Dataset Information

Cost-effective high-speed, three-dimensional live-cell imaging of HIV-1 transfer at the T cell virological synapse.

ABSTRACT: The availability of cost-effective, highly portable, and easy to use high-resolution live-cell imaging systems could present a significant technological break-through in challenging environments, such as high-level biosafety laboratories or sites where new viral outbreaks are suspected. We describe and demonstrate a cost-effective high-speed fluorescence microscope enabling the live tracking of virus particles across virological synapses that form between infected and uninfected T cells. The dynamics of HIV-1 proteins studied at the cellular level and the formation of virological synapses in living T cells reveals mechanisms by which cell-cell interactions facilitate infection between immune cells. Dual-color 3D fluorescence deconvolution microscopy of HIV-1 particles at frames rates of 100 frames per second allows us to follow the transfer of HIV-1 particles across the T cell virological synapse between living T cells. We also confirm the successful transfer of virus by imaging T cell samples fixed at specific time points during cell-cell virus transfer by super-resolution structured illumination microscopy.

SUBMITTER: Sandmeyer A

PROVIDER: S-EPMC9663902 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Cost-effective high-speed, three-dimensional live-cell imaging of HIV-1 transfer at the T cell virological synapse.

Sandmeyer Alice A Wang Lili L Hübner Wolfgang W Müller Marcel M Chen Benjamin K BK Huser Thomas T

iScience 20221029 11

The availability of cost-effective, highly portable, and easy to use high-resolution live-cell imaging systems could present a significant technological break-through in challenging environments, such as high-level biosafety laboratories or sites where new viral outbreaks are suspected. We describe and demonstrate a cost-effective high-speed fluorescence microscope enabling the live tracking of virus particles across virological synapses that form between infected and uninfected T cells. The dyn ...[more]

PMID: 36388970

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Project description:Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1. The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones, forming foci within 5-15 minutes. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis. In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

Dataset Information

Cost-effective high-speed, three-dimensional live-cell imaging of HIV-1 transfer at the T cell virological synapse.

Publications

Cost-effective high-speed, three-dimensional live-cell imaging of HIV-1 transfer at the T cell virological synapse.

Similar Datasets

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