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Purdue Team Develops Method For Detecting Kinase Translocation

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Chang Lu
Assistant Professor
Purdue University
Name: Chang Lu
 
Position: Assistant professor, biological engineering, biomedical engineering, chemical engineering, Purdue University, 2004 to present
 
Background: Postdoc, applied physics, Cornell University, 2002 to 2004; 2002 PhD, chemical engineering, University of Illinois, Urbana-Champaign, 2002
 
While the ability to directly localize kinases within cells is important for gaining knowledge about their role in signal transduction, existing methods for detecting such protein movements have had limitations.
 
For instance, subcellular fractionation and Western blots provide information on an individual cell level but offer no insight on a larger scale. And flow cytometry offers a more global view of cellular activity, but no single-cell information.
 
Chang Lu and colleagues at Purdue University have devised a high-throughput technique combining electroporation with flow cytometry that they say allows researchers to study the translocation of proteins and kinases “at the level of the cell population with information gathered from single cells.”
 
The technique, dubbed electroporative flow cytometry, is described in the current issue of Analytical Chemistry.
 
The university has filed a patent application with the US Patent and Trademark Office, and has received inquiries from several companies about commercializing the technique, Lu told ProteoMonitor this week.
 
Below is an edited version of the conversation.
 

 
Was this technique based on your own experience trying to localize kinases?
 
That’s actually from our collaborator, Bob Geahlen; he’s also a co-author on the paper. They have been working a lot on protein translocations, kinase translocation in particular for cell signaling studies.
 
I think he had this problem: You can use Western blot, you can use cell fractionation to look at the average behavior of the cell population in terms of translocations, or you can do imaging to look at localization and everything. But in the first case, you only look at the average behavior of the cell population, and in the second case you’re looking at a small number of cells, which may or may not be representative of the entire cell population. So there isn’t any technique in between [that allows you to look at both the entire population as well as individual cells].
 
We were trying to bridge that [gap]. That’s where we saw the need.
 
How did you get involved in this?
 
We wanted to do some research using microfluidics … to essentially look at some cells, signaling ones. And when I talked to Bob, we realized we could possibly solve [this predicament].
 
Certainly, we had previous research on electroporation phenomenon, and that laid the foundation for this current work. And we realized that it’s possible to combine electroporation and flow cytometry.
 
Can you describe any hurdle you encountered in localizing kinases with your technique as you were developing it, or things that will need to be addressed as you further develop it?
 
We essentially used the EGFP-tagged kinase … the cell line was created using molecular biology techniques. The kinase there was fluorescently labeled by EGFP already.
 
However, it’s probably not a realistic approach if you’re dealing with primary cells, say the cells derived from patients. In that case, you probably need to use an immunofluorescence method to label the particular kinase of interest. So we are still working on that because in terms of labeling, it’s certainly more challenging if you’re dealing with primary cells.
 
In this study, we used cell lines that contain EGFP tagged proteins. That’s an idealized situation.
 
What’s the challenge when you’re doing the labeling?
 
Essentially if we use the same approach … we need to label the proteins at the cell membrane surface. That’s challenging because the protein has to be attached to the surface receptor and the fluorescently labeled antibody at the same time. So your labeling cannot block that interaction between the surface receptor and the kinase.
 
And then you need to use a pretty big fluorescently labeled antibody or something to attach to the protein at the other end. We see that as a challenge [and] we’re still working on that.
 
We have some positive findings. It’s too early to tell if it’s working or not.
 
Are there indications that you can overcome this?
 
I think so. There are different ways we can do that. I do think that’s a solvable problem. Hopefully, we can come up with some creative ways of doing this.
 
Is there any specific reason you chose the Syk protein for your study?
 
As we indicated in the paper, it’s an important kinase, particularly for B cells. And we have stably created a cell line with this Syk protein, Syk kinase labeled by EGFP. That was something we could get our hands on, so that naturally [made it] the choice to start with.
 
Are there any conditions in which your technique would work best, for example, any cell types or processes?
 
I don’t think our technique is limited to certain cell types, but … translocation means that a fraction of the protein has moved from the cytoplasma, from the cytosome to the cellular membrane. And the fraction could be, say 10 percent or 5 percent or 20 percent.
 
It’s largely limited by how many surface receptors you have at the cell membrane. If we see a large fraction of the protein movement, then that’s easier for our technique to determine. If we are dealing with, say, 5 percent, then it’s harder because it’s just not that much significant movement to see.
 
In our current studies, there is about 20 percent [kinase translocation], so we were able to detect that.
 
Your paper says your technique can be used by drug developers. What about proteomic researchers? What’s the application for them?  
 
I think certainly as long as the researcher is dealing with [protein or kinase] translocation, our technique will be useful. And the good thing about this kind of translocation is that it’s normally not possible to detect the translocation using conventional techniques such as flow cytometry.
 
The reason is that as long as the total amount of proteins is in the cells, then you have the same signal. It doesn’t differentiate subcellular locations. That creates difficulties for people working on proteomics if [the work] involves kinase translocation, which is a fairly common phenomenon. That’s part of the activity they need to have in order to be activated, to carry out their later biochemical functions.
 
How reproducible is the technique?
 
I think we did three trials in the data. I think it’s fairly reproducible, but certainly I suspect, for example, if you change the system to another kinase, or [if you have different degrees of translocation], then you need to optimize the parameters a little bit differently.
 
What I mean by parameters is parameters for electroporation. Remember that we are essentially combining electroporation with flow cytometry, and for the electroporation, there [are parameters] for the intensity of the field and how long the cells are in the electroporation field. That determines how fast the proteins get released.
 
You don’t want to do it too late, you don’t want to overdo that either, so there is an optimal window that you’re working [with] … I imagine that window is dependent on the molecular species that you’re dealing with.
 
How do you see this being commercialized?
 
I think for flow cytometry, I think it’s probably easier for the industry to do a simple addition to the existing flow cytometer, to essentially incorporate the electroporation process into the flow cytometer, which will be as simple as adding two electrodes. That would give [researchers] the option of doing electroporative flow cytometry. That should be a fairly minor addition to the existing flow cytometry apparatus, and I imagine that’s an easier way to go about [commercializing] this technique.

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