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Histochemistry and Cell Biology

, Volume 139, Issue 1, pp 173–179 | Cite as

Single-molecule coordinate-based analysis of the morphology of HIV-1 assembly sites with near-molecular spatial resolution

  • Sebastian Malkusch
  • Walter Muranyi
  • Barbara Müller
  • Hans-Georg Kräusslich
  • Mike Heilemann
Original Paper

Abstract

We apply single-molecule super-resolution microscopy and coordinate-based cluster analysis to extract information on the distribution and on the morphology and size of clusters of the human immunodeficiency virus (HIV-1) Gag polyprotein in fixed cells. Three different patterns of Gag distribution could be distinguished. A major type of assembly observed was in accordance with previous electron microscopy analyses revealing ~140 nm-sized HIV-1 buds at the plasma membrane of virus-producing cells. The distribution of Gag molecules in the 2D projection at these sites was consistent with a semi-spherical 3D assembly. We compared different methods of cluster analysis and demonstrated that we can reliably distinguish different distribution patterns of the Gag polyprotein. These methods were applied to extract information on the properties of the different Gag clusters.

Keywords

Super-resolution microscopy Single-molecule fluorescence microscopy Cluster analysis Cellular structures Human immunodeficiency virus Gag 

Abbreviations

CA

Capsid domain

dSTORM

Direct stochastic optical reconstruction microscopy

EMCCD

Electron-multiplying charge device

HIV-1

Human immunodeficiency virus type 1

MA

Matrix domain

MEA

Mercaptoethylamine

NC

Nucleocapsid domains

ROI

Region of interest

Ryr

Ryanodine receptors

TIRF

Total internal reflection fluorescence

Notes

Acknowledgments

This work was supported by the Systems Biology Initiative (FORSYS) of the German Ministry of Research and Education (BMBF), Project VIROQUANT and Grant No. 0315262.

Supplementary material

418_2012_1014_MOESM1_ESM.docx (1.6 mb)
Supplementary material 1 (DOCX 1617 kb)

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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Sebastian Malkusch
    • 1
  • Walter Muranyi
    • 2
  • Barbara Müller
    • 2
  • Hans-Georg Kräusslich
    • 2
  • Mike Heilemann
    • 1
    • 3
  1. 1.Biotechnology and BiophysicsJulius-Maximilians-University WürzburgWürzburgGermany
  2. 2.Department of Infectious Diseases, VirologyUniversity Hospital HeidelbergHeidelbergGermany
  3. 3.BIOQUANT CentreUniversity of HeidelbergHeidelbergGermany

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