A5072470655- Cellular Mechanics and Interactions
- Genomics and Chromatin Dynamics
- Advanced Fluorescence Microscopy Techniques
- 3D Printing in Biomedical Research
- RNA Research and Splicing
- Force Microscopy Techniques and Applications
- Single-cell and spatial transcriptomics
- Astronomical Observations and Instrumentation
- Gamma-ray bursts and supernovae
- Photoreceptor and optogenetics research
- Luminescence and Fluorescent Materials
- Bacteriophages and microbial interactions
- Advanced Electron Microscopy Techniques and Applications
- Photosynthetic Processes and Mechanisms
- Cancer therapeutics and mechanisms
- Nanoplatforms for cancer theranostics
- Particle Detector Development and Performance
- RNA and protein synthesis mechanisms
- Advanced Scientific Research Methods
- Computational Drug Discovery Methods
- Chemokine receptors and signaling
- Fungal and yeast genetics research
- Bacterial Genetics and Biotechnology
- Microtubule and mitosis dynamics
- Collagen: Extraction and Characterization
Oncode Institute
2019-2024
The Netherlands Cancer Institute
2019-2024
Leiden University
2014-2020
Harvard University
2019-2020
Boston Children's Hospital
2019-2020
Huygens Institute for History and Culture of the Netherlands
2018-2020
Institute for Atomic and Molecular Physics
2013
Within cells, the spatial compartmentalization of thousands distinct proteins serves a multitude diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) can elucidate protein relationships to global ultrastructure, but has suffered from tradeoffs structure preservation, retention, resolution, field view. We developed platform for three-dimensional cryogenic SR focused ion beam-milled block-face EM across entire vitreously frozen cells. The...
Many transcription factors (TFs) localize in nuclear clusters of locally increased concentrations, but how TF clustering is regulated and it influences gene expression not well understood. Here, we use quantitative microscopy living cells to study the regulation function budding yeast Gal4 its endogenous context. Our results show that forms overlap with GAL loci. Cluster number, density size are different growth conditions by Gal4-inhibitor Gal80 concentration. truncation mutants reveal...
DNA supercoiling has emerged as a major contributor to gene regulation in bacteria, but how impacts transcription dynamics eukaryotes is unclear. Here, using single-molecule dual-color nascent imaging budding yeast, we show that transcriptional bursting of divergent and tandem GAL genes coupled. Temporal coupling neighboring requires rapid release supercoils by topoisomerases. When accumulate, one inhibits at its adjacent genes. Transcription inhibition the results from destabilized binding...
Light upconversion is a very powerful tool in bioimaging as it can eliminate autofluorescence, increase imaging contrast, reduce irradiation damage, and excitation penetration depth vivo. In particular, triplet–triplet annihilation upconverting (TTA‐UC) nanoparticles liposomes offer high efficiency at low power. However, TTA‐UC quenched air by oxygen, which also leads to the formation of toxic singlet oxygen. this work, polyisobutylene‐monomethyl polyethylene glycol block copolymers are...
(Right) Optical micrograph (TRITC–Phalloidin) of a fibroblastoid cell. (Left) Configuration obtained from theoretical model using the adhesion sites experimental data as input.
Light upconversion by triplet-triplet annihilation (TTA-UC) in nanoparticles has received considerable attention for bioimaging and light activation of prodrugs. However, the mechanism TTA-UC is inherently sensitive quenching molecular oxygen. A potential oxygen protection strategy coating with a layer oxygen-impermeable material. In this work, we explore if (organo)silica can fulfill protecting role. Three synthesis routes are described preparing water-dispersible (organo)silica-coated...
The spatiotemporal configuration of genes with distal regulatory elements is believed to be crucial for transcriptional control, but full mechanistic understanding lacking. We combine simultaneous live tracking pairs genomic loci and nascent transcripts molecular dynamics simulations assess the Sox2 gene its enhancer. find that both exhibit more constrained mobility than control sequences due stalled cohesin at CCCTC-binding factor sites. Strikingly, enhancer becomes on firing, homogenizing...
This protocol describes how to image fluorescently tagged proteins, RNA, or DNA inside living Saccharomyces cerevisiae cells at the single-molecule level. Imaging cells, as opposed fixed materials, gives access real-time kinetic information. Although various imaging applications are discussed, we focus on of gene transcription single-RNA To obtain best possible results, it is important that both parameters and yeast culture conditions optimized. Here, aspects described. For complete details...
We investigate the geometrical and mechanical properties of adherent cells characterized by a highly anisotropic actin cytoskeleton. Using combination theoretical work experiments on micropillar arrays, we demonstrate that shape cell edge is accurately described elliptical arcs, whose eccentricity expresses degree anisotropy internal stresses. This results in spatially varying tension along edge, significantly affects traction forces exerted substrate. Our highlights strong interplay between...
Abstract Single-molecule imaging inside living cells has revealed that transcription factors (TFs) bind to DNA transiently, but a long-standing question is how this transient binding related activation. Here, we devised microscopy method simultaneously measure TF at single locus and the effect of these events on transcription. We show yeast Gal4 activates target gene within few seconds, with least ∼ 20% efficiency high initiation rate 1 RNA/s. dissociation decreases rapidly. Moreover,...
Abstract DNA supercoiling has emerged as a major contributor to gene regulation in bacteria. The impact of on transcription dynamics eukaryotes is less clear. Here, using single-molecule dual-color RNA imaging budding yeast, we show that transcriptional bursting the divergent and tandem GAL genes coupled. Upon topoisomerase degradation, supercoils buildup from inhibit subsequent at neighboring genes, thereby reducing their simultaneous bursting. inhibited more by negative than positive...
Abstract Living cells function through the spatial compartmentalization of thousands distinct proteins serving a multitude diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) has emerged as pathway to directly view nanoscale protein relationships underlying global ultrastructure, but traditionally suffered from tradeoffs structure preservation, retention, resolution, field view. We developed platform for three-dimensional correlative...
SUMMARY Many transcription factors (TFs) localize in nuclear clusters of locally increased concentrations, but how TF clustering is regulated and it influences gene expression not well understood. Here, we use quantitative microscopy living cells to study the regulation function budding yeast Gal4 its endogenous context. Our results show that cluster formation facilitated by, does completely depend on DNA binding intrinsically disordered regions. properties are by Gal4-inhibitor Gal80...
We investigate the mechanical interplay between spatial organization of actin cytoskeleton and shape animal cells adhering on micropillar arrays. Using a combination analytical work, computer simulations in vitro experiments, we demonstrate that orientation stress fibers strongly influences geometry cell edge. In presence uniformly aligned cytoskeleton, edge can be well approximated by elliptical arcs, whose eccentricity reflects degree anisotropy cell's internal stresses. Upon modeling as...