Interaction Forces between F-Actin and Titin PEVK Domain Measured with Optical Tweezers

Abstract

Titin is a giant protein that determines the elasticity of striated muscle and is thought to play important roles in numerous regulatory processes. Previous studies have shown that titin's PEVK domain interacts with F-actin, thereby creating viscous forces of unknown magnitude that may modulate muscle contraction. Here we measured, with optical tweezers, the forces necessary to dissociate F-actin from individual molecules of recombinant PEVK fragments rich either in polyE or PPAK motifs. Rupture forces at a stretch rate of 250 nm/s displayed a wide, nonnormal distribution with a peak at approximately 8 pN in the case of both fragments. Dynamic force spectroscopy experiments revealed low spontaneous off-rates that were increased even by low forces. The loading-rate dependence of rupture force was biphasic for polyE in contrast with the monophasic response observed for PPAK. Analysis of the molecular lengths at which rupture occurred indicated that there are numerous actin-binding regions along the PEVK fragments' contour, suggesting that the PEVK domain is a promiscuous actin-binding partner. The complexity of PEVK-actin interaction points to an adaptable viscoelastic mechanism that safeguards sarcomeric structural integrity in the relaxed state and modulates thixotropic behavior during contraction.

Non-crossbridge stiffness in active muscle fibres

“…There are two possible mechanisms: the first is a direct effect of calcium on titin stiffness via calcium binding to the E-rich motifs (Tatsumi et al, 2001;Labeit et al, 2003;Cornachione and Rassier, 2012;Rassier et al, 2015), which could stabilize the PEVK segment, making it stiffer (Fig. 1C); the second involves an effect of calcium on titin-actin interactions that induces an increase in the binding between thin filament and titin, increasing sarcomere stiffness (Kellermayer and Granzier, 1996;Linke et al, 1997Linke et al, , 2002Bianco et al, 2007;Leonard and Herzog, 2010;Nishikawa et al, 2012). The first mechanism could lead to a reduction in the persistent length of titin and a potential increase in passive force and stiffness.…”

Exclusion-Zone Dynamics Explored with Microfluidics and Optical Tweezers

“…Our results indicate that near the Nafion surface the force calibration is indeed offset. Because our optical tweezers instrument measures force from changes in photonic momentum [21][22][23] rather than from bead-based calibration, we may conclude that the offset is indeed due to a local change in refraction. Since the offset exceeds 1 pN, measuring the force acting on a bead upon EZ formation is not possible with a precision better than a few piconewtons when using optical tweezers.…”

“…Manipulation of polystyrene beads was carried out with a custom-built dual-beam counter-propagating optical tweezers apparatus [21][22][23]. For force measurements the optical trap was formed with two 60× 1.2 NA water immersion objective lenses (Olympus, Tokyo, Japan) mounted on opposite sides of a Parafilm-based microfluidic device with coverslip walls.…”

The N-terminal Region of Twitchin Binds Thick and Thin Contractile Filaments

“…The PEVK domain of titin has been shown to interact with actin (28 -30, 36). There is differential binding to actin in different PEVK regions of titin, and Bianco et al (37) suggest that the PEVK region of titin is a "promiscuous actin-binding partner," with numerous actin binding regions, just as are present in the N-and C-terminal regions of the twitchin molecule. These actin binding sites in titin may provide a viscous load that resists the relative sliding of thick and thin filaments (36,37).…”

“…There is differential binding to actin in different PEVK regions of titin, and Bianco et al (37) suggest that the PEVK region of titin is a "promiscuous actin-binding partner," with numerous actin binding regions, just as are present in the N-and C-terminal regions of the twitchin molecule. These actin binding sites in titin may provide a viscous load that resists the relative sliding of thick and thin filaments (36,37). A "sticky spring" model for titin-induced force enhancement and force depression in striated muscle has been proposed in which the PEVK region interacts with the thin filament (38).…”