analysis-of-multidimensional-microscopy-data-using-cell-acdc

Analysis of Multidimensional Microscopy Data Using Cell-ACDC

Recent advances in quantitative microscopy for the life sciences have enabled experimental biologists to probe cells with unprecedented resolution and ...

Helmholtz Zentrum München

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.

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

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.

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

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.

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

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.

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

Mitochondrial DNA (mtDNA) is present in multiple copies within cells and is required for mitochondrial ATP generation. Even within individual cells, mtDNA copies can differ in their sequence, a state known as heteroplasmy. The principles underlying dynamic changes in the degree of heteroplasmy remain incompletely understood, due to the inability to monitor this phenomenon in real time. Here, we employ mtDNA-based fluorescent markers, microfluidics, and automated cell tracking, to follow mtDNA variants in live heteroplasmic yeast populations at the single-cell level. This approach, in combination with direct mtDNA tracking and data-driven mathematical modeling reveals asymmetric partitioning of mtDNA copies during cell division, as well as limited mitochondrial fusion and fission frequencies, as critical driving forces for mtDNA variant segregation. Given that our approach also facilitates assessment of segregation between intact and mutant mtDNA, we anticipate that it will be instrumental in elucidating the mechanisms underlying the purifying selection of mtDNA.

Real-time assessment of mitochondrial DNA heteroplasmy dynamics at the single-cell level.

To maintain protein homeostasis in changing nutrient environments, cells must precisely control the amount of their proteins, despite the accompanying changes in cell growth and biosynthetic capacity. As nutrients are major regulators of cell cycle length and progression, a particular challenge arises for the nutrient-dependent regulation of 'cell cycle genes', which are periodically expressed during the cell cycle. One important example are histones, which are needed at a constant histone-to-DNA stoichiometry. Here we show that budding yeast achieves histone homeostasis in different nutrients through a decoupling of transcript and protein abundance. We find that cells downregulate histone transcripts in poor nutrients to avoid toxic histone overexpression, but produce constant amounts of histone proteins through nutrient-specific regulation of translation efficiency. Our findings suggest that this allows cells to balance the need for rapid histone production under fast growth conditions with the tight regulation required to avoid toxic overexpression in poor nutrients.

Decoupled transcript and protein concentrations ensure histone homeostasis in different nutrients.

Chromatin architecture is a fundamental mediator of genome function. Fasting is a major environmental cue across the animal kingdom, yet how it impacts three-dimensional (3D) genome organization is unknown. Here we show that fasting induces an intestine-specific, reversible and large-scale spatial reorganization of chromatin in Caenorhabditis elegans. This fasting-induced 3D genome reorganization requires inhibition of the nutrient-sensing mTOR pathway, acting through the regulation of RNA Pol I, but not Pol II nor Pol III, and is accompanied by remodelling of the nucleolus. By uncoupling the 3D genome configuration from the animal's nutritional status, we find that the expression of metabolic and stress-related genes increases when the spatial reorganization of chromatin occurs, showing that the 3D genome might support the transcriptional response in fasted animals. Our work documents a large-scale chromatin reorganization triggered by fasting and reveals that mTOR and RNA Pol I shape genome architecture in response to nutrients.

Fasting shapes chromatin architecture through an mTOR/RNA Pol I axis.

Nutrient availability influences ribosome biogenesis, requiring dynamic regulation of RNA Pol I activity. In  , fasting reduces pre-rRNA levels. However, whether this reduction stems from a regulation of RNA Pol I expression remains unclear. Here, we examined how the nutritional status affects the localization and expression levels of RPOA-2 , a core subunit of RNA Pol I, in the intestine. We found that RPOA-2 retains its nucleolar localization regardless of animals being fed, fasted or fed after fasting. Interestingly, fasting reduces RPOA-2 protein amounts which are restored upon feeding. These findings suggest that the availability of RNA Pol I core subunits contributes to the regulation of rDNA transcription in response to nutrients.

Localization and expression dynamics of an RNA Pol I core subunit in response to fasting in .

The size of organelles and cellular structures needs to be tightly regulated and coordinated with overall cell size. A well-studied example is the Cdc42-driven polarization and subsequent septin ring formation in Saccharomyces cerevisiae, where the size of the resulting structures scales with cell size. However, the mechanisms underlying this scaling remain unclear. Here, we combine live-cell imaging, genetic perturbations, and three-dimensional mathematical modeling to investigate how septin ring size is controlled. Our integrative approach reveals that positive feedback in the polarization pathway, together with an increase of the amount of polarity proteins as cell size grows, can explain the scaling of the Cdc42 cluster and, consequently, septin ring diameter. Additionally, we show that in cells lacking the formin Bni1, where F-actin-cable assembly and directed polarization are disrupted, exocytosis becomes diffuse, leading to abnormally large septin rings. By integrating new experimental findings and mathematical modeling of yeast polarization, our study provides insights into the origin of septin ring size control.

Article	Year
Nanotechnological immunoassay for rapid label-free analysis of candidate malaria vaccines. Nanoscale\| PubMed ID: 33438712	2021
Multimodal real-time frequency tracking of cantilever arrays in liquid environment for biodetection: Comprehensive setup and performance analysis. The Review of scientific instruments\| PubMed ID: 34243575	2021
Single-molecule experiments reveal the elbow as an essential folding guide in SMC coiled-coil arms. Biophysical journal\| PubMed ID: 36242515	2022
Regulation with cell size ensures mitochondrial DNA homeostasis during cell growth. Nature structural & molecular biology\| PubMed ID: 37679564	2023
Real-time assessment of mitochondrial DNA heteroplasmy dynamics at the single-cell level. The EMBO journal\| PubMed ID: 39103491	2024
Decoupled transcript and protein concentrations ensure histone homeostasis in different nutrients. The EMBO journal\| PubMed ID: 39271795	2024
Fasting shapes chromatin architecture through an mTOR/RNA Pol I axis. Nature cell biology\| PubMed ID: 39300311	2024
Localization and expression dynamics of an RNA Pol I core subunit in response to fasting in . microPublication biology\| PubMed ID: 39975508	2025
The origin of septin ring size control in budding yeast. The EMBO journal\| PubMed ID: 41039155	2025

Francesco Padovani

Biography

JoVE Journal Publications

Other Publications