Calcium Dependent Interaction Between N2A-Halo and F-Actin: A Single Molecule Study

Abstract

In the last two decades, interactions between titin's I-band region and actin have been widely investigated for skeletal and cardiac isoforms. Previous studies suggested a calcium-dependent interaction between titin and actin. However, previous in vitro motility studies did not quantify the strength of interaction between titin's N2A region and actin in the presence of calcium. Here, we used AFM single molecule force spectroscopy (SMFS) to probe the interaction strength between a recombinant N2A-HALO construct and actin filaments (F-actin), in the presence (pCa = 4.0) and absence of calcium. The N2A-Halo construct consisted of the N2A region of titin (Ig80-IS-Ig81-Ig82-Ig83, where IS is the insertion sequence) with a C-terminal Halo-tag. The construct was immobilized on an AFM cantilever tip via its N-terminal cysteines. The interaction strength was determined by probing the molecule during multiple approach-retraction cycles at physiological pulling rates (300-1000 nm/sec) over various locations on a substrate functionalized with F-actin stabilized on a lipid bilayer with ATP. Preliminary data show that both the yield and strength of N2A-Halo interactions with F-actin nearly doubled in the presence of Ca2+, with multiple peaks in the rupture force distribution at 50 ± 25 pN, 75 ± 10 pN, and 100 ± 25 pN. The force extension curves also showed different rupture lengths, indicating multiple actin binding sites. No significant interactions occurred between N2A constructs and the charged lipid bi-layer surface in the absence of F-actin. These experiments show that calcium increases the interaction probability and binding strength between N2A-Halo and F-actin and that the binding strength between N2A titin and F-actin is nearly 4 times greater than previously reported for PEVK titin - F-actin interactions. These results support a physiological role for titin in active muscle contraction and force enhancement.