Segmentation, tracking and cell cycle analysis of live-cell imaging data with Cell-ACDC.

BACKGROUND: High-throughput live-cell imaging is a powerful tool to study dynamic cellular processes in single cells but creates a bottleneck at the stage of data analysis, due to the large amount of data generated and limitations of analytical pipelines. Recent progress on deep learning dramatically improved cell segmentation and tracking. Nevertheless, manual data validation and correction is typically still required and tools spanning the complete range of image analysis are still needed. RESULTS: We present Cell-ACDC, an open-source user-friendly GUI-based framework written in Python, for segmentation, tracking and cell cycle annotations. We included state-of-the-art deep learning models for single-cell segmentation of mammalian and yeast cells alongside cell tracking methods and an intuitive, semi-automated workflow for cell cycle annotation of single cells. Using Cell-ACDC, we found that mTOR activity in hematopoietic stem cells is largely independent of cell volume. By contrast, smaller cells exhibit higher p38 activity, consistent with a role of p38 in regulation of cell size. Additionally, we show that, in S. cerevisiae, histone Htb1 concentrations decrease with replicative age. CONCLUSIONS: Cell-ACDC provides a framework for the application of state-of-the-art deep learning models to the analysis of live cell imaging data without programming knowledge. Furthermore, it allows for visualization and correction of segmentation and tracking errors as well as annotation of cell cycle stages. We embedded several smart algorithms that make the correction and annotation process fast and intuitive. Finally, the open-source and modularized nature of Cell-ACDC will enable simple and fast integration of new deep learning-based and traditional methods for cell segmentation, tracking, and downstream image analysis. Source code: https://github.com/SchmollerLab/Cell_ACDC.

Single-molecule experiments reveal the elbow as an essential folding guide in SMC coiled-coil arms.

Structural maintenance of chromosome (SMC) complexes form ring-like structures through exceptional elongated coiled-coils (CCs). Recent studies found that variable CC conformations, including open and collapsed forms, which might result from discontinuities in the CC, facilitate the diverse functions of SMCs in DNA organization. However, a detailed description of the SMC CC architecture is still missing. Here, we study the structural composition and mechanical properties of SMC proteins with optical tweezers unfolding experiments using the isolated Psm3 CC as a model system. We find a comparatively unstable protein with three unzipping intermediates, which we could directly assign to CC features by crosslinking experiments and state-of-the-art prediction software. Particularly, the CC elbow is shown to be a flexible, potentially non-structured feature, which divides the CC into sections, induces a pairing shift from one CC strand to the other and could facilitate large-scale conformational changes, most likely via thermal fluctuations of the flanking CC sections. A replacement of the elbow amino acids hinders folding of the consecutive CC region and frequently leads to non-native misalignments, revealing the elbow as a guide for proper folding. Additional in vivo manipulation of the elbow flexibility resulted in impaired cohesin complexes, which directly link the sensitive CC architecture to the biological function of cohesin.

Microrheological Coagulation Assay Exploiting Micromechanical Resonators.

Rheological measurements in biological liquids yield insights into homeostasis and provide information on important molecular processes that affect fluidity. We present a fully automated cantilever-based method for highly precise and sensitive measurements of microliter sample volumes of human blood plasma coagulation (0.009 cP for viscosity range 0.5-3 cP and 0.0012 g/cm3 for density range 0.9-1.1 g/cm3). Microcantilever arrays are driven by a piezoelectric element, and resonance frequencies and quality factors of sensors that change over time are evaluated. A highly accurate approximation of the hydrodynamic function is introduced that correlates resonance frequency and quality factor of cantilever beams immersed in a fluid to the viscosity and density of that fluid. The theoretical model was validated using glycerol reference solutions. We present a surface functionalization protocol that allows minimization of unspecific protein adsorption onto cantilevers. Adsorption leads to measurement distortions and incorrect estimation of the fluid parameters (viscosity and density). Two hydrophilic terminated self-assembled monolayers (SAMs) sensor surfaces are compared to a hydrophobic terminated SAM coating. As expected, the hydrophobic modified surfaces induced the highest mass adsorption and could promote conformational changes of the proteins and subsequent abnormal biological activity. Finally, the activated partial thromboplastin time (aPTT) coagulation assay was performed, and the viscosity, density, and coagulation rate of human blood plasma were measured along with the standard coagulation time. The method could extend and improve current coagulation testing.

Regulation with cell size ensures mitochondrial DNA homeostasis during cell growth.

To maintain stable DNA concentrations, proliferating cells need to coordinate DNA replication with cell growth. For nuclear DNA, eukaryotic cells achieve this by coupling DNA replication to cell-cycle progression, ensuring that DNA is doubled exactly once per cell cycle. By contrast, mitochondrial DNA replication is typically not strictly coupled to the cell cycle, leaving the open question of how cells maintain the correct amount of mitochondrial DNA during cell growth. Here, we show that in budding yeast, mitochondrial DNA copy number increases with cell volume, both in asynchronously cycling populations and during G1 arrest. Our findings suggest that cell-volume-dependent mitochondrial DNA maintenance is achieved through nuclear-encoded limiting factors, including the mitochondrial DNA polymerase Mip1 and the packaging factor Abf2, whose amount increases in proportion to cell volume. By directly linking mitochondrial DNA maintenance to nuclear protein synthesis and thus cell growth, constant mitochondrial DNA concentrations can be robustly maintained without a need for cell-cycle-dependent regulation.

An mTOR/RNA pol I axis shapes chromatin architecture in response to fasting.

Chromatin architecture is a fundamental mediator of genome function. Fasting is a major environmental cue across the animal kingdom. Yet, how it impacts on 3D genome organization is unknown. Here, we show that fasting induces a reversible and large-scale spatial reorganization of chromatin in C. elegans . This fasting-induced 3D genome reorganization requires inhibition of the nutrient-sensing mTOR pathway, a major regulator of ribosome biogenesis. Remarkably, loss of transcription by RNA Pol I, but not RNA Pol II nor Pol III, induces a similar 3D genome reorganization in fed animals, and prevents the restoration of the fed-state architecture upon restoring nutrients to fasted animals. Our work documents the first large-scale chromatin reorganization triggered by fasting and reveals that mTOR and RNA Pol I shape genome architecture in response to nutrients.

Multimodal real-time frequency tracking of cantilever arrays in liquid environment for biodetection: Comprehensive setup and performance analysis.

We present a nanomechanical platform for real-time quantitative label-free detection of target biomolecules in a liquid environment with mass sensitivity down to few pg. Newly fabricated arrays of up to 18 cantilevers are integrated in a micromachined fluidic chamber, connected to software-controlled fluidic pumps for automated sample injections. We discuss two functionalization approaches to independently sensitize the interface of different cantilevers. A custom piezo-stack actuator and optical readout system enable the measurement of resonance frequencies up to 2 MHz. We implement a new measurement strategy based on a phase-locked loop (PLL), built via in-house developed software. The PLL allows us to track, within the same experiment, the evolution of resonance frequency over time of up to four modes for all the cantilevers in the array. With respect to the previous measurement technique, based on standard frequency sweep, the PLL enhances the estimated detection limit of the device by a factor of 7 (down to 2 pg in 5 min integration time) and the time resolution by more than threefold (below 15 s), being on par with commercial gold-standard techniques. The detection limit and noise of the new setup are investigated via Allan deviation and standard deviation analysis, considering different resonance modes and interface chemistries. As a proof-of-concept, we show the immobilization and label-free in situ detection of live bacterial cells (E. coli), demonstrating qualitative and quantitative agreement in the mechanical response of three different resonance modes.

Nanotechnological immunoassay for rapid label-free analysis of candidate malaria vaccines.

Malaria is a life-threatening epidemic disease with half of the world's population at risk. Although its incidence rate has fallen since 2010, this ratio dramatically stalled between 2014 and 2018. New fast and optimized tools in vaccine analysis and seroconversion testing are critically needed. We developed a clinical diagnostic device based on piezo-actuated nanoresonators that perform as quantitative in situ calibrated nano-bio sensors for specific detection of multiple target molecules in serum samples. The immunoassay successfully diagnoses humoral immune responses induced by malaria vaccine candidates and reveals the timeline and stage of the infection. We applied the newly developed strategy to a variety of different samples, from pure antibody/vaccine solutions, to blood samples from clinical trials on both naïve and pre-exposed malaria volunteers from sub-Saharan countries. Our nanomechanical assay provides a direct one-step label-free quantitative immunoassay that is on par with the gold-standard, multi-step enzyme-linked immunosorbent assay (ELISA). We achieve a limit of detection of few pg ml-1, or sub-pM concentrations. The 6 µl sample volume allows more than 50 experiments from one finger prick. Furthermore, we simultaneously detected multiple analytes by differential functionalization of multiple sensors in parallel. The inherent differential read-out with in situ controls reduces false positive results. Due to the faster turnaround time, the minimal volume required and the automatized handling system, this technique has great potential for miniaturization and routine diagnostics in pandemic emergencies.

Towards personalised rapid label free miRNA detection for cancer and liver injury diagnostics in cell lysates and blood based samples.

Advances in prevention, diagnosis and therapy are coupled to innovation and development of new medical tools, leading to improved patient prognosis. We developed an automatic biosensor platform that could provide a non-invasive, rapid and personalised diagnosis using nanomechanical cantilever sensors. miRNA are involved in gene expression and are extractable biomarkers for multiple diseases. We detected specific expression patterns of miRNA relevant to cancer and adverse drug effects directly in cell lysates or blood based samples using only a few microliters of sample within one hour. Specific miRNA hybridisation to the upper cantilever surface induces physical bending of the sensor which is detected by monitoring the position of a laser that reflects from the sensors surface. Internal reference sensors negate environmental and nonspecific effects. We showed that the sensitivity of label free cantilever nanomechanical sensing of miRNA surpasses that of surface plasmon resonance by more than three orders of magnitude. A cancer associated miRNA expression profile from cell lysates and one associated with hepatocytes derived from necrotic liver tissue in blood-based samples has been successfully detected. Our label free mechanical approach displays the capability to perform in relevant clinical samples while also obtaining comparable results to PCR based techniques. Without the need to individually extend, amplify or label each target allowing multitarget analysis from one sample.

Nanomechanical clinical coagulation diagnostics and monitoring of therapies.

Clinical coagulation diagnostics often requires multiple tests. Coagulation times are a first indication of an abnormal coagulation process, such as a coagulation factor deficiency. To determine the specific deficient factor, additional immuno- and/or enzyme assays are necessary. Currently, every clinical laboratory has to normalize their assays (international normalized ratio, INR), and therefore, certain variability within the clinical analytics exists. We report a novel strategy for a quick, reliable and quantitative diagnosis of blood coagulation diseases (e.g. haemophilia) and for monitoring factor replacement and anticoagulant therapies (e.g. heparin treatment). We exploit nano-oscillations of microcantilevers for real-time measurements of the evolving blood plasma clot strength (viscosity). The sensors are oscillated at multiple high resonance mode numbers, in order to minimise the oscillation amplitude (a few nanometers), to provide direct internal control and to increase the quality factor. Along with the activated thromboplastin time (aPTT) and prothrombin time (PT) other parameters important for thrombosis diagnostics can be obtained, including the final clot strength and the fibrinolysis time. We demonstrate the dependence of the parameters on factor deficiencies and we diagnose a specific factor deficiency through an integrated and quantitative in situ immunoassay. This approach does not require continuous calibration since it delivers an absolute quantity (clot strength). The low sample volume required (a few µl) and the ability to measure different parameters within the same test (PT, aPTT and global coagulation assay) make the presented technique a versatile point-of-care device for clinical coagulation diagnostics.

Energetics of surface confined ferritin during iron loading.

We report on the first quantitative picture on how iron loading inside ferritin molecules occurs when they are self-assembled onto solid surfaces. Recombinant human ferritin H-chain with ferroxidase activity was adsorbed onto microcantilever beams to form a stable close-packed thin film. The obtained nanomechanical system was used to track in real time the energetics of inter-ferritin surface interactions during incubation with Fe(II) for iron loading. We observed that iron loading is accompanied by increasing attractive in-plane inter-ferritin interactions able to perform a maximum surface work of 6.0±1.5mJ/m(2), corresponding to a surface energy variation per ferritin of about 40kbT. Unique to this protein surface transformation, part of the surface work is exerted by the attractive electrostatic forces arising among the new born nanosized iron cores inside the ferritin shells. The remaining work comes from subtle action of steric, bridging and depletion forces. These findings are of fundamental interest and add important information for the rational development of ferritin nanotechnology.