to correctly time the permeabilization so that the detergent solution leaves tiny holes throughout the membrane of the cell.

The tiny holes serve two purposes. Firstly, they allow excess dye to leave the cytosol or inner fluid of the cell. In my experiment, we were using Rhodamine 2-AM dye. This is a dye that is sensitive to calcium concentrations and will intensify when calcium is present. When staining the cell, the dye goes everywhere inside the cell. However we only want the dye inside the mitochondria because we want to measure the influx of calcium inside of the mitochondria. By creating tiny holes, we can force out the unnecessary dye that is surrounding the exterior of the mitochondria.

Secondly, the tiny holes allow the uptake of calcium to occur at a more reasonable rate. Without the permeabilization the calcium uptake would take minutes. With the permeabilization, the calcium uptake occurs in seconds, which allows us to produce many assays in one sitting.

Knowing when a cell is permeabilized was a huge challenge for me, because I had no idea what would a permeabilized cell looked like. On top of that, each cell line had different permeabilization time that was very inconsistent and would change every single time. With practice and much time, I was able to recognize and identify the signs of successful permeabilization.

From what i achieved, a successful permeabilization will result in a grainy texture of the cell fluid that surrounds the nucleus. The grainy texture is in fact small bubbles that are a result of the break down of the cell membrane. Once we reach this stage, the permeabilization has just begun. To make sure that it is the correct amount permeabilization, the bubbles will increase in size and become even more grainy. Eventually, there comes a point where the bubbles are just the right size so that it will not destroy the membrane and it’s big enough to allow dye to flow out and calcium to flow in. This point is up to the discretion of the researcher. There is not a justified size to the bubbles, but after many practice rounds, there is a preferred size for each researcher every time.

Once I accomplished the skill of permeabilization, I was able to gather data. The way we gathered data was to start the laser recording, and during the recording, we would add a measured calcium solution directly onto the cells. After, we would watch what happened to the intensity of the dye. An increasing intensity would result in a higher influx of calcium. This is what we are looking for when we analyze the recording, because it means the cell is working and the mitochondria is doing its job.

For the most part, the results have been extremely good, but I am still collecting data at this time. It has taken many hours of hard work and dedication to reach this point, but both my mentor and I agree that from this point, things will be picking up at an increasingly fast pace. From this experience, I learned never to underestimate the easy things because sometimes there the ones that are standing in your way.

Image Resources:

Permeabilized cell:
https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-analysis-learning-center/molecular-probes-school-of-fluorescence/imaging-basics/sample-considerations/preparing-fixed-cells-imaging.html

Confocal microscope:

https://itn-snal.net/2014/10/laser-scanning-confocal-microscopy-lscm/

Dyed cell:

Direct from lab data.

Introduction

My name is Tyler Pham, and I am currently a junior at Emory University. I am pursuing a degree in both Biology and Chemistry. This year, I will be researching in Dr. Jennifer Kwong’s Lab to better understand the mitochondrial calcium uniporter.

Research 

Dr. Kwong’s Lab primarly focuses on a specific calcium uniporter within mitchondria called the Mitochondrial Calcium Uniporter (MCU). MCU is located in the inner membrane of the mitochondria and is responsible for uptaking calcium. Calcium acts as a second messenger that is able to both increase ATP production and signal cell death during a calcium overload. Understanding how the MCU works will help prevent cell death during calcium overload in such events like a heart attack (ischemia-reperfussion). Not much information is known about the MCU, but it is fairly certain that the MCU is made up of 4 subunits that together form a tetramer pore. Dr. Kwong’s Lab conducts research to both better understand how the subunits function and to improve regulation methods to this uniporter.

Image from Kwong (2015)

Involvement

My research project primarly focuses on laying a foundational knowledge to better undestand how the MCU subunits function. I will be measuring and comparing the calcium influx between an untransfected MCU and genetically mutated MCU with variant subunits. The variant subunits will contain differnet selectivity filters embedded within them to alter the flow of calcium into the tetramer pore. By analyzing the effects of mutating the subunit, we can better understand how the MCU functions.

I have only started working in Dr. Kwong’s lab for less than 2 weeks, and I have already learned so much from working with her. She has been training me in workshop like sessions to teach me the various skills and techniques I would need to know to work in her lab. So far, I have learned how to properly grow and plate cell tissue cultures and how to isolate proteins by lysing cells. In addition, I am currently perfecting how to measure calcium influx using a confocal microscope that is able to detect fluorescent dyes with lasers. I only have a few more skills and techniques to learn, and I hope to finish in the next couple of days.

Future Plans

I hope to finish up learning all of the necessary skills required to work on this research project. In addition, I plan to calculate and collect my first data set from the intial untransfected cell lines. I am excited to see where this project leads and what potential can come out of it. I hope to see my efforts play a major role in the future studies to come.

References