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Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis

Nature Methods volume 8, pages 969–975 (2011)Cite this article

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Abstract

Photoactivated localization microscopy (PALM) is a powerful approach for investigating protein organization, yet tools for quantitative, spatial analysis of PALM datasets are largely missing. Combining pair-correlation analysis with PALM (PC-PALM), we provide a method to analyze complex patterns of protein organization across the plasma membrane without determination of absolute protein numbers. The approach uses an algorithm to distinguish a single protein with multiple appearances from clusters of proteins. This enables quantification of different parameters of spatial organization, including the presence of protein clusters, their size, density and abundance in the plasma membrane. Using this method, we demonstrate distinct nanoscale organization of plasma-membrane proteins with different membrane anchoring and lipid partitioning characteristics in COS-7 cells, and show dramatic changes in glycosylphosphatidylinositol (GPI)-anchored protein arrangement under varying perturbations. PC-PALM is thus an effective tool with broad applicability for analysis of protein heterogeneity and function, adaptable to other single-molecule strategies.

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Figure 1: Single molecules appear multiple times with variable blinking intervals.
Figure 2: PC-PALM distinguishes random versus clustered distributions.
Figure 3: Steady-state spatial distribution of plasma-membrane proteins assessed by PC-PALM.
Figure 4: Reorganization of PAGFP-GPI induced by perturbations of plasma membrane evaluated by PC-PALM.
Figure 5: Actin-PAmCh localizes with clusters of antibody–cross-linked PAGFP-GPI.

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Acknowledgements

We thank G. Patterson (US National Institute of Biomedical Imaging and Bioengineering) for providing plasmid constructs; E. Ambroggio (US National Institute of Child Health and Development) for providing purified PAGFP protein; H. Hess and G. Stengel for providing the Peak Selector software and valuable discussion; S. Manley for help in setting up the PALM microscope; B. Baird and D. Holowka for valuable discussions. S.L.V. was supported by US National Institutes of Health grant R00GM87810.

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Author notes

  1. Tijana Jovanovic-Talisman

    Present address: Present address: Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii, USA.,

  2. Prabuddha Sengupta and Tijana Jovanovic-Talisman: These authors contributed equally to this work.

Authors and Affiliations

  1. The Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA

    Prabuddha Sengupta, Tijana Jovanovic-Talisman, Dunja Skoko, Malte Renz & Jennifer Lippincott-Schwartz

  2. Department of Biophysics, University of Michigan, Ann Arbor, Michigan, USA

    Sarah L Veatch

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  1. Prabuddha Sengupta
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Contributions

P.S. and T.J-T. conceived and designed experiments, developed experimental techniques, performed experiments, developed the analytical method, analyzed data and wrote the paper; D.S. helped develop the analytical method; M.R. contributed to characterization of PA-FP and imaging regime; S.L.V. contributed equations used to analyze data; J.L-S. conceived and designed the experiments, and wrote the paper.

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Correspondence to Jennifer Lippincott-Schwartz.

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The authors declare no competing financial interests.

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Sengupta, P., Jovanovic-Talisman, T., Skoko, D. et al. Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis. Nat Methods 8, 969–975 (2011). https://doi.org/10.1038/nmeth.1704

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