A versatile high-vacuum cryo-transfer system for cryo-microscopy and analytics

Cryogenic microscopy methods have gained increasing popularity, as they offer an unaltered view on the architecture of biological specimens. As a prerequisite, samples must be handled under cryogenic conditions below their recrystallization temperature, and contamination during sample transfer and handling must be prevented. We present a high-vacuum cryo-transfer system that streamlines the entire handling of frozen-hydrated samples from the vitrification process to low temperature imaging for scanning transmission electron microscopy and transmission electron microscopy. A template for cryo-electron microscopy and multimodal cryo-imaging approaches with numerous sample transfer steps is presented

Parallel Acquisition of Plasma Membrane Ultrastructure and Cytosolic Protein Localisation in Cultured Cells via Correlated Immunogold SEM

Scanning electron microscopy (SEM) takes advantage of distinct detectors to visualise secondary and back-scattering electrons. Here, we report an integrated approach that relies on these two detection methods to simultaneously acquire correlated information on plasma membrane topography and curvature-sensitive cytosolic protein localization in intact cell samples. We further provide detailed preparation and staining protocols, as well as a thorough example-based discussion for imaging optimisation. Collectively, the presented method enables rapid and precise analysis of cytosolic proteins adjacent to cellular membranes with a resolution of ~100 nm, without time-consuming preparations or errors induced by sequential visualisation present in fluorescence-based correlative approaches

Can one determine the density of an individual synthetic macromolecule?

Dendronized polymers (DPs) are large and compact main-chain linear polymers with a cylindrical shape and cross-sectional diameters of up to ∼15 nm. They are therefore considered molecular objects, and it was of interest whether given their experimentally accessible, well-defined dimensions, the density of individual DPs could be determined. We present measurements on individual, deposited DP chains, providing molecular dimensions from scanning and transmission electron microscopy and mass-per-length values from quantitative scanning transmission electron microscopy. These results are compared with density values obtained from small-angle X-ray scattering on annealed bulk specimen and with classical envelope density measurements, obtained using hydrostatic weighing or a density gradient column. The samples investigated comprise a series of DPs with side groups of dendritic generations g = 1-8. The key findings are a very large spread of the density values over all samples and methods, and a consistent increase of densities with g over all methods. While this work highlights the advantages and limitations of the applied methods, it does not provide a conclusive answer to the question of which method(s) to use for the determination of densities of individual molecular objects. We are nevertheless confident that these first attempts to answer this challenging question will stimulate more research into this important aspect of polymer and soft matter science

Schuermann_Figure_3

Schuermann_Figure_3 - contains Excel-data on HhC85S multimerization and demonstrates that HhNC85S, a non-lipidated artificial Hh variant, does not act in dominant-negative manner. Contains Prize-Files to quantify unaffected wing patterning in three independently derived HhNC85S lines. The original wing patterns are shown in folders en>HhNC85S and ptc>HhNC85S

Schuermann_Figure_4

Schuermann_Figure_4 - contains files demonstrating that the consecutive N-terminal truncation of HhC85S reverses the dominant negative phenotype. Supplemental folders contain confirming data obtained from independent driver lines en(2)-Gal4 and Hh_Gal4. Unimpaired multimerization as a prerequisite for direct contact with, and dominant-negative suppression of Hh activity are shown as excel-files. HhC85SD86-100 denotes a non-palmitoylated Hh variant that lacks inhibitory N-terminal peptide amino acids 86-100

Data from: Proteolytic processing of palmitoylated Hedgehog peptides specifies the 3-4 intervein region of the Drosophila wing

Cell fate determination during development often requires morphogen transport from producing to distant responding cells. Hedgehog (Hh) morphogens present a challenge to this concept, as all Hhs are synthesized as terminally lipidated molecules that form insoluble clusters at the surface of producing cells. While several proposed Hh transport modes tie directly into these unusual properties, the crucial step of Hh relay from producing cells to receptors on remote responding cells remains unresolved. Using wing development in Drosophila melanogaster as a model, we show that Hh relay and direct patterning of the 3-4 intervein region strictly depend on proteolytic removal of lipidated N-terminal membrane anchors. Site-directed modification of the N-terminal Hh processing site selectively eliminated the entire 3-4 intervein region, and additional targeted removal of N-palmitate restored its formation. Hence, palmitoylated membrane anchors restrict morphogen spread until site-specific processing switches membrane-bound Hh into bioactive forms with specific patterning functions

Pathogenic variants in CLXN encoding the outer dynein arm docking–associated calcium-binding protein calaxin cause primary ciliary dyskinesia

Purpose: Primary ciliary dyskinesia (PCD) is a heterogeneous disorder that includes respiratory symptoms, laterality defects, and infertility caused by dysfunction of motile cilia. Most PCD-causing variants result in abnormal outer dynein arms (ODAs), which provide the generative force for respiratory ciliary beating and proper mucociliary clearance. Methods: In addition to studies in mouse and planaria, clinical exome sequencing and functional analyses in human were performed. Results: In this study, we identified homozygous pathogenic variants in CLXN (EFCAB1/ODAD5) in 3 individuals with laterality defects and respiratory symptoms. Consistently, we found that Clxn is expressed in mice left-right organizer. Transmission electron microscopy depicted ODA defects in distal ciliary axonemes. Immunofluorescence microscopy revealed absence of CLXN from the ciliary axonemes, absence of the ODA components DNAH5, DNAI1, and DNAI2 from the distal axonemes, and mislocalization or absence of DNAH9. In addition, CLXN was undetectable in ciliary axonemes of individuals with defects in the ODA-docking machinery: ODAD1, ODAD2, ODAD3, and ODAD4. Furthermore, SMED-EFCAB1-deficient planaria displayed ciliary dysmotility. Conclusion: Our results revealed that pathogenic variants in CLXN cause PCD with defects in the assembly of distal ODAs in the respiratory cilia. CLXN should be referred to as ODA-docking complex–associated protein ODAD5

General_information_readme

general description of project and description of manuscript file

Schuermann_Figure_1

Schuermann_Figure_1 - contains Prism-File "angles between nearest neighbours" to quantify patterns of immunoggold-labelling of cell-surface associated large Hedgehog (Hh) morphogen clusters. Contains Folder "IEM of Shh on Bosc23" showing original IEM data. Contains PyMol file "apbs_olig_hh.pse" showing a modelled fly Hh pentamer using crystal lattice interactions observed in the human Sonic Hh crystal structure pdb3m1n as a template

Schuermann_Figure_2

Schuermann_Figure_2 - contains Folders showing Drosophila wing patterning phenotypes as a consequence of Hh or dominant-negative HhC85S over expression under the control of engrailed-Gal4 or patched-Gal4 (described below). Contains Prizm files to quantify the intervein regions between longitudinal wing veins L3-L4 and L2-L3. The obtained ratio serves as a readout for Hh patterning activity. CD8-GFP serves as a control (wild-type patterning)