Biology subjects

Kreysing, J. P.

Publications and source records attributed to Kreysing, J. P..

6 recordsLinked to original sources

bioRxiv2024

Passage of the HIV capsid cracks the nuclear pore

Upon infection, human immunodeficiency virus (HIV-1) releases its cone-shaped capsid into the cytoplasm of infected T-cells and macrophages. As its largest known cargo, the capsid enters the nuclear pore complex (NPC), driven by interactions with numerous FG-repeat nucleoporins (FG-Nups). Whether NPCs structurally adapt to capsid passage and whether capsids are modified during passage remains unknown, however. Here, we combined super-resolution and correlative microscopy with cryo electron tomography and molecular simulations to study nuclear entry of HIV-1 capsids in primary human macrophages. We found that cytosolically bound cyclophilin A is stripped off capsids entering the NPC, and the capsid hexagonal lattice remains largely intact inside and beyond the central channel. Strikingly, the NPC scaffold rings frequently crack during capsid passage, consistent with computer simulations indicating the need for NPC widening. The unique cone shape of the HIV-1 capsid facilitates its entry into NPCs and helps to crack their rings.

biophysics↗

bioRxiv2024

Nuclear pores safeguard the integrity of the nuclear envelope

Nuclear pore complexes (NPCs) constitute giant channels within the nuclear envelope that mediate nucleocytoplasmic exchange. NPC diameter is thought to be regulated by nuclear envelope tension, but how such diameter changes are physiologically linked to cell differentiation, where mechanical properties of nuclei are remodeled and nuclear mechanosensing occurs, remains unstudied. Here we used cryo-electron tomography to show that NPCs dilate during differentiation of mouse embryonic stem cells into neural progenitors. In Nup133-deficient cells, which are known to display impaired neural differentiation, NPCs however fail to dilate. By analyzing the architectures of individual NPCs with template matching, we revealed that the Nup133-deficient NPCs are structurally heterogeneous and frequently disintegrate, resulting in the formation of large nuclear envelope openings. We propose that the elasticity of the NPC scaffold mechanically safeguards the nuclear envelope. Our studies provide a molecular explanation for how genetic perturbation of scaffolding components of macromolecular complexes causes tissue-specific phenotypes.

cell biology↗

bioRxiv2024

Nuclear pores as conduits for fluid flow during osmotic stress

Changing environmental conditions necessitate an immediate cellular adaptation to ensure survival. Dictyostelium discoideum, a bacteriovore slime mold present in the soil of most terrestrial ecosystems, is known for its ability to tolerate drastic changes in osmolarity. How the cells cope with the resulting mechanical stress remains understudied. Here we show that D. discoideum has extraordinarily elaborate and resilient nuclear pores that serve as conduits for massive fluid exchange between cytosol and nucleus. We capitalize on the unique properties of D. discoideum cells to quantify flow across the nuclear envelope that is necessitated by changing nuclear size in response to osmotic stress. Based on mathematical concepts adapted from hydrodynamics, we conceptualize this phenomenon as porous flow across nuclear pores. This type of fluid flow is distinct from the canonically characterized modes of nucleocytoplasmic transport, i.e. passive diffusion and active nuclear transport, because of its dependence on pressure. Our insights are relevant in any biological condition that necessitates rapid nuclear size changes, which includes metastasizing cancer cells squeezing through constrictions, migrating cells and differentiating tissues.

cell biology↗

bioRxiv2023

High-confidence 3D template matching for cryo-electron tomography

Cryo-electron tomography (CryoET) resolves individual macromolecules inside living cells. However, the complex composition and high density of cells challenge the faithful identification of features in tomograms. Here, we capitalize on recent advances in electron tomography and demonstrate that 3D template matching (TM) localizes a wide range of structures inside crowded eukaryotic cells with confidence 10 to 100-fold above the noise level. We establish a TM pipeline with systematically tuned parameters for automated, objective and comprehensive feature identification. High-fidelity and high-confidence localizations of nuclear pore complexes, vaults, ribosomes, proteasomes, lipid membranes and microtubules, and individual subunits, demonstrate that TM is generic. We resolve [~]100-kDa proteins, connect the functional states of complexes to their cellular localization, and capture vaults carrying ribosomal cargo in situ. By capturing individual molecular events inside living cells with defined statistical confidence, high-confidence TM greatly speeds up the CryoET workflow and sets the stage for visual proteomics.

molecular biology↗

bioRxiv2023

Thinner is not always better: Optimising cryo lamellae for subtomogram averaging

Cryo-electron tomography (cryo-ET) is a powerful method to elucidate subcellular architecture and to structurally analyse biomolecules in situ by subtomogram averaging (STA). Specimen thickness is a key factor affecting cryo-ET data quality. Cells that are too thick for transmission imaging can be thinned by cryo-focused-ion-beam (cryo-FIB) milling. However, optimal specimen thickness for cryo-ET on lamellae has not been systematically investigated. Furthermore, the ions used to ablate material can cause damage in the lamellae, thereby reducing STA resolution. Here, we systematically benchmark the resolution depending on lamella thickness and the depth of the particles within the sample. Up to ca. 180 nm, lamella thickness does not negatively impact resolution. This shows that there is no need to generate very thin lamellae and thickness can be chosen such that it captures major cellular features. Furthermore, we show that gallium-ion-induced damage extends to depths of up to 30 nm from either lamella surface.

biophysics↗

bioRxiv2023

Translation dynamics in human cells visualized at high-resolution reveal cancer drug action

Ribosomes catalyze protein synthesis by cycling through various functional states. These states have been extensively characterized in vitro, yet their distribution in actively translating human cells remains elusive. Here, we optimized a cryo-electron tomography-based approach and resolved ribosome structures inside human cells with a local resolution of up to 2.5 angstroms. These structures revealed the distribution of functional states of the elongation cycle, a Z tRNA binding site and the dynamics of ribosome expansion segments. In addition, we visualized structures of Homoharringtonine, a drug for chronic myeloid leukemia treatment, within the active site of the ribosome and found that its binding reshaped the landscape of translation. Overall, our work demonstrates that structural dynamics and drug effects can be assessed at near-atomic detail within human cells. One-Sentence SummarySnapshots of ribosome dynamics at near-atomic resolution within native and drug-treated human cells are revealed.

cell biology↗