T cell receptor (TcR)͞CD3 ligation initiates a signaling cascade that involves src kinases p56 lck and -associated protein 70, leading to the phosphorylation of substrates such as TcR, Vav, SH2-domain-containing leukocyte protein 76 (SLP-76), cbl, and p120͞130. FYN binding protein (FYB or p120͞130) associates with p59 fyn , the TcR͞ CD3 complex, and becomes tyrosine-phosphorylated in response to receptor ligation. In this study, we report the cDNA cloning of human and murine FYB and show that it is restricted in expression to T cells and myeloid cells and possesses an overall unique hydrophilic sequence with several tyrosine-based motifs, proline-based type I and type II SH3 domain binding motifs, several putative lysine͞glutamic acidrich nuclear localization motifs, and a SH3-like domain. In addition to binding the src kinase p59 fyn , FYB binds specifically to the hematopoietic signaling protein SLP-76, an interaction mediated by the SLP-76 SH2 domain. In keeping with this, expression of FYB augmented interleukin 2 secretion from a T cell hybridoma, DC27.10, in response to TcR͞CD3 ligation. FYB is therefore a novel hematopoietic protein that acts as a component of the FYN and SLP-76 signaling cascades in T cells.

To broaden the range of tools available for proteomic research, we generated a library of 16,368 unique full-length human ORFs that are expressible as N-terminal GST-His6 fusion proteins. Following expression in yeast, these proteins were then individually purified and used to construct a human proteome microarray. To demonstrate the usefulness of this reagent, we developed a streamlined strategy for the production of monospecific monoclonal antibodies that used immunization with live human cells and microarray-based analysis of antibody specificity as its central components. We showed that microarray-based analysis of antibody specificity can be performed efficiently using a two-dimensional pooling strategy. We also demonstrated that our immunization and selection strategies result in a large fraction of monospecific monoclonal antibodies that are both immunoblot and immunoprecipitation grade. Our data indicate that the pipeline provides a robust platform for the generation of monoclonal antibodies of exceptional specificity.

Tropomodulins are a family of proteins that cap the slow-growing end of actin filaments. Erythrocyte tropomodulin (E-Tmod) stabilizes short actin protofilaments in erythrocytes and caps longer sarcomeric actin filaments in striated muscles. We report the knockin of the beta-galactosidase gene (LacZ) under the control of the endogenous E-Tmod promoter and the knockout of E-Tmod in mouse embryonic stem cells. E-Tmod(-/-) embryos die around embryonic day 10 and exhibit a noncontractile heart tube with disorganized myofibrils and underdevelopment of the right ventricle, accumulation of mechanically weakened primitive erythroid cells in the yolk sac, and failure of primary capillary plexuses to remodel into vitelline vessels, all required to establish blood circulation between the yolk sac and the embryo proper. We propose a hemodynamic "plexus channel selection" mechanism as the basis for vitelline vascular remodeling. The defects in cardiac contractility, vitelline circulation, and hematopoiesis reflect an essential role for E-Tmod capping of the actin filaments in both assembly of cardiac sarcomeres and of the membrane skeleton in erythroid cells that is not compensated for by other proteins.

Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) can cause arrhythmias, heart failure, and cardiac death. Here, we functionally characterized the motor domains of five DCM-causing mutations in human β-cardiac myosin. Kinetic analyses of the individual events in the ATPase cycle revealed that each mutation alters different steps in this cycle. For example, different mutations gave enhanced or reduced rate constants of ATP binding, ATP hydrolysis, or ADP release or exhibited altered ATP, ADP, or actin affinity. Local effects dominated, no common pattern accounted for the similar mutant phenotype, and there was no distinct set of changes that distinguished DCM mutations from previously analyzed HCM myosin mutations. That said, using our data to model the complete ATPase contraction cycle revealed additional critical insights. Four of the DCM mutations lowered the duty ratio (the ATPase cycle portion when myosin strongly binds actin) because of reduced occupancy of the force-holding A·M·D complex in the steady state. Under load, the A·M·D state is predicted to increase owing to a reduced rate constant for ADP release, and this effect was blunted for all five DCM mutations. We observed the opposite effects for two HCM mutations, namely R403Q and R453C. Moreover, the analysis predicted more economical use of ATP by the DCM mutants than by WT and the HCM mutants. Our findings indicate that DCM mutants have a deficit in force generation and force-holding capacity due to the reduced occupancy of the force-holding state.

To investigate the nanomechanics of the erythrocyte membrane we developed a hybrid model that couples the actin-spectrin network to the lipid bilayer. This model features a Fourier space Brownian dynamics model of the bilayer, a Brownian dynamics model of the actin protofilament, and a modified wormlike-chain model of the spectrin (including a cable-dynamics model to predict the oscillation in tension). This model enables us to predict the nanomechanics of single or multiple units of the protein network, the lipid bilayer, and the effect of their interactions. The present work is focused on the attitude of the actin protofilament at the equilibrium states coupled with the elevations of the lipid bilayer through their primary linkage at the suspension complex in deformations. Two different actin-spectrin junctions are considered at the junctional complex. With a point-attachment junction, large pitch angles and bifurcation of yaw angles are predicted. Thermal fluctuations at bifurcation may lead to mode-switching, which may affect the network and the physiological performance of the membrane. In contrast, with a wrap-around junction, pitch angles remain small, and the occurrence of bifurcation is greatly reduced. These simulations suggest the importance of three-dimensional molecular junctions and the lipid bilayer/protein network coupling on cell membrane mechanics.

The erythrocyte membrane skeleton deforms constantly in circulation, but the mechanics of a junctional complex (JC) in the network is poorly understood. We previously proposed a 3-D mechanical model for a JC (Sung, L. A., and C. Vera. Protofilament and hexagon: A three-dimensional mechanical model for the junctional complex in the erythrocyte membrane skeleton. Ann Biomed Eng 31:1314-1326, 2003) and now developed a mathematical model to compute its equilibrium by dynamic relaxation. We simulated deformations of a single unit in the network to predict the tension of 6 alphabeta spectrin (Sp) (top, middle, and bottom pairs), and the attitude of the actin protofilament [pitch (theta), yaw (phi) and roll (psi) angles]. In equibiaxial deformation, 6 Sp would not begin their first round of "single domain unfolding in cluster" until the extension ratio (lambda) reach approximately 3.6, beyond the maximal sustainable lambda of approximately 2.67. Before Sp unfolds, the protofilament would gradually raise its pointed end away from the membrane, while phi and psi remain almost unchanged. In anisotropic deformation, protofilaments would remain tangent but swing and roll drastically at least once between lambda(i) = 1.0 and approximately 2.8, in a deformation angle- and lambda(i)-dependent fashion. This newly predicted nanomechanics in response to deformations may reveal functional roles previous unseen for a JC, and molecules associated with it, during erythrocyte circulation.

Background: R453C is a mutation in human cardiac myosin and is associated with a high incidence of sudden cardiac death. Results: R453C alters few kinetic parameters, except for the conformational changes associated with ATP binding and hydrolysis. Conclusion:The closure of switch-2 on ATP is disrupted by R453C. Significance: This is the first detailed kinetic analysis of the motor domain of the human ␤-cardiac myosin carrying the R453C mutation.

It is a long-standing mystery why erythrocyte actin filaments in the junctional complex (JC) are uniformly ~37 nm and the membrane skeleton consists of hexagons. We have previously proposed that a "molecular ruler" formed by Etropomodulin (E-Tmod) and tropomyosin (TM) 5 or 5b functions to generate protofilaments of 12 G actin under mechanical stress. Here we illustrate that intrinsic properties of actin filaments, e.g., turns, chemical bonds, and dimensions of the helix, also favor fragmentation into protofilaments under mechanical stress. We further construct a mechanical model in that a pair of G actin is wrapped around by a split α and β spectrin, which may spin to two potential positions, and stabilize to one when the tail end is restricted. A reinforced protofilament may function as a mechanical axis to anchor 3 (top, middle and bottom) pairs of Sp. Each Sp pair may wrap around the protofilament with a wide dihedral angle (~166.