Quantitative live-cell imaging reveals spatio-temporal dynamics and cytoplasmic assembly of the 26S proteasome

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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A live-cell imaging system for visualizing the transport of Marburg virus nucleocapsid-like structures

Virology Journal ◽

10.1186/s12985-019-1267-9 ◽

2019 ◽

Vol 16 (1) ◽

Cited By ~ 1

Author(s):

Yuki Takamatsu ◽

Olga Dolnik ◽

Takeshi Noda ◽

Stephan Becker

Keyword(s):

Live Cell Imaging ◽

Intracellular Transport ◽

Actin Polymerization ◽

Ebola Virus ◽

Cell Imaging ◽

Temporal Dynamics ◽

Imaging System ◽

Marburg Virus ◽

Live Cell ◽

Infected Cells

Abstract Background Live-cell imaging is a powerful tool for visualization of the spatio-temporal dynamics of moving signals in living cells. Although this technique can be 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. Methods 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. Results Our experiments revealed that nucleocapsid-like structures have similar transport characteristics to those of nucleocapsids observed in MARV-infected cells, both of which are mediated by actin polymerization. Conclusions We developed a non-infectious live cell imaging system to visualize intracellular transport of MARV nucleocapsid-like structures. This system provides a safe platform to evaluate antiviral drugs that inhibit MARV nucleocapsid transport.

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Spatio-temporal modeling and live-cell imaging of proteolysis in the 4D microenvironment of breast cancer

Cancer and Metastasis Reviews ◽

10.1007/s10555-019-09810-8 ◽

2019 ◽

Vol 38 (3) ◽

pp. 445-454 ◽

Cited By ~ 3

Author(s):

Kyungmin Ji ◽

Mansoureh Sameni ◽

Kingsley Osuala ◽

Kamiar Moin ◽

Raymond R. Mattingly ◽

...

Keyword(s):

Breast Cancer ◽

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell ◽

Temporal Modeling ◽

Spatio Temporal

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Live cell imaging of nuclear actin filaments and heterochromatic repair foci in Drosophila and mouse cells

10.7287/peerj.preprints.27900v1 ◽

2019 ◽

Cited By ~ 1

Author(s):

Colby See ◽

Deepak Arya ◽

Emily Lin ◽

Irene Chiolo

Keyword(s):

Image Processing ◽

Dna Sequences ◽

Actin Filaments ◽

Live Cell Imaging ◽

Cell Imaging ◽

Temporal Dynamics ◽

Nuclear Architecture ◽

Live Cell ◽

Nuclear Actin ◽

Mouse Cells

Pericentromeric heterochromatin largely comprises repeated DNA sequences prone to aberrant recombination during double-strand break (DSB) repair. Studies in Drosophila and mouse cells revealed that ‘safe’ homologous recombination (HR) repair of these sequences relies on the relocalization of repair sites to outside the heterochromatin domain before Rad51 recruitment. Relocalization requires a striking network of nuclear actin filaments (F-actin) and myosins generating directed motions. Understanding this pathway requires the ability to detect nuclear actin filaments that are significantly less abundant than cytoplasmic filaments, and to image and track repair sites for long time periods. Here we describe an optimized protocol for live cell imaging of nuclear F-actin in response to IR in Drosophila cells, and for repair focus tracking in mouse cells, including imaging setup, image processing approaches, and analytical methods. We emphasize approaches that can be applied to identify the most effective fluorescent markers for live cell imaging, strategies to minimize photobleaching and phototoxicity with a DeltaVision deconvolution microscope, and image processing and analysis methods using SoftWoRx and Imaris software. These approaches enable a deeper understanding of the spatial and temporal dynamics of heterochromatin repair and have broad applicability in the fields of nuclear architecture, nuclear dynamics, and DNA repair.

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Revealing spatio-temporal dynamics with long-term trypanosomatid live-cell imaging

10.1101/2021.09.15.460474 ◽

2021 ◽

Author(s):

Richard S Muniz ◽

Paul C Campbell ◽

Thomas E Sladewski ◽

Lars D Renner ◽

Christopher L de Graffenried

Keyword(s):

Cell Division ◽

Live Cell Imaging ◽

Cell Imaging ◽

Temporal Dynamics ◽

Live Cell ◽

Three Dimensions ◽

Imaging Method ◽

Differential Rate ◽

Pdms Stamp

Trypanosoma brucei, the causative agent of human African trypanosomiasis, employs a flagellum for dissemination within the parasite's mammalian and insect hosts. T. brucei cells are highly motile in culture and must be able to move in all three dimensions for reliable cell division. These characteristics have made long-term microscopic imaging of live T. brucei cells challenging, which has limited our understanding of a variety of important cell-cycle events. To address this issue, we have devised an imaging approach that confines cells to small volumes that can be imaged continuously for up to 24 h. This system employs cast agarose microwells generated using a PDMS stamp that can be made with different dimensions to maximize cell viability and imaging quality. Using this approach, we have imaged individual T. brucei through multiple rounds of cell division with high spatial and temporal resolution. We have employed this method to study the differential rate of T. brucei daughter cell division and show that the approach is compatible with loss-of-function experiments such as small molecule inhibition and RNAi. We have also developed a strategy that employs in-well "sentinel" cells to monitor potential toxicity due to imaging. This live-cell imaging method will provide a novel avenue for studying a wide variety of cellular events in trypanosomatids that have previously been inaccessible.

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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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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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Live cell imaging of nuclear actin filaments and heterochromatic repair foci in Drosophila and mouse cells

10.7287/peerj.preprints.27900 ◽

2019 ◽

Author(s):

Colby See ◽

Deepak Arya ◽

Emily Lin ◽

Irene Chiolo

Keyword(s):

Image Processing ◽

Dna Sequences ◽

Actin Filaments ◽

Live Cell Imaging ◽

Cell Imaging ◽

Temporal Dynamics ◽

Nuclear Architecture ◽

Live Cell ◽

Nuclear Actin ◽

Mouse Cells

Pericentromeric heterochromatin largely comprises repeated DNA sequences prone to aberrant recombination during double-strand break (DSB) repair. Studies in Drosophila and mouse cells revealed that ‘safe’ homologous recombination (HR) repair of these sequences relies on the relocalization of repair sites to outside the heterochromatin domain before Rad51 recruitment. Relocalization requires a striking network of nuclear actin filaments (F-actin) and myosins generating directed motions. Understanding this pathway requires the ability to detect nuclear actin filaments that are significantly less abundant than cytoplasmic filaments, and to image and track repair sites for long time periods. Here we describe an optimized protocol for live cell imaging of nuclear F-actin in response to IR in Drosophila cells, and for repair focus tracking in mouse cells, including imaging setup, image processing approaches, and analytical methods. We emphasize approaches that can be applied to identify the most effective fluorescent markers for live cell imaging, strategies to minimize photobleaching and phototoxicity with a DeltaVision deconvolution microscope, and image processing and analysis methods using SoftWoRx and Imaris software. These approaches enable a deeper understanding of the spatial and temporal dynamics of heterochromatin repair and have broad applicability in the fields of nuclear architecture, nuclear dynamics, and DNA repair.

Download Full-text