Acids, which, technically speaking, are molecules that give off positively charged hydrogen ions when dissolved in water, play a role in some of the most important functions in our bodies. Your stomach contains something very close to hydrochloric acid — a highly acidic substance that helps your body dissolve food and nearly bottoms out the pH scale, scientists’ measure of acidity. Amino acids and fatty acids are, respectively, the building blocks of proteins and lipids, two of your body’s most essential complex molecules. The A in DNA? That stands for acid. But even with all that acids do for us, Associate Professor of Biology Kalyan Kondapalli thinks we may still be underestimating their power. With a new National Institutes of Health-funded project, he’s investigating whether pH may be functioning like a cellular GPS, helping our cells route important cargo within their borders to perform functions that are fundamental to our health.
Kondapalli’s latest work focuses on immune cells called macrophages, which he says are sort of the paramedics of the immune system. When a bacteria or virus enters your body, the macrophage is often the first to arrive. It engulfs the invader, enveloping the bacteria or virus inside the borders of its cell membrane, where it is packaged into a transport vesicle called a phagosome. This transport vesicle then begins a journey away from the outer edge of the cell toward the interior of the cell, where it eventually merges with a lysosome — an acidic organelle that dissolves the bacteria or virus into its component parts. After this process is complete, the phagosome reverses course, transporting the bits and pieces back to the edge of the macrophage, where they are attached to the outside of the macrophage's membrane. Here, more specialized immune cells interpret these components of the virus or bacteria and start the process of manufacturing antibodies — kicking off a counterattack by the immune system that’s tailored to the specific disease.
This process is a classic showcase of the body’s use of acids to break things down. But Kondapalli says that when you start looking at the finer details of the phagosome’s journey within the cell, it’s not the only role that acids appear to be playing. One of the most peculiar things is that as the phagosome begins its journey of ferrying the bacteria or virus from the cell membrane to the interior part of the cell, the environment inside the phagosome gradually gets more acidic. What’s especially weird, Kondapalli says, is that the change in acidity doesn’t appear to be strictly associated with breaking things down. So what exactly is going on?
Kondapalli had encountered similar phenomena when he was doing his postdoc work at Johns Hopkins, where he studied a type of specialized protein that are found on endosomes — the class of subcellular transport vesicles of which phagosomes are one type. He says the proteins function sort of like a fine-tuning knob for controlling the pH within endosomes by constantly allowing more or less acid to leave the vesicle. Changes in how this protein behaves, or having too much or too little of the protein, is consequential across an array of organisms. In some plants, for example, fiddling with this knob, and hence fiddling with the acidity of the transport vesicles, can change the color of flowers. One of Kondapalli’s studies found that some people with autism didn’t have enough of these knobs, which meant that a transport vesicle responsible for disposing of excess neurotransmitters was failing to do so, leaving the brain flooded with chemicals. A significant number of people with glioblastoma, one of the most lethal forms of brain cancer, appeared to have too much of this knob. “With this cancer, one of the proteins that is supposed to be transported to the lysosome to be degraded instead gets delivered to the surface of the cell. And that cargo is a receptor that signals the cell to grow and divide. That can be a big factor in developing cancer,” Kondapalli says.
Kondapalli knew that this knob was associated with the regulation of pH within endosomes. And he also knew that irregularities with this knob often meant transport vesicles were struggling to deliver their cargo to the proper places. So he began formulating a bold hypothesis: What if pH was somehow regulating the directional movement of the transport vesicles? What if acid was functioning not just as a fundamental building block of something else or something that dissolves other things, but as a sort of GPS within the cell?
To test his hypothesis, Kondapalli first formulated a way to fiddle with the knob so he could manipulate the acidity of the endosomes and see what happened to their behavior under different conditions. Using a genetically engineered virus that triggered the endosomes to make more or less of these proteins, he could make the environment inside the endosome either more acidic or more alkaline. But then he needed some way to precisely measure how this influenced the endosomes’ directional movement. Assistant Professor of Physics Suvranta Tripathy, who specializes in experimental biophysics, still remembers Kondapalli’s very specific line of questioning on the day Tripathy was interviewing for his position at UM-Dearborn in 2020. “I was doing my presentation on my research, and I remember Kalyan said, basically, ‘Do you think you can solve this problem?’ And I said, ‘Yes, I think we can definitely do that,’” Tripathy recalls. “Then, of course, after formally joining UM-Dearborn, I sent him an email reminding him that he asked me this question and that’s how we started working on it.”
Tripathy, it turned out, was also very interested in how phagosomes move through the cell. He was particularly focused on two types of proteins located on the outside of the phagosome that function like motors, propelling the phagosome either toward or away from the center of the cell, depending on which type of motor protein was more abundant. He had been studying exactly how these motor proteins know where to go, and he found Kondapalli’s hypothesis that pH was potentially driving their directional movement to be surprising and exciting. On their first project, Tripathy used a 2018 Nobel-prize-winning technique called optical tweezers to track the movement of phagosomes within the macrophage, while Kondapalli manipulated the phagosomes’ pH using his genetic engineering process. To do this, Tripathy attached a tiny plastic bead to a bacteria, which was then engulfed by the macrophage and packaged, along with the bead, inside a phagosome. Using a tightly focused laser, Tripathy was then able to measure the mechanical parameters related to the movement of the bead inside the phagosome under different pH conditions as it traveled throughout the cell. The results confirmed Kondapalli’s general hypothesis. If the maturing phagosome never got acidic enough, it wouldn’t reach its intended meet-up point with the lysosome, the invader would not be dissolved and the rest of the immune system would never be alerted. pH, it seemed, was indeed linked to the directional movement of phagosome transport — and, fundamentally, to our health.
The results were exciting, but Kondapalli and Tripathy say they’re hoping their new NIH-funded project will reveal many more details of how exactly pH is regulating phagosome transport. Their general working hypothesis is that changes in acidity inside the phagosome drive changes in the kind of lipids that make up the phagosome’s membrane. These lipids then “recruit” corresponding motor proteins, which, again, come in two varieties — one that drives movement away from the center of the cell and one that drives movement toward it. So, stitching it all together: An increasingly acidic environment inside the phagosome means the phagosome’s membrane will contain more of a kind of lipid that attracts motor proteins that move the phagosome to the center of the cell. A more alkaline phagosome creates a cell membrane full of different kinds of lipids, which attract the other kind of motor protein and propel it in the other direction. And Tripathy says the process may be more complicated than that. For example, it appears that motor proteins may require a binding protein to attach to the lipid, and that binding protein can be pH-sensitive.
Kondapalli says if they can more thoroughly document this connection between pH and directional movement in phagosomes, it could have a variety of implications. On the practical side, it could provide a path to new types of therapeutics. He says the bacteria that cause tuberculosis and Legionnaires' disease, for example, have defenses that specifically disrupt the phagosome’s acidification process in an effort to keep from being destroyed. So if scientists could discover chemical pharmaceuticals that do what Kondapalli currently does with genetic engineering, we could also control acidity — countering the effects of the bacteria’s defenses and steering phagosomes to their proper destinations.
Bigger picture: Establishing this new role for pH would be a pretty big-deal revelation in the world of cell biology, given that acidity is generally thought to only perform certain kinds of functions in the body. Moreover, what would make this finding so interesting is macrophages aren’t the only type of cells that have transport systems. Many types of cells depend on similar endosomes to ferry cargo, so it’s entirely possible that pH is functioning as a GPS all over the body, though that would take more research to fully establish. Indeed, Kondapalli thinks there may still be other roles for pH that scientists have yet to explore. “If you think of the different organelles in the cell as rooms within the house, what’s really fascinating is that each room has a unique pH,” he explains. “You take mitochondria, where the cell’s energy is made, the pH is slightly alkaline. The lysosome is acidic, around 4.5 or 5. The nucleus is neutral, it’s around 7. The cytosol, the media in the cell, is also close to neutral. Why is this? We still don’t have the answer. So we may really just be scratching the surface with this new work. Acidification may be doing a lot more in the body than we ever thought.”
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Story by Lou Blouin
