Ahead of our 2022 Virtual KAND Family & Scientific Engagement Conference on August 13, 2022, KIF1A.ORG interviewed core KIF1A Research Network members to discuss their #relentless efforts to understand KIF1A and help KAND patients in this special “Meet the Research Network” series on the KIF1A.ORG blog.
KIF1A.ORG’s Research Engagement Director Dylan Verden, PhD, had the pleasure of talking with Will Hancock, PhD, professor of bioengineering at Pennsylvania State University. Dr. Hancock tells us about KIF1A’s walking patterns and the molecular techniques he uses to study them.
Dr. Hancock: My name is Will Hancock and I am a Professor of Biomedical Engineering at Penn State University. I’ve been here since the year 2000 and I’ve been studying kinesins since 1994. I got into them because I was studying muscle contraction for my PhD, understanding heart muscle, and these were a different type of motor. It was very new and exciting then. So I’ve been working on kinesins since then. I’ve been working on KIF1A maybe almost 15 years, 12 years, and quite intensively for the last 4 or 5.
Dylan: At a broad scale within the scope of kinesins, what is it that your lab is studying now?
Dr. Hancock: Well, one of the things that I’ve been interested in for the whole time of my career studying kinesins is what we call the inter-head coordination. Basically if we think of them as walking along the microtubule, the fact that they have two feet, and these feet coordinate their activities, and they need to go through cycles of attaching to the microtubule and detaching, but doing that out of phase, so there’s always one head bound at all times, that’s how they carry their cargo. The timing of that and the communication, which we call mechano-chemical communication, between the two heads, is just an incredibly fascinating question, because it’s just an enzyme but the two domains are communicating with each other, and that’s really integral to their function. So that’s just one of the big things that we’re studying with all kinesins. We want to understand fundamentally how, when they use ATP and they go through their enzymatic cycle, how they’re able to produce conformational changes that directs the walking and how different kinesins, like kinesins like KIF5B, KIF1A, KIF3A, there’s 45 in the genome, how they’re tuned differently. So for KIF1A, it’s interesting that they’re particularly fast and they’re very processive, meaning they stay on the microtubule really well, and so we’re doing a lot of biochemical experiments to try to understand that and in collaboration with Serapion Pyrpassopoulos and Mike Ostap at University of Pennsylvania about 3 hours away, we’re studying the mechanical properties of these same KIF1A motors using optical tweezers.
Dylan: That’s a great lead-in to my next question, which is about tools and techniques you’re using. Could you briefly describe how optical tweezers work, what that technique entails?
Dr. Hancock: So optical tweezers are a way of manipulating molecules. The idea is that light, photons, have momentum. We don’t really think of photons that way normally, but if they are focused in such a way that they’re traveling through a bead, and the bead is acting as a lens, because they have momentum, the lens bead is changing their direction, and that requires a force. So that means, just from basic principles, that the light is causing a force back onto the bead or the particle. Because the forces that these motors generate are very small, in the piconewton range, the forces that light can generate, as long as you have light from a laser and it’s coupled properly, are in the same regime. The best analogy is a tractor beam from Star Trek where the ship sends out a beam and it traps another ship. Conceptually, that’s what they’re doing. So we’re trapping a bead, and generating force on it, so the motor is generating force against it, and we can measure those forces, we can measure those displacements. It’s a sophisticated way to measure the stepping dynamics and to measure the amount of force that can be generated by these motors.
Dylan: What other techniques do you use to study KIF1A?
Dr. Hancock: Another technique that we use is what’s called gold nanoparticle tracking. The question that we’re asking is we want to understand how the heads are interacting with one another and how their cycle occurs as they’re walking along the microtubule. They take more than a hundred steps per second, so that means each steps takes just a few milliseconds, so it’s all very fast, and because they’re small, then the displacements that we want to see are very small, like nanometers, 10^-9 of a meter. What we’ve been able to do, building on work that was developed by other people, is to attach a really small gold nanoparticle, it’s a 20-30 nm gold nanoparticle, so it’s actually bigger than the head, but because of the scaling of forces down to the nanoscale, it doesn’t perturb the head. There’s a property of these gold nanoparticles in that they scatter light really well, so by shining light on the gold nanoparticle-labeled motor, we can visualize these gold nanoparticles. We can do so at 10,000 frames per second, so we can measure the position at one of the heads 10,000 times per second as it’s stepping along the microtubule, and we can do that at the precision of a couple nanometers, which is the size of the displacement. That has really opened up new ways of understanding the hydrolysis cycle, how the two heads coordinate their activities, and really understanding the guts of the motors and how they work. That’s a really exciting technique that we use in my lab quite a bit. We’ve done it for other kinesins; we’re currently working on it with KIF1A. We have some preliminary data but we’re still working out the kinks. Hopefully over the next year or so we’ll generate that data and that work will come out.
Dylan: How do you see these kinds of biomechanistic questions fitting into the larger questions of how do we find treatments or understand diseases like KIF1A-Associated Neurological Disorder?
Will: The work that we’re doing is really down at the molecular level and understanding these motor mechanisms. We want to understand the specialties of this motor and understand the subtleties of how they interact with microtubules and how they work with other motors to carry cargo, because in the end, the mutations that lead to KAND disorders are affecting the motors and how they’re walking, how they’re interacting with the microtubule. So we’re trying to understand how it normally works, and we can make those mutations in the motors we use, using recombinant DNA technology to make recombinant motors, so then we can understand how those mutations change the motor properties. Then we want to go one level up to the cell and say “if this is what’s wrong, then when they’re carrying a cargo through the web-like networks of the cytoplasm and the axon and working with other motors, how is that going to affect thow they carry their cargo, and then how do changes in how they carry their cargo affect the state of the neurons, the health of the neurons, and then how is that going to affect the body, and over time, over years of development”. So it’s really a multiple scale problem from the molecular, to the cellular, to the tissue, to the organism. And then it’s multiple time, so we’re looking at things down at the millisecond scale, then you have seconds, hours, days, weeks, months, years. So it takes a village, research-wise, it’s really important that there’s a community of researchers studying, using different techniques at different scales, and to bring that all together. We hope that by understanding it at the different levels, we can really get a clear picture that will give us new ideas about how would we design drugs that would try to correct these defects or compensate for them in some way? I think taking that holistic picture and trying things with different compounds, that’s a really important part of it too, to see how we can perturb things.