University of Minnesota
School of Physics & Astronomy

Physics and Astronomy Calendar

Thursday, October 17th 2019
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Jared Hennen, UMN, Mueller Lab
Subject: Protein mobility and local volume fluctuations identify membrane binding of proteins within the sub-resolution structure of the nuclear envelope

The nuclear envelope (NE) consists of two concentric nuclear membranes that surrounds the nucleus from the cytoplasm in eukaryotic cells. The two nuclear membranes are separated by a thin fluid layer, referred to as the lumen, which is densely packed with proteins. These proteins are implicated in important cellular processes, such as mechano-regulated gene expression. The molecular details of these regulation processes, which typically require the formation of protein complexes, are not well understood due to a lack of techniques for the physical characterization of protein assembly in the NE of living cells. While recent progress has been made using fluorescence fluctuation spectroscopy (FFS) to quantify the assembly states of NE proteins in their native environment, monitoring the interaction of proteins with the membrane during assembly remains an unsolved problem. This is a significant shortcoming because membrane binding is often associated with conformational changes in proteins that are critical to cellular signaling pathways. Particularly vexing for studying membrane association is the close proximity of the nuclear membranes, which are only separated by the approximately 40 nm thick lumen. Thus, optical resolution is insufficient to directly distinguish luminal and membrane-bound NE proteins by fluorescence imaging methods.

To overcome this obstacle, we examined protein mobility within the NE by FFS methods. While membrane association has been detected using mobility in other regions of the cell, the confined spatial structure of the NE requires us to check the validity of the Stokes-Einstein relation for luminal proteins. In addition, we explore the temperature-dependent mobility of soluble and membrane-bound proteins in the NE as a potential marker for differentiating these populations. Finally, we look at the undulations of the nuclear membranes, which introduce local volume changes. This process is detected by FFS as an additional fluctuation signal for luminal proteins, but is absent for membrane-bound proteins. We harness this difference to provide a second, independent tool for identifying membrane-bound NE proteins. These new techniques are then applied to investigate the membrane association of two proteins native to the NE: SUN2, a constituent protein of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, and the AAA+ ATPase torsinA.

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