φ::pif - Phytochrome Interaction Movies
Abbreviations
ΦB - A. thaliana PhytochromeB (residues 1-908 = CBD+PHY+2xPAS domains)
pif6 - A. thaliana PIF6 (residues 1-100 = APB domain)


Movies Demonstrating Temporal Control (Spinning Disk Confocal)


1 YFP membrane recruitment Cytosolic pif6YFP is recruited onto the membrane by ΦB under the action of 650nm light. A confocal slice of a NIH3T3 cell is shown.


2 YFP membrane dissociation Membrane-bound pif6YFP is released back to the cytosol by ΦB under the action of >750nm light. A confocal slice of a NIH3T3 cell is shown.


3 oscillating YFP translocation The robustness of the system is shown by repeated rounds of recruitment and release of pif6YFP to and from the membrane by ΦB under action of oscillating 650nm and IR (>750nm) light.


Movies Demonstrating Spatiotemporal Control (TIRF Microscopy)


4 YFP point recruitment pif6YFP is recruited to a single, moving point by action of a focused 650nm laser with global inhibition by >750nm irradiation. The membrane of a NIH3T3 cell is shown using TIRF microscopy.


5 patterned recruitment of YFP pif6YFP is recruited in a patterned fashion to the plasma membrane. Sequential frames of a movie of a “glider” pattern from the “game of life” cellular automaton is projected onto the membrane as an inverse R/IR light distribution. The membrane of a NIH3T3 cell is shown using TIRF microscopy.


6 Titration of pif-YFP recruitment by tuning 650nm ratio By "dithering" the red pattern used to activate a small patch of phytochromes on a NIH3T3 cell we are able to tune the relative amount of activated phytochrome and thus tunably regulate the relative degree of recruitment of pifYFP. (Note that 20s is given between frames to ensure diffusive binding equilibrium is reached.)


Movies Demonstrating Local Perturbation of Actin Cytoskeleton via GEF Recruitment (Widefield Epi Microscopy)


7 cell extrusion by Tiam(Rac GEF) recruitment An extended process from a cell is induced and guided by locally stimulating Rac1 via membrane-recruited Tiam(Rac GEF) DHPH-YFP-pif6. A NIH3T3 cell is shown using standard epifluorescence microscopy.


8 dynamic lamellipodia by Tiam(Rac GEF) recruitment Lamellipodia are induced and released on a physiological timescale by local Tiam(Rac GEF) recruitment, showing the quick responsiveness of the system. A NIH3T3 cell is shown using standard epifluorescence microscopy.


9 periodic contractions by Tim(Rho GEF) recruitment Alternating global irradiation of NIH3T3 cells transfected with a Tim (Rho GEF) DHPH recruitee construct causes rhythmic contractions of the cell tightly coupled to the light condition. A NIH3T3 cell is shown using standard epifluorescence microscopy.


10 pif-YFP Only Recruitment Control This movie shows the typical result that pif-YFP recruitment alone does not seem to produce any detectable polymerization or ruffling activity in the cytoskeleton. (Such spurious morphological effects have not been seen in any of our YFP-only system characterization experiments.)


Movies Demonstrating Downstream Signalling Activity by TIRF Recruitment Biosensors (TIRF Microscopy)


11 Intersectin(Cdc42 GEF) induces WASP-GBD recruitment φ-pif recruitment of IntersectinDHPH to the plasma membrane causes it to bind and activate Cdc42 by inducing nucleotide exchange to the GTP bound form. The WASP GBD domain specifically recognizes this form and is preferentially recruited to regions of high Cdc42-GTP content. By explointing this fact in TIRF mode we can monitor the downstream effects of GEF activity in NIH3T3 cells.


12 Tiam(Rac GEF) induces PAK-GBD recruitment φ-pif recruitment of TiamDHPH to the plasma membrane causes it to bind and activate Rac1 by inducing nucleotide exchange to the GTP bound form. The PAK GBD domain specifically recognizes this form and is preferentially recruited to regions of high Rac1-GTP content (albeit more weakly than in the above case). By explointing this fact in TIRF mode we can monitor the downstream effects of the GEF activity in NIH3T3 cells.