Biology subjects

Elferich, J.

Publications and source records attributed to Elferich, J..

11 recordsLinked to original sources

bioRxiv2026

Mapping AMPA and NMDA receptors at mouse brain hippocampal synapses by cryo-ET

AMPA and NMDA receptors are fundamental to synaptic transmission through the detection of released neurotransmitter. These ion channels open in response to glutamate binding, resulting in depolarization of the postsynaptic membrane and influx of calcium. Receptor localization plays a key role in synaptic strength, and changes in receptor number and location underpin synaptic plasticity, learning and memory. Here we apply receptor subunit-specific antibody fragments and multiplexed gold nanoparticle conjugates, together with cryo-electron tomography (cryo-ET), to elucidate the in-situ molecular organization of AMPA and NMDA receptors at hippocampal synapses in adult mouse brain tissue. AMPA receptors are predominantly found in densely packed liquid-like clusters, with interspersed NMDA receptors and adjacent NMDA receptor clusters. By direct localization of individual receptors, we provide molecular-resolution insight into the distribution of AMPA and NMDA receptors across the synapse, at peri and extrasynaptic sites, and within endocytosed vesicles. Visualization of multiple presynaptic vesicle fusion events suggests that receptors are near, but not always directly adjacent to, transmitter release sites. By mapping AMPA and NMDA receptors, and glutamate release sites, we provide receptor-resolution insight into the structure and function of brain hippocampal synapses.

neuroscience↗

bioRxiv2026

Programmable gold nanoparticle conjugates enable precise AMPA receptor localization at brain synapses by cryo-ET

Cryo-electron tomography (cryo-ET) is a powerful approach to image cells, organelles, viruses and molecular assemblies. However, resolving smaller complexes and molecules is challenging and presents a roadblock to the broad application of cryo-ET. Gold nanoparticles (AuNPs) have been used to localize otherwise unidentifiable molecules and complexes in tomograms but are typically tethered to the target of interest via a flexible linker. As a result, the position of the AuNP does not directly define the location of the target. Here we attach AuNPs to Fab antibody fragments via cysteine residues on defined elements of secondary structure and show that the AuNPs are rigidly bound to the Fab. We use these Fab-AuNP conjugates to locate AMPA receptors and probe their conformation upon desensitization with sub nanometer resolution in tomograms derived from brain slice lamella. By harnessing small, homogeneous AuNPs with precise conjugation to a Fab, we have developed reagents that enable direct investigation of conformational states of previously unidentifiable biomolecules and complexes in tomograms.

biochemistry↗

bioRxiv2025

Tolerance to the antifungal drug fluconazole is mediated by tuning cytoplasmic fluidity

Treatment failure rates for fungal infections cannot be explained simply by increased rates of drug resistance. Antifungal drug tolerance, the ability of a susceptible isolate to grow in the presence of inhibitory drug concentrations, relies upon a broad set of stress response pathways and can contribute to antifungal drug treatment failures. As the physical properties of the cytoplasm are critical for diverse cellular processes, we investigated whether cytoplasmic mesoscale fluidity is linked to antifungal tolerance, taking advantage of a fluorescent microrheological probe. Here, we show either using fluconazole, a common drug that inhibits ergosterol biosynthesis, or mutants in the ergosterol biosynthesis pathway, that cytoplasmic fluidity decreased and could be reversed upon drug removal. Reducing ribosome concentration decreased drug tolerance and restored cytoplasmic fluidity, highlighting a link between tolerance and cytoplasmic crowding and/or viscosity. However, growth in fluconazole did not increase ribosome concentrations; rather growth in fluconazole increased the number of dormant, hibernating ribosomes, cytoplasmic protein concentration, viscosity and condensate formation. Specifically, growth in fluconazole resulted in a substantial increase in processing bodies (P-bodies), whose presence correlated with azole tolerance. Furthermore, we found a substantial increase in cell-cell heterogeneity in all biophysical, biochemical and molecular outputs analyzed. Together, our results reveal that changes in the physical properties of the cytoplasm occur in response to antifungal drug and suggest that these changes, as well as increased cell-cell heterogeneity, are crucial for survival in fungistatic drugs.

microbiology↗

bioRxiv2025

Unbend: Correction of local beam-induced sample motion in cryo-EM images using a 3D spline model

The exposure of frozen biological samples to the high-energy electron beam in a cryo-electron microscope commonly leads to beam-induced sample motion and distortions. Previously, we described Unblur, which is part of our cisTEM software to correct for beam-induced motion based on the alignment of full frames in a movie collected during the beam exposure (Grant et al., 2015). However, Unblur cannot accommodate motion due to more localized sample bending and distortions. Here, we present Unbend, extending Unblur by incorporating local motion correction using a three-dimensional cubic spline model. The 3D spline model is constructed using cubic B-splines along the exposure time axis, and bicubic B-splines within movie frames. Unbend is integrated into our cisTEM software with a new local motion visualization panel within the cisTEM graphical user interface. We processed movie frames from various in-situ sample types, including whole cells, lamellae, and cell lysates, to analyze motion behavior across different specimen types. To quantify the improvement in high-resolution signal, we utilized the 2D template matching method, which operates independently of the motion correction process, to search large ribosomal subunits from the motion-corrected micrographs. Overall, the signal-to-noise ratio of detected particles improved by 3-8% across different samples compared with full-frame aligned micrographs, while the number of detected target particles increased by up to [~]300%. The total and Von Mises equivalent strain shows a deformation scale of less than 1% in most of the samples, confirming that our model induces minimal additional distortion. Furthermore, we processed micrograph montages to study motion patterns across an entire sample, revealing considerable variance in distortion scale within the same sample, suggesting a complex underlying mechanism.

biophysics↗

bioRxiv2025

In-Situ High-Resolution Cryo-EM Reconstructions from CEMOVIS

Cryo-electron microscopy can be used to image cells and tissue at high resolution. To ensure electron transparency, sample thickness must not exceed 500 nm. Focused-ion-beam (FIB) milling has become the standard method to prepare thin samples (lamellae), however, the material removed by the milling process is lost, the imageable area is usually limited to a few square microns, and the surface layers sustain damage from the ion beam. We have examined cryo-electron microscopy of vitreous sections (CEMOVIS), a preparation technique based on cutting thin sections with a knife, as an alternative to FIB-milling. CEMOVIS sections also sustain damage, including compression, shearing and cracks. However, samples can be sectioned in series, producing many orders of magnitude more imageable area compared to lamellae making CEMOVIS an alternative to FIB-milling with distinct advantages. Using 2-dimensional template matching on images of CEMOVIS sections of Saccharomyces cerevisiae cells, we reconstructed the 60S ribosomal subunit at near-atomic resolution, demonstrating that, in many regions of the sections, the molecular structure of these subunits is largely intact, comparable to FIB-milled lamellae.

biophysics↗

bioRxiv2024

Dynamic cytoplasmic fluidity during morphogenesis in a human fungal pathogen

The molecular crowding of the cytoplasm impacts a range of cellular processes. Using a fluorescent microrheological probe (GEMs), we observed a striking decrease in molecular crowding during the yeast to filamentous growth transition in the human fungal pathogen Candida albicans. This decrease in crowding is due to a decrease in ribosome concentration that results in part from an inhibition of ribosome biogenesis, combined with an increase in cytoplasmic volume; leading to a dilution of the major cytoplasmic crowder. Moreover, our results suggest that inhibition of ribosome biogenesis is a trigger for C. albicans morphogenesis.

cell biology↗

bioRxiv2024

Cryo-electron tomographic investigation of native hippocampal glutamatergic synapses

Chemical synapses are the major sites of communication between neurons in the nervous system and mediate either excitatory or inhibitory signaling [1]. At excitatory synapses, glutamate is the primary neurotransmitter and upon release from presynaptic vesicles, is detected by postsynaptic glutamate receptors, which include ionotropic AMPA and NMDA receptors [2, 3]. Here we have developed methods to identify glutamatergic synapses in brain tissue slices, label AMPA receptors with small gold nanoparticles (AuNPs), and prepare lamella for cryo-electron tomography studies. The targeted imaging of glutamatergic synapses in the lamella is facilitated by fluorescent pre- and postsynaptic signatures, and the subsequent tomograms allow for identification of key features of chemical synapses, including synaptic vesicles, the synaptic cleft and AuNP-labeled AMPA receptors. These methods pave the way for imaging brain regions at high resolution, using unstained, unfixed samples preserved under near-native conditions.

biochemistry↗

bioRxiv2024

CTFFIND5 provides improved insight into quality, tilt and thickness of TEM samples

Images taken by transmission electron microscopes are usually affected by lens aberrations and image defocus, among other factors. These distortions can be modeled in reciprocal space using the contrast transfer function (CTF). Accurate estimation and correction of the CTF is essential for restoring the high-resolution signal in cryogenic electron microscopy (cryoEM). Previously, we described the implementation of algorithms for this task in the cisTEM software package (Grant et al., 2018). Here we show that taking sample characteristics, such as thickness and tilt, into account can improve CTF estimation. This is particularly important when imaging cellular samples, where measurement of sample thickness and geometry derived from accurate modeling of the Thon ring pattern helps judging the quality of the sample. This improved CTF estimation has been implemented in CTFFIND5, a new version of the cisTEM program CTFFIND. We evaluated the accuracy of these estimates using images of tilted aquaporin crystals and eukaryotic cells thinned by focused ion beam milling. We estimate that with micrographs of sufficient quality CTFFIND5 can measure sample tilt with an accuracy of 3{degrees} and sample thickness with an accuracy of 5 nm.

biophysics↗

bioRxiv2023

Single molecule studies of the native hair cell mechanosensory transduction complex

Hearing and balance rely on the conversion of a mechanical stimulus into an electrical signal, a process known as mechanosensory transduction (MT). In vertebrates, this process is accomplished by an MT complex that is located in hair cells of the inner ear. While the past three decades of research have identified many subunits that are important for MT and revealed interactions between these subunits, the composition and organization of a functional complex remains unknown. The major challenge associated with studying the MT complex is its extremely low abundance in hair cells; current estimates of MT complex quantity range from 3-60 attomoles per cochlea or utricle, well below the detection limit of most biochemical assays that are used to characterize macromolecular complexes. Here we describe the optimization of two single molecule assays, single molecule pull-down (SiMPull) and single molecule array (SiMoA), to study the composition and quantity of native mouse MT complexes. We demonstrate that these assays are capable of detecting and quantifying low attomoles of the native MT subunits protocadherin-15 (PCDH15) and lipoma HMGIC fusion partner-like protein 5 (LHFPL5). Our results illuminate the stoichiometry of PCDH15- and LHFPL5-containing complexes and establish SiMPull and SiMoA as productive methods for probing the abundance, composition, and arrangement of subunits in the native MT complex. Impact StatementIn the present work, the authors develop and employ single molecule methods to detect, characterize, and quantitate attomole quantities of the hair cell mechanosensory transduction complex.

biochemistry↗

bioRxiv2022

Defocus Corrected Large Area Cryo-EM (DeCo-LACE) for Label-Free Detection of Molecules across Entire Cell Sections

A major goal of biological imaging is localization of biomolecules inside a cell. Fluorescence microscopy can lo-calize biomolecules inside whole cells and tissues, but its ability to count biomolecules and accuracy of the spatial coordinates is limited by the wavelength of visible light. Cryo-electron microscopy (cryo-EM) provides highly accurate position and orientation information of biomolecules but is often confined to small fields of view inside a cell, limiting biological context. In this study we use a new data-acquisition scheme called "Defocus-Corrected Large-Area cryo-EM" (DeCo-LACE) to collect high-resolution images of entire sections (100 - 200 nm thick lamel-lae) of neutrophil-like mouse cells, representing 1-2% of the total cellular volume. We use 2D template matching (2DTM) to determine localization and orientation of the large ribosomal subunit in these sections. These data provide "maps" of ribosomes across entire sections of mammalian cells. This high-throughput cryo-EM data collection approach together with 2DTM will advance visual proteomics and provide biological insight that cannot be obtained by other methods.

cell biology↗

bioRxiv2021

Molecular structure and conformation of stereocilia tip-links elucidated by cryo-electron tomography

Mechanosensory transduction (MT), the conversion of mechanical stimuli into electrical signals, underpins hearing and balance and is carried out within hair cells in the inner ear. Hair cells harbor actin-filled stereocilia, arranged in rows of descending heights, where the tips of stereocilia are connected to their taller neighbors by a filament composed of protocadherin 15 (PCDH15) and cadherin 23 (CDH23), deemed the tip-link. Tension exerted on the tip-link opens an ion channel at the tip of the shorter stereocilia, thus converting mechanical force into an electrical signal. While biochemical and structural studies have provided insights into the molecular composition and structure of isolated portions of the tip-link, the architecture, location and conformational states of intact tip-links, on stereocilia, remains unknown. Here we report in situ cryo-electron microscopy imaging of the tip-link in mouse stereocilia. We observe individual PCDH15 molecules at the tip and shaft of stereocilia and determine their stoichiometry, conformational heterogeneity, and their complexes with CDH23. The PCDH15/CDH23 complexes occur in clusters, frequently with more than one copy of PCDH15 at the tip of stereocilia, suggesting that tip-links might consist of more than one copy of the PCDH15/CDH23 heterotetramer and by extension, might include multiple MT complexes.

neuroscience↗