Dr. Hisham Mazal

High-resolution studies in structural biology are often limited by the challenges of crystallization and low contrast in the cellular native environment. The exquisite labeling specificity of fluorescence microscopy gets around these issues and allows superresolution microscopy, but to date, these works have used chemically fixed samples. To establish light microscopy as a workhorse in structural biology, two main requirements must be fulfilled: near-native sample preservation and near-atomic optical resolution. Here, we introduce single-particle cryogenic light microscopy (spCryo-LM) as a technique that satisfies these key criteria. We adapt established protocols from cryogenic electron microscopy (Cryo-EM) for shock-freezing samples and use a high-vacuum cryogenic shuttle system to transfer them in and out of a liquid-helium cryostat that houses a superresolution fluorescence microscope. By exploiting the enhanced photophysics at low temperature, angstrom precision can be achieved in localizing several fluorophores attached to proteins separated by a few hundred nanometers. We present various characterization studies on vitreous ice, single-molecule photoblinking behavior, and the effects of laser intensity and benchmark our method by resolving the heptameric membrane protein alpha-hemolysin in a synthetic lipid membrane. Additionally, we report on the technique’s capability to resolve membrane proteins in their native cellular membrane environment. spCryo-LM enables structural studies of proteins in their native environment without chemical fixation or protein isolation, and can be integrated with other superresolution or spectroscopic techniques. We believe our approach establishes light microscopy as a powerful tool in structural biology and sets the stage for correlative microscopy with Cryo-EM and related techniques.

Investigations based on cryo–electron microscopy (cryo-EM), atomic force microscopy, and super-resolution microscopy reveal a symmetric trimer with propeller-like blades for the mechanosensitive ion channel PIEZO. However, a conclusive understanding of its conformations in the cell membrane is lacking. Here, we implement a high-vacuum cryogenic shuttle to transfer shock-frozen cell membranes in and out of a cryostat designed for single-particle cryo–light microscopy (spCryo-LM). By localizing fluorescent labels placed at the extremities of the blades of the mouse PIEZO1 protein in unroofed cell membranes, we ascertain three configurations with radii of 6, 12, and 20 nanometers as projected onto the membrane plane. We elaborate on the correspondence of these data with previous reports in the literature. The combination of spCryo-LM with cryo-EM promises to provide quantitative insights into the structure and function of biomolecular complexes in their native environments without the need for chemical fixation or protein isolation, ushering in a new regime of correlative studies in structural biology.

Aggregation intermediates play a pivotal role in the assembly of amyloid fibrils, which are central to the pathogenesis of neurodegenerative diseases. The structures of filamentous intermediates and mature fibrils are now efficiently determined by single-particle cryo-electron microscopy. By contrast, smaller pre-fibrillar α-Synuclein (αS) oligomers, crucial for initiating amyloidogenesis, remain largely uncharacterized. We report an atomic-resolution structural characterization of a toxic pre-fibrillar aggregation intermediate (I1) on pathway to the formation of lipidic fibrils, which incorporate lipid molecules on protofilament surfaces during fibril growth on membranes. Super-resolution microscopy reveals a tetrameric state, providing insights into the early oligomeric assembly. Time-resolved nuclear magnetic resonance (NMR) measurements uncover a structural reorganization essential for the transition of I1 to mature lipidic L2 fibrils. The reorganization involves the transformation of anti-parallel β-strands during the pre-fibrillar I1 state into a β-arc characteristic of amyloid fibrils. This structural reconfiguration occurs in a conserved structural kernel shared by a vast number of αS-fibril polymorphs, including extracted fibrils from Parkinson’s and Lewy Body Dementia patients. Consistent with reports of anti-parallel β-strands being a defining feature of toxic αS pre-fibrillar intermediates, I1 impacts the viability of neuroblasts and disrupts cell membranes, resulting in increased calcium influx. Our results integrate the occurrence of anti-parallel β-strands as salient features of toxic oligomers with their significant role in the amyloid fibril assembly pathway. These structural insights have implications for the development of therapies and biomarkers.