Determining in Vivo Protein Complex Stoichiometry from Superresolution Microscopy

Arizona State University, Scottsdale AZ

Investigators

Abstract

Determining in vivo protein complex stoichiometry from superresolution microscopy. Proteins form one of the basic building blocks of life. In living cells, proteins typically assemble into macromolecular complexes in order to perform targeted tasks. Knowing the location and exact composition of protein assemblies enables investigators to understand the molecular mechanisms underlying their activity during key cellular events such as division or movement. Unfortunately, relatively little is known about the composition of such complexes in living cells, and it is clear that composition can vary depending on the cellular environment. This project will develop an optical/computational method to count subunits in protein assemblies in living cells, and enable exceptionally detailed information to be gathered about biological processes in native, non-invasive environments. This project combines expertise from the biological, mathematical, and physical sciences and as such provides exceptional interdisciplinary training opportunities for students and postdocs. Moreover, the project will yield methods that will have broad applicability in the optical biological sciences. This project will extract protein complex stoichiometry directly from the superresolution microscopy technique called PALM (PhotoActivated Localization Microscopy). PALM works by genetically encoding proteins of interest with photoswitchable fluorescing protein (FP) tags. Under sufficiently low light, each FP photoactivates and subsequently photobleaches. Thus, the number of fluorescence spikes over one small region of space should coincide with the number of protein subunits confined to that region (and normally confined to a specific protein complex). In practice, FPs fluoresce on and off (i.e. they 'blink') with blinking properties dependent on the local cellular environment. This blinking has been a major challenge in using PALM as a quantitative tool. The project will adapt the mathematical and statistical inference tools used in the analysis of ion channel patch clamp experiments to determine protein complex stoichiometry directly from PALM data (Goal 1). To determine the complex stoichiometry more accurately, the FP's blinking properties will be simultaneously extracted while determining the complex stoichiometry (Goal 2). The method will be tested on synthetic as well as real data sets where the protein complex stoichiometry is already known. The analysis will then be extended to protein complexes where the stoichiometry is still under current investigation (SpoIIIE and the kinetochore). If successful, this counting method will have several decisive advantages over existing counting methods, which assume specific FP photophysical properties or do not treat FP properties stochastically. This grant is funded jointly by the Cellular Dynamics Program in the Division of Molecular and Cellular Biosciences and the Physics of Living Systems Program in the Division of Physics.