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Biology subjects

Goepfrich, K.

Publications and source records attributed to Goepfrich, K..

6 recordsLinked to original sources

Volume and surface methods for microparticle traction force microscopy: a computational and experimental comparison

It is an essential element of mechanobiology to measure the forces of biological cells. In microparticle traction force microscopy, they are inferred from the deformation of elastic microparticles. Two complementary variants have been introduced before: the volume method, which reconstructs surface stresses from the displacements of fiducial markers embedded inside the particles, and the surface method, which infers stresses directly from the deformation of the particle surface. However, a systematic comparison of the two methods has been lacking. Here, we quantitatively compare both approaches using simulated traction fields representing biologically relevant loading scenarios. We find that the surface method consistently reconstructs traction profiles with substantially lower errors than the volume method, which suffers from displacement tracking and stress calculation at the surface. At high noise levels, however, the performance gap becomes smaller. To compare the performance of the two methods in a realistic experimental setting, we developed DNA-based hydrogel microparticles equipped with both fluorescent surface labels and embedded fluorescent nanoparticles, enabling the direct comparison of the two methods within the same system. Compression experiments produced traction profiles consistent with Hertzian contact mechanics and confirmed the trends observed in the simulations. We also show that despite large experimental deformations and strains (both up to 20 percent), linear elasticity theory should still be valid. While our computational workflow establishes a framework to apply both methods, our experimental workflow establishes DNA microparticles as versatile and biocompatible probes for measuring cellular forces.

biophysics↗

Nonstop nanometric resolution of randomly moving point scatterers with focused light

In established super-resolution fluorescence microscopy, resolving multiple fluorescent molecules at sub-diffraction distances requires the molecules to emit sequentially so that they become discernible from their neighbors one after another. Simultaneous tracking of multiple fluorophores that are only a few nanometers apart is thus conceptually and practically impossible. We have recently shown that probing a sub-diffraction region with an excitation beam featuring an intensity zero, i.e., MINFLUX, super-resolves and tracks closely packed fluorophores without interruption. Here, we provide a conceptual framework for resolving and tracking constantly emitting fluorophores - more generally, point scatterers - that undergo random changes in position. In particular, we show that the detection rates available in fluorescence microscopy are sufficient to track sub-10 nm distance changes within micro-to milliseconds. By using a DNA origami construct with a fixed and a movable fluorophore as a proxy, we prove the concept that thermally driven conformational changes of biomolecules are continuously detectable with visible light. Conformational changes of the DNA nanostructure leading to random jumps in distance of about 10 nm between two labels are registered within about a millisecond. Our work paves the way towards super-resolving complex conformational transitions of individual biomolecules with focused light.

biophysics↗

Fast Single-Cell MALDI Imaging of Low-Mass Metabolites Reveals Cellular Activation Markers

Single-cell MALDI mass spectrometry imaging (MSI) of lipids and metabolites >200 Da has recently come to the forefront of biomedical research and chemical biology, but fast metabolome-preserving methods without paraformaldehyde fixation for analysis of low mass, hydrophilic metabolites (<200 Da) in large cell populations are lacking. Introducing giant unilamellar vesicles (GUVs) as MSI ground truth for cell-sized objects and Monte Carlo reference-based consensus clustering for data-dependent identification of cell subpopulations. The PRISM-MS (PRescan Imaging for Small Molecule - Mass Spectrometry) dual-scan MSI workflow is presented, enabling space-efficient and therefore faster lipid analysis in single GUVs and cells. Beyond lipids, PRISM-MS enables MSI and on-cell MS2-based identification of low-mass metabolites like amino acids or Krebs cycle intermediates involved in stimulus-dependent cell activation. The utility of PRISM-MS is demonstrated through the characterization of complex metabolome changes in lipopolysaccharide (LPS)-stimulated microglial cells and human-induced pluripotent stem cell-derived microglia. Translation of single cell results to endogenous microglia in organotypic hippocampal slice cultures indicates that LPS-activation involves changes of the itaconate-to-taurine ratio and alterations in neuron-to-glia glutamine-glutamate shuttling. The data suggests that PRISM-MS could serve as a standard method in single cell metabolomics, given its capability to characterize larger cell populations and low-mass metabolites.

bioinformatics↗

Minimal-invasive 3D laser printing of microimplants in organismo

Multi-photon 3D laser printing has gathered much attention in recent years as a means of manufacturing biocompatible scaffolds that can modify and guide cellular behavior in vitro. However, in vivo tissue engineering efforts have been limited so far to the implantation of beforehand 3D printed biocompatible scaffolds and in vivo bioprinting of tissue constructs from bioinks containing cells, biomolecules, and printable hydrogel formulations. Thus, a comprehensive 3D laser printing platform for in vivo and in situ manufacturing of microimplants raised from synthetic polymer-based inks is currently missing. Here we present a platform for minimal-invasive manufacturing of microimplants directly in the organism by one-photon photopolymerization and multi-photon 3D laser printing. Employing a commercially available elastomeric ink giving rise to biocompatible synthetic polymer-based microimplants, we demonstrate first applicational examples of biological responses to in situ printed microimplants in the teleost fish Oryzias latipes and in embryos of the fruit fly Drosophila melanogaster. This provides a framework for future studies addressing the suitability of inks for in vivo 3D manufacturing. Our platform bears great potential for the direct engineering of the intricate microarchitectures in a variety of tissues in model organisms and beyond.

bioengineering↗

Amphiphiles formed from synthetic DNA-nanomotifs mimic the dispersal of transcriptional clusters in the cell nucleus

Stem cells exhibit prominent clusters controlling the transcription of genes into RNA. These clusters form by a phase-separation mechanism, and their size and shape are controlled via an amphiphilic effect of transcribed genes. Here, we construct amphiphile-nanomotifs purely from DNA, and achieve similar size and shape control for phase-separated droplets formed from fully synthetic, self-interacting DNA-nanomotifs. Low amphiphile concentrations induce rounding of droplets, followed by splitting and, ultimately, full dispersal at higher concentrations. Super-resolution microscopy data obtained from zebrafish embryo stem cells reveal a comparable transition for transcriptional clusters with increasing transcription levels. Brownian dynamics and lattice simulations further confirm that addition of amphiphilic particles is sufficient to explain the observed changes in shape and size. Our work reproduces key aspects of the complex organization of transcription in biological cells using relatively simple, DNA sequence-programmable nanostructures, opening novel ways to control mesoscopic organization of synthetic nanomaterials. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/525851v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1a8cc4borg.highwire.dtl.DTLVardef@fc6651org.highwire.dtl.DTLVardef@a17c74org.highwire.dtl.DTLVardef@1f1cf14_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

Tuning epithelial cell-cell adhesion and collective dynamics with functional DNA-E-cadherin hybrid linkers

The binding strength between epithelial cells is crucial for tissue integrity, signal transduction and collective cell dynamics. However, there is no experimental approach to precisely modulate cell-cell adhesion strength at the cellular and molecular level. Here, we establish DNA nanotechnology as tool to control cell-cell adhesion of epithelial cells. We designed a DNA-E-cadherin hybrid system consisting of complementary DNA strands covalently bound to a truncated E-cadherin with a modified extracellular domain. DNA sequence design allows to tune the DNA-E-cadherin hybrid molecular binding strength, while retaining its cytosolic interactions and downstream signaling capabilities. The DNA-E-cadherin hybrid facilitates strong and reversible cell-cell adhesion in E-cadherin deficient cells by forming mechanotransducive adherens junctions. We assess the direct influence of cell-cell adhesion strength on intracellular signaling and collective cell dynamics. This highlights the scope of DNA nanotechnology as a precision technology to study and engineer cell collectives.

bioengineering↗