Biology subjects

Mund, M.

Publications and source records attributed to Mund, M..

14 recordsLinked to original sources

bioRxiv2026

CRISPR-engineered inducible flocculation in Komagataella phaffii enables enhanced biomass separation for biopharmaceutical production

Biomass separation represents a critical bottleneck in Komagataella phaffii-based biopharmaceutical processes, as typically high cell densities of 40 - 50 % create significant operational, technical and economic challenges for harvest operations. Yeast cell aggregation (flocculation) provides a solution to accelerate cell sedimentation by increasing particle size, thus allowing to improve biomass-supernatant separation efficiency during both natural gravity settling and (continuous) centrifugation operations. This study demonstrates successful engineering of K. phaffii strains with an inducible flocculation phenotype using CRISPR/Cas9-based genome editing to integrate the Saccharomyces cerevisiae FLO1 (ScFLO1) gene under control of various regulatory elements, including methanol-inducible and derepressible promoters. Flocculation strength could be enhanced by implementing transcriptional positive feedback circuits based on the methanol-inducible AOX1 promoter. To address methanol-free production requirements, we developed alternative systems to retrofit PAOX1-based ScFLO1 expression and exploited the derepressible PDF promoter, offering broader compatibility with biopharmaceutical manufacturing facilities. Flocculating cells cultivated in a bioreactor demonstrated significantly improved sedimentation behavior, with considerably lower supernatant turbidity after short low-speed centrifugation compared to non-flocculating controls. Crucially, cell flocculation had no negative impact on product amount and quality when expressing a multivalent NANOBODY(R) VHH molecule with pharmaceutical relevance. Thus, this work establishes the first genetically engineered flocculation system in K. phaffii compatible with recombinant protein production, providing the basis for an innovative approach to streamline harvest operations in biopharmaceutical processes.

bioengineering↗

bioRxiv2025

Isohydric species show earlier drought-induced declines in stem water content, rehydration, sap flow, and growth than anisohydric species

O_LIIsohydric species reduce water potential fluctuations through more stringent stomatal regulation. It is unclear whether isohydric behavior leads to smaller reversible diurnal stem shrinkage (i.e. greater nocturnal stem rehydration) during drought and a lower drought sensitivity of radial growth compared to anisohydric behavior. C_LIO_LIWith synchronous high-resolution sap flow and dendrometer measurements in mature anisohydric European beech and isohydric Douglas fir trees in pure and mixed stands, we quantified declines in stem water content (SWC), stem rehydration, sap flow and radial growth during soil dry-down and determined the critical soil moisture levels (expressed as Relative Extractable Water, REW) and elapsed desiccation time until 30-90% reductions in these traits. C_LIO_LISap flow and growth started to decline in both species at REW [~]0.6, preceding declines in stem rehydration. Water-spending Douglas fir approached 50% drops in SWC, sap flow, stem rehydration and growth during soil dry-down faster than beech, indicating higher drought sensitivity. In mixture, both species reached these reduction levels later than in monoculture, suggesting positive mixing effects on the species drought resistance. C_LIO_LIOur findings demonstrate that SWC, sap flow and radial growth decrease earlier than nocturnal stem rehydration, with isohydric and anisohydric species exhibiting different timelines of physiological downregulation during soil dry-down. C_LI

physiology↗

bioRxiv2025

Continuum architecture dynamics of vesicle tethering in exocytosis

Essential for eukaryotic organisms, multiple copies of the exocyst complex tether each secretory vesicle to the plasma membrane (PM) in constitutive exocytosis. The exocyst higher-order structure (ExHOS) that coordinates the action of these multiple exocysts remains unexplored. We integrated particle-tracking, super-resolution microscopy and cryo-electron tomography in a model that time-resolves the continuum conformational landscape of the ExHOS and functionally annotates its different conformations. We found that 7 exocysts form flexible ring-shaped ExHOS that tether vesicles at <45 nm from the PM. The ExHOS, initially 19 nm in radius, rapidly expands while it pulls the vesicle towards the PM. Subsequently, the ExHOS stabilizes, securing the vesicle at [~]4 nm from the PM. After fusion, Sec18 mediates the ExHOS disassembly when its radius is 38 nm. By resolving the fundamental biophysical principles of tethering we bridged the gap between static isolated structures and the dynamic and multimeric nature of exocytosis.

cell biology↗

bioRxiv2023

Metabolization of alpha-D-carba-glucosamine in vivo generates antimetabolites of cell wall precursors

-D-Carba-glucosamine (CGlcN) is a carbocyclic analog of -D-glucosamine that inhibits growth of Bacillus subtilis and Staphylococcus aureus. CGlcN is internalized and concomitantly phosphorylated via the phosphotransferase system yielding -D-carba-glucosamine-6-phosphate (CGlcN6P), which interferes with expression of the glutamine-fructose-6-phosphate amidotransferase (GlmS; glucosamine synthase) by activating the glmS riboswitch. Herein, we report that CGlcN6P is efficiently metabolized to carbasugar nucleotides along the peptidoglycan biosynthetic route. Mass spectrometric analysis confirmed the occurrence of carbocyclic peptidoglycan nucleotides UDP-carba-D-N-acetyl-glucosamine (UDP-CGlcNAc) and UDP-carba-D-N-acetylmuramic acid-pentapeptide (UDP-CMurNAc-5P) in the presence of CGlcN and revealed accumulation of these carba-metabolites upon antibiotic treatment interfering with biosynthetic enzyme functions. Thus, carbocyclic carbohydrates and nucleotide analogs are generated by the promiscuous bacterial cell wall biosynthetic enzymes and act as antimetabolites, causing bacterial growth inhibition by interference with cell wall synthesis. Our findings reveal CGlcN not only as putative antibiotic molecule with previously unknown antimetabolite mode of action, but also as tool to study the bacterial cell wall metabolism, e.g., in synergy with other antibiotics.

microbiology↗

bioRxiv2022

A MAD7-based genome editing system for E. coli

A broad variety of biomolecules is industrially produced in bacteria and yeasts. These microbial expression hosts can be optimized through genomic engineering using CRISPR tools. Here, we designed and characterized such a modular genome editing system based on the Cas12a-like RNA guided nuclease MAD7 in E. coli. Our system enables the efficient generation of single nucleotide polymorphisms (SNPs) or gene deletions and can directly be used with donor DNA from benchtop DNA assembly to increase throughput. We combined multiple edits to engineer an E. coli strain with reduced overflow metabolism and increased plasmid yield, highlighting the versatility and industrial applicability of our approach.

bioengineering↗

bioRxiv2021

Superresolution microscopy reveals partial preassembly and subsequent bending of the clathrin coat during endocytosis

Eukaryotic cells use clathrin-mediated endocytosis to take up a large range of extracellular cargos. During endocytosis, a clathrin coat forms on the plasma membrane, but it remains controversial when and how it is remodeled into a spherical vesicle. Here, we use 3D superresolution microscopy to determine the precise geometry of the clathrin coat at large numbers of endocytic sites. Through pseudo-temporal sorting, we determine the average trajectory of clathrin remodeling during endocytosis. We find that clathrin coats assemble first on flat membranes to 50% of the coat area, before they become rapidly and continuously bent, and confirm this mechanism in three cell lines. We introduce the cooperative curvature model, which is based on positive feedback for curvature generation. It accurately describes the measured shapes and dynamics of the clathrin coat and could represent a general mechanism for clathrin coat remodeling on the plasma membrane.

cell biology↗

bioRxiv2021

Maximum-likelihood model fitting for quantitative analysis of SMLM data

Quantitative analysis is an important part of any single-molecule localization microscopy (SMLM) data analysis workflow to extract biological insights from the coordinates of the single fluorophores, but current approaches are restricted to simple geometries or do not work on heterogenous structures. Here, we present LocMoFit (Localization Model Fit), an open-source framework to fit an arbitrary model directly to the localization coordinates in SMLM data. Using maximum likelihood estimation, this tool extracts the most likely parameters for a given model that best describe the data, and can select the most likely model from alternative models. We demonstrate the versatility of LocMoFit by measuring precise dimensions of the nuclear pore complex and microtubules. We also use LocMoFit to assemble static and dynamic multi-color protein density maps from thousands of snapshots. In case an underlying geometry cannot be postulated, LocMoFit can perform single-particle averaging of super-resolution structures without any assumption about geometry or symmetry. We provide extensive simulation and visualization routines to validate the robustness of LocMoFit and tutorials based on example data to enable any user to increase the information content they can extract from their SMLM data.

biophysics↗

bioRxiv2020

Autoregulation clamps the synaptic membrane-remodeling machinery and promotes productive actin-dependent endocytosis

Synaptic membrane-remodeling events such as endocytosis require force-generating actin assembly. The endocytic machinery that regulates these actin and membrane dynamics localizes at high concentrations to large areas of the presynaptic membrane, but actin assembly and productive endocytosis are far more restricted in space and time. Here we describe a mechanism whereby autoinhibition clamps the presynaptic endocytic machinery to limit actin assembly to discrete functional events. We found that collective interactions between the Drosophila endocytic proteins Nwk/FCHSD2, Dap160/Intersectin, and WASp relieve Nwk autoinhibition and promote robust membrane-coupled actin assembly in vitro. Using automated particle tracking to quantify synaptic actin dynamics in vivo, we discovered that Nwk-Dap160 interactions constrain spurious assembly of WASp-dependent actin structures. These interactions also promote synaptic endocytosis, suggesting that autoinhibition both clamps and primes the synaptic endocytic machinery, thereby constraining actin assembly to drive productive membrane remodeling in response to physiological cues.

cell biology↗

bioRxiv2019

Nuclear pores as versatile reference standards for quantitative superresolution microscopy

Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions.\n\nHere we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag or HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use a) as 3D resolution standards for calibration and quality control, b) to quantify absolute labeling efficiencies and c) as precise reference standards for molecular counting.\n\nThese cell lines will enable the broad community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements.

biophysics↗

bioRxiv2019

Depth-dependent PSF calibration and aberration correction for 3D single-molecule localization

3D Single molecule localization microscopy relies on fitting of the individual molecules with a point spread function (PSF) model. The reconstructed images often show local squeezing or expansion in z. A common cause are depth-induced aberrations in conjunction with an imperfect PSF model calibrated from beads on a coverslip, resulting in a mismatch between measured PSF and real PSF. Here, we developed a strategy for accurate z-localization in which we use the imperfect PSF model for fitting, determine the fitting errors and correct for them in a post-processing step. We present an open-source software tool and a simple experimental calibration procedure that allow retrieving accurate z-positions in any PSF engineering approach or fitting modality, even at large imaging depths.

biophysics↗

bioRxiv2018

Type-I myosins promote actin polymerization to drive membrane bending in endocytosis

Clathrin-mediated endocytosis in budding yeast requires the formation of a dynamic actin network that produces the force to invaginate the plasma membrane against the intracellular turgor pressure. The type-I myosins Myo3 and Myo5 are important for endocytic membrane reshaping, but mechanistic details of their function remain scarce. Here, we studied the function of Myo3 and Myo5 during endocytosis using quantitative live-cell imaging and genetic perturbations. We show that the type-I myosins promote, in a dose-dependent way, the growth and expansion of the actin network, which controls the speed of membrane and coat internalization. We found that this myosin-activity is independent of the actin nucleation promoting activity of myosins, and cannot be compensated for by increasing actin nucleation. Our results suggest a new mechanism for type-I myosins to produce force by promoting actin filament polymerization.

cell biology↗

bioRxiv2018

Topological data analysis quantifies biological nano-structure from single molecule localization microscopy

The study of complex molecular organisation and nano-structure by localization based microscopy is limited by the available analysis tools. We present a segmentation protocol which, through the application of persistence based clustering, is capable of probing densely packed structures which vary in scale. An increase in segmentation performance over state-of-the-art methods is demonstrated. Moreover we employ persistent homology to move beyond clustering, and quantify the topological structure within data. This provides new information about the preserved shapes formed by molecular architecture. Our methods are flexible and we demonstrate this by applying them to receptor clustering in platelets, nuclear pore components and endocytic proteins. Both 2D and 3D implementations are provided within RSMLM, an R package for pointillist based analysis and batch processing of localization microscopy data.

bioinformatics↗

bioRxiv2017

Systematic analysis of the molecular architecture of endocytosis reveals a nanoscale actin nucleation template that drives efficient vesicle formation

Clathrin-mediated endocytosis is an essential cellular function in all eukaryotes that is driven by a self-assembled macromolecular machine of over 50 different proteins in tens to hundreds of copies. How these proteins are organized to produce endocytic vesicles with high precision and efficiency is not understood. Here, we developed high-throughput superresolution microscopy to reconstruct the nanoscale structural organization of 23 endocytic proteins from over 100,000 endocytic sites in yeast. We found that proteins assemble by radially-ordered recruitment according to function. WASP family proteins form a circular nano-scale template on the membrane to spatially control actin nucleation during vesicle formation. Mathematical modeling of actin polymerization showed that this WASP nano-template creates sufficient force for membrane invagination and substantially increases the efficiency of endocytosis. Such nanoscale pre-patterning of actin nucleation may represent a general design principle for directional force generation in membrane remodeling processes such as during cell migration and division.

cell biology↗

bioRxiv2017

Fast, robust and precise 3D localization for arbitrary point spread functions

We present a fitter for 3D single-molecule localization of arbitrary, experimental point spread functions (PSFs) that reaches minimum uncertainty for EMCCD and sCMOS cameras, and achieves more than 105 fits/s. We provide tools to robustly model experimental PSFs and correct for depth induced aberrations, which allowed us to achieve an unprecedented 3D resolution with engineered astigmatic PSFs, and acquire high quality 3D superresolution images even on standard microscopes without 3D optics.

biophysics↗