Biology subjects

Raedler, J. O.

Publications and source records attributed to Raedler, J. O..

12 recordsLinked to original sources

bioRxiv2026

siRNA/mRNA payload of lipid nanoparticles quantified using fluorescence microscopy and correlation spectroscopy

Lipid nanoparticles (LNPs) have emerged as the most promising vectors to deliver nucleic acids to mammalian cells. Advanced delivery strategies involving multiple nucleic acid species require a reliable determination of RNA payload and LNP size. Here, we use total internal reflection fluorescence (TIRF) microscopy and fluorescence correlation spectroscopy (FCS) to determine mRNA payload and siRNA/mRNA codelivered payload. Particularly, FCS proves to be a reliable method for payload determination, independently of fluorophore properties that can be altered during encapsulation. By varying the PEG-lipid fraction relative to the reference ONPATTRO formulation, we find that mRNA payload of enhanced green fluorescent protein (eGFP) coding mRNA increases from [~]10 with LNP size according to a power law with an exponent 1.3. Cryogenic electron microscopy (Cryo-EM) reveals that in this case blebs occur more frequently in small LNPs. For siRNA/mRNA coencapsulation the siRNA fraction within LNPs exceeds that in the initial RNA mixture, suggesting that coencapsulation is competitive and constrained by nucleic acid diffusion. Together, our findings highlight the importance of particle size and encapsulation kinetics in controlling the loading and composition of RNA payloads in LNPs.

biophysics↗

bioRxiv2024

Less is more: Slow-codon windows enhance eGFP mRNA resilience against RNA interference

Extensive efforts have been devoted to enhance the translation efficiency of mRNA delivered to mammalian cells via codon optimization. However, the impact of codon choice on mRNA stability remains underexplored. In this study, we investigated the influence of codon usage on mRNA degradation kinetics in cultured human cell lines using live-cell imaging on single-cell arrays (LISCA). By measuring mRNA lifetimes at the single-cell level for synthetic mRNA constructs, we confirmed that mRNAs containing slowly translated codon windows have shorter lifetimes. Unexpectedly, these mRNAs did not exhibit decreased stability in the presence of siRNA compared to the unmutated sequence, suggesting an interference of different concurrent degradation mechanisms. We employed stochastic simulations to predict ribosome density along the open reading frame, revealing that the ribosome densities correlated with mRNA stability in a cell-type- and codon-position-specific manner. In summary, our results suggest that the effect of codon choice and its influence on mRNA lifetime is context dependent with respect to cell type, codon position, and RNA interference.

biophysics↗

bioRxiv2023

Unravelling the metastasis-preventing effect of miR-200c in vitro and in vivo

Advanced breast cancer as well as insufficient treatment can lead to the dissemination of malignant cells from the primary tumor to distant organs. Recent research has shown that miR-200c can hamper certain steps of the invasion-metastasis cascade. However, it is still unclear, whether sole miR-200c expression is sufficient to prevent breast cancer cells from metastasis formation. Hence, we performed a xenograft mouse experiment with inducible miR-200c expression in MDA-MB 231 cells. The ex vivo analysis of metastatic sites in a multitude of organs including lung, liver, brain, and spleen has revealed a dramatically reduced metastatic burden of mice with miR-200c expressing tumors. A fundamental prerequisite for metastasis formation is the motility of cancer cells and, therefore, their migration. Consequently, we analyzed the effect of miR-200c on collective and single cell migration in vitro, utilizing MDA-MB 231 and MCF7 cell systems with genetically modified miR-200c expression. Analysis of collective cell migration has resulted in confluence dependent motility of cells with altered miR-200c expression. Additionally, scratch assays have shown enhanced predisposition of miR-200c negative cells to leave cell clusters. The in-between stage of collective and single cell migration was validated using transwell assays, which have displayed reduced migration of miR-200c positive cells. Finally, to measure migration on single cell level, a novel assay on dumbbell shaped micropatterns was performed, which revealed that miR-200c critically determines confined cell motility. All of these results demonstrate that exclusive expression of miR-200c impedes metastasis formation in vivo and migration in vitro and highlight miR-200c as metastatic suppressor in breast cancer.

cancer biology↗

bioRxiv2023

Nuclear deformation and dynamics of migrating cells in 3D confinement reveal adaptation of pulling and pushing forces

Eukaryotic cells show an astounding ability to migrate through pores and constrictions smaller than their nuclear diameter. However, the forces engaged in nuclear deformation and their effect on confined cell dynamics remain unclear. Here, we study the mechanics and dynamics of nuclei of mesenchymal cancer cells as they spontaneously and repeatedly transition through 3D compliant hydrogel channels. We find a biphasic dependence of migration speed and transition frequency on channel width, revealing maximal transition rates at widths comparable to the nuclear diameter. Using confocal imaging and hydrogel bead displacement, we determine the nuclear deformation and corresponding forces during spontaneous confined migration. We find the nucleus to reversibly deform with an elastic modulus not adapting to the confinement. Instead, with decreasing channel width, the nuclear shape during transmigration changes biphasically concomitant with the transitioning dynamics. The nucleus exhibits a prolate form along the migration direction in wide channels and a more compressed oblate shape in narrow channels. We propose a physical model for confined cell migration that explains the observed nuclear shapes and slowing down in terms of the cytoskeletal force-generation adapting from a pulling-to a pushing-dominated mechanism with increasing nuclear confinement.

biophysics↗

bioRxiv2023

Combining molecular dynamics simulations and X-ray scattering techniques for the accurate treatment of protonation degree and packing of ionizable lipids in monolayers

The pH-dependent change in protonation of ionizable lipids is crucial for the success of lipid-based nanoparticles as mRNA delivery systems. Despite their widespread application in vaccines, the structural changes upon acidification are not well understood. Molecular dynamics simulations support structure prediction but require an a-priori knowledge of the lipid packing and protonation degree. The presetting of the protonation degree is a challenging task in the case of ionizable lipids since it depends on pH and on the local lipid environment and often lacks experimental validation. Here, we introduce a methodology of combining all-atom molecular dynamics simulations with experimental total-reflection X-ray fluorescence and scattering measurements for the ionizable lipid Dlin-MC3-DMA (MC3) in POPC monolayers. This joint approach allows us to simultaneously determine the lipid packing and the protonation degree of MC3. The consistent parameterization is expected to be useful for further predictive modeling of the action of MC3-based lipid nanoparticles.

biophysics↗

bioRxiv2022

Self-Assembly of Tunable Intrinsically DisorderedPeptide Amphiphiles

Intrinsically disordered peptide amphiphiles (IDPAs) present a novel class of synthetic conjugates that consist of short hydrophilic polypeptides anchored to hydrocarbon chains. These hybrid polymer-lipid block constructs spontaneously self-assemble into dispersed nanoscopic aggregates or ordered mesophases in aqueous solution due to hydrophobic interactions. Yet, the possible sequence variations and their influence on the self-assembly structures is vast and have hardly been explored. Here, we measure the nanoscopic self-assembled structures of four IDPA systems that differ by their amino acid sequence. We show that permutations in the charge pattern along the sequence remarkably alter the headgroup conformation and consequently alters the pH-triggered phase transitions between spherical, cylindrical micelles and hexagonal condensed phases. We demonstrate that even a single amino acid mutation is sufficient to tune structural transitions in the condensed IDPA mesophases, while peptide conformations remain unfolded and disordered. Furthermore, alteration of the peptide sequence can render IDPAs to become susceptible to enzymatic cleavage and induces enzymatically activated phase transitions. These results hold great potential for embedding multiple functionalities into lipid nanoparticle delivery systems by incorporating IDPAs with desired properties.

biophysics↗

bioRxiv2022

3D printed SAXS chamber for controlled in-situ dialysis and optical characterization

3D printing changes the scope of how samples can be mounted for small angle X-ray scattering (SAXS). In this paper we present a 3D printed X-ray chamber, which allows for in-situ exchange of buffer and in-situ optical transmission spectroscopy. The chamber is made of cyclic olefin copolymers (COC), including COC X-ray windows providing ultra low SAXS background. The design integrates a membrane insert for in-situ dialysis of the 100 {micro}l sample volume against a reservoir, which enables measurements of the same sample under multiple conditions using an in-house X-ray setup equipped with a 17.4 keV molybdenum source. We demonstrate the designs capabilities by measuring reversible structural changes in lipid and polymer systems as a function of salt concentration and pH. In the same chambers optical light transmission spectroscopy was carried out measuring optical turbidity of the mesophases and local pH values using pH-responsive dyes. Microfluidic exchange and optical spectroscopy combined with in-situ X-ray scattering enables vast applications for the study of responsive materials.

biophysics↗

bioRxiv2020

Nanoscale Organization of FasL on DNA Origami as a Versatile Platform to Tune Apoptosis Signaling in Cells

Nanoscale probes with fine-tunable properties are of key interest in cell biology and nanomedicine to elucidate and eventually control signaling processes in cells. A critical, still challenging issue is to conjugate these probes with molecules in a number- and spatially-controlled manner. Here, DNA origami-based nanoagents as nanometer precise scaffolds presenting Fas ligand (FasL) in well-defined arrangements to cells are reported. These nanoagents activate receptor molecules in the plasma membrane initiating apoptosis signaling in cells. Signaling for apoptosis depends sensitively on FasL geometry: fastest time-to-death kinetics are obtained for FasL nanoagents representing predicted structure models of hexagonal receptor ordering with 10 nm inter-molecular spacing. Slower kinetics are observed for one to two FasL on DNA origami or FasL coupled with higher flexibility. Nanoagents with FasL arranged in hexagons with small (5 nm) and large (30 nm) spacing impede signal transduction. Moreover, for predicted hexagonal FasL nanoagents, signaling efficiency is faster and 100× higher compared to naturally occurring soluble FasL. Incubation of the FasL-origami nanoagent in solution exhibited an EC50 value of only 90 pM. These studies present DNA origami as versatile signaling platforms to probe the significance of molecular number and nanoscale ordering for signal initiation in cells.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2019

Area and Geometry Dependence of Cell Migration in Asymmetric Two-State Micropatterns

Micro-structured surfaces provide a unique framework to probe cell migration and cytoskeletal dynamics in a standardized manner. Here, we report on the steady-state occupancy probability of cells in asymmetric two-state microstructures that consist of two fibronectin-coated adhesion sites connected by a thin guidance cue. In these dumbbell-like structures, cells transition between the two sites in a repeated and stochastic manner and average dwell times in the respective microenvironments are determined from the cell trajectories. We study the dynamics of human breast carcinoma cells (MDA-MB-231) in these microstructures as a function of area, shape and orientation of the adhesion sites. On square adhesive sites with different areas, we find that the occupancy probability ratio is directly proportional to the ratio of corresponding adhesion site areas. Sites of equal area but different shape lead to equal occupancy, if shapes are isotropic, e.g. squared or circular. In contrast, an asymmetry in the occupancy is induced by anisotropic shapes like rhombi, triangles or rectangles that enable motion in the direction perpendicular to the transition axis. Analysis of the 2D motion of cells between two rectangles with orthogonal orientation suggests that cellular transition rates depend on the cell polarisation induced by anisotropic micropatterns. Taken together, our results illustrate how two-state-micropatterns provide a dynamic migration assay with distinct dwell times and relative cell occupancy as readouts, which may potentially be useful to probe cell-microenvironment interactions.

biophysics↗

bioRxiv2019

Quasi-periodic migration of single cells on short microlanes

Cell migration on microlanes represents a suitable and simple platform for the exploration of the molecular mechanisms underlying cell cytoskeleton dynamics. Here, we report on the quasi-periodic movement of cells confined in stripe-shaped microlanes. We observe persistent polarized cell shapes and directed pole-to-pole motion within the microlanes. Cells depolarize at one end of a given microlane, followed by delayed repolarization towards the opposite end. We analyze cell motility via the spatial velocity distribution, the velocity frequency spectrum and the reversal time as a measure for depolarization and spontaneous repolarization of cells at the microlane ends. The frequent encounters of a boundary in the stripe geometry provides a robust framework for quantitative investigations of the cytoskeleton protrusion and repolarization dynamics. In a first advance to rigorously test physical models of cell migration, we find that the statistics of the cell migration is recapitulated by a Cellular Potts model with a minimal description of cytoskeleton dynamics. Using LifeAct-GFP transfected cells and microlanes with differently shaped ends, we show that the local deformation of the leading cell edge in response to the tip geometry can locally either amplify or quench actin polymerization, while leaving the average reversal times unaffected.

biophysics↗

bioRxiv2019

Disentangling the Behavioural Variability of Confined Cell Migration

Cell-to-cell variability is inherent to numerous biological processes, including cell migration. Quantifying and characterizing the variability of migrating cells is challenging, as it requires monitoring many cells for long time windows under identical conditions. Here, we observe the migration of single human breast cancer cells (MDA-MB-231) in confining two-state micropatterns. To describe the stochastic dynamics of this confined migration, we employ a dynamical systems approach. We identify statistics to measure the behavioural variance of the migration, which significantly exceed those predicted by a population-averaged stochastic model. This additional variance can be explained by the combination of an aging process and population heterogeneity. To quantify population heterogeneity, we decompose the cells into subpopulations of slow and fast cells, revealing the presence of distinct classes of dynamical systems describing the migration, ranging from bistable to limit cycle behaviour. Our findings highlight the breadth of migration behaviours present in cell populations.

biophysics↗

bioRxiv2019

Correlation of mRNA delivery timing and protein expression in lipid-based transfection

Non-viral gene delivery is constrained by the dwell time that most synthetic nucleic acid nanocarriers spend inside endosomal compartments. In order to overcome this endosomal-release bottleneck, methods are required that measure nanocarrier uptake kinetics and transfection efficiency simultaneously. Here, we employ live-cell imaging on single-cell arrays (LISCA) to study the delivery-time distribution of lipid-based mRNA complexes under varied serum conditions. By fitting a translation-maturation model to hundreds of individual eGFP reporter fluorescence time courses, the protein expression onset times and the expression rates after transfection are determined. Using this approach, we find that delivery timing and protein expression rates are not intrinsically correlated at the single-cell level, even though population-averaged values of both parameters conjointly change as a function of increasing external serum protein fraction. Lipofectamine mediated delivery showed decreased transfection efficiency and longer delivery times with increasing serum protein concentration. This is in contrast to ionizable lipid nanoparticles (LNPs) mediated transfer, which showed increased efficiency and faster uptake in the presence of serum. In conclusion, the interdependences of single-cell expression rates and onset timing provide additional clues on uptake and release mechanisms, which are useful for improving nucleic acid delivery.

systems biology↗