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Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface
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Adhesion force and attachment lifetime of the KIF16B-PX domain interaction with lipid membranes

Serapion Pyrpassopoulos1, Henry Shuman1, E Michael Ostap2

  • 1Pennsylvania Muscle Institute, Department of Physiology, and Center for Engineering Mechanobiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104.

Molecular Biology of the Cell
|September 22, 2017
PubMed

Insights

The PX domain of KIF16B acts as a robust mechanical anchor, mediating strong adhesion to early endosomes via PI(3)P interactions. This binding is crucial for the motor protein

Area of Science:

  • Cell Biology
  • Molecular Motors
  • Membrane Trafficking

Background:

  • Kinesin family member 16B (KIF16B) is essential for early endosome trafficking and tubulation.
  • KIF16B utilizes its C-terminal PX domain for attachment to lipid cargoes, specifically binding phosphatidylinositol-3-phosphate (PI[3]P) with nanomolar affinity.

Purpose of the Study:

  • To quantify the adhesion forces and detachment kinetics of the KIF16B PX domain interacting with PI[3]P-containing membranes.
  • To determine the mechanical properties of the PX domain as a cargo-binding anchor for KIF16B.

Main Methods:

  • Utilized optical tweezers to measure single PX domain adhesion forces and detachment kinetics.
  • Studied interactions with artificial membranes composed of PI[3]P and phosphatidylcholine at varying loading rates.

Main Results:

  • Single PX domain adhesion forces ranged from 19 to 54 pN across loading rates of 80–1500 pN/s.
  • Adhesion strength exceeded that of pleckstrin homology domains binding to PI(4,5)P2, attributed to hydrophobic residue membrane insertion and electrostatic PI[3]P interactions.
  • Attachment lifetime decreased with increasing force, consistent with slip-bond behavior.

Conclusions:

  • The KIF16B PX domain exhibits strong membrane adhesion, facilitated by both electrostatic and hydrophobic interactions.
  • The measured attachment lifetimes are well-matched to KIF16B's processive motility, confirming its role as an effective mechanical anchor for intracellular transport.

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