Biology subjects

Jahnke, K.

Publications and source records attributed to Jahnke, K..

12 recordsLinked to original sources

bioRxiv2024

Engineering liposomes with cell membrane proteins to disrupt melanosome transfer

Cells communicate by transporting vesicles and organelles, essential for maintaining cellular homeostasis. However, excessive vesicle transfer can cause several diseases and medical conditions like hyperpigmentation due to an unregulated intercellular transfer of melanosomes. Current treatments often focus on eliminating the compartment contents with drugs but can cause significant side effects. Here, we engineer liposomes with cell membrane proteins to directly disrupt intercellular transport without specialized therapeutics. We demonstrate this approach by reducing melanosome transfer from melanocytes to keratinocytes. To achieve this, we incorporate keratinocyte cell membrane proteins into liposomes using microfluidics, which can enhance uptake by melanocytes while reducing uptake by keratinocytes. We also show that these engineered liposomes reduce melanosome transfer because they attach to the surface of pigment globules, impeding pigment globule uptake by keratinocytes. Our findings provide an effective strategy for reducing melanosome transfer to treat hyperpigmentation and introduce a drug-free approach for regulating cellular communication via extracellular vesicles and organelles.

biophysics↗

bioRxiv2024

Engineering asymmetric nanoscale lipid vesicles for drug delivery

The delivery of therapeutics to cells enables both the treatment and the prevention of diseases. To protect therapeutics from degradation and enable cell-specific targeting, they are often encapsulated into drug delivery vehicles such as lipid nanoparticles, viral vectors or lipid vesicles. These delivery vehicles have been extremely successful in delivering small molecules, nucleic acids or proteins. However, there is no universal drug delivery vehicle that can deliver therapeutics irrespective of the choice of cargo. Here, we present a method to engineer lipid vesicles with asymmetric leaflets and show that they can deliver mRNA and proteins to cells. We also find that the leaflet asymmetry can increase the lipid vesicle uptake by cells. When we load asymmetric vesicles with mRNA, we observe a 5-fold increase in the transfection efficiency indicative of an improved uptake and release by asymmetric vesicles. Moreover, our findings extend beyond mRNA cargos by showcasing the effectiveness of asymmetric vesicles in delivering a wide range of proteins to cells, including the promising CRISPR/Cas9 gene editing system. Our method and findings expand the parameter space for engineering drug delivery vehicles and demonstrate the pivotal role of leaflet asymmetry in determining the performance of drug delivery vehicles. Consequently, our work leads to many applications, including the formation of more efficient universal drug carriers and the delivery of gene-editing proteins to cells.

biophysics↗

bioRxiv2023

UPP affects chloroplast development by interfering with chloroplast proteostasis

Arabidopsis uracil phosphoribosyltransferase (UPP) is an essential enzyme which appears to have a previously unknown, moonlighting activity. Our analysis of UPP amiRNA mutants has confirmed that this vital function is crucial for chloroplast development and growth. Interestingly, this function appears to be unrelated to nucleotide homeostasis since nucleotide levels were not altered in the studied mutants. Transcriptomics and proteomic analysis suggest that UPP plays a role in chloroplast proteostasis, especially under high light (HL). Immunoblots of mature plants and a de-etiolation experiment with young seedlings revealed PetC, the iron-sulfur protein of the cytochrome b6f complex, as a putative UPP target. In addition, UPP and PetC were identified in a high molecular weight complex. Consistently, we show that PetC is massively reduced in UPP knock-down plants. The block observed in photosynthetic electron transport, as evidenced by reduced high light-induced non-photochemical quenching (NPQ) but increased unregulated energy dissipation (NO), might be therefore a consequence of reduced PetC. After HL treatment, UPP amiRNA mutants showed impaired photosynthesis and reduced carbohydrate contents, resulting in an inability to induce flavonoid biosynthesis. In addition, the levels of the osmoprotectants raffinose, proline and fumarate were found to be reduced. Proteases, including thylakoid filamentation temperature-sensitive 1, 5 (FtsH), caseinolytic protease proteolytic subunit 1 (ClpP1), and processing peptidases, as well as components of the chloroplast protein import machinery, were up-regulated. In sum, our work suggests that UPP assists in stabilizing and protection of PetC during the assembly of the Fe-S cluster and targeting to the thylakoid.

molecular biology↗

bioRxiv2023

Self-assembly and contraction of micron-scale DNA rings

Contractile rings formed from cytoskeletal filaments mediate the division of cells. The reverse-engineering of synthetic contractile rings could shed light on fundamental physical principles of the ring self-assembly and dynamics independent of the natural protein-based compounds. Here, we engineer DNA nanotubes and crosslink them with a synthetic peptide-functionalized star-PEG construct. The star-PEG construct induces the formation of DNA nanotube bundles composed of several tens of individual DNA nanotubes. Importantly, the DNA nanotube bundles curve into closed micron-scale DNA rings in a high-yield one-pot self-assembly process resulting in several thousand rings per microliter. The crosslinked DNA rings can undergo contraction to less than half of their initial diameter by two distinct mechanisms, triggered by increasing molecular crowding or temperature. DNA-based contractile rings expand the toolbox of DNA nanotechnology and could be a future element of an artificial division machinery in synthetic cells.

biophysics↗

bioRxiv2022

A DNA segregation module for synthetic cells

The bottom-up construction of an artificial cell requires the realization of synthetic cell division. Significant progress has been made towards reliable compartment division, yet mechanisms to segregate the DNA-encoded informational content are still in their infancy. Herein, droplets of DNA Y-motifs are formed by liquid-liquid phase separation (LLPS). Entropy-driven DNA droplet segregation is obtained by cleaving the linking component between two populations of DNA Y-motifs. In addition to enzymatic cleavage, photolabile sites are introduced for spatio-temporally controlled DNA segregation in bulk as well as in cell-sized water-in-oil droplets and giant unilamellar lipid vesicles (GUVs). Notably, the segregation process is slower in confinement than in bulk. The ionic strength of the solution and the nucleobase sequences are employed to regulate the segregation dynamics. The experimental results are corroborated in a lattice-based theoretical model which mimics the interactions between the DNA Y-motif populations. Altogether, engineered DNA droplets, reconstituted in GUVs, could represent a strategy towards an entropy-driven DNA segregation module within bottom-up assembled synthetic cells. Table of Contents O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY An entropy-driven DNA segregation module for bottom-up assembled synthetic cells is realized. It is based on DNA droplets that are engineered to segregate upon enzymatic or photocleavage inside giant unilamellar lipid vesicles (GUVs). The segregation kinetics is altered by the confinement, as confirmed by lattice-based numerical simulations. DNA segregation is further controlled by temperature, ionic strengths and nucleobase sequence. C_FIG_DISPLAY

biophysics↗

bioRxiv2022

Mechanochemical signal transduction in synthetic cells

Mechanotransduction determines the adaptive response of natural cells via transmem-brane proteins1. The incorporation of membrane-spanning structures to guide cellular function and to enable transmembrane signalling is therefore a critical aim for bottom-up synthetic biology2,3,4. Here, we design membrane-spanning DNA origami signalling units (DOSUs) and mechanically couple them to DNA cytoskeletons5 encapsulated within giant unilamellar vesicles (GUVs). We verify the assembly and incorporation of the DOSUs into the GUV membranes and achieve their clustering upon external stimulation. The transmembrane-spanning DOSUs act as a pore to allow for the transport of single-stranded DNA into the GUVs. We employ this to externally trigger the reconfiguration of DNA cytoskeletons within GUVs using strand displacement reactions. In addition to chemical signalling, we achieve the mechanical coupling of the externally added DOSUs and the internal DNA cytoskeletons. We induce clustering of the DOSUs, which triggers a symmetry break in the organization of the DNA cytoskeleton which is mechanically coupled to the DOSU.Our work thus provides a mechanical and chemical transmembrane signaling module towards the assembly of stimuli-responsive and adaptive synthetic cells.

biophysics↗

bioRxiv2022

Simultaneous adjustments of major mitochondrial pathways through redox regulation of dihydrolipoamide dehydrogenase (mtLPD1)

Thioredoxins (TRX) are pivotal for the redox regulation of enzyme activities to adjust metabolic fluxes towards environmental changes. Previous reports demonstrated TRX o1 and h2 impact on mitochondrial metabolism including photorespiration and the tricarboxylic acid (TCA) cycle. Here, we aimed to unravel potential specificities between regulation modes of both TRXs, especially under conditions with short-term changes in photorespiration. Therefore, short-term metabolite responses of single TRX mutants were analyzed after exposure to altered CO2/O2 ratios during darkness and illumination. This approach was complemented by comprehensive characterization of multiple Arabidopsis mutants lacking either one or both TRX in the wild-type Arabidopsis or the glycine decarboxylase (GDC) T-protein knock down line (gldt1). The results provided evidence for additive effects of combined TRX o1 and h2 deficiency to suppress growth, photosynthesis and mitochondrial metabolism. Quantification of pyrimidine nucleotides in conjunction with metabolite and 13C-labelling approaches revealed a rather uniform impact on mitochondrial dihydrolipoamide dehydrogenase (mtLPD1) dependent pathways. Biochemical analysis of recombinant mtLPD1 demonstrated its inhibition by NADH, pointing at an additional measure to fine-tune its in vivo activity. Collectively, we propose that TRX o1 and h2 contribute to the communication of altered subcellular redox-states through direct and indirect regulation of mtLPD1. This regulation module might represent a common intercept for simultaneous adjustments in the operation of photorespiration, the TCA-cycle and the degradation of branched chain amino acids. One-sentence summaryRedox regulation of mitochondrial dihydrolipoamide dehydrogenase (mtLPD1) simultaneously modulates photorespiration, the tricarboxylic acid (TCA)-cycle and branched chain amino acid (BCAA) degradation in response to rapid environmental changes.

plant biology↗

bioRxiv2022

Triosephosphate export from chloroplasts regulates flavonoid biosynthesis and permits high light acclimation through the inactivation of SnRK1

Plants evolved multiple strategies to cope with rapid changes in the environment. During high light acclimation, biosynthesis of photoprotective flavonoids, such as anthocyanins, is induced. However, the exact nature of the signal and downstream factors for high light induction of flavonoid biosynthesis (FB) are still under debate. Here we show that carbon-fixation in chloroplasts, subsequent export of photosynthates by TRIOSEPHOSPHATE/PHOSPHATE TRANSLOCATOR (TPT), and the rapid increase in cellular sugar contents permit the transcriptional activation of FB during high light acclimation. In combination with genetic and physiological analysis, targeted and whole transcriptome gene expression studies showed that reactive oxygen species and phytohormones play only a minor role for rapid HL-induction of the anthocyanin branch of FB. In addition to FB, sugar-responsive genes were late-repressed or induced in tpt-2 in the course of the high light treatment and a significant overlap with transcripts regulated by SNF1-RELATED PROTEIN KINASE 1 (SnRK1) was found. Analysis of mutants with increased and repressed SnRK1 activity revealed that inactivation of SnRK1 is required for the rapid induction of FB during high light acclimation. Our study underlines the central role of chloroplasts as sensors for environmental changes and emphasizes the vital function of sugar-signalling in plant acclimation, even beyond the regulation of FB.

plant biology↗

bioRxiv2021

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↗

bioRxiv2021

pH-Triggered Assembly of Endomembrane Multicompartments in Synthetic Cells

Bottom-up synthetic biology thrives to reconstruct basic cellular processes into a minimalist cellular replica to foster their investigation in greater details with a reduced number of variables. Among these cellular features, the endomembrane system is an important aspect of cells which is at the origin of many of their functions. Still, the reconstruction of these inner compartments within a lipid-based vesicle remains challenging and poorly controlled. Herein, we report the use of pH as external trigger to self-assemble compartmentalized giant unilamellar vesicles (GUVs) by either bulk, or droplet-based microfluidics. By co-encapsulating pH sensitive small unilamellar vesicles (SUVs), negatively charged SUVs and/or proteins, we show that acidification of the droplets efficiently produces GUVs while sequestrating the co-encapsulated material with flexibility and robustness. The method enables the simultaneous reconstruction of more than a single cellular phenotype from the bottom-up, corresponding to an important advancement in the current status quo of bottom-up synthetic biology.

synthetic biology↗

bioRxiv2020

Choice of fluorophore affects dynamic DNA nanostructures

The ability to dynamically remodel DNA origami structures or functional nanodevices is highly desired in the field of DNA nanotechnology. Concomitantly, the use of fluorophores to track and validate the dynamics of such DNA-based architectures is commonplace and often unavoidable. It is therefore crucial to be aware of the side effects of popular fluorophores, which are often exchanged without considering the potential impact on the system. Here, we show that the choice of fluorophore can strongly affect the reconfiguration of DNA nanostructures. To this end, we encapsulate a triple-stranded DNA (tsDNA) into water-in-oil compartments and functionalize their periphery with a single-stranded DNA handle (ssDNA). Thus, the tsDNA can bind and unbind from the periphery by reversible opening of the triplex and subsequent strand displacement. Using a combination of experiments, molecular dynamics (MD) simulations, and reaction-diffusion modeling, we demonstrate for twelve different fluorophore combinations that it is possible to alter or even inhibit the DNA nanostructure formation - without changing the DNA sequence. Besides its immediate importance for the design of pH-responsive switches and fluorophore labelling, our work presents a strategy to precisely tune the energy landscape of dynamic DNA nanodevices.

biophysics↗

bioRxiv2019

An integrated microfluidic platform for quantifying drug permeation across biomimetic vesicle membranes

The low membrane permeability of candidate drug molecules is a major challenge in drug development and insufficient permeability is one reason for the failure of antibiotic treatment against bacteria. Quantifying drug transport across specific pathways in living systems is challenging since one typically lacks knowledge of the exact lipidome and proteome of the individual cells under investigation. Here, we quantify drug permeability across biomimetic liposome membranes, with comprehensive control over membrane composition. We integrate the microfluidic octanol-assisted liposome assembly platform with an optofluidic transport assay to create a complete microfluidic total analysis system for quantifying drug permeability. Our system enables us to form liposomes with charged lipids mimicking the negative charge of bacterial membranes at physiological salt and pH levels, which proved difficult with previous liposome formation techniques. Furthermore, the microfluidic technique yields an order of magnitude more liposomes per experiment than previous assays. We demonstrate the feasibility of the assay by determining the permeability coefficient of norfloxacin across biomimetic liposomes.

biophysics↗