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Tunable Substrates Improve Imaging of Viruses and Cancer Proteins


Carly E. Winton , 1 Brian L. Gilmore , 1 Justin R. Tanner , 1 A. Cameron Varano , 1 , 2 Zhi Sheng , 1


and Deborah F. Kelly 1 * 1 Virginia Tech Carilion Research Institute , Virginia Tech , Roanoke , VA 24016 2 Translational Biology , Medicine , and Health Graduate Program , Virginia Tech , Blacksburg , VA 24061


* debkelly@vt.edu


Abstract: Recent breakthroughs in cryo-electron microscopy imaging technology provide an unprecedented view of biology at the nanoscale. To complement these technical advances, we demonstrate here the use of tunable substrates to streamline the isolation of biological entities from human cells. We have tested the capacity of tunable microchip devices using a variety of samples including virus assemblies and the breast cancer susceptibility protein (BRCA1) produced in cancer cells. Future microchip applications may shed light on ill-defi ned clinical issues related to molecular disease mechanisms.


Introduction


Understanding the properties of molecular assemblies can provide important insight to decode human health and disease processes. Transmission electron microscopes (TEMs) are important tools to view the intricate details of disease-related tissues, cells, and proteins. By preparing EM specimens under frozen-hydrated conditions, we can preserve biological features in a near-native environment [ 1 ]. T is form of preservation embeds the starting material in thin fi lms of vitreous ice. By convention, amorphous carbon is the most common support fi lm used to prepare specimens for cryogenic (cryo)-EM imaging. Micron-sized holes engineered into carbon support fi lms provide a transparent background to see individual proteins contained in the surrounding ice [ 2 ]. Factors that limit spatial resolution in ice are specimen charging, beam-induced movements, and other noise- producing artifacts [ 3 ]. Recent advances in the EM fi eld are aimed at minimizing resolution-limiting eff ects. Devices that spurred these eff orts include automated freezing units, phase- plates, direct electron detectors, and in-column energy fi lters. Each of these tools off ers improved technology for recording images of biological and clinical samples. Correspondingly, a new generation of specimen support materials also must be developed to best utilize these tools.


With the progression of the U.S. Materials Genome Initiative and other worldwide ventures to produce new substances, the EM fi eld is presented with a prime opportunity to test alternative substrates for EM support fi lms. Some examples of alternative substrates include conductive titanium-silicon metal glass (Ti 88 Si 12 ) [ 4 ], silicon carbide (cryomesh) [ 5 ], graphene [ 6 ], and silicon nitride (SiN) [ 7 ]. A major benefi t of using alternative substrates is their versatile surface properties in comparison to carbon-based fi lms. One example of this versatility for the SiN microchip is the ability to modify its surface and tether biological complexes to the pristine, fl at microchip prior to specimen preservation. Taking this process a step further, by decorating microchips with specifi c adaptor molecules, we can create “tunable” devices for the intended purpose of harvesting proteins from human cells for clinical studies. Here, tunable devices are


22


engineered before use to target a particular protein or process under investigation, rather than being tuned during use. Traditionally, protein isolation schemes involve lengthy steps that employ harsh chemicals to isolate fragile proteins. During the several days required to complete chromatographic separations, multi-subunit protein assemblies can easily dissociate, which is detrimental to studying their structural attributes. In an eff ort to minimize these negative eff ects and to streamline the isolation of native assemblies from human cells, we tested the capacity of SiN microchips decorated with specifi c antibody adaptors. In each test case, the microchips eff ectively isolated the target proteins from human cells, nuclear material, or pre-fractionated samples. For cryo-EM applications, we refer to SiN microchips as “Cryo-SiN” [ 7 ]. Recently developed protocols for using Cryo-SiN permitted us to recruit protein assemblies from patient-derived cancer cells under mild conditions [ 8 , 9 ]. T is approach represents a viable alternative to extensive protein purifi cation techniques and can be applied to a multitude of proteins. T erefore, a major advantage to using SiN microchips is the cost and time savings in comparison to conventional pursuits. To demonstrate these advances, we describe in this article cryo-EM information recently published for both viruses and protein assemblies isolated from eukaryotic cells using tunable microchip devices [ 7 – 9 ].


Materials and Methods Rotavirus preparation . Rotavirus (strain SA11-4F) double-layered particles (DLPs) were prepared as described previously [ 7 ] by the laboratory of Dr. Sarah M. McDonald at the Virginia Tech Carilion Research Institute. Aliquots (3 μ L each) of purifi ed DLPs (0.1 mg/mL) contained in 50 mM HEPES buff er (pH 7.5) supplemented with 150 mM NaCl, 10 mM CaCl 2 , and 10 mM MgCl 2 were applied to antibody-decorated EM grids ( Figures 1 a and 1 b) or to SiN microchips ( Figure 1c ) and incubated for 2 minutes prior to plunge-freezing.


BRCA1 protein assemblies isolated from breast cancer


cells . We separated the nuclear contents of primary ductal carcinoma cells that express wild type BRCA1 (HCC70 line; ATCC) using the commercially available NE-PER extraction kit (T ermo Scientifi c). Transcriptional assemblies from the cells’ nuclei can be naturally separated by interacting with immobilized- metal affi nity matrices [ 8 – 10 ]. T e following components were contained in the separated nuclear material and were collectively enriched by incubating with nickel-nitrilotriacetic acid (Ni-NTA) agarose beads: (1) active RNA Polymerase II (RNAP II);


doi: 10.1017/S1551929517000657 www.microscopy-today.com • 2017 July


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