Frank Waggoner

Protein Building and Kinase Control in Yeast.

Abstract: In living cells, Kinases are the primary catalysts in many cellular behaviors and the ability to control their activation would be extremely beneficial in understanding how they work. For my project, I worked on building two protein complexes that would have the ability to bind to kinases and regulate their interactions. By integrating the TULIP System and a drug called Rapamycin, the activation of these protein complexes will be dependant on both the presence of light and drug, allowing for spatial and temporal control of the kinases they are bound to. Once the plasmids for the protein complexes are complete they will be transformed into yeast, which will in turn be observed under a microscope. Additionally, the DNA sequences for these proteins will be mutated, in an attempt to make activation almost entirely light dependent. Another project I worked on involved staining for bud scars in yeast, using calcofluor and different fixing solutions. By looking at bud scars, it is easy to diagnose the type of budding the yeast are experiencing, making experiments that involve budding mutation more efficient.

The research performed by the Glotzer Lab revolves around cytokenesis, specifically the physical separation of the cell towards the end of its division cycle. Some of the main questions asked are “How is the cleavage furrow positioned?” “How does the contractile ring assemble and function?” and “How does the central spindle assemble and function?”i To address these questions, the lab uses C. Elegans, cultured human cells and yeast as models when conducting experiments. A recent tool that has been developed by the Glotzer Lab is the TULIP (tunable, light-controlled interacting protein tags) system. The system involves two protein domains, called ePDZ and LOVpep, which are fused to the two proteins in question respectively. In the presence of light, the two domains interact, forcing interaction between the proteins bound to each domain. This is a powerful tool that gives The Glotzer Lab the ability to control the location of interaction of two proteins that can help them answer further questions. Further details of this can be found in the paper, TULIPs: tunable, light-controlled interacting protein tags for cell biology, published by members of the lab in 2012.ii

Frank Waggoner

Most of the time I spent in the lab was spent making these protein constructs:

The following are pictures of some of the results from the steps I took to create these constructs.

DNA Digest of plasmid DS282 using the restriction enzyme BSM1

Frank Waggoner

A double digest of plasmid DS195 using restriction enzymes BAMH1 and Nde1

The Mid2-GFP with TEF promoter, PCR product

Frank Waggoner

For the yeast staining experiment, these are pictures of stained yeast using the procedure that worked best.

YLS, random budding pattern

W303alpha, semi-random budding

MGY50, patterned budding

Frank Waggoner

Throughout my time in the lab, I took part in many experimental protocols and procedures. My first few weeks were spent learning the basic techniques used for the types of experiments I would be doing. I learned how to sent up digests, run PCR, purify DNA from gels, transforming DNA into bacteria and yeast and much more. After I became comfortable performing these tasks, I was let to do them on my own, receiving only minimal guidance from other members of the lab. I additionally learned how to use the microscopes and micropoints in the lab and even worked on writing some programs in imageJ, a tool used for image analyses.

During my last few weeks at the lab, I was able to successfully clone both of the desired constructs and transform them into yeast for further observation and experimentation. The next steps would be to look for recruitment near the cell membrane in the yeast cells when both drug and light is present. Once it is established that activation of the constructs is dependant on both light and drug, mutations will be induced in order to lower the amount of drug that is needed. This will allow activation to be almost entirely light dependent, but still require both light and drug for activation.iii The next steps in the yeast staining experiment is to write a program that will take images like the ones shown above and analyze them, telling us if the yeast strain’s budding is random, patterned or somewhere in between.

i Glotzer Lab website, Introduction, http://glotzerlab.uchicago.edu/ii Strickland D, Lin Y, Wagner E, Hope CM, Zayner J, Antoniou C, Sosnick TR, Weiss EL, Glotzer M. (2012) TULIPs: tunable, light-controlled interacting protein tags for cell biology. Nat Methods. 2012 Mar 4.iii Project proposal, Description of Proposed Research.