Biology subjects

Walther, N.

Publications and source records attributed to Walther, N..

10 recordsLinked to original sources

bioRxiv2025

Distinct Sox9 single-molecule dynamics characterize adult differentiation and fetal-like reprogrammed states in intestinal organoids

Transcription factors (TFs) mediate gene expression changes during differentiation and development. However, how TF biophysical properties and abundance dynamically regulate specific cell state transitions remains poorly understood. Using automated live-cell single-molecule tracking (SMT) in intestinal organoid models, we revealed an expression level-independent decrease in the fraction of immobile Sox9 molecules during differentiation from [~]48% to [~]38%, largely dependent on DNA binding. Strikingly, long-term Sox9 overexpression caused organoids to transition from budding to spheroid morphology accompanied by increased proliferation and a loss in gene expression signatures for intestinal identity and function. In this fetal-like reprogrammed state, a larger fraction of partially self- interacting Sox9 molecules ([~]61%) binds to DNA. Our results suggest context-dependent Sox9 single-molecule dynamics during adult intestinal differentiation and fetal-like reversion in consequence to long-term Sox9 overexpression. Our work underpins the power of our automated live-cell SMT framework to generate testable hypotheses towards unraveling molecular mechanisms underlying tissue-level phenotypes. Highlights- Heterogenous diffusion behavior of Sox9 across intestinal differentiation states - Concentration-independent decrease in chromatin-bound Sox9 upon differentiation - Sox9 overexpression leads to fetal-like reversion and more immobile Sox9 molecules - Automated SMT in 2D monolayers reveals TF dynamics underlying organoid phenotypes

developmental biology↗

bioRxiv2024

Automated live-cell single-molecule tracking in enteroid monolayers reveals transcription factor dynamics probing lineage-determining function

Lineage transcription factors (TFs) provide one regulatory level of differentiation crucial for the generation and maintenance of healthy tissues. To probe TF function by measuring their dynamics during adult intestinal homeostasis, we established HILO-illumination-based live-cell single-molecule tracking (SMT) in mouse small intestinal enteroid monolayers recapitulating tissue differentiation hierarchies in vitro. To increase the throughput, capture cellular features, and correlate morphological characteristics with diffusion parameters, we developed an automated imaging and analysis pipeline, broadly applicable to 2D culture systems. Studying two absorptive lineage-determining TFs, we find an expression level-independent contrasting diffusive behavior: While Hes1, key determinant of absorptive lineage commitment, displays a large cell-to-cell variability and an average fraction of DNA-bound molecules of [~]32%, Hnf4g, conferring enterocyte identity, exhibits more uniform dynamics and a bound fraction of [~]56%. Our results suggest that TF diffusive behavior can indicate the progression of differentiation and modulate early versus late differentiation within a lineage. Highlights- Automated live-cell single-molecule tracking records hundreds of cells in enteroid monolayers - Cellular diffusion clustering and morphological feature correlation reveals subpopulations - Transcription factor dynamics regulate differentiation independent of expression level - Hes1 and Hnf4g display contrasting dynamics assisting early vs. late absorptive differentiation

developmental biology↗

bioRxiv2023

Transgene Expression in Cultured Cells Using Unpurified Recombinant Adeno-Associated Viral Vectors

Recombinant adeno-associated viral vectors (rAAV) can achieve potent and durable transgene expression without integration in a broad range of tissue types, making them a popular choice for gene delivery in animal models and in clinical settings. In addition to therapeutic applications, rAAVs are a useful laboratory tool for delivering transgenes tailored to the researchers experimental needs and scientific goals in cultured cells. Some examples include exogenous reporter genes, overexpression cassettes, RNA interference, and CRISPR-based tools including those for genome-wide screens. rAAV transductions are less harmful to cells than electroporation or chemical transfection and moreover do not require any special equipment or expensive reagents to produce. Crude lysates or conditioned media containing rAAVs can be added directly to cultured cells without further purification to transduce many cell types - an underappreciated feature of rAAVs. Here, we provide protocols for basic transgene cassette cloning and demonstrate how to produce and apply crude rAAV preparations to cultured cells. As proof-of-principle, we demonstrate transduction of three cell types that have not yet been reported in rAAV applications. We discuss appropriate uses for crude rAAV preparations, the limitations of rAAVs for gene delivery, and considerations for capsid choice. The simplicity of production, exceedingly low cost, and often potent results make crude rAAV a primary choice for researchers to achieve effective DNA delivery. SummaryRecombinant adeno-associated virus (rAAV) is widely used for clinical and preclinical gene delivery. An underappreciated use for rAAVs is the robust transduction of cultured cells without the need for purification. For researchers new to rAAV, we provide a protocol for transgene cassette cloning, crude vector production, and cell culture transduction.

molecular biology↗

bioRxiv2019

Absolute quantification of cohesin, CTCF and their regulators in human cells

The organisation of mammalian genomes into loops and topologically associating domains (TADs) contributes to chromatin structure, gene expression and recombination. Loops and TADs are formed by cohesin and positioned by CTCF. In proliferating cells, cohesin also mediates sister chromatid cohesion, which is essential for chromosome segregation. Current models of chromatin folding and cohesion are based on assumptions of how many cohesin and CTCF molecules organise the genome. Here we have measured absolute copy numbers and dynamics of cohesin, CTCF, NIPBL, WAPL and sororin by mass spectrometry, fluorescence-correlation spectroscopy and fluorescence recovery after photobleaching in HeLa cells. In G1-phase there are ~245,000 cohesin complexes, of which ~139,000 are on chromatin. Comparison with chromatin immunoprecipitation-sequencing data implies that some genomic cohesin and CTCF enrichment sites are unoccupied in single cells at any one time. We discuss the implications of these findings for how cohesin can contribute to genome organisation and cohesion.

cell biology↗

bioRxiv2018

A quantitative map of human Condensins provides new insights into mitotic chromosome architecture

The two Condensin complexes in human cells are essential for mitotic chromosome structure. We used homozygous genome editing to fluorescently tag Condensin I and II subunits and mapped their absolute abundance, spacing and dynamic localization during mitosis by fluorescence correlation spectroscopy-calibrated live cell imaging and super-resolution microscopy. While [~]35,000 Condensin II complexes are stably bound to chromosomes throughout mitosis, [~]195,000 Condensin I complexes dynamically bind in two steps, in prometaphase and early anaphase. The two Condensins rarely co-localize at the chromatid axis, where Condensin II is centrally confined but Condensin I reaches [~]50% of the chromatid diameter from its center. Based on our comprehensive quantitative data, we propose a three-step hierarchical loop model of mitotic chromosome compaction: Condensin II initially fixes loops of a maximum size of [~]450 kb at the chromatid axis whose size is then reduced by Condensin I binding to [~]90 kb in prometaphase and [~]70 kb in anaphase, achieving maximum chromosome compaction upon sister chromatid segregation.

cell biology↗

bioRxiv2017

An experimental and computational framework to build a dynamic protein atlas of human cell division

Essential biological functions, such as mitosis, require tight coordination of hundreds of proteins in space and time. Localization, timing of interactions and changes in cellular structure are all crucial to ensure correct assembly, function and regulation of protein complexes1-4. Live cell imaging can reveal protein distributions and dynamics but experimental and theoretical challenges prevented its use to produce quantitative data and a model of mitosis that comprehensively integrates information and enables analysis of the dynamic interactions between the molecular parts of the mitotic machinery within changing cellular boundaries.\n\nTo address this, we generated a 4D image data-driven, canonical model of the morphological changes during mitotic progression of human cells. We used this model to integrate dynamic 3D concentration data of many fluorescently knocked-in mitotic proteins, imaged by fluorescence correlation spectroscopy-calibrated microscopy5. The approach taken here in the context of the MitoSys consortium to generate a dynamic protein atlas of human cell division is generic. It can be applied to systematically map and mine dynamic protein localization networks that drive cell division in different cell types and can be conceptually transferred to other cellular functions.

systems biology↗

bioRxiv2017

Generation and validation of homozygous fluorescent knock-in cells using genome editing

Gene tagging with fluorescent proteins is essential to investigate the dynamic properties of cellular proteins. CRISPR/Cas9 technology is a powerful tool for inserting fluorescent markers into all alleles of the gene of interest (GOI) and permits functionality and physiological expression of the fusion protein. It is essential to evaluate such genome-edited cell lines carefully in order to preclude off-target effects caused by either (i) incorrect insertion of the fluorescent protein, (ii) perturbation of the fusion protein by the fluorescent proteins or (iii) non-specific genomic DNA damage by CRISPR/Cas9. In this protocol1, we provide a step-by-step description of our systematic pipeline to generate and validate homozygous fluorescent knock-in cell lines.\n\nWe have used the paired Cas9D10A nickase approach to efficiently insert tags into specific genomic loci via homology-directed repair with minimal off-target effects. It is time- and cost-consuming to perform whole genome sequencing of each cell clone. Therefore, we have developed an efficient validation pipeline of the generated cell lines consisting of junction PCR, Southern Blot analysis, Sanger sequencing, microscopy, Western blot analysis and live cell imaging for cell cycle dynamics. This protocol takes between 6-9 weeks. Using this protocol, up to 70% of the targeted genes can be tagged homozygously with fluorescent proteins and result in physiological levels and phenotypically functional expression of the fusion proteins.\n\nEditorial SummaryThis protocol provides a detailed workflow describing how to insert fluorescent markers into all alleles of a gene of interest using CRISPR/Cas 9 technology and how to generate and validate homozygous fluorescent knock-in cell lines.

molecular biology↗

bioRxiv2017

Quantitative mapping of fluorescently tagged cellular proteins using FCS-calibrated four dimensional imaging

EDITORIAL SUMMARYThis protocol describes how to estimate and spatially resolve the concentration and copy number of fluorescently tagged proteins in live cells using fluorescence imaging and fluorescence correlation spectroscopy (FCS).\n\nTWEETDetermining protein concentrations and copy numbers in live cells using fluorescence correlation spectroscopy (FCS)-calibrated imaging.\n\nCOVER TEASER Map protein concentrations with FCS-calibrated imagingUp to four primary research articles where the protocol has been used and/or developed:\n\nO_LIWalther, N., Hossain, M. J., Politi, A. Z., Koch, B., Kueblbeck, M., Oedegaard-Fougner, O., Lampe, M. and J. Ellenberg (2018). A quantitative map of human Condensins provides new insights into mitotic chromosome architecture. bioRxiv, 237834. https://doi.org/10.1101/2378342.\nC_LIO_LICai, Y., Hossain, M. J., Heriche, J.-K., Politi, A. Z., Walther, N., Koch, B., Wachsmuth, M., Nijmeijer, B., Kueblbeck, M., Martinic, M., Ladurner, R., Peters, J.M. and J. Ellenberg (2017). An experimental and computational framework to build a dynamic protein atlas of human cell division. bioRxiv, 227751 https://doi.org/10.1101/227751\nC_LIO_LIGermier, T., Kocanova, S., Walther, N., Bancaud, A., Shaban, H.A., Sellou, H., Politi, A.Z., Ellenberg, J., Gallardo, F. and K. Bystricky (2017). Real-Time Imaging of a Single Gene Reveals Transcription-Initiated Local Confinement. Biophysical Journal, 113(7), 1383-1394, https://doi.org/10.1016/j.bpj.2017.08.014.\nC_LIO_LICuylen, S., Blaukopf, C., Politi, A. Z., Muller-Reichert, T., Neumann, B., Poser, I., Ellenberg, J., Hyman, A.A., and D.W. Gerlich (2016). Ki-67 acts as a biological surfactant to disperse mitotic chromosomes. Nature, 535(7611), 308-312. http://doi.org/10.1038/nature18610.\nC_LI\n\nAbstractThe ability to tag a protein at its endogenous locus with a fluorescent protein (FP) enables the quantitative understanding of protein dynamics at the physiological level. Genome editing technology has now made this powerful approach routinely applicable to mammalian cells and many other model systems, opening up the possibility to systematically and quantitatively map the cellular proteome in four dimensions. 3D time-lapse confocal microscopy (4D imaging) is an essential tool to investigate spatial and temporal protein dynamics, however it lacks the required quantitative power to make absolute and comparable measurements required for systems analysis. Fluorescence correlation spectroscopy (FCS) on the other hand provides quantitative proteomic and biophysical parameters such as protein concentration, hydrodynamic radius and oligomerization but lacks the ability for high-throughput application in 4D spatial and temporal imaging. Here, we present an automated experimental and computational workflow that integrates both methods and delivers quantitative 4D imaging data in high-throughput. These data is processed to yield a calibration curve relating the fluorescence intensities of image voxels to absolute protein abundance. The calibration curve allows the conversion of the arbitrary fluorescence intensities to protein amounts for all voxels of 4D imaging stacks. With our workflow the users can acquire and analyze hundreds of FCS-calibrated image series to map their proteins of interest in four dimensions. Compared to other protocols, the current protocol does not require additional calibration standards and provides an automated acquisition pipeline for FCS and imaging data. The protocol can be completed in 1 day.

molecular biology↗

bioRxiv2017

CTCF, WAPL and PDS5 proteins control the formation of TADs and loops by cohesin

Mammalian genomes are organized into compartments, topologically-associating domains (TADs) and loops to facilitate gene regulation and other chromosomal functions. Compartments are formed by nucleosomal interactions, but how TADs and loops are generated is unknown. It has been proposed that cohesin forms these structures by extruding loops until it encounters CTCF, but direct evidence for this hypothesis is missing. Here we show that cohesin suppresses compartments but is essential for TADs and loops, that CTCF defines their boundaries, and that WAPL and its PDS5 binding partners control the length of chromatin loops. In the absence of WAPL and PDS5 proteins, cohesin passes CTCF sites with increased frequency, forms extended chromatin loops, accumulates in axial chromosomal positions (vermicelli) and condenses chromosomes to an extent normally only seen in mitosis. These results show that cohesin has an essential genome-wide function in mediating long-range chromatin interactions and support the hypothesis that cohesin creates these by loop extrusion, until it is delayed by CTCF in a manner dependent on PDS5 proteins, or until it is released from DNA by WAPL.

genomics↗

bioRxiv2017

Real-time chromatin dynamics at the single gene levelduring transcription activation

Genome dynamics relate to regulation of gene expression, the most fundamental process in biology. Yet we still do not know whether the very process of transcription drives spatial organization and chromatin conformation at specific gene loci. To address this issue, we have optimized the ANCHOR/ParB DNA labeling system for real-time imaging and quantitative analysis of the dynamics of a single-copy transgene in human cells. Transcription of the transgene under the control of the endogenous Cyclin D1 promoter was induced by addition of 17{beta}-estradiol. Motion of the ANCHOR3-tagged DNA locus was recorded in the same cell prior to and during appearance of nascent mRNA visualized using the MS2 system. We found that transcription initiation resulted in rapid confinement of the mRNA-producing gene. The confinement was maintained even upon inhibition of pol2 elongation. It did not occur when recruitment of pol2 or transcription initiation was blocked by anti-estrogens or Triptolide. These results suggest that preinitiation complex formation and concomitant reorganization of the chromatin domain constrains freedom of movement of an induced genes promoter within minutes. Confined diffusion reflects assembly of functional protein hubs and DNA processing during the rate-limiting steps of transcription.

cell biology↗