研究目的
Investigating the dynamics and mechanisms of HIV-1 Gag protein assembly at the plasma membrane of host CD4+ T cells using single-molecule localisation microscopy.
研究成果
The study provides quantitative insights into the dynamics of HIV-1 Gag protein assembly at the plasma membrane of host CD4+ T cells. It was found that VLP formation lasts roughly 15 minutes with an assembly time of 5 minutes. The viral RNA genome acts as a spatiotemporal coordinator of the membrane assembly process rather than increasing the attraction of Gag at the membrane towards the assembling site.
研究不足
The study is limited by the technical constraints of live PALM imaging, including the spatial and temporal resolution. Additionally, the analysis is focused on membrane-associated Gag proteins, and cytosolic Gag proteins could not be monitored by this approach.
1：Experimental Design and Method Selection:
Live photoactivated localisation microscopy (PALM) was used to monitor VLP formation at the plasma membrane of host CD4+ T cells. The study involved the analysis of long duration recordings of single-molecule Gag protein localisation and movement.
2：Sample Selection and Data Sources:
Jurkat T cells (a human T-cell leukaemia cell line) were used as the host cells. HIV-1 Gag and assembly defective mutants that harbour the mEOS2 fluorescent protein were generated and characterised for cell expression and VLP production.
3：List of Experimental Equipment and Materials:
Inverted motorised microscope (Nikon Ti) equipped with a 100 × 1.45 NA PL-APO objective, 405 nm laser (Omicron), 561 nm laser (Cobolt JiveTM), EMCCD camera (Evolve, Photometric), and MetaMorph software (Molecular Devices).
4：45 NA PL-APO objective, 405 nm laser (Omicron), 561 nm laser (Cobolt JiveTM), EMCCD camera (Evolve, Photometric), and MetaMorph software (Molecular Devices).
Experimental Procedures and Operational Workflow:
4. Experimental Procedures and Operational Workflow: Cells expressing mEOS2-tagged Gag molecules were photoactivated using a 405 nm laser, and the resulting photoconverted single-molecule fluorescence was excited with a 561 nm laser. Fluorescence signals were collected and analysed online with laser feedback to ensure the optimal and constant number of localisations during acquisition.
5：Data Analysis Methods:
The data were analysed using Bayesian inference and the modified Langevin equation to quantify the motion of individual Gag molecules. Temporal changes in VLP dynamics were monitored by time windowing.
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