Dataset Information

Modeling capping protein FRAP and CALI experiments reveals in vivo regulation of actin dynamics.

ABSTRACT: 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.

SUBMITTER: Kapustina M

PROVIDER: S-EPMC2929670 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Modeling capping protein FRAP and CALI experiments reveals in vivo regulation of actin dynamics.

Kapustina Maryna M Vitriol Eric E Elston Timothy C TC Loew Leslie M LM Jacobson Ken K

Cytoskeleton (Hoboken, N.J.) 20100801 8

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 ...[more]

PMID: 20623665

Similar Datasets

Project description:The actin capping protein (CP) tightly binds to the barbed end of actin filaments, thus playing a key role in actin-based lamellipodial dynamics. V-1 and CARMIL proteins directly bind to CP and inhibit the filament capping activity of CP. V-1 completely inhibits CP from interacting with the barbed end, whereas CARMIL proteins act on the barbed end-bound CP and facilitate its dissociation from the filament (called uncapping activity). Previous studies have revealed the striking functional differences between the two regulators. However, the molecular mechanisms describing how these proteins inhibit CP remains poorly understood. Here we present the crystal structures of CP complexed with V-1 and with peptides derived from the CP-binding motif of CARMIL proteins (CARMIL, CD2AP, and CKIP-1). V-1 directly interacts with the primary actin binding surface of CP, the C-terminal region of the alpha-subunit. Unexpectedly, the structures clearly revealed the conformational flexibility of CP, which can be attributed to a twisting movement between the two domains. CARMIL peptides in an extended conformation interact simultaneously with the two CP domains. In contrast to V-1, the peptides do not directly compete with the barbed end for the binding surface on CP. Biochemical assays revealed that the peptides suppress the interaction between CP and V-1, despite the two inhibitors not competing for the same binding site on CP. Furthermore, a computational analysis using the elastic network model indicates that the interaction of the peptides alters the intrinsic fluctuations of CP. Our results demonstrate that V-1 completely sequesters CP from the barbed end by simple steric hindrance. By contrast, CARMIL proteins allosterically inhibit CP, which appears to be a prerequisite for the uncapping activity. Our data suggest that CARMIL proteins down-regulate CP by affecting its conformational dynamics. This conceptually new mechanism of CP inhibition provides a structural basis for the regulation of the barbed end elongation in cells.

Dataset Information

Modeling capping protein FRAP and CALI experiments reveals in vivo regulation of actin dynamics.

Publications

Modeling capping protein FRAP and CALI experiments reveals in vivo regulation of actin dynamics.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets