Figure 6.

PHB and ZPR3 Form Tetrameric Complexes.

(A) Overlaid TIRF images of YFP (green) and mCherry (magenta) channels taken from the same region of the slide (left column) show pronounced colocalization of PHB-YFP and ZPR3-mCherry (white spots in Overlay panels), compared with overlaid TIRF images obtained from random, nonoverlapping regions of the slide (middle column) or the same region of an AS2-YFP + ZPR3-mCherry SiMPull slide (right column). Bars = 5 µm.

(B) The frequency of PHB-YFP and ZPR3-mCherry colocalization is significantly higher (P < 10⁻²⁴) for overlaid TIRF images taken from the same region of a PHB-YFP + ZPR3-mCherry SiMPull slide compared with controls. Colocalization frequencies (means ± se) are calculated from at least 30 images, from three independent biological replicates. Student’s t test: ***P < 0.001.

(C) Photobleaching analysis of 2006 single PHB-YFP:ZPR3-mCherry complexes reveals photobleaching step distributions occur at frequencies expected for a tetrameric complex comprised of two YFP and two mCherry fluorophores (black dots). Values (means ± se) are calculated from six independent biological replicates.