Super-Resolution Microscopy and Particle-Tracking Approaches for the Study of Vesicular Trafficking in Primary Neutrophils

  • Jennifer L. Johnson
  • Kersi Pestonjamasp
  • William B. Kiosses
  • Sergio D. CatzEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2233)


Neutrophils are short-lived cells after isolation. The analysis of neutrophil vesicular trafficking requires rapid and gentle handling. Recently developed super-resolution microscopy technologies have generated unparalleled opportunities to help understand the molecular mechanisms regulating neutrophil vesicular trafficking, exocytosis, and associated functions at the molecular level. Here, we describe super-resolution and total internal reflection fluorescence (TIRF) microscopy approaches for the analysis of vesicular trafficking and associated functions of primary neutrophils.

Key words

Total internal reflection fluorescence (TIRF) Vesicular trafficking Neutrophils 


  1. 1.
    Ramadass M, Catz SD (2016) Molecular mechanisms regulating secretory organelles and endosomes in neutrophils and their implications for inflammation. Immunol Rev 273:249–265CrossRefGoogle Scholar
  2. 2.
    Johnson JL, Ramadass M, Haimovich A, McGeough MD, Zhang J, Hoffman HM, Catz SD (2017) Increased neutrophil secretion induced by NLRP3 mutation links the Inflammasome to Azurophilic granule exocytosis. Front Cell Infect Microbiol 7:507CrossRefGoogle Scholar
  3. 3.
    Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3:793–795CrossRefGoogle Scholar
  4. 4.
    Huang B, Jones SA, Brandenburg B, Zhuang X (2008) Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution. Nat Methods 5:1047–1052CrossRefGoogle Scholar
  5. 5.
    Huang B, Wang W, Bates M, Zhuang X (2008) Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319:810–813CrossRefGoogle Scholar
  6. 6.
    Johnson JL, Monfregola J, Napolitano G, Kiosses WB, Catz SD (2012) Vesicular trafficking through cortical actin during exocytosis is regulated by the Rab27a effector JFC1/Slp1 and the RhoA-GTPase-activating protein Gem-interacting protein. Mol Biol Cell 23:1902–1916CrossRefGoogle Scholar
  7. 7.
    Johnson JL, He J, Ramadass M, Pestonjamasp K, Kiosses WB, Zhang J, Catz SD (2016) Munc13-4 is a Rab11-binding protein that regulates Rab11-positive vesicle trafficking and docking at the plasma membrane. J Biol Chem 291:3423–3438CrossRefGoogle Scholar
  8. 8.
    Zhang J, Johnson JL, He J, Napolitano G, Ramadass M, Rocca C, Kiosses WB, Bucci C, Xin Q, Gavathiotis E, Cuervo AM, Cherqui S, Catz SD (2017) Cystinosin, the small GTPase Rab11, and the Rab7 effector RILP regulate intracellular trafficking of the chaperone-mediated autophagy receptor LAMP2A. J Biol Chem 292:10328–10346CrossRefGoogle Scholar
  9. 9.
    Johnson JL, Hong H, Monfregola J, Kiosses WB, Catz SD (2011) Munc13-4 restricts motility of Rab27a-expressing vesicles to facilitate lipopolysaccharide-induced priming of exocytosis in neutrophils. J Biol Chem 286:5647–5656CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  • Jennifer L. Johnson
    • 1
  • Kersi Pestonjamasp
    • 1
  • William B. Kiosses
    • 2
  • Sergio D. Catz
    • 1
    Email author
  1. 1.Department of Molecular MedicineThe Scripps Research InstituteLa JollaUSA
  2. 2.Department of Vascular and Cell BiologyLa Jolla Institute for ImmunologyLa JollaUSA

Personalised recommendations