Biology subjects

Volkmann, N.

Publications and source records attributed to Volkmann, N..

9 recordsLinked to original sources

bioRxiv2022

MEPSi: A tool for simulating tomograms of membrane-embedded proteins

The throughput and fidelity of cryogenic cellular electron tomography (cryo-ET) is constantly increasing through advances in cryogenic electron microscope hardware, direct electron detection devices, and powerful image processing algorithms. However, the need for careful optimization of sample preparations and for access to expensive, high-end equipment, make cryo-ET a costly and time-consuming technique. Generally, only after the last step of the cryo-ET workflow, when reconstructed tomograms are available, it becomes clear whether the chosen imaging parameters were suitable for a specific type of sample in order to answer a specific biological question. Tools for a-priory assessment of the feasibility of samples to answer biological questions and how to optimize imaging parameters to do so would be a major advantage. Here we describe MEPSi (Membrane Embedded Protein Simulator), a simulation tool aimed at rapid and convenient evaluation and optimization of cryo-ET data acquisition parameters for studies of transmembrane proteins in their native environment. We demonstrate the utility of MEPSi by showing how to detangle the influence of different data collection parameters and different orientations in respect to tilt axis and electron beam for two examples: (1) simulated plasma membranes with embedded single-pass transmembrane IIb{beta}3 integrin receptors and (2) simulated virus membranes with embedded SARS-CoV-2 spike proteins. HIGHLIGHTSO_LITool to simulate tomograms of membrane-embedded proteins C_LIO_LIDetangles influence of data acquisition parameters from sample quality issues C_LIO_LIRapid evaluation and optimization of cryo-ET data acquisition parameters C_LIO_LIProof-of-concept provided with integrins and SARS-CoV-2 spike simulations C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/501771v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@120b44eorg.highwire.dtl.DTLVardef@1d9056org.highwire.dtl.DTLVardef@ef327aorg.highwire.dtl.DTLVardef@1daf773_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Characterization of heterogeneity in nanodisc samples using Feret signatures

Nanodiscs have become a popular tool in structure determination of membrane proteins using cryogenic electron microscopy and single particle analysis. However, the structure determination of small membrane proteins remains challenging. When the embedded protein is in the same size range as the nanodisc, the nanodisc can significantly contribute to the alignment and classification during the structure determination process. In those cases, it is crucial to minimize the heterogeneity in the nanodisc preparations to assure maximum accuracy in the classification and alignment steps of single particle analysis. Here, we introduce a new in-silico method for the characterization of nanodisc samples that is based on analyzing the Feret diameter distribution of their particle projection as imaged in the electron microscope. We validated the method with comprehensive simulation studies and show that Feret signatures can detect subtle differences in nanodisc morphologies and composition that might otherwise go unnoticed. We used the method to identify a specific biochemical nanodisc preparation with low size variations, allowing us to obtain a structure of the 23-kDa single-span membrane protein Bcl-xL while embedded in a nanodisc. Feret signature analysis can steer experimental data collection strategies, allowing more efficient use of high-end data collection hardware, as well as image analysis investments in studies where nanodiscs significantly contribute to the total volume of the full molecular species. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/501900v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@19ea37aorg.highwire.dtl.DTLVardef@1fed1aeorg.highwire.dtl.DTLVardef@781962org.highwire.dtl.DTLVardef@ca7859_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LINew methodology to characterize nanodiscs based on Feret signatures C_LIO_LIFeret signatures distinguish nanodisc morphologies and compositions C_LIO_LIAnalysis is highly sensitive to sample quality C_LIO_LIMethod selected condition to solve structure of small membrane protein Bcl-xL C_LI

molecular biology↗

bioRxiv2022

ACE2 nanoparticles prevent cell entry of SARS-CoV-2

The continual evolution of SARS-CoV-2 has challenged the efficacy of many COVID19 vaccines and treatment options. One strategy that evades viral escape is using the entry receptor, human Angiotensin-Converting Enzyme 2 (hACE2). Soluble hACE2 receptor domains show potential as decoys but genetic modifications are necessary to provide sufficient efficacy. However, these engineered constructs are potentially susceptible to viral escape. We combined native hACE2 with viral vectors to form nanoparticles presenting hACE2 analogous to human cells. Cell-based viral infection assays and cryogenic in-situ tomography show that hACE2 nanoparticles sequester viruses through aggregation, efficiently blocking entry of SARS-CoV-2 and its variants in model cell systems and human respiratory tract explants using native hACE2. Thus, we show that hACE2 nanoparticles have high potential as pan-variant COVID19 therapeutics.

cell biology↗

bioRxiv2021

ECM dimensionality tunes actin tension to modulate the endoplasmic reticulum and spheroid phenotype

Primary tissue organoids and cell spheroids recapitulate tissue physiology with remarkable fidelity. We investigated how engagement with a three dimensional laminin-rich extracellular matrix supports the polarized, stress resilient spheroid phenotype of mammary epithelial cells. Cells within a three dimensional laminin-rich extracellular matrix decreased and redistributed the actin crosslinker filamin to reduce their cortical actin tension. Cells with low cortical actin tension had increased plasma membrane protrusions that promoted negative plasma membrane curvature and fostered protein associations with the plasma membrane, consistent with efficient protein secretion. By contrast, cells engaging a laminin-rich extracellular matrix in two dimensions had high filamin-dependent cortical actin tension, exhibited compromised endoplasmic reticulum function including increased expression of PKR-like Endoplasmic Reticulum Kinase signaling effectors, and had compromised protein secretion. Cells with low filamin-mediated cortical actin tension and reduced endoplasmic reticulum stress response signaling secreted, and assembled, a polarized endogenous basement membrane and survived better, and their spheroids were more resistant to exogenous stress. The findings implicate filamin-dependent cortical actin tension in endoplasmic reticulum function and highlight a role for mechanics in organoid homeostasis.

cell biology↗

bioRxiv2020

Structural basis of αE-catenin-F-actin catch bond behavior

Cell-cell and cell-matrix junctions transmit mechanical forces during tissue morphogenesis and homeostasis. -Catenin links cell-cell adhesion complexes to the actin cytoskeleton, and mechanical load strengthens its binding to F-actin in a direction-sensitive manner. This so-called catch bond behavior is described by a model in which force promotes a transition between weak and strong actin-bound states. We describe the cryo-electron microscopy structure of the F-actin-bound E-catenin actin-binding domain, which in solution forms a 5-helix bundle. Upon binding to actin, the first helix of the bundle dissociates and the remaining four helices and connecting loops rearrange to form the interface with actin. Deletion of the N-terminal helix produces strong actin binding in the absence of force. Our analysis explains how mechanical force applied to E-catenin or its homolog vinculin favors the strongly bound state, and the dependence of catch bond strength on the direction of applied force.

biophysics↗

bioRxiv2020

Factor VIII exhibits chaperone-dependent and glucose-regulated reversible amyloid formation in the endoplasmic reticulum

Factor VIII (FVIII) is the coagulation factor deficient in hemophilia A, which is treated by protein replacement. Unfortunately, this regimen is costly due to the expense of producing recombinant FVIII as a consequence of its low level secretion. FVIII expression activates the endoplasmic reticulum (ER) stress response, causes oxidative stress and induces apoptosis. Importantly, little is known about the factors that cause protein misfolding and aggregation in metazoans. Here we identified intrinsic and extrinsic factors that cause FVIII to form aggregates in the ER. We show that FVIII forms amyloid-like fibrils within the ER upon increased FVIII synthesis or inhibition of glucose metabolism. Significantly, FVIII amyloids can be dissolved upon restoration of glucose metabolism to produce functional secreted FVIII. Two ER chaperones and their co-chaperones, BiP and CANX/CRT, promote FVIII solubility in the ER, where the former is also required for disaggregation. A short aggregation motif in the FVIII A1 domain (termed Aggron) is necessary and sufficient to seed {beta}-sheet polymerization and BiP binding to this Aggron prevents amyloidogenesis. Our findings provide novel insight into mechanisms that limit FVIII secretion and ER protein folding in general and have implication for ongoing hemophilia A gene therapy clinical trials. Key Points- FVIII forms amyloid aggregates in the ER that are dissolved in a chaperone- and glucose-dependent manner to produce secreted active FVIII. - A short amino acid sequence in the A1 domain causes {beta} sheet polymerization and ER chaperone BiP binding to this site prevents aggregation.

molecular biology↗

bioRxiv2019

Extracellular matrix micropatterning technology for whole cell cryogenic electron microscopy studies

Cryogenic electron tomography is the highest resolution tool available for structural analysis of macromolecular organization inside cells. Micropatterning of extracellular matrix (ECM) proteins is an established in vitro cell culture technique used to control cell shape. Recent traction force microscopy studies have shown correlation between cell morphology and the regulation of force transmission. However, it remains unknown how cells sustain increased strain energy states and localized stresses at the supramolecular level. Here, we report a technology to enable direct observation of mesoscale organization in epithelial cells under morphological modulation, using a maskless protein photopatterning method to confine cells to ECM micropatterns on electron microscopy substrates. These micropatterned cell culture substrates can be used in mechanobiology research to correlate changes in nanometer-scale organization at cell-cell and cell-ECM contacts to strain energy states and traction stress distribution in the cell.

bioengineering↗

bioRxiv2018

Electron cryo-tomography of vestibular hair-cell stereocilia

High-resolution imaging of hair-cell stereocilia of the inner ear has contributed substantially to our understanding of auditory and vestibular function. To provide three-dimensional views of the structure of stereocilia cytoskeleton and membranes, we developed a method for rapidly freezing unfixed stereocilia on electron microscopy grids, which allowed subsequent 3D imaging by electron cryo-tomography. Structures of stereocilia tips, shafts, and tapers were revealed, demonstrating that the actin paracrystal was not perfectly ordered. This sample-preparation and imaging procedure will allow for examination of structural features of stereocilia in a near-native state.

neuroscience↗

bioRxiv2017

Accurate alignment between correlated light, cryo-light, and electron cryo-microscopy data using sample support features

Combining fluorescence microscopy with electron cryo-tomography allows, in principle, spatial localization of tagged macromolecular assemblies and structural features within the cellular environment. To allow precise localization and scale integration between the two disparate imaging modalities, accurate alignment procedures are needed. Here, we describe a marker-free method for aligning images from light or cryo-light fluorescence microscopy and from electron cryo-microscopy that takes advantage of sample support features, namely the holes in the carbon film. We find that the accuracy of this method, as judged by prediction errors of the hole center coordinates, is better than 100 nm.

biophysics↗