Dr. Hisham Mazal

Cryogenic light microscopy of vitrified samples with Ångstrom precision

Hisham Mazal, Franz Wieser, Daniel Bollschweiler, Vahid Sandoghdar

High-resolution studies in structural biology are commonly based on diffraction methods and on electron microscopy. However, these approaches are limited by the difficulty in crystallization of biomolecules or by a low contrast that makes high-resolution measurements very challenging in crowded samples such as a cell membrane. The exquisite labeling specificity of fluorescence microscopy gets around these issues. Indeed, several recent reports have reached resolutions down to the Ångstrom level in super-resolution microscopy, but to date, these works used 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 demonstrate a technique that satisfies these key criteria with particular promise for conformational studies on membrane proteins and their complexes. To prepare cell membranes in their near-native state, we adapt established protocols from cryogenic electron microscopy (Cryo-EM) for shock-freezing and transfer of samples. We developed a high-vacuum cryogenic shuttle system that allows us to transfer vitrified samples in and out of a liquid-helium cryostat that houses a super-resolution fluorescence microscope. Sample temperatures below 10 K help dissipate the heat from laser illumination, thus maintaining intact vitreous ice. We utilize the photoblinking of organic dye molecules attached to well-defined positions of a protein to localize one label fluorophore at a time. We present various characterization studies of the vitreous ice, photoblinking behavior, and the effects of the laser intensity. Moreover, we benchmark our method by demonstrating Ångstrom precision in resolving the full assembled configuration of the heptameric membrane protein alpha-hemolysin (αHL) in a synthetic lipid membrane as a model system. Additionally, we report on the technique’s capability to resolve membrane proteins in their native cellular membrane environment. Our method, which we term single-particle cryogenic light microscopy (spCryo-LM), enables structural studies of membrane protein tertiary and quaternary conformations without the need for chemical fixation or protein isolation. The approach can also integrate other super-resolution or spectroscopic techniques with particular promise in correlative microscopy with images from Cryo-EM and related techniques.

Lipidic folding pathway of α-Synuclein via a toxic oligomer

Vrinda Sant, Dirk Matthes, Hisham Mazal, Leif Antonschmidt, Franz Wieser, Kumar Tekwani Movellan, Kai Xue, Evgeny Nimerovsky, Marianna Stampolaki, et al.

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.

Higher order transient membrane protein structures

Yuxi Zhang, Hisham Mazal, Venkata Shiva Mandala, Gonzalo Perez-Mitta, Vahid Sandoghdar, Christoph A. Haselwandter, Roderick McKinnon

This study shows that five membrane proteins—three GPCRs, an ion channel, and an enzyme—form self-clusters under natural expression levels in a cardiac-derived cell line. The cluster size distributions imply that these proteins self-oligomerize reversibly through weak interactions. When the concentration of the proteins is increased through heterologous expression, the cluster size distributions approach a critical distribution at which point a phase transition occurs, yielding larger bulk phase clusters. A thermodynamic model like that explaining micellization of amphiphiles and lipid membrane formation accounts for this behavior. We propose that many membrane proteins exist as oligomers that form through weak interactions, which we call higher-order transient structures (HOTS). The key characteristics of HOTS are transience, molecular specificity, and a monotonically decreasing size distribution that may become critical at high concentrations. Because molecular specificity invokes self-recognition through protein sequence and structure, we propose that HOTS are genetically encoded supramolecular units.

Cryogenic light microscopy with Ångstrom precision deciphers structural conformations of PIEZO1

Hisham Mazal, Alexandra Schambony, Vahid Sandoghdar

Despite the impressive progress in molecular biochemistry and biophysics, many questions regarding the conformational states of large (transmembrane) protein complexes persist. In the case of the PIEZO protein, investigations by cryogenic electron microscopy (Cryo-EM) and atomic force microscopy (AFM) have established a symmetric trimer structure with three long-bladed domains in a propeller-like configuration. A transition of PIEZO protein from curved to flat conformation is hypothesized to actuate closed and open channels for the flow of ions. However, conclusive high-resolution data on the molecular organization of PIEZO in its native form are lacking. To address this shortcoming, we exploit single-particle cryogenic light microscopy (spCryo-LM) to decipher the conformational states of the mouse PIEZO1 protein (mPIEZO1) in the cell membrane. Here, we implement a high-vacuum cryogenic shuttle to transfer shock-frozen unroofed cell membranes in and out of a cryostat for super-resolution microscopy at liquid helium temperature. By localizing fluorescent labels placed at the extremities of the three blades with Ångstrom precision, we ascertain three configurations of the protein with radii of 6, 12, and 20 nm as projected onto the membrane plane. Our data suggest that in the smallest configuration, the blades form a nano-dome structure that is more strongly curved than previously observed and predicted by AlphaFold-3. In the largest conformation, we believe the structure must fully unbend in an anticlockwise manner to form a flat extended state. We attribute the 12 nm conformation, the most frequently occupied state, to an intermediate state and discuss our results in the context of the findings from other groups. Combination of spCryo-LM and Cryo-EM measurements together with in situ photothermal stimulation promises to provide quantitative insight into the interplay between structure and function of PIEZO and other biomolecular complexes in their native environments.

Insights into protein structure using cryogenic light microscopy

Hisham Mazal, Franz Wieser, Vahid Sandoghdar

Fluorescence microscopy has witnessed many clever innovations in the last two decades, leading to new methods such as structured illumination and super-resolution microscopies. The attainable resolution in biological samples is, however, ultimately limited by residual motion within the sample or in the microscope setup. Thus, such experiments are typically performed on chemically fixed samples. Cryogenic light microscopy (Cryo-LM) has been investigated as an alternative, drawing on various preservation techniques developed for cryogenic electron microscopy (Cryo-EM). Moreover, this approach offers a powerful platform for correlative microscopy. Another key advantage of Cryo-LM is the strong reduction in photobleaching at low temperatures, facilitating the collection of orders of magnitude more photons from a single fluorophore. This results in much higher localization precision, leading to Angstrom resolution. In this review, we discuss the general development and progress of Cryo-LM with an emphasis on its application in harnessing structural information on proteins and protein complexes.

Self-supervised machine learning pushes the sensitivity limit in label-free detection of single proteins below 10 kDa

Mahyar Dahmardeh, Houman Mirzaalian Dastjerdi, Hisham Mazal, Harald Köstler, Vahid Sandoghdar

Interferometric scattering (iSCAT) microscopy is a label-free optical method capable of detecting single proteins, localizing their binding positions with nanometer precision, and measuring their mass. In the ideal case, iSCAT is limited by shot noise such that collection of more photons should extend its detection sensitivity to biomolecules of arbitrarily low mass. However, a number of technical noise sources combined with speckle-like background fluctuations have restricted the detection limit in iSCAT. Here, we show that an unsupervised machine learning isolation forest algorithm for anomaly detection pushes the mass sensitivity limit by a factor of 4 to below 10 kDa. We implement this scheme both with a user-defined feature matrix and a self-supervised FastDVDNet and validate our results with correlative fluorescence images recorded in total internal reflection mode. Our work opens the door to optical investigations of small traces of biomolecules and disease markers such as α-synuclein, chemokines and cytokines.<br><br>

Deciphering a hexameric protein complex with Angstrom optical resolution

Hisham Mazal, Franz Wieser, Vahid Sandoghdar

Cryogenic optical localization in three dimensions (COLD) was recently shown to resolve up to four binding sites on a single protein. However, because COLD relies on intensity fluctuations that result from the blinking behavior of fluorophores, it is limited to cases where individual emitters show different brightness. This significantly lowers the measurement yield. To extend the number of resolved sites as well as the measurement yield, we employ partial labeling and combine it with polarization encoding in order to identify single fluorophores during their stochastic blinking. We then use a particle classification scheme to identify and resolve heterogenous subsets and combine them to reconstruct the three-dimensional arrangement of large molecular complexes. We showcase this method (polarCOLD) by resolving the trimer arrangement of proliferating cell nuclear antigen (PCNA) and six different sites of the hexamer protein Caseinolytic Peptidase B (ClpB) of Thermus thermophilus in its quaternary structure, both with Angstrom resolution. The combination of polarCOLD and single-particle cryogenic electron microscopy (cryoEM) promises to provide crucial insight into intrinsic heterogeneities of biomolecular structures. Furthermore, our approach is fully compatible with fluorescent protein labeling and can, thus, be used in a wide range of studies in cell and membrane biology.