Biology subjects

Endesfelder, U.

Publications and source records attributed to Endesfelder, U..

9 recordsLinked to original sources

bioRxiv2026

Multi-foci replication domains in Haloferax volcanii visualised by super-resolution microscopy

The archaeon Haloferax volcanii is a unique model organism to investigate replication dynamics and dissect the interplay between multiple origins, alternative replication pathways, and polyploidy. The combination of variable chromosome copy number and asynchronous replication across the cell population gives rise to pronounced phenotypic heterogeneity, further increasing system complexity. To investigate these mechanisms, we implemented (spt-)PALM and STORM microscopy to probe replication dynamics in different cell lines and growth conditions. We established the first STORM-based super-resolution imaging approach of a protein in Haloferax volcanii. Altogether, our results support a model in which each H. volcanii cell replicates a constant fraction of its chromosome copies, irrespective of total ploidy. Consequently, cells with a higher number of chromosomes exhibit proportionally more replication forks, resulting in pronounced ploidy heterogeneity across the population. Moreover, we show that replication foci are not randomly distributed but cluster into discrete Replication Domains containing multiple active sites. This organization persists across different growth conditions and is independent of canonical replication origins, as demonstrated in a strain lacking all four origins. Our findings suggest that replication coordination in H. volcanii is governed by spatial architecture rather than origin-dependent initiation, providing new insights into the evolutionary diversity of replication control mechanisms.

microbiology↗

bioRxiv2025

A novel expression system for imaging single-molecule fluorescence in Haloferax volcanii WR806 enables visualization of Cas1 involvement in UV-light associated DNA repair

Fluorescence microscopy has become an indispensable tool in biological research, offering powerful approaches to study protein dynamics and molecular biochemistry in vivo. Among archaea, Haloferax volcanii has emerged as a particularly well-suited model organism for imaging studies, with a growing toolkit of established fluorescent markers, plasmids, and promoter systems. Recent advances in single-molecule imaging techniques have created new opportunities through WR806, a carotenoid-free strain providing reduced autofluorescence background. However, existing plasmid-based expression systems in WR806 show critical limitations in protein expression control and challenges with protein aggregation. To address these limitations, we developed pUE001, a novel expression system specifically designed for WR806. This system achieves precise expression control by decoupling selection and induction through strategic implementation of the trpA selection marker. Through comprehensive characterization, we demonstrate that pUE001 provides superior control over protein expression compared to the previously established pTA962 system. It enables linear, titratable expression of diverse proteins -- from the highly regulated CRISPR-Cas component Cas1 to the abundant structural protein FtsZ1 -- while preventing protein aggregation that could compromise native cellular functions. Additionally, we performed a comprehensive analysis of WR806 to show that carotenoid depletion does not affect native cellular physiology. Finally, to demonstrate the systems utility, we investigated the role of Cas1 in UV-induced DNA repair using single-particle tracking photoactivated localization microscopy (sptPALM). Our findings reveal significant, dose-dependent changes in Cas1 mobility following UV-light induced damage, providing evidence for its involvement in DNA repair processes and offering new insights into the expanding roles of CRISPR-Cas systems beyond adaptive immunity.

microbiology↗

bioRxiv2024

EVE is an open modular data analysis software for event-based localization microscopy

Event-based sensors (EBS), or neuromorphic vision sensors, offer a novel approach to imaging by recording light intensity changes asynchronously, unlike conventional cameras that capture light over fixed exposure times. This capability results in high temporal resolution, reduced data redundancy, and a wide dynamic range. This makes EBS ideal for Single-Molecule Localization Microscopy (SMLM) as SMLM relies on the sequential imaging of sparse, blinking fluorescent emitters to achieve super-resolution. Recent studies have shown that EBS can effectively capture these emitters, achieving spatial resolution comparable to traditional cameras. However, existing analyses of event-based SMLM (eveSMLM) data have relied on converting event lists into image frames for conventional analysis, limiting the full potential of the technology. To overcome this limitation, we developed EVE, a specialized software for analyzing eveSMLM data. EVE offers an integrated platform for detection, localization, and post-processing, with various algorithmic options tailored for the unique structure of eveSMLM data. EVE is user-friendly and features an open, modular infrastructure that supports ongoing development and optimization. EVE is the first dedicated tool for event-based SMLM, transforming the analysis process to fully utilize the spatiotemporal data generated by EBS. This allows researchers to explore the full potential of eveSMLM and encourages the development of new analytical methods and experimental improvements.

biophysics↗

bioRxiv2023

Temporal analysis of relative distances (TARDIS) is a robust, parameter-free alternative to single-particletracking

In single-particle tracking (spt), individual particles are localized and tracked over time to probe their diffusion and molecular interactions. Temporal crossing of trajectories, blinking particles, and false-positive localizations present computational challenges that have remained difficult to overcome. Here, we introduce a robust, parameter-free alternative to spt: TARDIS. In TARDIS, an all-to-all distance analysis between localizations is performed with increasing temporal shifts. These pairwise distances fall in two categories: inter-particle distances not originating from the same particle, and intra-particle distances originating from the same particle. Since the distribution of inter-particle distances is unaffected by temporal shifts, the distribution of particle jump distances can be analytically fitted by analysing multiple temporal shifts. TARDIS outperforms existing tracking algorithms especially in complex conditions, and is even robust in scenarios that exceed the capabilities of current localization algorithms. Using TARDIS, we further show that measurements can be five-fold shorter without loss of information.

biophysics↗

bioRxiv2021

Unraveling the kinetochore nanostructure in Schizosaccharomyces pombe using multi-color single-molecule localization microscopy

The key to ensuring proper chromosome segregation during mitosis is the kinetochore complex. This large and tightly regulated multi-protein complex links the centromeric chromatin to the microtubules attached to the spindle pole body and as such leads the segregation process. Understanding the architecture, function and regulation of this vital complex is therefore essential. However, due to its complexity and dynamics, only its individual subcomplexes could be studied in high-resolution structural detail so far. In this study we construct a nanometer-precise in situ map of the human-like regional kinetochore of Schizosaccharomyces pombe (S. pombe) using multi-color single-molecule localization microscopy (SMLM). We measure each kinetochore protein of interest (POI) in conjunction with two reference proteins, cnp1CENP-A at the centromere and sad1 at the spindle pole. This arrangement allows us to determine the cell cycle and in particularly the mitotic plane, and to visualize individual centromere regions separately. From these data, we determine protein distances within the complex using Bayesian inference, establish the stoichiometry of each POI for individual chromosomes and, consequently, build an in situ kinetochore model for S.pombe with so-far unprecedented precision. Being able to quantify the kinetochore proteins within the full in situ kinetochore structure, we provide valuable new insights in the S.pombe kinetochore architecture.

microbiology↗

bioRxiv2020

CAP1 and cofilin1 cooperate in neuronal actin dynamics, growth cone function and neuron connectivity

Neuron connectivity depends on growth cones that navigate axons through the developing brain. Growth cones protrude and retract actin-rich structures to sense guidance cues. These cues control local actin dynamics and steer growth cones towards attractants and away from repellents, thereby directing axon outgrowth. Hence, actin binding proteins (ABPs) moved into the focus as critical regulators of neuron connectivity. We found cyclase-associated protein 1 (CAP1), an ABP with unknown brain function, abundant in growth cones. Super-resolution microscopy and live cell imaging combined with pharmacological approaches on hippocampal neurons from gene-targeted mice revealed a crucial role for CAP1 in actin dynamics that is critical for growth cone morphology and function. Growth cone defects in mutant neurons compromised neuron differentiation and was associated with impaired neuron connectivity in CAP1 mutant brains. Mechanistically, we found that CAP1 and cofilin1 synergistically control growth cone actin dynamic and morphology. Together, we identified CAP1 as a novel actin regulator in growth cone that is relevant for neuron connectivity.

neuroscience↗

bioRxiv2020

Establishing live-cell single-molecule localization microscopy imaging and single-particle tracking in the archaeon Haloferax volcanii

In recent years, fluorescence microscopy techniques for the localization and tracking of single molecules in living cells have become well-established and indispensable tools for the investigation of cellular biology and in vivo biochemistry of many bacterial and eukaryotic organisms. Nevertheless, these techniques are still not established for imaging archaea. Their establishment as a standard tool for the study of archaea will be a decisive milestone for the exploration of this branch of life and its unique biology. Here we have developed a reliable protocol for the study of the archaeon Haloferax volcanii. We have generated an autofluorescence-free H. volcanii strain, evaluated several fluorescent proteins for their suitability to serve as single-molecule fluorescence markers and codon-optimized them to work under optimal H. volcanii cultivation conditions. We found that two of them, Dendra2Hfx and PAmCherry1Hfx, provide state-of-the-art single-molecule imaging. Our strategy is quantitative and allows dual-color imaging of two targets in the same field of view as well as DNA co-staining. We present the first single-molecule localization microscopy (SMLM) images of the subcellular organization and dynamics of two crucial intracellular proteins in living H. volcanii cells, FtsZ1, which shows complex structures in the cell division ring, and RNA polymerase, which localizes around the periphery of the cellular DNA. This work should provide incentive to develop SMLM strategies for other archaeal organisms in the near future.

microbiology↗

bioRxiv2020

Post-mitotic expansion of cell nuclei requires ACTN4-mediated nuclear actin filament bundling

The actin cytoskeleton operates in a multitude of cellular processes including cell shape and migration, mechanoregulation, as well as membrane or organelle dynamics. However, its filamentous properties and functions inside the mammalian cell nucleus are less well explored. We previously described transient actin assembly at mitotic exit that promotes nuclear expansion during chromatin decondensation. Here, we identify non-muscle ACTN4 as a critical regulator to facilitate F-actin formation, reorganization and bundling during postmitotic nuclear expansion. ACTN4 binds to nuclear actin filaments and ACTN4 clusters associate with nuclear F-actin in a highly dynamic fashion. ACTN4 but not ACTN1 is required for proper postmitotic nuclear volume expansion, mediated by its actin binding domain. Using super-resolution imaging to quantify actin filament numbers and widths in individual nuclei we find that ACTN4 is necessary for postmitotic nuclear actin assembly and actin filament bundling. Our findings uncover a nuclear cytoskeletal function for ACTN4 to control nuclear size during mitotic cell division.

cell biology↗

bioRxiv2019

Dynamic relocalization of the cytosolic type III secretion system components ensures specific protein secretion

Many bacterial pathogens use a type III secretion system (T3SS) to manipulate host cells. Protein secretion by the T3SS injectisome is activated upon contact to any host cell, and it has been unclear how premature secretion is prevented during infection. We found that in gastrointestinal pathogens, cytosolic injectisome components are temporarily released from the proximal interface of the injectisome at low external pH, preventing protein secretion in acidic environments, such as the stomach. In Yersinia enterocolitica, low external pH is detected in the periplasm and leads to a partial dissociation of the inner membrane injectisome component SctD, which in turn causes the dissociation of the cytosolic T3SS components. This effect is reversed upon restoration of neutral pH, allowing a fast activation of the T3SS at the native target regions within the host. These findings indicate that the cytosolic components form an adaptive regulatory interface, which regulates T3SS activity in response to environmental conditions.

microbiology↗