Forced apart: a microtubule-based mechanism for equidistant positioning of multiple nuclei in single cells

AbstractCells can position multiple copies of components like carboxysomes, nucleoids, and nuclei at regular intervals. By controlling positions, cells, for example, ensure equal partitioning of organelles over daughter cells and, in the case of nuclei, control cell sizes during cellularization. Mechanisms that generate regular patterns are as yet poorly understood. We used fission yeast cell cycle mutants to investigate the dispersion of multiple nuclei by microtubule-generated forces in single cells. After removing internuclear attractive forces by microtubule-based molecular motors, we observed the establishment of regular patterns of nuclei. Based on live-cell imaging, we hypothesized that microtubule growth within internuclear spaces pushes neighbouring nuclei apart. In the proposed mechanism, which was validated by stochastic simulations, the repulsive force weakens with increasing separation because stochastic shortening events limit the extent over which microtubules generate forces. Our results, therefore, demonstrate how cells can exploit the dynamics of microtubule growth for the equidistant positioning of organelles.

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Regeneration of the Eyespot and Flagellum in Euglena gracilis during Cell Division

Plants ◽

10.3390/plants10102004 ◽

2021 ◽

Vol 10 (10) ◽

pp. 2004

Author(s):

Kazunari Ozasa ◽

Hyunwoong Kang ◽

Simon Song ◽

Shun Tamaki ◽

Tomoko Shinomura ◽

...

Keyword(s):

Cell Division ◽

Life Span ◽

Euglena Gracilis ◽

Live Cell Imaging ◽

Cell Tracking ◽

Cell Imaging ◽

Early Stage ◽

Single Cells ◽

Wild Type ◽

Daughter Cells

Cell division of unicellular microalgae is a fascinating process of proliferation, at which whole organelles are regenerated and distributed to two new lives. We performed dynamic live cell imaging of Euglena gracilis using optical microscopy to elucidate the mechanisms involved in the regulation of the eyespot and flagellum during cell division and distribution of the organelles into the two daughter cells. Single cells of the wild type (WT) and colorless SM-ZK cells were confined in a microfluidic device, and the appearance of the eyespot (stigma) and emergent flagellum was tracked in sequential video-recorded images obtained by automatic cell tracking and focusing. We examined 12 SM-ZK and 10 WT cells and deduced that the eyespot diminished in size and disappeared at an early stage of cell division and remained undetected for 26–97 min (62 min on average, 22 min in deviation). Subsequently, two small eyespots appeared and were distributed into the two daughter cells. Additionally, the emergent flagellum gradually shortened to zero-length, and two flagella emerged from the anterior ends of the daughter cells. Our observation revealed that the eyespot and flagellum of E. gracilis are degraded once in the cell division, and the carotenoids in the eyespot are also decomposed. Subsequently, the two eyespots/flagella are regenerated for distribution into daughter cells. As a logical conclusion, the two daughter cells generated from a single cell division possess the equivalent organelles and each E. gracilis cell has eternal or non-finite life span. The two newly regenerated eyespot and flagellum grow at different rates and mature at different timings in the two daughter cells, resulting in diverse cell characteristics in E. gracilis.

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Long-term fate of etoposide-induced micronuclei and micronucleated cells in Hela-H2B-GFP cells

Archives of Toxicology ◽

10.1007/s00204-020-02840-0 ◽

2020 ◽

Vol 94 (10) ◽

pp. 3553-3561

Author(s):

Hauke Reimann ◽

Helga Stopper ◽

Henning Hintzsche

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell ◽

Cellular Structures ◽

Death Rates ◽

Daughter Cells

Abstract Micronuclei are small nuclear cellular structures containing whole chromosomes or chromosomal fragments. While there is a lot of information available about the origin and formation of micronuclei, less is known about the fate of micronuclei and micronucleated cells. Possible fates include extrusion, degradation, reincorporation and persistence. Live cell imaging was performed to quantitatively analyse the fates of micronuclei and micronucleated cells occurring in vitro. Imaging was conducted for up to 96 h in HeLa-H2B-GFP cells treated with 0.5, 1 and 2 µg/ml etoposide. While a minority of micronuclei was reincorporated into the main nucleus during mitosis, the majority of micronuclei persisted without any alterations. Degradation and extrusion were observed rarely or never. The presence of micronuclei affected the proliferation of the daughter cells and also had an influence on cell death rates. Mitotic errors were found to be clearly increased in micronucleus-containing cells. The results show that micronuclei and micronucleated cells can, although delayed in cell cycle, sustain for multiple divisions.

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Microfluidic platform enables live-cell imaging of signaling and transcription combined with multiplexed secretion measurements in the same single cells

Integrative Biology ◽

10.1093/intbio/zyz013 ◽

2019 ◽

Vol 11 (4) ◽

pp. 142-153 ◽

Cited By ~ 1

Author(s):

Ramesh Ramji ◽

Amanda F Alexander ◽

Andrés R Muñoz-Rojas ◽

Laura N Kellman ◽

Kathryn Miller-Jensen

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Nuclear Translocation ◽

Single Cells ◽

Live Cell ◽

Relative Level ◽

Fluorescent Reporter ◽

Cytokines And Chemokines ◽

Cell Processes ◽

Cell To Cell Variability

Abstract Innate immune cells, including macrophages and dendritic cells, protect the host from pathogenic assaults in part through secretion of a program of cytokines and chemokines (C/Cs). Cell-to-cell variability in C/C secretion appears to contribute to the regulation of the immune response, but the sources of secretion variability are largely unknown. To begin to track the biological sources that control secretion variability, we developed and validated a microfluidic device to integrate live-cell imaging of fluorescent reporter proteins with a single-cell assay of protein secretion. We used this device to image NF-κB RelA nuclear translocation dynamics and Tnf transcription dynamics in macrophages in response to stimulation with the bacterial component lipopolysaccharide (LPS), followed by quantification of secretion of TNF, CCL2, CCL3, and CCL5. We found that the timing of the initial peak of RelA signaling in part determined the relative level of TNF and CCL3 secretion, but not CCL2 and CCL5 secretion. Our results support evidence that differences in timing across cell processes partly account for cell-to-cell variability in downstream responses, but that other factors introduce variability at each biological step.

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C. elegans chromosomes connect to centrosomes by anchoring into the spindle network

10.1101/060855 ◽

2016 ◽

Cited By ~ 3

Author(s):

Stefanie Redemann ◽

Johannes Baumgart ◽

Norbert Lindow ◽

Sebastian Fürthauer ◽

Ehssan Nazockdast ◽

...

Keyword(s):

Light Microscopy ◽

Mitotic Spindle ◽

Live Cell Imaging ◽

Large Scale ◽

Cell Imaging ◽

Electron Tomography ◽

Mitotic Spindles ◽

C Elegans ◽

Microtubule Growth ◽

Segregation Of Chromosomes

AbstractThe mitotic spindle ensures the faithful segregation of chromosomes. To discover the nature of the crucial centrosome-to-chromosome connection during mitosis, we combined the first large-scale serial electron tomography of whole mitotic spindles in early C. elegans embryos with live-cell imaging. Using tomography, we reconstructed the positions of all microtubules in 3D, and identified their plus- and minus-ends. We classified them as kinetochore (KMTs), spindle (SMTs), or astral microtubules (AMTs) according to their positions, and quantified distinct properties of each class. While our light microscopy and mutant studies show that microtubules are nucleated from the centrosomes, we find only a few KMTs are directly connected to the centrosomes. Indeed, by quantitatively analysing several models of microtubule growth, we conclude that minus-ends of KMTs have selectively detached and depolymerized from the centrosome. In toto, our results show that the connection between centrosomes and chromosomes is mediated by an anchoring into the entire spindle network and that any direct connections through KMTs are few and likely very transient.

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Live cell imaging of the hyperthermophilic archaeon Sulfolobus acidocaldarius identifies complementary roles for two ESCRTIII homologues in ensuring a robust and symmetric cell division

10.1101/2020.02.18.953042 ◽

2020 ◽

Cited By ~ 2

Author(s):

Andre Arashiro Pulschen ◽

Delyan R. Mutavchiev ◽

Kim Nadine Sebastian ◽

Jacques Roubinet ◽

Marc Roubinet ◽

...

Keyword(s):

Cell Division ◽

Cell Biology ◽

Live Cell Imaging ◽

Cell Imaging ◽

Halophilic Archaea ◽

Live Cell ◽

Tight Coupling ◽

Dna Compaction ◽

Cellular Processes ◽

Daughter Cells

Live-cell imaging has revolutionized our understanding of dynamic cellular processes in bacteria and eukaryotes. While similar techniques have recently been applied to the study of halophilic archaea, our ability to explore the cell biology of thermophilic archaea is limited, due to the technical challenges of imaging at high temperatures. Here, we report the construction of the Sulfoscope, a heated chamber that enables live-cell imaging on an inverted fluorescent microscope. Using this system combined with thermostable fluorescent probes, we were able to image Sulfolobus cells as they divide, revealing a tight coupling between changes in DNA compaction, segregation and cytokinesis. By imaging deletion mutants, we observe important differences in the function of the two ESCRTIII proteins recently implicated in cytokinesis. The loss of CdvB1 compromises cell division, causing occasional division failures and fusion of the two daughter cells, whereas the deletion of cdvB2 leads to a profound loss of division symmetry, generating daughter cells that vary widely in size and eventually generating ghost cells. These data indicate that DNA separation and cytokinesis are coordinated in Sulfolobus, as is the case in eukaryotes, and that two contractile ESCRTIII polymers perform distinct roles to ensure that Sulfolobus cells undergo a robust and symmetrical division. Taken together, the Sulfoscope has shown to provide a controlled high temperature environment, in which cell biology of Sulfolobus can be studied in unprecedent details.

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Connecting the dots across time: reconstruction of single-cell signalling trajectories using time-stamped data

Royal Society Open Science ◽

10.1098/rsos.170811 ◽

2017 ◽

Vol 4 (8) ◽

pp. 170811 ◽

Cited By ~ 4

Author(s):

Sayak Mukherjee ◽

David Stewart ◽

William Stewart ◽

Lewis L. Lanier ◽

Jayajit Das

Keyword(s):

Single Cell ◽

Live Cell Imaging ◽

Cell Imaging ◽

Single Cells ◽

Cell Signalling ◽

Live Cell ◽

Multidimensional Space ◽

Matching Problems ◽

Wide Range ◽

Slow Variables

Single-cell responses are shaped by the geometry of signalling kinetic trajectories carved in a multidimensional space spanned by signalling protein abundances. It is, however, challenging to assay a large number (more than 3) of signalling species in live-cell imaging, which makes it difficult to probe single-cell signalling kinetic trajectories in large dimensions. Flow and mass cytometry techniques can measure a large number (4 to more than 40) of signalling species but are unable to track single cells. Thus, cytometry experiments provide detailed time-stamped snapshots of single-cell signalling kinetics. Is it possible to use the time-stamped cytometry data to reconstruct single-cell signalling trajectories? Borrowing concepts of conserved and slow variables from non-equilibrium statistical physics we develop an approach to reconstruct signalling trajectories using snapshot data by creating new variables that remain invariant or vary slowly during the signalling kinetics. We apply this approach to reconstruct trajectories using snapshot data obtained from in silico simulations, live-cell imaging measurements, and, synthetic flow cytometry datasets. The application of invariants and slow variables to reconstruct trajectories provides a radically different way to track objects using snapshot data. The approach is likely to have implications for solving matching problems in a wide range of disciplines.

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Endomitotic MKs Undergo Cortical Contraction during Anaphase but Do Not Complete Furrow Ingression.

Blood ◽

10.1182/blood.v108.11.1124.1124 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1124-1124

Author(s):

Amy Geddis ◽

Norma Fox ◽

Katherine Sear ◽

Eugene Tkachenko ◽

Kenneth Kaushansky

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Shape Change ◽

Control Cell ◽

Live Cell ◽

Cell Cortex ◽

Multipolar Spindle ◽

Dynamic Events ◽

Cell Shape Change ◽

Cortical Contractility

Abstract Megakaryocytes (MKs) develop high degrees of polyploidy in the course of normal maturation through a process called endomitosis. Previous studies have shown that endomitotic MKs abort anaphase after sister chromatid separation, and studies using fixed cells have shown that a midzone is formed in anaphase but that a well-developed cleavage furrow is not. However, studies in fixed cells may miss transient dynamic events that are critical for evaluating potential mechanisms of failed cytokinesis in endomitosis. Therefore we have used live cell imaging as a tool to further define the ability of primary endomitotic MKs to furrow during anaphase. We imaged mouse marrow MKs expressing tubulin fused to YFP, acquiring brightfield and fluorescent images every 2 minutes. Endomitotic cells were identified by the formation of a multipolar spindle, and anaphase was defined as the point at which separation of spindle poles was visible. The extent of cell shape change following anaphase was determined visually and also by measuring the circularity of the cell, with a perfectly round cell having a circularity value of 1. We found that 56% of endomitotic MKs underwent some shape change (n=25), visible as localized indentation, protrusion or elongation of the cell cortex, with an average reduction in circularity of 0.043 occurring a mean of 14.6 minutes after the initiation of anaphase. After an average of 7.8 minutes the cells generally reverted to a round shape without completing furrow ingression. In contrast, a typical mitotic control cell showed a reduction in circularity of 0.443 occurring 8 minutes after the start of anaphase and resulting in the formation of two cells (see Figure 1, representative cells). To determine if the shape change seen in endomitosis after anaphase is dependent on RhoA, we imaged MKs in the presence of the RhoA inhibitor C3 toxin. Although 2 μg/ml C3 toxin blocked cytokinesis in diploid control cells, it did not measurably alter the frequency (44%, n=16) or degree of shape change (0.039) in endomitotic MKs following anaphase, although treatment did appear to delay shape change (onset occurring a mean of 24.8 min after anaphase) and prolong the period of cortical contractility. Therefore although endomitotic MKs exhibit a phase of increased cortical contractility following anaphase, it is not solely dependent on the activity of RhoA and it does not result in sustained furrow ingression. Live cell imaging represents a powerful new tool for dissecting the mechanisms of endomitosis. Figure 1. Endomitotic MKs undergo shape change following anaphase Figure 1. Endomitotic MKs undergo shape change following anaphase

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4D Analyses Show That Replication Compartments Are Clonal Factories in Which Epstein–Barr Viral DNA Amplification Is Coordinated

Proceedings ◽

10.3390/proceedings2020050140 ◽

2020 ◽

Vol 50 (1) ◽

pp. 140

Author(s):

Thejaswi Nagaraju ◽

Arthur Sugden ◽

Bill Sugden

Keyword(s):

Dna Synthesis ◽

Live Cell Imaging ◽

Cell Imaging ◽

Single Cells ◽

Dna Amplification ◽

Live Cell ◽

Computational Simulations ◽

Viral Dna ◽

Barr Virus ◽

Epstein Barr

Most DNA viruses must amplify their DNA to form new viral particles. To kickstart their DNA amplification, herpesviruses alter the host cell cycle dynamics by halting G1/S progression. Soon after, the viruses begin amplifying their DNA and halt any detectable cellular DNA synthesis. Viral DNA amplification takes place in specialized regions of the cell known as replication compartments. The genesis and maturation of replication compartments are not well understood. While replication compartments can only be visualized via microscopy, examining DNA synthetic events requires ensemble approaches. We have therefore exploited single-cell assays, including live-cell imaging, fluorescence in situ hybridization (FISH), and EdU-pulse labeling, in combination with computational simulations and ensemble approaches, to study the role of replication compartments in the DNA amplification of the Epstein–Barr virus (EBV). FISH revealed that each replication compartment initially contained a single DNA molecule which did not travel between compartments. DNA amplification lasted for 13–14 h in single cells, as shown by live cell imaging. Replication compartments eventually grew to occupy 30% of the nucleus, which itself grew by 50%. We found that early in the lytic phase, the availability of DNA templates limited DNA synthesis, while late in the lytic phase, the majority of viral DNA molecules no longer served as templates, which correlated with a drop in the levels of the replication protein. The eventual decline in DNA synthesis did not result from encapsidation; only 1–2% of the viral DNA was encapsidated. The levels of viral DNA synthesis in each compartment were similar. Therefore, the number of compartments determined the total amount of DNA synthesized and, consequently, the levels of amplified DNA. This finding was predicted by computational simulations of the amplification of the two distinct EBV derived replicons that we studied. Our results establish that replication compartments represent clonal factories for DNA amplification that are regulated coordinately during the lytic phase.

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Accurate cell tracking and lineage construction in live-cell imaging experiments with deep learning

10.1101/803205 ◽

2019 ◽

Cited By ~ 8

Author(s):

Erick Moen ◽

Enrico Borba ◽

Geneva Miller ◽

Morgan Schwartz ◽

Dylan Bannon ◽

...

Keyword(s):

Deep Learning ◽

Single Cell ◽

Live Cell Imaging ◽

Cell Tracking ◽

Cell Imaging ◽

Single Cells ◽

Live Cell ◽

Imaging Data ◽

Cell Nuclei ◽

Data Types

AbstractLive-cell imaging experiments have opened an exciting window into the behavior of living systems. While these experiments can produce rich data, the computational analysis of these datasets is challenging. Single-cell analysis requires that cells be accurately identified in each image and subsequently tracked over time. Increasingly, deep learning is being used to interpret microscopy image with single cell resolution. In this work, we apply deep learning to the problem of tracking single cells in live-cell imaging data. Using crowdsourcing and a human-in-the-loop approach to data annotation, we constructed a dataset of over 11,000 trajectories of cell nuclei that includes lineage information. Using this dataset, we successfully trained a deep learning model to perform cell tracking within a linear programming framework. Benchmarking tests demonstrate that our method achieves state-of-the-art performance on the task of cell tracking with respect to multiple accuracy metrics. Further, we show that our deep learning-based method generalizes to perform cell tracking for both fluorescent and brightfield images of the cell cytoplasm, despite having never been trained on those data types. This enables analysis of live-cell imaging data collected across imaging modalities. A persistent cloud deployment of our cell tracker is available at http://www.deepcell.org.

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Cell-ACDC: a user-friendly toolset embedding state-of-the-art neural networks for segmentation, tracking and cell cycle annotations of live-cell imaging data

10.1101/2021.09.28.462199 ◽

2021 ◽

Author(s):

Francesco Padovani ◽

Benedikt Mairhoermann ◽

Pascal Falter-Braun ◽

Jette Lengefeld ◽

Kurt M Schmoller

Keyword(s):

Cell Cycle ◽

Image Analysis ◽

Deep Learning ◽

Live Cell Imaging ◽

Cell Imaging ◽

State Of The Art ◽

Single Cells ◽

Live Cell ◽

Cell Segmentation ◽

User Friendly

Live-cell imaging is a powerful tool to study dynamic cellular processes on the level of single cells with quantitative detail. Microfluidics enables parallel high-throughput imaging, creating a downstream bottleneck at the stage of data analysis. Recent progress on deep learning image analysis dramatically improved cell segmentation and tracking. Nevertheless, manual data validation and correction is typically still required and broadly used tools spanning the complete range of live-cell imaging analysis, from cell segmentation to pedigree analysis and signal quantification, are still needed. Here, we present Cell-ACDC, a user-friendly graphical user-interface (GUI)-based framework written in Python, for segmentation, tracking and cell cycle annotation. We included two state-of-the-art and high-accuracy deep learning models for single-cell segmentation of yeast and mammalian cells implemented in the most used deep learning frameworks TensorFlow and PyTorch. Additionally, we developed and implemented a cell tracking method and embedded it into an intuitive, semi-automated workflow for label-free cell cycle annotation of single cells. 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 or downstream image analysis. Source code: https://github.com/SchmollerLab/Cell_ACDC

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