Modeling capping protein FRAP and CALI experiments reveals in vivo regulation of actin dynamics.
To gain insights on cellular mechanisms regulating actin polymerization, we used the Virtual Cell to model fluorescence recovery after photobleaching (FRAP) and chromophore-assisted laser inactivation (CALI) experiments on EGFP-capping protein (EGFP-CP). Modeling the FRAP kinetics demonstrated that the in vivo rate for the dissociation of CP from actin filaments is much faster (approximately 0.1 s(-1)) than that measured in vitro (0.01-0.0004 s(-1)). The CALI simulation revealed that in order to induce sustainable changes in cell morphology after CP inactivation, the cells should exhibit anticapping ability. We included the VASP protein as the anticapping agent in the modeling scheme. The model predicts that VASP affinity for barbed ends has a cooperative dependence on the concentration of VASP-barbed end complexes. This dependence produces a positive feedback that stabilizes the complexes and allows sustained growth at clustered filament tips. We analyzed the range of laser intensities that are sufficient to induce changes in cell morphology. This analysis demonstrates that FRAP experiments with EGFP-CP can be performed safely without changes in cell morphology, because, the intensity of the photobleaching beam is not high enough to produce the critical concentration of free barbed ends that will induce filament growth before diffusional replacement of EGFP-CP occurs.
Kapustina
Maryna
M
Department of Cell and Developmental Biology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7090, USA.
Vitriol
Eric
E
Elston
Timothy C
TC
Loew
Leslie M
LM
Jacobson
Ken
K
D051339:Actin Depolymerizing Factors / Q000378:metabolism*; D000199:Actins / Q000096:biosynthesis / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D007700:Kinetics; D007834:Lasers; D011485:Protein Binding
D051339:Actin Depolymerizing Factors; D000199:Actins