Genetically encoded multimeric tags for intracellular protein localisation in cryo-EM
Posted on: 16 January 2023
Preprint posted on 10 December 2022
A new development of a small molecule-inducible approach to label proteins, enables nanometer-scale localization of molecules via cryo-ET combined with fluorescence microscopy.
Selected by Martyna Kosno-Vega
Categories: biochemistry, bioengineering, biophysics, cell biology, molecular biology, synthetic biology
Background:
To understand how cells function in a given process, we need to know the precise sub-cellular localization of all relevant molecules. However, the crowded cellular environment makes this a difficult task. Cryo-electron tomography (cryo-ET) and cryo-correlative light and electron microscopy (CLEM) are some of the techniques that can help address this problem [1, 2]. While much progress has been made in recent years, uncovering fine details using cellular imaging remains very difficult due to the fluorescence imaging resolution limit and potential distortion of samples during the preparation process. Thus, further developments of these imaging methods are needed.
In this collaborative study, the Mahamid and Culyen-Haering research groups from EMBL developed a novel strategy to tag proteins in cells in order to precisely identify their location. To do so, they designed a genetically encoded multimeric (GEM) protein tag, which binds to GFP upon small molecule treatment, through an FRB-FKBP interaction. They wanted the tag to not only provide fluorescent signal, but also be visible when using cryo-ET. Thus, the scientists based their labeling system on encapsulins – 25-42 nm proteins from archaea and bacteria with an unusual, icosahedral shape, which can be easily seen via electron microscopy [3].
Preprint Figure 1a: Schematic of the system designed by the authors.
Key Findings
GEM2 is the best candidate to tag proteins of interest
The group performed a library screen of multiple GEMs, including 10 natural encapsulins and 3 engineered proteins in HeLa cells. They found that one of the candidates exhibited uniform distribution both in the cytoplasm and the nucleus of mammalian cells (GEM2). Next, the authors used the GEM2 tag with several known targets, to assess its accuracy of labeling, its applicability to natural cellular systems and visibility in cryo-ET.
Functional assays confirm GEM2’s utility in structural and cellular studies
The scientists chose three known and diverse targets: the mitotic chromosome surface, nuclear pore, and endoplasmic reticulum-lipid droplet junction. In all cases they observed small molecule-inducible colocalization of the GEM tag with the chosen target protein. However, the dynamics of recruitment were dependent on the target protein’s abundance (the more abundant the protein, the faster the dynamics of recruitment). The icosahedral particles of the GEM tag were visible in cryo-ET, a majority within ~50nm of the expected location, making this system very accurate. Due to the multimeric nature of the GEMs, each particle comes with 60 Halo tags to provide strong fluorescence and thus help guide cryo-ET data collection.
Preprint Figure 2e: From left to right: cryo-TEM image overlaid with a fluorescent image. Center and right images show tomographic slice and itssegmentation with insets showing details of the ER-LD contact site with the GEM tags in close proximity.
In summary, this study provides a novel, small molecule-inducible approach to label proteins, allowing nanometer-scale localization of molecules. The method seems promising for many types of experiments, with some additional optimization required depending on the application. The authors claim that it is easy to implement and optimize for any system and that it should help expand our structural knowledge of diverse subcellular compartments.
Why I chose this preprint
I have been working towards my PhD in the Department of Biophysics at UT Southwestern, where a large focus of many labs is on structural biology. Nonetheless, I am a cell biologist at heart. This preprint provides a very interesting method to combine the two: structural biology and cell biology, promising to unravel fine details of subcellular organization.
I am also incredibly impressed when scientists decide to utilize a system that nature already came up with and optimize it for scientific purposes. We have seen a similar thing with CRISPR, back in 2012 [4]. Here, the two labs “borrowed” encapsulin proteins, which – in bacteria and archaea – serve as structural blocks to build protein cages. These cages provide a means of compartmentalization for an otherwise organelle-less organism. The scientists decided to utilize the large size and unique shape of these proteins to help guide cryo-ET in human cells.
Future directions and questions for the authors
It will be fascinating to see where this technology could lead structural studies in the coming years and how much it can be optimized. It would be great – for example – if the technology could be less dependent on protein abundance and potential protein shape. It will also be important to determine if in fact the applicability of this system is as wide as the authors claim.
- Could GEM tags affect the function of the target protein in systems other than the three tested by the authors?
- Due to their size, could GEMs sterically hinder the proper protein localization?
References
- Dahlberg, P.D., et al., Cryogenic single-molecule fluorescence annotations for electron tomography reveal in situ organization of key proteins in Caulobacter. Proc Natl Acad Sci U S A, 2020. 117(25): p. 13937-13944.
- Rigort, A., et al., Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography. Proc Natl Acad Sci U S A, 2012. 109(12): p. 4449-54.
- Andreas, M.P. and T.W. Giessen, Large-scale computational discovery and analysis of virus-derived microbial nanocompartments. Nat Commun, 2021. 12(1): p. 4748.
- Jinek, M., et al., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012. 337(6096): p. 816-21.
doi: https://doi.org/10.1242/prelights.33493
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