Super-resolution imaging helps identify new proteins associated with neuronal MPS structures

Ruobo Zhou, assistant professor of chemistry and of biochemistry and molecular biology, recently had a paper published by the journal Nature Communications titled “Proteomic and functional analyses of the periodic membrane skeleton in neurons”. This is Zhou’s first published research paper since joining Penn State in 2021.  

The paper was a collaboration among multiple research labs including the Zhou lab at Penn State, the Zhuang lab at Harvard University, the Fowler lab at the University of Delaware, and the Chishti lab at Tufts University School of Medicine. The research was supported by the U.S. National Institutes of Health (NIH) and Howard Hughes Medical Institute (HHMI). 

The paper systematically determined the cellular proteins that are associated with a recently discovered cytoskeleton structure in nerve fibers, through the combined use of super-resolution fluorescence nanoscopy with mass-spectrometry-based proteomics. These newly identified proteins led to the discoveries of previously unknown cellular functions for this recently discovered cytoskeleton structure in nerve fibers.  

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In 2013, Dr. Xiaowei Zhuang's lab, the lab where Zhou received his postdoctoral training, reported the discovery of a novel cytoskeleton structure in the axonal fibers of nerve cells. In this cytoskeleton structure, actin, the cytoskeleton protein that usually forms long filaments to provide the mechanical support in all mammalian cells, was found to form periodically distributed "rings" along the nerve fibers. This novel cytoskeleton structure was then named the membrane-associated periodic skeleton (MPS).  

However, since the discovery of the MPS nine years ago, the cellular functions of MPS remained largely mysterious. This evolving view of MPS structures was the forefront of Zhou’s paper. Though it is believed that for all the cellular structures, cells organize their proteins to form certain cellular structures for a reason and the structures often dictate their cellular functions.   

Based on the newly identified proteins associated with the MPS structures the research team further inferred and identified several important cellular functions of the MPS structure.  

Zhou explained “among the two techniques we used, the super-resolution fluorescence nanoscopy had a spatial resolution 10-20 times better than the resolution of conventional fluorescence microscopy limited by the diffraction of light.” This allowed the researchers to see a lot more molecular details inside biological specimens than could be seen before. Mass spectrometry, a technology to determine the mass of ionized biomolecules, has been broadly used to identify protein components from a complex mixture. 

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By combining these two powerful techniques, the team biochemically isolated the proteins that are associated with the MPS structure from the nerve cells, and successfully identified hundreds of MPS-interacting proteins that were previously unknown to be able to be associated with the MPS structure; this includes many membrane proteins and enzymes spanning diverse functional categories. Based on the newly identified list of MPS-interacting proteins, the team further demonstrated two important cellular functions of the MPS structure: 1) MPS can control the diameter of the nerve fiber; and 2) MPS can control the interaction between two bundled nerve fibers, which is important for neurite-neurite fasciculations (i.e., bundling) during brain development. A few proteins that are responsible for the MPS to achieve these controls at the molecular level were also identified. 

When asked why this research is important to recognize now, Zhou said “our work not only reveals a comprehensive interactome of the neuronal MPS, providing new insights into how the MPS functions as an organizer and mediator for membrane proteins and enzymes, but can open therapeutic avenues into nerve regeneration as well as some neurodevelopmental diseases.”