Biology subjects

Geertsema, H.

Publications and source records attributed to Geertsema, H..

5 recordsLinked to original sources

bioRxiv2026

Mechanically induced pre-mitotic nuclear deformations promote nuclear rupture through lamin B1 depletion

Disassembly of the nuclear lamina is essential for successful mitosis in eukaryotic cells, but the mechanism that triggers the lamina breakdown remains unclear. Using 3D fluorescence microscopy imaging combined with 3D curvature analysis, we found that microtubule-dependent invaginations trigger lamina remodelling and lamin B1 network disruption. Monte Carlo simulations provide mechanistic insight into how those invaginations induce a mechanical discontinuity between isotropic expansion and anisotropic shrinking forces at the rim of the nucleus, which correlated with lamin B1 depletion sites and the location of cytoplasmic protein influx within the nucleus. Our findings suggest that pre-mitotic modifications of the lamina function as a mechanical trigger to facilitate nuclear rupture and are thereby a key driver of mitotic entry, complementing established biochemical pathways.

cell biology↗

bioRxiv2026

Expression levels and dimer abundance of lamin A/C direct nuclear shape integrity in malignant cancer cells

Abnormalities in nuclear morphology are an important diagnostic tool to determine malignancy in cancer cells and are characterised by nuclear blebbing and deformations. Nuclear shape is mostly maintained by a dense protein meshwork of lamins, consisting of 4 lamin subtypes, of which the individual contribution to nuclear shape maintenance remains elusive. In this study, we decouple the roles of lamin A, C, and B1 across cancer cell lines with varying malignant potential (HeLa, HT1080, and MDA-MB-231). Using single-cell correlation analysis, we directly link reduced lamin A/C, and not lamin B1, expression levels to nuclear deformability. We found that the nuclear shape of the more malignant MDA-MB-231 cells is approximately 4-fold more sensitive to lamin A/C than HeLa and HT1080 cells. Biochemical analyses reveal cell-type-specific variation in lamin A/C interactions and homodimer formation that correlates with nuclear shape deformations. In contrast to healthy mouse embryonic fibroblast cells, malignant cells exhibit reduced dimerisation, which correlates with nuclear deformability. As such, our study links, for the first time, the lamin A/C dimerisation state to nuclear abnormalities, thereby providing new avenues for investigating cancer progression.

cell biology↗

bioRxiv2024

High-throughput single molecule microscopy with adaptable spatial resolution using exchangeable oligonucleotide labels

Super-resolution microscopy facilitates the visualization of cellular structures at resolutions approaching the molecular level. Especially, super-resolution techniques based on the localization of single-molecules have relatively modest instrument requirements and are thus good candidates for adoption in bioimaging. However, their low throughput nature hampers their applicability in biomolecular research and screening. Here, we propose a workflow for more efficient data collection, starting with scanning of large areas using fast fluctuation based imaging, followed by single-molecule localization microscopy of selected cells. To achieve this workflow, we exploit the versatility of DNA oligo hybridization kinetics with DNA-PAINT probes to tailor the fluorescent blinking towards high-throughput and high-resolution imaging. Additionally, we employ super-resolution optical fluctuation imaging (SOFI) to analyze statistical fluctuations in the DNA-PAINT binding kinetics, thereby tolerating much denser blinking and facilitating accelerated imaging speeds. Thus, we demonstrate 30-300-fold faster imaging of different cellular structures compared to conventional DNA-PAINT imaging, albeit at a lower resolution. Notably, by tuning image medium and data processing though, we can flexibly switch between high-throughput SOFI (scanning a FOV of 0.65mm x 0.52mm within 4 minutes of total acquisition time) and super-resolution DNA-PAINT microscopy and thereby demonstrate that combining DNA-PAINT and SOFI enables to adapt image resolution and acquisition time based to the imaging needs. We envision this approach to be especially powerful when combined with multiplexing and 3D imaging.

biophysics↗

bioRxiv2020

Left-handed DNA-PAINT for improved superresolution imaging in the nucleus.

DNA point accumulation in nanoscale topography (DNA-PAINT) advances super-resolution microscopy with superior resolution and multiplexing capabilities. However, cellular DNA may interfere with this single-molecule localization technique based on DNA-DNA hybridization. Here, we introduce left-handed DNA (L-DNA) oligomers that do not hybridize to naturally present R-DNA and demonstrate that L-DNA PAINT has the same specificity and multiplexing capability as R-DNA PAINT, but greatly improves specific visualization of nuclear target molecules.

biophysics↗

bioRxiv2018

Rapid and efficient C-terminal labeling of nanobodies for DNA-PAINT

Single molecule localization-based approaches to superresolution microscopy (SMLM) create images that resolve features smaller than the diffraction limit of light by rendering them from the sequentially measured positions of thousands of individual molecules. New SMLM approaches based on the transient binding of very bright dyes via DNA-DNA interaction (DNA-PAINT) allow the resolution of dyes only a few nanometers apart in vitro. This imaging of cellular structures requires the specific association of dyes to their targets, which results in an additional \"linkage error\". This error can be minimized by using extremely small, single-domain antibody-based binders such as nanobodies, but the DNA-oligomers used in DNA-PAINT are of significant size in comparison to nanobodies and may interfere with binding. We have here developed an optimized procedure based on enzymatic labeling and click-chemistry for the coupling of DNA oligomers to the nanobody C-terminus, which is located on the opposite side of the epitope-binding domain. Our approach allows for straightforward labeling, purification and DNA-PAINT imaging. We performed high efficiency labeling of two different nanobodies and show dual color multiplexed SMLM to demonstrate the general applicability of our labeling scheme.

biophysics↗