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Jasmin Mertins

Jasmin Mertins


New labeling strategies
for quantitative single-molecule imaging for membrane proteins

Principal Investigator: PD Dr. Maximilian Ulbrich

 

Abstract

Deciphering the exact stoichiometry of multi-complex receptor protein complexes can provide pivotal insights about their function. In order to determine their subunit composition, I will use a novel approach based on single-molecule microscopy of fluorescently labeled proteins. This single-molecule subunit counting approach uses the irreversible loss of fluorescence, the so-called photobleaching. The stepwise decrease in fluorescence intensity can be counted, making it possible to deduce the quantity and stoichiometry of receptor subunits [1][2]. The protein of interest which is genetically fused to a fluorescent protein will be expressed in Xenopus laevis oocytes and visualized via total internal reflection fluorescence microscopy (TIRFM). Due to the high illumination power which is used in single-molecule imaging, the choice of fluorescent dyes for labeling is restricted [3].
During my PhD thesis, I will examine the ionotropic glutamate AMPA receptor, which is responsible for the fast excitatory transmission in the central nervous system. The heteromeric AMPA receptor consists of four pore-forming GluA 1-4 subunits and at least three types of auxiliary subunits. I will be focusing on the interaction of the receptor core with its auxiliary subunits because different assemblies can alter the properties of the AMPA receptor and hence can influence the synaptic transmission [4]. Given the many possible co-assemblies of the AMPA receptor core- with its regulatory subunits, a broader choice of fluorescent labels is required to image the different compositions. It is my goal to expand the labeling strategies that are suitable for single-molecule imaging and improve the currently existing dual-color-imaging method to a reliable three- or four-color imaging. Besides using fluorescent proteins, I am also going to improve the use of organic dyes to label membrane proteins because they provide a wider range into the far red fluorescence emission, also expanding the choice of available dyes.

Methods

  • Single-molecule subunit counting approach by observing bleaching steps
  • Total internal reflection fluorescence microscopy (TIRFM) on Xenopus laevis oocytes
  • Molecular cloning of fluorescent fusion proteins
  • SNAP-tag labeling for organic dyes
  • Synthesis of RNA via in-vitro transcription

References

[1] Ulbrich MH. and Isacoff EY. (2007). Subunit counting in membrane-bound proteins. Nat. 
Methods. Apr;4(4), 319-21.


[2] Arant RJ. and Ulbrich MH. (2014). Deciphering the subunit composition of multimeric proteins by 
counting photobleaching steps. Chemphyschem. Mar 17;15(4), 600-5. 


[3] Johnson DS., Jaiswal JK., Simon S. (2012). Total internal reflection fluorescence (TIRF) microscopy illuminator for improved imaging of cell surface events. Curr. Protoc. Cytom. Jul; Chapter 12:Unit 12.29. 


[4] Schwenk J., Harmel N., Brechet A., Zolles G., Berkefeld H., Müller CS., Bildl W., Baehrens D., Hüber B., Kulik A., Klöcker N., Schulte U., Fakler B. (2012). High-resolution proteomics unravel architecture and molecular diversity of native AMPA receptor complexes. Neuron. May 24;74(4), 621-33.