Single-cell Tracking Research Articles

Resolving immune cells with patrolling behaviour by magnetic resonance time-lapse single cell tracking.

BackgroundImmune cells show distinct motion patterns that change upon inflammatory stimuli. Monocytes patrol the vasculature to screen for pathogens, thereby exerting an early task of innate immunity. Here, we aimed to non-invasively analyse single patrolling monocyte behaviour upon inflammatory stimuli.MethodsWe used time-lapse Magnetic Resonance Imaging (MRI) of the murine brain to dynamically track single patrolling monocytes within the circulation distant to the actual site of inflammation in different inflammatory conditions, ranging from a subcutaneous pellet model to severe peritonitis and bacteraemia.FindingsSingle patrolling immune cells with a velocity of <1 µm/s could be detected and followed dynamically using time-lapse MRI. We show, that due to local and systemic stimuli the slowly patrolling behaviour of monocytes is altered systemically and differs with type, duration and strength of the underlying stimulus.InterpretationUsing time-lapse MRI, it is now possible to investigate the behaviour of single circulating monocytes over the course of the systemic immune response. Monocyte patrolling behaviour is altered systemically even before the onset of clinical symptoms distant to and depending on the underlying inflammatory stimulus.FundingThis study was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - CRC 1009 - 194468054 to AZ, CF and - CRC 1450 - 431460824 to MM, SN, HB, AZ, CF, the Joachim Herz Foundation (Add-on Fellowship for Interdisciplinary Life Sciences to MM), the Interdisciplinary Centre for Clinical Research (IZKF, core unit PIX) and the Medical Faculty of the University of Muenster (MEDK fellowship to FF and IF).

Monitoring single-cell dynamics of entry into quiescence during an unperturbed life cycle.

The life cycle of microorganisms is associated with dynamic metabolic transitions and complex cellular responses. In yeast, how metabolic signals control the progressive choreography of structural reorganizations observed in quiescent cells during a natural life cycle remains unclear. We have developed an integrated microfluidic device to address this question, enabling continuous single-cell tracking in a batch culture experiencing unperturbed nutrient exhaustion to unravel the coordination between metabolic and structural transitions within cells. Our technique reveals an abrupt fate divergence in the population, whereby a fraction of cells is unable to transition to respiratory metabolism and undergoes a reversible entry into a quiescence-like state leading to premature cell death. Further observations reveal that nonmonotonous internal pH fluctuations in respiration-competent cells orchestrate the successive waves of protein superassemblies formation that accompany the entry into a bona fide quiescent state. This ultimately leads to an abrupt cytosolic glass transition that occurs stochastically long after proliferation cessation. This new experimental framework provides a unique way to track single-cell fate dynamics over a long timescale in a population of cells that continuously modify their ecological niche.

Automated Deep Lineage Tree Analysis Using a Bayesian Single Cell Tracking Approach

Single-cell methods are beginning to reveal the intrinsic heterogeneity in cell populations, arising from the interplay of deterministic and stochastic processes. However, it remains challenging to quantify single-cell behaviour from time-lapse microscopy data, owing to the difficulty of extracting reliable cell trajectories and lineage information over long time-scales and across several generations. Therefore, we developed a hybrid deep learning and Bayesian cell tracking approach to reconstruct lineage trees from live-cell microscopy data. We implemented a residual U-Net model coupled with a classification CNN to allow accurate instance segmentation of the cell nuclei. To track the cells over time and through cell divisions, we developed a Bayesian cell tracking methodology that uses input features from the images to enable the retrieval of multi-generational lineage information from a corpus of thousands of hours of live-cell imaging data. Using our approach, we extracted 20,000 + fully annotated single-cell trajectories from over 3,500 h of video footage, organised into multi-generational lineage trees spanning up to eight generations and fourth cousin distances. Benchmarking tests, including lineage tree reconstruction assessments, demonstrate that our approach yields high-fidelity results with our data, with minimal requirement for manual curation. To demonstrate the robustness of our minimally supervised cell tracking methodology, we retrieve cell cycle durations and their extended inter- and intra-generational family relationships in 5,000 + fully annotated cell lineages. We observe vanishing cycle duration correlations across ancestral relatives, yet reveal correlated cyclings between cells sharing the same generation in extended lineages. These findings expand the depth and breadth of investigated cell lineage relationships in approximately two orders of magnitude more data than in previous studies of cell cycle heritability, which were reliant on semi-manual lineage data analysis.

MicroRNA-124 Alleviates Retinal Vasoregression via Regulating Microglial Polarization.

Microglial activation is implicated in retinal vasoregression of the neurodegenerative ciliopathy-associated disease rat model (i.e., the polycystic kidney disease (PKD) model). microRNA can regulate microglial activation and vascular function, but the effect of microRNA-124 (miR-124) on retinal vasoregression remains unclear. Transgenic PKD and wild-type Sprague Dawley (SD) rats received miR-124 at 8 and 10 weeks of age intravitreally. Retinal glia activation was assessed by immunofluorescent staining and in situ hybridization. Vasoregression and neuroretinal function were evaluated by quantitative retinal morphometry and electroretinography (ERG), respectively. Microglial polarization was determined by immunocytochemistry and qRT-PCR. Microglial motility was examined via transwell migration assays, wound healing assays, and single-cell tracking. Our data showed that miR-124 inhibited glial activation and improved vasoregession, as evidenced by the reduced pericyte loss and decreased acellular capillary formation. In addition, miR-124 improved neuroretinal function. miR-124 shifted microglial polarization in the PKD retina from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype by suppressing TNF-α, IL-1β, CCL2, CCL3, MHC-II, and IFN-γ and upregulating Arg1 and IL-10. miR-124 also decreased microglial motility in the migration assays. The transcriptional factor of C/EBP-α-PU.1 signaling, suppressed by miR-124 both in vivo (PKD retina) and in vitro (microglial cells), could serve as a key regulator in microglial activation and polarization. Our data illustrate that miR-124 regulates microglial activation and polarization. miR-124 inhibits pericyte loss and thereby alleviates vasoregression and ameliorates neurovascular function.

A review for cell and particle tracking on microscopy images using algorithms and deep learning technologies

Time-lapse microscopy images generated by biological experiments have been widely used for observing target activities, such as the motion trajectories and survival states. Based on these observations, biologists can conclude experimental results or present new hypotheses for several biological applications, i.e. virus research or drug design. Many methods or tools have been proposed in the past to observe cell and particle activities, which are defined as single cell tracking and single particle tracking problems, by using algorithms and deep learning technologies. In this article, a review for these works is presented in order to summarize the past methods and research topics at first, then points out the problems raised by these works, and finally proposes future research directions. The contributions of this article will help researchers to understand past development trends and further propose innovative technologies.

P35.01 YB-1 is a Key Player in Aggressive Behaviour and Chemoresistance in Mesothelioma

Malignant pleural mesothelioma (MPM) is characterised by aggressive growth and frequent resistance to chemotherapy, poor prognosis for patients and limited therapeutic options. To establish potential new therapy targets, a better understanding of the MPM biology underlying these malignant behaviours is crucial. One potential candidate is the multifunctional oncoprotein YB-1, which is often overexpressed in various cancers and associated with aggressiveness, metastasis and poor outcome. YB-1 expression was analysed by qPCR or western blot in cell lines, and in patient material by immunohistochemistry. YB-1 overexpression was achieved by a doxycycline-inducible, retroviral construct, which was stably introduced into MPM cell lines. YB-1 knockdown was performed using YB-1 specific siRNA. For inhibition of YB-1 phosphorylation, the RSK inhibitor BI-D1870 was used. Effects of drug interaction (CI values) were calculated using the CompuSyn software. Cell migration was assessed by live cell videomicroscopy followed by manual single cell tracking and analysis using ImageJ and DiPer software, respectively. For the zebrafish model, RFP-expressing MPM cells were injected into 48 hour old larvae, imaged after 1 and 2 days and the number of cells in the tail was manually counted. We previously reported that YB-1 is up-regulated in MPM cell lines compared to non-malignant controls. In this study we analysed MPM tissue (n>120) and normal pleura (n=3) specimens for YB-1 expression. While all control samples were negative, tumours showed a heterogeneous YB-1 expression. YB-1 knockdown decreases MPM cell migration and invasion, hence we evaluated the impact of YB-1 overexpression on these phenotypes. MPM cell lines (n=6) which overexpress YB-1 in a doxycycline-inducible manner showed significantly increased cell scattering and migration. Furthermore, YB-1 overexpression lead to significantly higher migratory capacity in vivo using a zebrafish xenograft model. Additionally, when tumour spheroids were co-cultured with endothelial cells, YB-1 overexpression led to a significantly more extensive formation of gaps in the endothelial layer. Finally, since we found that YB-1 knockdown not only decreases cell growth in vitro and in vivo but also decreases the expression of LRP1 and ABCC1, two genes involved in drug resistance, we combined YB-1 knockdown as well as inhibition of YB-1 phosphorylation via an RSK inhibitor with cisplatin chemotherapy. Our data showed highly synergistic combination effects (CI values: 0.1 – 0.5) for several cisplatin doses in combination with either YB-1 siRNA or the RSK inhibitor. In this study we report a deregulated YB-1 expression in MPM tumor specimens compared to non-malignant controls. Our data show an important role of YB-1 in the regulation of cell migration and invasion in vitro and in vivo, which are key characteristics of MPM. Additionally, YB-1 knock down as well as pharmacological inhibition of YB-1 phosphorylation not only reduce MPM cell growth but also sensitise cells to cisplatin chemotherapy. These findings contribute to a better understanding of the biology of MPM and highlight YB-1’s potential as a therapeutic target.

Single platelet and megakaryocyte morpho-dynamics uncovered by multicolor reporter mouse strains in vitro and in vivo.

Visualizing cell behavior and effector function on a single cell level has been crucial for understanding key aspects of mammalian biology. Due to their small size, large number and rapid recruitment into thrombi, there is a lack of data on fate and behavior of individual platelets in thrombosis and hemostasis. Here we report the use of platelet lineage restricted multi-color reporter mouse strains to delineate platelet function on a single cell level. We show that genetic labeling allows for single platelet and megakaryocyte (MK) tracking and morphological analysis in vivo and in vitro, while not affecting lineage functions. Using Cre-driven Confetti expression, we provide insights into temporal gene expression patterns as well as spatial clustering of MK in the bone marrow. In the vasculature, shape analysis of activated platelets recruited to thrombi identifies ubiquitous filopodia formation with no evidence of lamellipodia formation. Single cell tracking in complex thrombi reveals prominent myosin-dependent motility of platelets and highlights thrombus formation as a highly dynamic process amenable to modification and intervention of the acto-myosin cytoskeleton. Platelet function assays combining flow cytrometry, as well as in vivo, ex vivo and in vitro imaging show unaltered platelet functions of multicolor reporter mice compared to wild-type controls. In conclusion, platelet lineage multicolor reporter mice prove useful in furthering our understanding of platelet and MK biology on a single cell level.

Single-cell tracking reveals super-spreading brain cancer cells with high persistence

Cell migration is a fundamental characteristic of vital processes such as tissue morphogenesis, wound healing and immune cell homing to lymph nodes and inflamed or infected sites. Therefore, various brain defect diseases, chronic inflammatory diseases as well as tumor formation and metastasis are associated with aberrant or absent cell migration. We embedded multicellular brain cancer spheroids in Matrigel™ and utilized single-particle tracking to extract the paths of cells migrating away from the spheroids. We found that – in contrast to local invasion – single cell migration is independent of Matrigel™ concentration and is characterized by high directionality and persistence. Furthermore, we identified a subpopulation of super-spreading cells with >200-fold longer persistence times than the majority of cells. These results highlight yet another aspect of cell heterogeneity in tumors.

Photothermal Agarose Microfabrication Technology for Collective Cell Migration Analysis.

Agarose photothermal microfabrication technology is one of the micropatterning techniques that has the advantage of simple and flexible real-time fabrication even during the cultivation of cells. To examine the ability and limitation of the agarose microstructures, we investigated the collective epithelial cell migration behavior in two-dimensional agarose confined structures. Agarose microchannels from 10 to 211 micrometer width were fabricated with a spot heating of a focused 1480 nm wavelength infrared laser to the thin agarose layer coated on the cultivation dish after the cells occupied the reservoir. The collective cell migration velocity maintained constant regardless of their extension distance, whereas the width dependency of those velocities was maximized around 30 micrometer width and decreased both in the narrower and wider microchannels. The single-cell tracking revealed that the decrease of velocity in the narrower width was caused by the apparent increase of aspect ratio of cell shape (up to 8.9). In contrast, the decrease in the wider channels was mainly caused by the increase of the random walk-like behavior of component cells. The results confirmed the advantages of this method: (1) flexible fabrication without any pre-designing, (2) modification even during cultivation, and (3) the cells were confined in the agarose geometry.

Linoleic and oleic acids enhance cell migration by altering the dynamics of microtubules and the remodeling of the actin cytoskeleton at the leading edge

Fatty acids (FA) have a multitude of biological actions on living cells. A target of their action is cell motility, a process of critical importance during cancer cell dissemination. Here, we studied the effect of unsaturated FA on ovarian cancer cell migration in vitro and its role in regulating cytoskeleton structures that are essential for cell motility. Scratch wound assays on human ovary cancer SKOV-3 cell monolayers revealed that low doses (16 μM) of linoleic acid (LA, 18:2 ω6) and oleic acid (OA; 18:1 ω9) promoted migration, while α-linolenic acid (ALA, 18:3 ω3), showed a migration rate similar to that of the control group. Single cell tracking demonstrated that LA and OA-treated cells migrated faster and were more orientated towards the wound closure than control. In vitro addition of those FA resulted in an increased number, length and protrusion speed of filopodia and also in a prominent and dynamic lamellipodia at the cell leading edge. Using time-lapse video-microscopy and FRAP we observed an increase in both the speed and frequency of actin waves associated with more mobile actin and augmented Rac1 activity. We also observed that FA induced microtubule-organizing center (MTOC)-orientation towards the cell front and affected the dynamics of microtubules (MT) in the direction of cell migration. We propose that environmental cues such as OA and LA present in ascitic fluid, should be taken into account as key factors for the regulation of cell migration.

Crash landing of Vibrio cholerae by MSHA pili-assisted braking and anchoring in a viscoelastic environment.

Mannose-sensitive hemagglutinin (MSHA) pili and flagellum are critical for the surface attachment of Vibrio cholerae, the first step of V. cholerae colonization on host surfaces. However, the cell landing mechanism remains largely unknown, particularly in viscoelastic environments such as the mucus layers of intestines. Here, combining the cysteine-substitution-based labeling method with single-cell tracking techniques, we quantitatively characterized the landing of V. cholerae by directly observing both pili and flagellum of cells in a viscoelastic non-Newtonian solution consisting of 2% Luria-Bertani and 1% methylcellulose (LB+MC). The results show that MSHA pili are evenly distributed along the cell length and can stick to surfaces at any point along the filament. With such properties, MSHA pili are observed to act as a brake and anchor during cell landing which includes three phases: running, lingering, and attaching. Importantly, loss of MSHA pili results in a more dramatic increase in mean path length in LB+MC than in 2% LB only or in 20% Ficoll solutions, indicating that the role of MSHA pili during cell landing is more apparent in viscoelastic non-Newtonian fluids than viscous Newtonian ones. Our work provides a detailed picture of the landing dynamics of V. cholerae under viscoelastic conditions, which can provide insights into ways to better control V. cholerae infections in a real mucus-like environment.

Abstract 3188: β-catenin in metastatic ovarian cancer cells mediates polyploidy induced by tumor-associated macrophages

Abstract Tumor-associated macrophages (TAMs) closely associate with tumor progression and could promote metastatic spread. However, how TAMs interact with individual cancer cell in the heterogeneous cancer cell population is unknown. Here, using an isogenic cell line pair with different metastatic potentials, we showed that highly metastatic (HM) cells, but not non-metastatic (NM) cells, could skew the co-cultured macrophages to TAM-like phenotypes. Single-cell tracking by time lapse imaging revealed that during co-culture, macrophages could induce failed cytokinesis in a subset of HM cells. Subsequent analyses suggested that this induction could be facilitated by direct cell-cell contact. We further identified that the cell-surface metadherin expression induced by β-catenin in HM cells is involved in this induction, while surface CEACAM1 expressed on macrophages could be responsible for the interaction with metadherin. HM-macrophage interaction promotes macrophage secretion of the cytokine CCL3, a strong chemoattractant for monocytes, which may further amplify the interaction. In an orthotopic humanized mice model, knockdown of β-catenin in HM cells reduced tumor burden as well as the number of tumor-infiltrating CD163+ TAMs. These findings demonstrate a positive feedback loop between macrophages and a subpopulation of cancer cells mediated by β-catenin to enhance the metastatic cascade (This work is supported by RGC GRF grant 17141216). Citation Format: Sally Kit Yan To, Jue Shi, Alice Sze Tsai Wong. β-catenin in metastatic ovarian cancer cells mediates polyploidy induced by tumor-associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3188.

Abstract 1954: Identifying synthetic lethality with novel CDK2 small molecule inhibitors via integration of high-content microscopy and -omics level platforms

Abstract CDK4/6 small molecule inhibitors represent a milestone in treating hormone receptor-positive breast cancer. PALOMA (Palbociclib: Ongoing Trials in the Management of Breast Cancer) studies have confirmed prolonged progression-free survival after CDK4/6 inhibitor and anti-estrogen therapy. Notably, disease progression is observed in a subset of patients receiving Palbociclib-Fulvestrant treatment (Turner et al., NEJM 2018). Circulating tumor DNA from PALOMA-3 patients revealed genetic mutations correlating with patient response (O'Leary et al., Cancer Discov, 2018). Compensations in CDK4/6i resistance include circumvention of CDK4/6 substrate regulation, CCNE1/2 amplification (Herrera-Abreu et al., Cancer Res. 2016), and compensatory CDK2 activity. The importance of compensatory CDK2 cancer re-wiring is highlighted by recently reported, highly efficacious CDK2/4/6i (Freeman-Cook et al., in review). Thus, mechanistic understanding of cancer cell and compensatory CDK2 activity is imperative to improve patient outcome. Acute, temporal phenotypes in CDK2-signaling have been observed by live single-cell tracking microscopy in human breast epithelial and ER+ breast cancer cells (Arora &amp; Moser et al., in review). In extending these studies, we utilize first-in-class small molecule inhibitors against CDK2 combined with high content imaging approaches to illuminate cancer cell CDK2 signaling and identify potential synthetic lethality. We report biomarkers parsed in cycling populations as quantifiable phenotypes and identify disengaged control of several processes including S- and G2/M licensing after CDK2i. Combined with internal -omics studies, CDK2i selective molecules support a positive regulatory role of CDK2 on CCNB1-PLK1 signaling before mitotic entry. We combine our high-content techniques with proximity labeling to quantify alternative CDK-Cyclin complex formation following pharmacologic perturbation. We hypothesize that dysregulated CDK-Cyclin complex formation elicits unintended CDK2 kinase activity, revealing potential synthetic lethality with inhibition of centrosome duplication pathways in ER+ breast cancers. Citation Format: Tim Sen Wang, Jon Almaden, Chen Shen, John Lapek, Sherry Niessen, Nichol Miller, Stephen Dann, Todd VanArsdale. Identifying synthetic lethality with novel CDK2 small molecule inhibitors via integration of high-content microscopy and -omics level platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1954.

Interaction between the type 4 pili machinery and a diguanylate cyclase fine-tune c-di-GMP levels during early biofilm formation

To initiate biofilm formation, it is critical for bacteria to sense a surface and respond precisely to activate downstream components of the biofilm program. Type 4 pili (T4P) and increasing levels of c-di-GMP have been shown to be important for surface sensing and biofilm formation, respectively; however, mechanisms important in modulating the levels of this dinucleotide molecule to define a precise output response are unknown. Here, using macroscopic bulk assays and single-cell tracking analyses of Pseudomonas aeruginosa, we uncover a role of the T4P alignment complex protein, PilO, in modulating the activity of the diguanylate cyclase (DGC) SadC. Two-hybrid and bimolecular fluorescence complementation assays, combined with genetic studies, are consistent with a model whereby PilO interacts with SadC and that the PilO-SadC interaction inhibits SadC's activity, resulting in decreased biofilm formation and increased motility. Using single-cell tracking, we monitor both the mean c-di-GMP and the variance of this dinucleotide in individual cells. Mutations that increase PilO-SadC interaction modestly, but significantly, decrease both the average and variance in c-di-GMP levels on a cell-by-cell basis, while mutants that disrupt PilO-SadC interaction increase the mean and variance of c-di-GMP levels. This work is consistent with a model wherein P. aeruginosa uses a component of the T4P scaffold to fine-tune the levels of this dinucleotide signal during surface commitment. Finally, given our previous findings linking SadC to the flagellar machinery, we propose that this DGC acts as a bridge to integrate T4P and flagellar-derived input signals during initial surface engagement.

Sulfation of Glycosaminoglycans Modulates the Cell Cycle of Embryonic Mouse Spinal Cord Neural Stem Cells.

In the developing spinal cord neural stem and progenitor cells (NSPCs) secrete and are surrounded by extracellular matrix (ECM) molecules that influence their lineage decisions. The chondroitin sulfate proteoglycan (CSPG) DSD-1-PG is an isoform of receptor protein tyrosine phosphatase-beta/zeta (RPTPβ/ζ), a trans-membrane receptor expressed by NSPCs. The chondroitin sulfate glycosaminoglycan chains are sulfated at distinct positions by sulfotransferases, thereby generating the distinct DSD-1-epitope that is recognized by the monoclonal antibody (mAb) 473HD. We detected the epitope, the critical enzymes and RPTPβ/ζ in the developing spinal cord. To obtain insight into potential biological functions, we exposed spinal cord NSPCs to sodium chlorate. The reagent suppresses the sulfation of glycosaminoglycans, thereby erasing any sulfation code expressed by the glycosaminoglycan polymers. When NSPCs were treated with chlorate and cultivated in the presence of FGF2, their proliferation rate was clearly reduced, while NSPCs exposed to EGF were less affected. Time-lapse video microscopy and subsequent single-cell tracking revealed that pedigrees of NSPCs cultivated with FGF2 were strongly disrupted when sulfation was suppressed. Furthermore, the NSPCs displayed a protracted cell cycle length. We conclude that the inhibition of sulfation with sodium chlorate interferes with the FGF2-dependent cell cycle progression in spinal cord NSPCs.

Viscoelastically tunable substrates elucidate the interface-relaxation-dependent adhesion and assembly behaviors of epithelial cells

Recent progress in mechanobiology sheds light on the regulation of cellular phenotypes by dissipative property of matrices, i.e., viscosity, fluidity, and stress relaxation, in addition to extensively studied elasticity. However, most researches have focused on bulk mechanics, despite cells in 2D culture can only interact with matrix interface directly. Here, we studied the impact of interfacial viscosity as well as elasticity of substrates on the early stage of adhesion behaviors of epithelial cells through new material design and mechanical characterization. The materials are copolymers of ε-caprolactone and d,l-lactide photocrosslinked by benzophenone. The substrate viscoelasticity changes depending on the polymer molecular weight and irradiation time. The interfacial elasticity and relaxation were determined by atomic force microscopy with modes of nanoindentation and tip-dwelling, respectively. MDCK cells changed morphologically, ranging from loose beaded assembly to more compact spheroids and eventual spread monolayer clusters, in response to the interfacial viscoelasticity change. Such morphological changes were mainly determined by substrate interfacial relaxation, rather than interfacial elasticity. Single-cell tracking identified biphasic motility with the minimum speed at intermediate relaxation time (~350 ms), where cells showed transitional morphologies between epithelial and mesenchymal traits. In that relaxation level, partially deformed cells moved around to coalesce with surrounding cells, eventually assembling into compact cellular aggregates. These results highlight, unlike the conventional hanging-drop technique, an appropriate level of interfacial relaxation is critical for efficient cell aggregate maturation on adhesive viscoelastic matrices. This work not only elucidates that the interfacial relaxation as the essential mechanical parameter for epithelial cell adhesion and migration, but also gives useful tips for creating physiologically relevant drug screening platform.

Multinucleation associated DNA damage blocks proliferation in p53-compromised cells

Nuclear atypia is one of the hallmarks of cancers. Here, we perform single-cell tracking studies to determine the immediate and long-term impact of nuclear atypia. Tracking the fate of newborn cells exhibiting nuclear atypia shows that multinucleation, unlike other forms of nuclear atypia, blocks proliferation in p53-compromised cells. Because ~50% of cancers display compromised p53, we explored how multinucleation blocks proliferation. Multinucleation increases 53BP1-decorated nuclear bodies (DNA damage repair platforms), along with a heterogeneous reduction in transcription and protein accumulation across the multi-nucleated compartments. Multinucleation Associated DNA Damage associated with 53BP1-bodies remains unresolved for days, despite an intact NHEJ machinery that repairs laser-induced DNA damage within minutes. Persistent DNA damage, a DNA replication block, and reduced phospho-Rb, reveal a novel replication stress independent cell cycle arrest caused by mitotic lesions. These findings call for segregating protective and prohibitive nuclear atypia to inform therapeutic approaches aimed at limiting tumour heterogeneity.

Effects of fixation on bacterial cellular dimensions and integrity

SummaryFixation facilitates imaging of subcellular localization and cell morphology, yet it remains unknown how fixation affects cellular dimensions and intracellular fluorescence patterns, particularly during long-term storage. Here, we characterized the effects of multiple fixatives on several bacterial species. Fixation generally reduced cell length by 5–15%; single-cell tracking in microfluidics revealed that the length decrease was an aggregate effect of many steps in the fixation protocol and that fluorescence of cytoplasmic GFP but not membrane-bound MreB-msfGFP was rapidly lost with formaldehyde-based fixatives. Cellular dimensions were preserved in formaldehyde-based fixatives for ≥4 days, but methanol caused length to decrease. Although methanol preserved cytoplasmic fluorescence better than formaldehyde-based fixatives, some Escherichia coli cells were able to grow directly after fixation. Moreover, methanol fixation caused lysis in a subpopulation of cells, with virtually all Bacillus subtilis cells lysing after one day. These findings highlight tradeoffs between maintenance of fluorescence and membrane integrity for future applications of fixation.

Cell kinetics of auxin transport and activity in Arabidopsis root growth and skewing

Auxin is a key regulator of plant growth and development. Local auxin biosynthesis and intercellular transport generates regional gradients in the root that are instructive for processes such as specification of developmental zones that maintain root growth and tropic responses. Here we present a toolbox to study auxin-mediated root development that features: (i) the ability to control auxin synthesis with high spatio-temporal resolution and (ii) single-cell nucleus tracking and morphokinetic analysis infrastructure. Integration of these two features enables cutting-edge analysis of root development at single-cell resolution based on morphokinetic parameters under normal growth conditions and during cell-type-specific induction of auxin biosynthesis. We show directional auxin flow in the root and refine the contributions of key players in this process. In addition, we determine the quantitative kinetics of Arabidopsis root meristem skewing, which depends on local auxin gradients but does not require PIN2 and AUX1 auxin transporter activities. Beyond the mechanistic insights into root development, the tools developed here will enable biologists to study kinetics and morphology of various critical processes at the single cell-level in whole organisms.

Alkaline pH Increases Swimming Speed and Facilitates Mucus Penetration for Vibrio cholerae.

Intestinal mucus is the first line of defense against intestinal pathogens. It acts as a physical barrier between epithelial tissues and the lumen that enteropathogens must overcome to establish a successful infection. We investigated the motile behavior of two Vibrio cholerae strains (El Tor C6706 and Classical O395) in mucus using single-cell tracking in unprocessed porcine intestinal mucus. We determined that V. cholerae can penetrate mucus using flagellar motility and that alkaline pH increases swimming speed and, consequently, improves mucus penetration. Microrheological measurements indicate that changes in pH between 6 and 8 (the physiological range for the human small intestine) had little effect on the viscoelastic properties of mucus. Finally, we determined that acidic pH promotes surface attachment by activating the mannose-sensitive hemagglutinin (MshA) pilus in V. cholerae El Tor C6706 without a measurable change in the total cellular concentration of the secondary messenger cyclic dimeric GMP (c-di-GMP). Overall, our results support the hypothesis that pH is an important factor affecting the motile behavior of V. cholerae and its ability to penetrate mucus. Therefore, changes in pH along the human small intestine may play a role in determining the preferred site for V. cholerae during infection.IMPORTANCE The diarrheal disease cholera is still a burden for populations in developing countries with poor sanitation. To develop effective vaccines and prevention strategies against Vibrio cholerae, we must understand the initial steps of infection leading to the colonization of the small intestine. To infect the host and deliver the cholera toxin, V. cholerae has to penetrate the mucus layer protecting the intestinal tissues. However, the interaction of V. cholerae with intestinal mucus has not been extensively investigated. In this report, we demonstrated using single-cell tracking that V. cholerae can penetrate intestinal mucus using flagellar motility. In addition, we observed that alkaline pH improves the ability of V. cholerae to penetrate mucus. This finding has important implications for understanding the dynamics of infection, because pH varies significantly along the small intestine, between individuals, and between species. Blocking mucus penetration by interfering with flagellar motility in V. cholerae, reinforcing the mucosa, controlling intestinal pH, or manipulating the intestinal microbiome will offer new strategies to fight cholera.