ac501127r_si_002.avi (7.72 MB)
Dynamic Submicroscopic Signaling Zones Revealed by Pair Correlation Tracking and Localization Microscopy
media
posted on 2014-09-02, 00:00 authored by Changjiang You, Christian P. Richter, Sara Löchte, Stephan Wilmes, Jacob PiehlerUnraveling the spatiotemporal organization
of signaling complexes
within the context of plasma membrane nanodomains has remained a highly
challenging task. Here, we have applied super-resolution image correlation
based on tracking and localization microscopy (TALM) for probing transient
confinement as well as ligand binding and intracellular effector recruitment
of the type I interferon (IFN) receptor in the plasma membrane of
live cells. Ligand and receptor were labeled with monofunctional quantum
dots, thus allowing long-term tracking with very high spatial and
temporal resolution without an artificial receptor cross-linking at
the cell surface. Dual-color TALM was employed for visualizing protein–protein
interactions involved in IFN signaling at both sides of the plasma
membrane with high spatial and temporal resolution. By pair correlation
analyses based on time-lapse TALM images (pcTALM), complex assembly
within dynamic submicroscopic zones was identified. Strikingly, recruitment
of the IFN effector protein signal transducer and activator of transcription
2 (STAT2) into these dynamic signaling zones could be observed. The
results suggest that confined diffusion zones in the plasma membrane
are employed as transient platforms for the assembly of signaling
complexes.