Biology subjects

Szwedziak, P.

Publications and source records attributed to Szwedziak, P..

6 recordsLinked to original sources

bioRxiv2026

Automated LN2 refill device for uninterrupted cryoFIB-SEM operations.

Due to recent technological advances, in situ structural cell biology is becoming a high throughput microscopy technique as all the steps of the workflow, from sample preparation to data analysis, are executed faster, more reliable and more reproducible. Sample thinning by cryoFIB-SEM is an essential tool in preparing electron transparent lamellae of biological specimens suitable for further characterization by cryoET. Modern cryoFIB-SEM instruments can be operated remotely and are capable of automated and unsupervised lamellae preparation. To take full advantage of these developments they need a constant supply of LN2 to maintain cryogenic conditions inside the microscope chamber. Here, we introduce a custom automated LN2 refill system that is compatible with gas cooled cryostages, supports long-term cryoFIB-SEM operations and liberates the user from highly repetitive and manual work. We believe this solution can be utilized with other cryoSEM or cryoFIB-SEM devices requiring N2 gas-flow cooling.

biophysics↗

bioRxiv2024

In situ structural analysis of mammalian cells using a 200 kV electron cryomicroscope: implications for research infrastructure.

BackgroundElectron cryotomography is a powerful imaging technique allowing for studying functional cellular modules in their native environment with macromolecular resolution. However, it requires access to complex and expensive instrumentation, typically a 300 kV electron cryomicroscope equipped with an energy filter. Simpler and cheaper 200 and 100 kV instruments have been successfully used for single particle cryoEM analyses, which has helped to democratize the technique and broaden access. It has not been systematically studied if 200 kV electron cryomicroscopes can deliver meaningful and interpretable data with respect to electron cryotomography applications. MethodsHere, we set out to investigate if a 200 kV electron cryomicroscope without an energy filter can be utilized for in situ structural studies of mammalian cells by electron cryotomography of thin cell edges followed by extensive image analysis including segmentations, subtomogram averaging and molecular sociology studies of lipid droplets. ResultsWe demonstrate that the resulting tomograms of thin edges of U2OS cells are of sufficient quality to annotate the contents of the cell and observe spatial inter-relationships among macromolecules. In particular, we undertook a molecular sociology analysis of lipid droplets and addressed their subcellular distribution and interactions with other organelles. Additionally, we performed subtomogram averaging of purified 70S ribosomes that resulted in [~]15 [A] resolution 3D reconstruction. Finally, we examined geographical distribution and scientific output of the two most common electron cryomicroscopy platforms and deduced that 200 kV instruments are heavily underutilized with respect to electron cryotomography applications. DiscussionThis study demonstrates that 200 kV electron cryomicroscopes can be utilized for structural cell biology studies by electron cryotomography. Given the favorable ratio of their versatility versus costs we foresee that 200 kV electron cryomicroscopes will become workhorses of local electron cryomicroscopy facilities.

biophysics↗

bioRxiv2024

Structural architecture of amyloid-β oligomers, curvilinear protofibrils and annular assemblies, imaged by cryo-EM and cryo-ET

Small prefibrillar structures of the amyloid-{beta} peptide (A{beta}) are believed to be central to cytotoxicity in Alzheimers disease, the most common form of dementia. A snapshot of these prefibrillar assemblies has therefore been characterized using a combination of cryo-ET and cryo-EM single particle analysis. This has facilitated an understanding of the relationship between, oligomers, curvilinear protofibrils and annular assemblies. A highly consistent diameter for all curvilinear protofibrils and oligomers of 28 [A], indicates that these assemblies are simply structural extensions from the smaller oligomers. Furthermore, their basic crosssection suggests mature amyloid fibrils might be initiated by the lateral binding of two curvilinear protofibrils. Ab-initio 3D reconstruction also reveals ring-shaped annular assemblies. These possess a central internal channel, ca. 14 [A] in diameter and 54 [A] long, which is capable of traversing lipid membranes. Large conductance recorded using patch-clamp electrophysiology, match the internal diameter of the A{beta} annular architecture.

biophysics↗

bioRxiv2020

Cytoplasmic polyadenylation by TENT5A is required for proper bone formation

Osteoblasts orchestrate bone formation by secreting dense, highly cross-linked type I collagen and other proteins involved in osteogenesis. Mutations in Col11, Col12, or collagen biogenesis factors lead to the human genetic disease, osteogenesis imperfecta (OI). Herein, we show that the TENT5A gene, whose mutation is responsible for poorly characterized type XVIII OI, encodes an active cytoplasmic poly(A) polymerase regulating osteogenesis. TENT5A is induced during osteoblast differentiation and TENT5A KO osteoblasts are defective in mineralization. The TENT5A KO mouse recapitulates OI disease symptoms such as bone fragility and hypomineralization. Direct RNA sequencing revealed that TENT5A polyadenylates and increases expression of Col11 and Col12 RNAs, as well as those of other genes mutated in OI, resulting in lower production and improper folding of collagen chains. Thus, we have identified the specific pathomechanism of XVIII OI and report for the first time a biologically relevant post-transcriptional regulator of collagen production. We further postulate that TENT5A, possibly together with its paralogue TENT5C, is responsible for the wave of cytoplasmic polyadenylation of mRNAs encoding secreted proteins occurring during bone mineralization.

genetics↗

bioRxiv2020

3D-Visualization of Amyloid-β Oligomer and Fibril Interactions with Lipid Membranes by Cryo-Electron Tomography

Amyloid-{beta} (A{beta}) monomers assemble into mature fibrils via a range of metastable oligomeric and protofibrillar intermediates. These A{beta} assemblies have been shown to bind to lipid bilayers. This can disrupt membrane integrity and cause a loss of cellular homeostasis, that triggers a cascade of events leading to Alzheimers disease. However, molecular mechanisms of A{beta} cytotoxicity and how the different assembly forms interact with the membrane remain enigmatic. Here we use cryo-electron tomography (cryoET) to obtain three-dimensional nano-scale images of various A{beta} assembly types and their interaction with liposomes. A{beta} oligomers bind extensively to the lipid vesicles, inserting and carpeting the upper-leaflet of the bilayer. Furthermore, curvilinear protofibrils also insert into the bilayer, orthogonally to the membrane surface. A{beta} oligomers concentrate at the interface of vesicles and form a network of A{beta}-linked liposomes. While crucially, monomeric and fibrillar A{beta} have relatively little impact on the membrane. Changes to lipid membrane composition highlights a significant role for GM1-ganglioside in promoting A{beta}-membrane interactions. The different effects of A{beta} assembly forms observed align with the highlighted cytotoxicity reported for A{beta} oligomers. The wide-scale incorporation of A{beta} oligomers and curvilinear protofibrils into the lipid bilayer suggests a mechanism by which membrane integrity is lost.

biophysics↗

bioRxiv2017

MreB Filaments Create Rod Shape By Aligning Along Principal Membrane Curvature.

MreB is essential for rod shape in many bacteria. Membrane-associated MreB filaments move around the rod circumference, helping to insert cell wall in the radial direction to reinforce rod shape. To understand how oriented MreB motion arises, we altered the shape of Bacillus subtilis. MreB motion is isotropic in round cells, and orientation is restored when rod shape is externally imposed. Stationary filaments orient within protoplasts, and purified MreB tubulates liposomes in vitro, orienting within tubes. Together, this demonstrates MreB orients along the greatest principal membrane curvature, a conclusion supported with biophysical modeling. We observed that spherical cells regenerate into rods in a local, self-reinforcing manner: rapidly propagating rods emerge from small bulges, exhibiting oriented MreB motion and increased glycan crosslinking. We propose that the coupling of MreB filament alignment to shape-reinforcing peptidoglycan synthesis creates a locally-acting, self-organizing mechanism allowing the rapid establishment and stable maintenance of emergent rod shape.

microbiology↗