Dynamics and Patterning of 5-Hydroxytryptamine 2 Subtype Receptors in JC Polyomavirus Entry
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells, Viruses, Antibodies, and Reagents
2.2. Generation of 5-HT2R-Dendra2 Fusion Constructs
2.3. Generation of HEK293A Stable Cell Lines for 5-HT2R-Dendra2
2.4. Indirect Immunofluorescence Detection and Quantification of JCPyV Infection
2.5. Cell-Surface Expression of 5-HT2R-Dendra2 Fusion Proteins in HEK293A Stable Cells
2.6. Analysis of JCPyV Attachment in Stable Cells
2.7. Quantification of JCPyV Entry in Stable Cells
2.8. Preparing Samples for Two-Color FPALM Experiment
2.9. Super-Resolution Fluorescence Photoactivation Localization Microscopy (FPALM)
2.10. Glucose Oxidase Imaging Buffer
2.11. Multicolor Widefield FPALM Imaging of JCPyV and Mock Infected Samples
3. Results
3.1. 5-HT2R-Dendra2 Forms Clusters on the Cell Surface of Stably Transfected Cells
3.2. Cell-Surface Characterization of 5-HT2Rs Using FPALM-TIRF Microscopy
3.3. Manders’ Colocalization Analysis: 5-HT2Rs Colocalize with JCPyV during Virus Entry
3.4. JCPyV Attachment Is Not Enhanced in Cells Expressing 5-HT2 Receptors
3.5. JCPyV Entry Is Enhanced in 5-HT2R-Expressing Cells
3.6. JCPyV Changes Cluster Properties for 5-HT2 Receptor Subtypes in Infected Cells
3.7. Radial Distribution Function Analysis: JCPyV Aggregation within and Adjacent to 5-HT2 Receptor Clusters
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Time Point | Cell Line | Manders’ Coefficient | Standard Error of Mean | % Difference with DRD2 Dendra2 (± Error) | p Value |
---|---|---|---|---|---|
5 mpi | 2A Dendra2 | 0.766 | 0.020 | 58 (±6) | <0.001 |
2B Dendra2 | 0.703 | 0.024 | 45 (±6) | <0.001 | |
2C Dendra2 | 0.823 | 0.022 | 69 (±6) | <0.001 | |
DRD2 Dendra2 | 0.486 | 0.026 | |||
15 mpi | 2A Dendra2 | 0.810 | 0.025 | 0.9 (±5) | >0.05 |
2B Dendra2 | 0.756 | 0.024 | −6 (±5) | >0.05 | |
2C Dendra2 | 0.886 | 0.016 | 10 (±4) | >0.05 | |
DRD2 Dendra2 | 0.803 | 0.027 |
Percent Difference | 2A Dendra2 | 2B Dendra2 | 2C Dendra2 | DRD2 Dendra2 | |
---|---|---|---|---|---|
5 mpi | 2A Dendra2 | 9% (±4%) | −7% (±4%) | 58% (±6%) | |
2B Dendra2 | −8% (±4%) | −14% (±4%) | 45% (±6%) | ||
2C Dendra2 | 7% (±4%) | 17% (±4%) | 69% (±6%) | ||
DRD2 Dendra2 | −36% (±6%) | −31% (±6%) | −41% (±6%) | ||
15 mpi | 2A Dendra2 | 7% (±4%) | −9% (±4%) | 0.9% (±5%) | |
2B Dendra2 | −7% (±4%) | −14% (±4%) | −6% (±5%) | ||
2C Dendra2 | 9% (±5%) | 17% (±4%) | 10% (±4%) | ||
DRD2 Dendra2 | −1% (± 5%) | 6% (±5%) | −9% (±4%) |
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Mehmood, K.; Wilczek, M.P.; DuShane, J.K.; Parent, M.T.; Mayberry, C.L.; Wallace, J.N.; Levasseur, F.L.; Fong, T.M.; Hess, S.T.; Maginnis, M.S. Dynamics and Patterning of 5-Hydroxytryptamine 2 Subtype Receptors in JC Polyomavirus Entry. Viruses 2022, 14, 2597. https://doi.org/10.3390/v14122597
Mehmood K, Wilczek MP, DuShane JK, Parent MT, Mayberry CL, Wallace JN, Levasseur FL, Fong TM, Hess ST, Maginnis MS. Dynamics and Patterning of 5-Hydroxytryptamine 2 Subtype Receptors in JC Polyomavirus Entry. Viruses. 2022; 14(12):2597. https://doi.org/10.3390/v14122597
Chicago/Turabian StyleMehmood, Kashif, Michael P. Wilczek, Jeanne K. DuShane, Matthew T. Parent, Colleen L. Mayberry, Jaqulin N. Wallace, Francois L. Levasseur, Tristan M. Fong, Samuel T. Hess, and Melissa S. Maginnis. 2022. "Dynamics and Patterning of 5-Hydroxytryptamine 2 Subtype Receptors in JC Polyomavirus Entry" Viruses 14, no. 12: 2597. https://doi.org/10.3390/v14122597
APA StyleMehmood, K., Wilczek, M. P., DuShane, J. K., Parent, M. T., Mayberry, C. L., Wallace, J. N., Levasseur, F. L., Fong, T. M., Hess, S. T., & Maginnis, M. S. (2022). Dynamics and Patterning of 5-Hydroxytryptamine 2 Subtype Receptors in JC Polyomavirus Entry. Viruses, 14(12), 2597. https://doi.org/10.3390/v14122597