Time series modeling of live-cell shape dynamics for image-based phenotypic profiling

AbstractLive-cell imaging is a powerful tool for visualization of the spatio-temporal dynamics of living organisms. Although this technique is utilized to visualize nucleocapsid transport in Marburg virus (MARV)- or Ebola virus-infected cells, the experiments require biosafety level-4 (BSL-4) laboratories, which are restricted to trained and authorized individuals. To overcome this limitation, we developed a live-cell imaging system to visualize MARV nucleocapsid-like structures using fluorescence-conjugated viral proteins, which can be conducted outside BSL-4 laboratories. Our experiments revealed that nucleocapsid-like structures have similar transport characteristics to nucleocapsids observed in MARV-infected cells. This system provides a safe platform to evaluate antiviral drugs that inhibit MARV nucleocapsid transport.

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Live cell imaging of meiosis in Arabidopsis thaliana

eLife ◽

10.7554/elife.42834 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 15

Author(s):

Maria A Prusicki ◽

Emma M Keizer ◽

Rik P van Rosmalen ◽

Shinichiro Komaki ◽

Felix Seifert ◽

...

Keyword(s):

Cell Shape ◽

Live Cell Imaging ◽

Cell Imaging ◽

Imaging System ◽

Chromatin Condensation ◽

Live Cell ◽

Meiotic Progression ◽

Cohesin Subunit ◽

Nucleus Position ◽

Imaging Setup

To follow the dynamics of meiosis in the model plant Arabidopsis, we have established a live cell imaging setup to observe male meiocytes. Our method is based on the concomitant visualization of microtubules (MTs) and a meiotic cohesin subunit that allows following five cellular parameters: cell shape, MT array, nucleus position, nucleolus position, and chromatin condensation. We find that the states of these parameters are not randomly associated and identify 11 cellular states, referred to as landmarks, which occur much more frequently than closely related ones, indicating that they are convergence points during meiotic progression. As a first application of our system, we revisited a previously identified mutant in the meiotic A-type cyclin TARDY ASYNCHRONOUS MEIOSIS (TAM). Our imaging system enabled us to reveal both qualitatively and quantitatively altered landmarks in tam, foremost the formation of previously not recognized ectopic spindle- or phragmoplast-like structures that arise without attachment to chromosomes.

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Long-term single-cell imaging and simulations of microtubules reveal principles behind wall patterning during proto-xylem development

Nature Communications ◽

10.1038/s41467-021-20894-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

René Schneider ◽

Kris van’t Klooster ◽

Kelsey L. Picard ◽

Jasper van der Gucht ◽

Taku Demura ◽

...

Keyword(s):

Cell Walls ◽

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell ◽

Quantitative Microscopy ◽

Microtubule Severing ◽

Single Cell Imaging ◽

Spatio Temporal

AbstractPlants are the tallest organisms on Earth; a feature sustained by solute-transporting xylem vessels in the plant vasculature. The xylem vessels are supported by strong cell walls that are assembled in intricate patterns. Cortical microtubules direct wall deposition and need to rapidly re-organize during xylem cell development. Here, we establish long-term live-cell imaging of single Arabidopsis cells undergoing proto-xylem trans-differentiation, resulting in spiral wall patterns, to understand microtubule re-organization. We find that the re-organization requires local microtubule de-stabilization in band-interspersing gaps. Using microtubule simulations, we recapitulate the process in silico and predict that spatio-temporal control of microtubule nucleation is critical for pattern formation, which we confirm in vivo. By combining simulations and live-cell imaging we further explain how the xylem wall-deficient and microtubule-severing KATANIN contributes to microtubule and wall patterning. Hence, by combining quantitative microscopy and modelling we devise a framework to understand how microtubule re-organization supports wall patterning.

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Faculty Opinions recommendation of Time series modeling of live-cell shape dynamics for image-based phenotypic profiling.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726010020.793520747 ◽

2016 ◽

Author(s):

Cynthia Reinhart-King

Keyword(s):

Time Series ◽

Cell Shape ◽

Live Cell ◽

Time Series Modeling ◽

Shape Dynamics

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Fast, volumetric live-cell imaging using high-resolution light-field microscopy

10.1101/439315 ◽

2018 ◽

Author(s):

Haoyu Li ◽

Changliang Guo ◽

Deborah Kim-Holzapfel ◽

Weiyi Li ◽

Yelena Altshuller ◽

...

Keyword(s):

High Resolution ◽

Live Cell Imaging ◽

Light Field ◽

Cell Imaging ◽

Spatial Scales ◽

Functional Brain Imaging ◽

Live Cell ◽

Three Dimensions ◽

Acquisition Time ◽

Spatio Temporal

AbstractVisualizing diverse anatomical and functional traits that span many spatial scales with high spatio-temporal resolution provides insights into the fundamentals of living organisms. Light-field microscopy (LFM) has recently emerged as a scanning-free, scalable method that allows for high-speed, volumetric functional brain imaging. Given those promising applications at the tissue level, at its other extreme, this highly-scalable approach holds great potential for observing structures and dynamics in single-cell specimens. However, the challenge remains for current LFM to achieve subcellular level, near-diffraction-limited 3D spatial resolution. Here, we report high-resolution LFM (HR-LFM) for live-cell imaging with a resolution of 300-700 nm in all three dimensions, an imaging depth of several micrometers, and a volume acquisition time of milliseconds. We demonstrate the technique by imaging various cellular dynamics and structures and tracking single particles. The method may advance LFM as a particularly useful tool for understanding biological systems at multiple spatio-temporal levels.

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The VersaLive platform enables pipette-compatible microfluidic mammalian cell culture for versatile applications

10.1101/2021.07.02.450869 ◽

2021 ◽

Author(s):

Giovanni Marco Nocera ◽

Gaetano Viscido ◽

Simona Brillante ◽

Sabrina Carrella ◽

Diego di Bernardo

Keyword(s):

Cell Culture ◽

Mammalian Cell ◽

Live Cell Imaging ◽

Cell Imaging ◽

Mammalian Cell Culture ◽

Time Lapse ◽

Live Cell ◽

Cell Recovery ◽

Mammalian Cell Lines ◽

Spatio Temporal

Microfluidic-based cell culture allows for precise spatio-temporal regulation of microenvironment, live cell imaging and better recapitulation of physiological conditions, while minimizing reagents consumption. Despite their usefulness, most microfluidic systems are designed with one specific application in mind and require specialized equipment and expertise for their operation. All these requirements prevent microfluidic- based cell culture to be widely adopted. Here, we designed and implemented a versatile and easy-to-use perfusion cell culture microfluidic platform for multiple application (VersaLive) requiring only standard pipettes. Here, we showcase the multiple uses of VersaLive (e.g., time-lapse live cell imaging, immunostaining, cell recovery, cell lysis) on mammalian cell lines and primary cells. VersaLive can replace standard cell culture formats in several applications, thus decreasing costs and increasing reproducibility across laboratories. The layout, documentation and protocols are open-source and available online at https://versalive.tigem.it/.

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Plasmodium berghei kinesin-5 associates with the spindle apparatus during cell division and is important for efficient production of infectious sporozoites

10.1101/2020.07.03.186031 ◽

2020 ◽

Cited By ~ 2

Author(s):

Mohammad Zeeshan ◽

Declan Brady ◽

Rebecca R. Stanway ◽

Carolyn A. Moores ◽

Anthony A. Holder ◽

...

Keyword(s):

Cell Division ◽

Plasmodium Berghei ◽

Live Cell Imaging ◽

Cell Imaging ◽

Subcellular Location ◽

Live Cell ◽

Efficient Production ◽

Spindle Apparatus ◽

Partial Overlap ◽

Fixed Cell

AbstractKinesin-5 motors play essential roles in spindle apparatus assembly during cell division, by generating forces to establish and maintain the spindle bipolarity essential for proper chromosome segregation. Kinesin-5 is largely conserved structurally and functionally in model eukaryotes, but its role is unknown in the Plasmodium parasite, an evolutionarily divergent organism with several atypical features of both mitotic and meiotic cell division. We have investigated the function and subcellular location of kinesin-5 during cell division throughout the Plasmodium berghei life cycle. Deletion of kinesin-5 had little visible effect at any proliferative stage except sporozoite production in oocysts, resulting in a significant decrease in the number of motile sporozoites in mosquito salivary glands, which were able to infect a new vertebrate host. Live-cell imaging showed kinesin-5-GFP located on the spindle and at spindle poles during both atypical mitosis and meiosis. Fixed-cell immunofluorescence assays revealed kinesin-5 co-localized with α-tubulin and centrin-2 and a partial overlap with kinetochore marker NDC80 during early blood stage schizogony. Dual-colour live-cell imaging showed that kinesin-5 is closely associated with NDC80 during male gametogony, but not with kinesin-8B, a marker of the basal body and axonemes of the forming flagella. Treatment of gametocytes with microtubule-specific inhibitors confirmed kinesin-5 association with nuclear spindles and not cytoplasmic axonemal microtubules. Altogether, our results demonstrate that kinesin-5 is associated with the spindle apparatus, expressed in proliferating parasite stages, and important for efficient production of infectious sporozoites.

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Live cell imaging of meiosis in Arabidopsis thaliana - a landmark system

10.1101/446922 ◽

2018 ◽

Cited By ~ 4

Author(s):

Maria Ada Prusicki ◽

Emma Mathilde Keizer ◽

Rik Peter van Rosmalen ◽

Shinichiro Komaki ◽

Felix Seifert ◽

...

Keyword(s):

Cell Shape ◽

Live Cell Imaging ◽

Cell Imaging ◽

Chromatin Condensation ◽

Live Cell ◽

Meiotic Progression ◽

Novel Method ◽

New Criteria ◽

Nucleus Position ◽

Imaging Setup

AbstractMeiosis is essential for sexual reproduction and key to the generation of genetic diversity. To reveal the robustness of meiocyte differentiation and progression through meiosis, we have here established a live cell imaging setup to follow the dynamics of individual male meiocytes in Arabidopsis. Our method is based on the concomitant visualization of microtubules and a meiotic cohesion subunit that allowed following five cellular parameters: cell shape, nucleus position, nucleolus position, chromatin condensation and microtubule array. We find that the states of these parameters are not randomly associated and identify 11 states, referred to as landmarks, that occur much more frequently than closely related states, indicating that they are convergent points of meiotic progression. With this, the here-presented landmark system represents a novel method to analyze meiosis not only allowing a high-temporal dissection but also providing new criteria to evaluate mutants or environmental effects on meiosis.

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