Explaining my research, part 2: The importance of calcium in the diet of a neutrophil
In my first “explaining my research” blog post, I gave some background on the importance of neutrophil chemotaxis (the process of crawling in the direction of pathogens) and phagocytosis (the process of engulfing pathogens) in defending your body against invading pathogens (please take a look at the post if you haven’t had a chance yet). As a fun refresher, here’s a popular video of a neutrophil chasing after a small cluster of bacteria; I always enjoy watching this one (not our video, this one is from the 1950s, David Rogers [1])
As I mentioned, I am particularly interested in how chemical input to the cell elicits a very specific mechanical output. In this video, some chemical (chemoattractant) is released from the surface of the bacteria, and the neutrophil not only senses this chemical, but steers towards wherever the bacteria happen to move! And towards the end of the video, the neutrophil engulfs the bacteria, which looks simple enough, but again requires it to recognize molecules on the surface of the bacteria and enclose them in a little package of cell membrane. Remarkable! In my project, I’m focusing on a potential key player in transducing chemical input to mechanical output: calcium.
I’m sure you’ve heard of calcium, probably in the context of it being good for your bones. Here, we’re talking about the same element, calcium, but in a completely different context. Calcium exists not only in solid structures like bones, but also in aqueous (water-based) solutions, as a positively charged ion (Ca2+). As you may have heard, the body is mostly (about 60%) water, and this water contains some specific mixture of charged ions (K+, Na+, Cl-, Ca2+, Mg2+, and more). Just like if you stir table salt into a glass of water, it will dissolve, these ions easily mix into water. The amount of calcium in the water-based solution changes dramatically depending on which fluid we are talking about. For instance, blood contains about 0.1 grams for every liter (other bodily fluids contain a similar amount), while the fluid inside cells has more than 10,000 times less, about 4 millionths of a gram for every liter (for perspective, that mass is about 1,000 times less than the mass of a single grain of sand). This concentration difference is huge, and it’s fundamental to the role that calcium ions play inside of cells.
A cartoon depiction of different calcium concentrations inside and outside the cell.
Calcium is, in fact, important for all the cells in your body. Because of this huge discrepancy between the calcium concentration inside the cell vs. outside the cell, if the cell opens up any channels in its membrane, calcium will come flooding in. Kind of like how if you have a crowd of people enclosed in a small space, and you open a door to the outside, people will spill out the door and begin to spread out more (as they certainly should in this world of physical distancing). Similarly, there are compartments inside the cell that contain high concentrations of calcium (see my cartoon), so if any of the “doors” to these compartments are opened, calcium will flood out and the calcium concentration inside the cell as a whole will be higher. Why does this matter? Well, when the calcium concentration inside the cell changes, certain processes inside the cell also change dramatically. Perhaps the best known example of this is in muscle cells. When calcium is released from their internal stores, this actually leads to muscle contraction! Generally, calcium is referred to as a second messenger because it relays signals sensed at the cell surface to other regions of the cell.
Now let’s bring it back to immune cells, how does this relate to chemotaxis and phagocytosis? Well, let’s start with some videos from my research (these videos and more are on the Heinrich Lab YouTube channel, you can also refer to our 2018 paper [2]). In this first video, we show what we call a “calcium burst”, which is just our way of saying that the calcium concentration inside the cell changes rapidly and dramatically over a matter of seconds. The brightness of the green in the video indicates how high the calcium concentration is (in these experiments, I’ve loaded the cell with a fluorescent indicator that glows brighter when there are more calcium ions present). In this example of phagocytosis, we see the cell light up early during the process, and it’s hard to miss! Remarkably, we observe calcium bursts in practically 100% of the cases of phagocytosis, but it’s not exactly clear why they are important.
It’s not that there are no ideas out there, it’s just much less understood in this case of phagocytosis than it is for the case of contracting muscle cells. This is a knowledge gap, which is what you need when you’re building up a research project. Before I was brought into the world of scientific research, I don’t know how much I appreciated that scientific knowledge is a dynamic, changing entity. When we learn science in high school or even in college, it’s often taught as a set, established list of rules, but the exciting thing about science is that it is always in progress. Even what we view as well-established laws could be overturned with a new body of experimental work. But okay, that’s a subject for another time. The point here is that we found that calcium bursts in phagocytosis occur consistently, but researchers don’t exactly agree on why they are important (or even if they are), especially when we start talking about the mechanical behavior of the cell (forces, shape changes, etc.)
When I first started researching in the Heinrich Lab as an undergraduate, I investigated whether these bursts also occur during chemotaxis. There was some conflicting evidence out there, but with our experiments, we were uniquely posed to answer this question definitively. Our experiments are a little different than the one shown up top, because in our case, the neutrophil is not crawling on a surface, but just changing shape as we hold it in a micropipette (the tiny glass straw you see in our videos). Therefore, rather than observing the response of the cell to the surface it’s on plus the chemoattractant molecules it senses, we are observing just the response to chemoattractant. Therefore, we call the response we observe “pure chemotaxis”. While others had seen calcium bursts during chemotaxis when the cell spreads on a surface, we found (somewhat surprisingly) that they almost never occur during pure chemotaxis, as shown in the video here. Only once the cell makes contact with the particle and starts eating it (phagocytosis) do we observe the calcium burst.
In both cases, pure chemotaxis and phagocytosis, the cell senses chemical cues and deforms in a highly controlled and specific manner. So why do we see calcium bursts in one case (phagocytosis) and not in the other (pure chemotaxis)? Answering this question is central to my PhD work. More generally, I want to understand 1) What determines the onset of calcium bursts in neutrophils? and 2) What happens inside the cell as a result of the calcium burst? Is it actually important for the process of phagocytosis, and if so, why?
One partial answer to these questions can be constructed from the experiments I’ve shown and discussed so far. In these cases, calcium bursts only occurs when a neutrophil is adherent; that is, it sticks to either a surface (when it is crawling) or a particle (phagocytosis). So maybe adhesion is what triggers the calcium burst, or, vice-versa, a calcium burst is required to stabilize cell adhesion.
Indeed, this idea of the importance of adhesion brings us to the central hypothesis of my PhD project. Without stating a specific and testable hypothesis, scientific research can lack direction. The key is to make an educated guess about what you think the answer is to the research questions you posed, but be willing to discard your guess and form a new one if the evidence points in another direction! I’ll state my hypothesis in general terms for now, but more of the specifics will come out as I discuss my project further in the rest of these blog posts.
A simplified graphical version of my hypothesis.
Hypothesis: Calcium bursts in immune cells are triggered by a combination of chemical (e.g. chemoattractants or molecules on pathogen surfaces) and mechanical (e.g. adhesion or applied force) cues, and they play a key role in regulating the direction and timing of protrusion during phagocytosis.
In the following three posts of this blog series, I will discuss the three central aims of my PhD project one by one, which will bring us to some very interesting and exciting science (okay, I’m biased)! Written as questions, my aims could be written as
What triggers calcium bursts?
How does altering or eliminating calcium bursts affect phagocytosis?
Do my findings from aim 1 and 2 agree with a computational model of cell movement and calcium signaling during phagocytosis?
I’ll admit now that I don’t have complete answers to these questions, but my intention is to describe some of my key findings thus far and what work remains to be done. Rather than go in depth explaining all of my experiments and data, I’d like to use these posts as an opportunity to take a brief look at what I’m testing and how I’m testing it and definitely look at some exciting images and videos along the way. I hope you’ll join me for the ride!
References:
Thomas P. Stossel, Thomas D. Coates, George McNamara 2010 David Rogers 1950s panorama movie of human polymorphonuclear leukocyte (PMN, neutrophil) chasing 3 bacteria. Background found at https://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.369.1468&rep=rep1&type=pdf
2018. Francis, E.A., and V. Heinrich. Extension of chemotactic pseudopods by nonadherent human neutrophils does not require or cause calcium bursts. Science Signaling 11(521):eaal4289. doi.org/10.1126/scisignal.aal4289