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MIT Assay Measures Cytoskeletal Forces; Observed Protein Bonds May Be Rx Targets

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Researchers at the Massachusetts Institute of Technology have used optical tweezers to develop a native-like assay that can test the rupture force of a complex formed by an actin-binding protein linking two quasiparallel actin filaments.
 
The assay enabled the researchers to observe ABP unfolding and conformational transition events, suggesting that both may play a significant role in the temporal regulation of the mechanical properties of the actin cytoskeleton.
 
In turn, the bond between ABPs and actin filaments represents a potential druggable target, the researchers said.
 
To demonstrate the adaptability of this assay, the investigators tested it with two different ABPs: α-actinin and filamin. They immobilized actin filaments on the surface of a flow channel, introduced either filamin or α-actinin, and formed tethers with actin filaments bound to beads.
 
They then performed force-induced unbinding experiments at different loading rates to better characterize the dynamic behavior of these interactions.
 
For actin/filamin and actin/α-actinin, the researchers measured similar rupture forces of 40-80 pN for loading rates between 4-50 pN/s.
 
Writing in a study published in the advance online issue of the Proceedings of the National Academy of Sciences, the investigators said their assay can be used to study a wide range of ABP/actin interactions.
 
Matthew Lang, an assistant professor of biological and mechanical engineering at MIT and senior author on the paper, spoke with CBA News last week about his lab’s work, the reason it is important to study the role of physical forces in biology, and his assay’s applicability in drug discovery.
 
Last fall, Lang spoke with CBA News about how his lab used optical tweezers to manipulate cells (see CBA News, 11/9/07).
 

 
Can you give me a little background on the work that appears in the PNAS paper?
 
We are interested in understanding the role of physical forces in biology — sort of a systems-level approach that you might call the mechanome. How biology uses physical structures to cause cells to move around, contract, and sense things.
 
We are really taking a physical approach to biology. One of the most important structures is the cytoskeleton, and actin is one of its main components.
 
Our methods are really in the realm of single-molecule biophysics. Typically they have been applied to biological motors. What we did was sort of turn them around and develop a generalized assay for probing actin filament and actin-binding protein interactions.
 
This paper probes two of these interactions, actin/α-actinin and actin/filamin, although there are hundreds of these types of interactions. I think we are only just scratching the surface of what there may be in terms of the richness of this type of machinery.
 
There are a wealth of ways that these filaments can be configured that the cell uses generally to move and sustain load, et cetera.  
 
Why are people interested in understanding the role of physical forces in biology?
 
At a fundamental level, forces are required for things to move, and movement is a hallmark of life. These structures are active and very precisely organized, and we want to better understand that design.
 
From a disease perspective, these protein interactions in the cytoskeleton may be good targets for shutting down diseases. One example is how [the commonly used chemotherapy] paclitaxel will stabilize microtubules to shut down the machinery of cell division.
 
There are other physical things down the road; you might find ways of promoting cell motility or interrupting cell motility by better understanding these structures, for example.
 
Can you further describe the assay described in the paper?
 
We isolate single filaments, and we take one of these filaments, and lash it to the cover-slip surface. Then we take a second filament and connect it to beads.
 
We use these beads to pick the filaments up and exert forces on them. We can grab these beads with our optical trap, or tweezers.
 
Now that we have one filament attached to the surface, we can flow in any number of actin-binding proteins, such as α-actinin. We also flow in our second actin filament that is attached to a bead. What that does is form a bond, a linkage, between the lower filament and the upper filament, and we can interrogate that linkage with force.
 
We basically come in with the light, find the bead, and tweeze it. We apply forces to that linkage, and we look at how much force it takes before something happens, such as everything falling apart. We have just measured the strength of that bond.
 
Another thing that could happen is that we may have some reconfiguration of the actin-binding protein. It might have some kind of conformational change or unfolding event. We can also see that when we pull on the bead.   
 
So that is the basic assay, setting up the system with two filaments lashed together by an actin-binding protein. The nice thing about this assay is that it is fairly general, and we can use it to look at a whole series of actin-binding proteins.
 
What do you see as the appropriate applications for this technique?  
 
I think the main application is trying to understand cell machinery. It’s an extremely well-controlled probe, because we can probe a single interaction, and we can probe it in terms of direction and how rapidly we apply the force.
 
So we have extreme control over interrogating these bonds. This is really important, because it is one of the only ways that you are going to get the molecular details that underlie all of these connections that you would see within a cell.
 
Can you talk more about your assay’s applicability in drug discovery?  
 
For example, if cytoskeletal machinery is used to give the cell motility, as in cancer metastasis, one thing you might want to do to shut down that motility is to shut down the structures and the machinery that is allowing for that motility.
 
So by understanding that machinery better, we may find new targets for fighting disease. These are fundamental structures, so there are many ways you could disrupt the machinery.
 
Or maybe there are diseases that arise from problems with some of these structures, and we may understand how these diseases come about a little better.
 
I think it’s more a basic understanding of what has really been a ‘black box,’ so to speak.
 
What is the next step in this project?
 
I think what we want to do is explore a couple of other actin-binding proteins. Another thing we want to explore is the directional dependence of the rupture of the filament/actin-binding protein bond. In general, we pulled with parallel filaments, but we think that there may be a strong dependence on direction.
 
We are also going to work more on visualizing these structures while we are pulling them; so we will watch the filaments and the actin-binding protein, and see whether it dissociates from the upper filament or the lower filament.

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