Biology subjects

Schnorrenberg, S.

Publications and source records attributed to Schnorrenberg, S..

6 recordsLinked to original sources

bioRxiv2025

Hydrophobic interactions of FG-nucleoporins are required for dilating nuclear membrane pores into selective transport channels after mitosis

Nuclear envelope (NE) reformation after mitosis is essential for daughter cell viability and requires tightly coordinated nuclear pore complex (NPC) assembly and nuclear membrane reformation. To reveal how these processes are mechanistically linked, we combined acute molecule perturbations in live cells with correlative 3D electron tomography or MINFLUX super-resolution microscopy. We show that degrading Nup62 during mitosis arrests NPC assembly at an intermediate step with smaller membrane pores and removes the whole central transport channel. Molecular dynamics simulations predicted that 32 copies of the central channel subcomplex, recruited into the previously unoccupied pore center, can self-associate via hydrophobic interactions to occupy the volume required for full pore size and exert an outward pushing force; indeed, disrupting these interactions during NPC assembly blocked pore dilation. Later in mitotic exit, perturbed cells exhibited impaired nuclear import, smaller nuclei, and looser NE spacing. Acute inhibition of nuclear import recapitulated these NE defects without affecting NPC assembly. Together, our findings reveal a new, two-step molecular mechanism linking NPC assembly and NE reformation. First, hydrophobic FG-nucleoporins dilate the assembling nuclear pore to its full width by forming the central transport channel, which then allows nuclear import-driven nuclear expansion leading to tight, regular NE membrane spacing.

cell biology↗

bioRxiv2025

Clustering within a single-component biomolecular condensate

Biomolecular condensates (BMCs) are assemblies of hundreds to many thousands of macromolecules within cells that are organized without physical barriers. Condensate function is dictated not only by its molecular composition, but also by substructural organization and molecular mobility. One hypothesis for the onset of multiple protein aggregation diseases is that the increased densities of specific proteins within BMCs promotes the formation of solid inclusions. However, deciphering the internal structural and functional properties of BMCs at the nanoscale and identifying the initiating events of inclusion formation requires tools with high spatiotemporal precision. Here we show using single molecule and other microscopy approaches that single component Fused in Sarcoma (FUS) condensates exhibit confinement and contain clusters with higher FUS density even at early timepoints. Upon aging, condensates displayed altered physical properties and reduced monomer mobility, and yet most FUS monomers diffused throughout the condensate within seconds. While an increase in connectivity over time explains reduced mobility, the large fraction of molecules retaining high mobility suggests a sponge-like structure rather than a system-spanning network. These findings indicate that a pseudo-equilibrium between distinct structural connectivities can exist within simple condensates, suggesting the potential for substantial structural and functional complexity of BMCs at the nanoscale.

biophysics↗

bioRxiv2022

Direct observation of motor protein stepping in living cells using MINFLUX

Dynamic measurements of molecular machines can provide invaluable insights into their mechanism, but have been challenging in living cells. Here, we developed live-cell tracking of single fluorophores with nanometer spatial and millisecond temporal resolution in 2D and 3D using the recently introduced super-resolution technique MINFLUX. This allowed us to resolve the precise stepping motion of the motor protein kinesin-1 as it walks on microtubules in living cells. In addition, nanoscopic tracking of motors on microtubule of fixed cells enabled us to resolve their spatial organization with protofilament resolution. Our approach will enable future in vivo studies of motor protein kinetics in complex environments and super-resolution mapping of dense microtubule arrays, and pave the way towards monitoring functional conformational changes of protein machines at high spatiotemporal resolution in living systems.

biophysics↗

bioRxiv2022

CSPP1 stabilizes growing microtubule ends and damaged lattices from the luminal side.

Microtubules are dynamic cytoskeletal polymers, and their organization and stability are tightly regulated by numerous cellular factors. While regulatory proteins controlling formation of interphase microtubule arrays and mitotic spindles have been extensively studied, the biochemical mechanisms responsible for generating stable microtubule cores of centrioles and cilia are poorly understood. Here, we used in vitro reconstitution assays to investigate microtubule-stabilizing properties of CSPP1, a centrosome and cilia-associated protein mutated in the neurodevelopmental ciliopathy Joubert syndrome. We found that CSPP1 preferentially binds to polymerizing microtubule ends that grow slowly or undergo growth perturbations and, in this way, resembles microtubule-stabilizing compounds such as taxanes. Fluorescence microscopy and cryo-electron tomography showed that CSPP1 is deposited in the microtubule lumen and inhibits microtubule growth and shortening through two separate domains. CSPP1 also specifically recognizes and stabilizes damaged microtubule lattices. These data help to explain how CSPP1 regulates elongation and stability of ciliary axonemes and other microtubule-based structures.

cell biology↗

bioRxiv2021

Fluorescence-based 3D targeting of FIB-SEM acquisition of small volumes in large samples

Cells are three dimensional objects. Therefore, 3D electron microscopy is often crucial for correct interpretation of ultrastructural data. Today samples are frequently imaged in 3D at ultrastructural resolution using volume Scanning Electron Microscopy (SEM) methods such as Focused Ion Beam (FIB) SEM and Serial Block face SEM. While these imaging modalities allow for automated data acquisition, precise targeting of (small) volumes of interest within a large sample remains challenging. Here, we provide an easy and reliable approach to target FIB-SEM acquisition of fluorescently labelled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeting based on confocal acquisition of the fluorescence signal in the resin block. Targeted trimming of the block exposes the cell of interest and laser branding of the surface after trimming creates landmarks to precisely position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as a 3D culture of mouse primary mammary gland organoids, tracheal terminal cells in Drosophila melanogaster larvae and ovarian follicular cells in adult Drosophila, discovering ultrastructural details that could not be appreciated before. SummaryRonchi et al. present a workflow to facilitate the precise targeting of three-dimensional (3D) Electron Microscopy acquisitions, guided by fluorescence. This method allows ultrastructural visualization of single cells within a millimeter-range large specimen, based on molecular identity characterized by fluorescence.

cell biology↗

bioRxiv2020

Overcoming efflux of fluorescent probes for actin imaging in living cells

Actin cytoskeleton is crucial for endocytosis, intracellular trafficking, cell shape maintenance and a wide range of other cellular functions. Recently introduced cell-permeable fluorescent actin probes suffer from poor membrane permeability and stain some cell populations inhomogeneously due to the active efflux by the plasma membrane pumps. We addressed this issue by constructing a series of probes which employ modified rhodamine fluorophores. We found that the best performing probes are based on 6-carboxy-carbopyronine scaffold. These probes show preferential binding to F-actin, do not require efflux pumps inhibitors for staining and can be used for 2D and 3D fluorescence nanoscopy at high nanomolar concentrations without significant cytotoxicity. We demonstrate their excellent performance in multiple organisms and cell types: human cell lines, frog erythrocytes, fruit fly tissues and primary neurons.

biochemistry↗