Investigation of podosome ring protein arrangement using localization microscopy images

Methods. 2017 Feb 15:115:9-16. doi: 10.1016/j.ymeth.2016.11.005. Epub 2016 Nov 10.

Abstract

Podosomes are adhesive structures formed on the plasma membrane abutting the extracellular matrix of macrophages, osteoclasts, and dendritic cells. They consist of an f-actin core and a ring structure composed of integrins and integrin-associated proteins. The podosome ring plays a major role in adhesion to the underlying extracellular matrix, but its detailed structure is poorly understood. Recently, it has become possible to study the nano-scale structure of podosome rings using localization microscopy. Unlike traditional microscopy images, localization microscopy images are reconstructed using discrete points, meaning that standard image analysis methods cannot be applied. Here, we present a pipeline for podosome identification, protein position calculation, and creating a podosome ring model for use with localization microscopy data.

Keywords: Image analysis; Localization microscopy; Pattern recognition; Podosomes; Super-resolution microscopy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actin Cytoskeleton / metabolism
  • Actin Cytoskeleton / ultrastructure*
  • Carbocyanines / chemistry
  • Cell Movement
  • Cells, Cultured
  • Dendritic Cells / metabolism
  • Dendritic Cells / ultrastructure
  • Extracellular Matrix / metabolism
  • Extracellular Matrix / ultrastructure*
  • Fibroblasts / metabolism
  • Fibroblasts / ultrastructure
  • Fluorescent Dyes / chemistry
  • Gene Expression
  • Genes, Reporter
  • Humans
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Macrophages / metabolism
  • Macrophages / ultrastructure*
  • Microscopy, Fluorescence / methods*
  • Osteoclasts / metabolism
  • Osteoclasts / ultrastructure
  • Paxillin / genetics
  • Paxillin / metabolism
  • Podosomes / metabolism
  • Podosomes / ultrastructure*
  • Red Fluorescent Protein
  • Staining and Labeling / methods
  • Talin / genetics
  • Talin / metabolism
  • Vinculin / genetics
  • Vinculin / metabolism

Substances

  • Alexa Fluor 647
  • Carbocyanines
  • Fluorescent Dyes
  • Luminescent Proteins
  • PXN protein, human
  • Paxillin
  • Talin
  • VCL protein, human
  • cyanine dye 3
  • Vinculin