Macropinosome formation by tent pole ruffling in macrophages

Nicholas D Condon1, John M Heddleston2, Teng-Leong Chew2

  • 1Institute for Molecular Bioscience (IMB) and IMB Centre for Inflammation and Disease Research, The University of Queensland, Brisbane, Australia.

Insights

Macrophages use F-actin tent poles to form large macropinosomes during innate immune responses. This process, crucial for pathogen detection, involves Rab13 and is visualized with lattice light sheet microscopy.

Area of Science:

  • Cell Biology
  • Immunology
  • Microscopy

Background:

  • Macrophages are key innate immune cells involved in pathogen recognition and clearance.
  • Macropinocytosis, a process involving large membraneപ്പം engulfments, is critical for macrophage environmental sampling.
  • Bacterial lipopolysaccharide (LPS) is a potent activator of macrophages, inducing surface ruffling and macropinosome formation.

Purpose of the Study:

  • To redefine the mechanisms of dorsal ruffle formation and macropinosome closure in LPS-activated macrophages.
  • To investigate the role of F-actin and Rab13 in macropinosome biogenesis.
  • To leverage high-resolution live imaging to visualize dynamic cellular processes.

Main Methods:

  • Lattice light sheet microscopy (LLSM) for high-resolution, live imaging of macrophages.
  • Bacterial lipopolysaccharide (LPS) stimulation of primary macrophages.
  • CRISPR-Cas9 gene editing to delete Rab13.
  • Fluorescence microscopy to visualize F-actin dynamics and Rab13 localization.

Main Results:

  • LLSM revealed F-actin tent poles supporting dorsal ruffles that constrict to form macropinosomes.
  • A novel model of macropinosome formation involving F-actin tent pole crossover and twisting was proposed.
  • Active Rab13 was enriched on tent pole ruffles.
  • CRISPR deletion of Rab13 disrupted tent pole ruffles and blocked large LPS-induced macropinosome formation.

Conclusions:

  • The study redefines macropinosome formation, highlighting the role of F-actin tent poles and Rab13.
  • LPS-induced macropinosome formation relies on a dynamic interplay of cytoskeletal rearrangements and Rab13.
  • LLSM provides unprecedented insights into the spatiotemporal dynamics of innate immune cell processes.

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