Troponin Structural Dynamics in the Native Cardiac Thin Filament Revealed by Cryo Electron Microscopy.

Cardiac muscle contraction occurs due to repetitive interactions between myosin thick and actin thin filaments (TF) regulated by Ca levels, active cross-bridges, and cardiac myosin-binding protein C (cMyBP-C). The cardiac TF (cTF) has two nonequivalent strands, each comprised of actin, tropomyosin (Tm), and troponin (Tn). Tn shifts Tm away from myosin-binding sites on actin at elevated Ca levels to allow formation of force-producing actomyosin cross-bridges.

Cardiac troponin T N-domain variant destabilizes the actin interface resulting in disturbed myofilament function.

Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and sudden cardiac arrest in juveniles. cTnT-I79N is located in the cTnT N-terminal (TnT1) loop region and is known for its pathological and prognostic relevance. A recent structural study revealed that I79 is part of a hydrophobic interface between the TnT1 loop and actin, which stabilizes the relaxed (OFF) state of the cardiac thin filament.

High-resolution cryo-EM structure of the junction region of the native cardiac thin filament in relaxed state.

Cardiac contraction depends on molecular interactions among sarcomeric proteins coordinated by the rising and falling intracellular Ca levels. Cardiac thin filament (cTF) consists of two strands composed of actin, tropomyosin (Tm), and equally spaced troponin (Tn) complexes forming regulatory units. Tn binds Ca to move Tm strand away from myosin-binding sites on actin to enable actomyosin cross-bridges required for force generation.

Cryo-Electron Microscopy Reveals Cardiac Myosin Binding Protein-C M-Domain Interactions with the Thin Filament.

Cardiac myosin binding protein C (cMyBP-C) modulates cardiac contraction via direct interactions with cardiac thick (myosin) and thin (actin) filaments (cTFs). While its C-terminal domains (e.g.

Actomyosin Complex.

Formation of cross-bridges between actin and myosin occurs ubiquitously in eukaryotic cells and mediates muscle contraction, intracellular cargo transport, and cytoskeletal remodeling. Myosin motors repeatedly bind to and dissociate from actin filaments in a cycle that transduces the chemical energy from ATP hydrolysis into mechanical force generation. While the general layout of surface elements within the actin-binding interface is conserved among myosin classes, sequence divergence within these motifs alters the specific contacts involved in the actomyosin interaction as well as the kinetics of mechanochemical cycle phases.

Ca attenuates nucleation activity of leiomodin.

A transient increase in Ca concentration in sarcomeres is essential for their proper function. Ca drives striated muscle contraction via binding to the troponin complex of the thin filament to activate its interaction with the myosin thick filament. In addition to the troponin complex, the myosin essential light chain and myosin-binding protein C were also found to be Ca sensitive.

Optimization of small extracellular vesicle isolation from expressed prostatic secretions in urine for in-depth proteomic analysis.

The isolation and subsequent molecular analysis of extracellular vesicles (EVs) derived from patient samples is a widely used strategy to understand vesicle biology and to facilitate biomarker discovery. Expressed prostatic secretions in urine are a tumor proximal fluid that has received significant attention as a source of potential prostate cancer (PCa) biomarkers for use in liquid biopsy protocols. Standard EV isolation methods like differential ultracentrifugation (dUC) co-isolate protein contaminants that mask lower-abundance proteins in typical mass spectrometry (MS) protocols.

Interaction of the C2 Ig-like Domain of Cardiac Myosin Binding Protein-C with F-actin.

Cardiac muscle contraction depends on interactions between thick (myosin) and thin (actin) filaments (TFs). TFs are regulated by intracellular Ca levels. Under activating conditions Ca binds to the troponin complex and displaces tropomyosin from myosin binding sites on the TF surface to allow actomyosin interactions.

Anomalous structural dynamics of minimally frustrated residues in cardiac troponin C triggers hypertrophic cardiomyopathy.

Cardiac TnC (cTnC) is highly conserved among mammals, and genetic variants can result in disease by perturbing Ca-regulation of myocardial contraction. Here, we report the molecular basis of a human mutation in cTnC's αD-helix (-p.C84Y) that impacts conformational dynamics of the D/E central-linker and sampling of discrete states in the N-domain, favoring the "primed" state associated with Ca binding.

The structure of the native cardiac thin filament at systolic Ca levels.

Every heartbeat relies on cyclical interactions between myosin thick and actin thin filaments orchestrated by rising and falling Ca levels. Thin filaments are comprised of two actin strands, each harboring equally separated troponin complexes, which bind Ca to move tropomyosin cables away from the myosin binding sites and, thus, activate systolic contraction. Recently, structures of thin filaments obtained at low (pCa ∼9) or high (pCa ∼3) Ca levels revealed the transition between the Ca-free and Ca-bound states.

High-Resolution Cryo-EM Structure of the Cardiac Actomyosin Complex.

Heart contraction depends on a complicated array of interactions between sarcomeric proteins required to convert chemical energy into mechanical force. Cyclic interactions between actin and myosin molecules, controlled by troponin and tropomyosin, generate the sliding force between the actin-based thin and myosin-based thick filaments. Alterations in this sophisticated system due to missense mutations can lead to cardiovascular diseases.

Effects of cardiomyopathy-linked mutations K15N and R21H in tropomyosin on thin-filament regulation and pointed-end dynamics.

Missense mutations K15N and R21H in striated muscle tropomyosin are linked to dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), respectively. Tropomyosin, together with the troponin complex, regulates muscle contraction and, along with tropomodulin and leiomodin, controls the uniform thin-filament lengths crucial for normal sarcomere structure and function. We used Förster resonance energy transfer to study effects of the tropomyosin mutations on the structure and kinetics of the cardiac troponin core domain associated with the Ca-dependent regulation of cardiac thin filaments.

N-Terminal Domains of Cardiac Myosin Binding Protein C Cooperatively Activate the Thin Filament.

Muscle contraction relies on interaction between myosin-based thick filaments and actin-based thin filaments. Myosin binding protein C (MyBP-C) is a key regulator of actomyosin interactions. Recent studies established that the N'-terminal domains (NTDs) of MyBP-C can either activate or inhibit thin filaments, but the mechanism of their collective action is poorly understood.

Accurate flexible refinement of atomic models against medium-resolution cryo-EM maps using damped dynamics.

Background: Dramatic progress has recently been made in cryo-electron microscopy technologies, which now make possible the reconstruction of a growing number of biomolecular structures to near-atomic resolution. However, the need persists for fitting and refinement approaches that address those cases that require modeling assistance.

Methods: In this paper, we describe algorithms to optimize the performance of such medium-resolution refinement methods.

Ca-induced movement of tropomyosin on native cardiac thin filaments revealed by cryoelectron microscopy.

Muscle contraction relies on the interaction of myosin motors with F-actin, which is regulated through a translocation of tropomyosin by the troponin complex in response to Ca The current model of muscle regulation holds that at relaxing (low-Ca) conditions tropomyosin blocks myosin binding sites on F-actin, whereas at activating (high-Ca) conditions tropomyosin translocation only partially exposes myosin binding sites on F-actin so that binding of rigor myosin is required to fully activate the thin filament (TF). Here we used a single-particle approach to helical reconstruction of frozen hydrated native cardiac TFs under relaxing and activating conditions to reveal the azimuthal movement of the tropomyosin on the surface of the native cardiac TF upon Ca activation. We demonstrate that at either relaxing or activating conditions tropomyosin is not constrained in one structural state, but rather is distributed between three structural positions on the surface of the TF.

Filaments and fingers: Novel structural aspects of the single septin from .

Septins are filament-forming GTP-binding proteins involved in many essential cellular events related to cytoskeletal dynamics and maintenance. Septins can self-assemble into heterocomplexes, which polymerize into highly organized, cell membrane-interacting filaments. The number of septin genes varies among organisms, and although their structure and function have been thoroughly studied in opisthokonts (including animals and fungi), no structural studies have been reported for other organisms.

Multimodal and Polymorphic Interactions between Anillin and Actin: Their Implications for Cytokinesis.

Cytokinesis of animal cells requires the assembly of a contractile ring, which promotes daughter cell splitting. Anillin is a conserved scaffold protein involved in organizing the structural components of the contractile ring including filamentous actin (F-actin), myosin, and septins and in forming the subsequent midbody ring. Like other metazoan homologs, Drosophila anillin contains a conserved domain that can bind and bundle F-actin, but the importance and molecular details of its interaction with F-actin remain unclear.

C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation.

Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g.

Septin 9 Exhibits Polymorphic Binding to F-Actin and Inhibits Myosin and Cofilin Activity.

Septins are a highly conserved family of proteins in eukaryotes that is recognized as a novel component of the cytoskeleton. Septin 9 (SEPT9) interacts directly with actin filaments and functions as an actin stress fiber cross-linking protein that promotes the maturation of nascent focal adhesions and cell migration. However, the molecular details of how SEPT9 interacts with F-actin remain unknown.

Near-atomic resolution for one state of F-actin.

Actin functions as a helical polymer, F-actin, but attempts to build an atomic model for this filament have been hampered by the fact that the filament cannot be crystallized and by structural heterogeneity. We have used a direct electron detector, cryo-electron microscopy, and the forces imposed on actin filaments in thin films to reconstruct one state of the filament at 4.7 Å resolution, which allows for building a reliable pseudo-atomic model of F-actin.

Septins promote stress fiber-mediated maturation of focal adhesions and renal epithelial motility.

Organogenesis and tumor metastasis involve the transformation of epithelia to highly motile mesenchymal-like cells. Septins are filamentous G proteins, which are overexpressed in metastatic carcinomas, but their functions in epithelial motility are unknown. Here, we show that a novel network of septin filaments underlies the organization of the transverse arc and radial (dorsal) stress fibers at the leading lamella of migrating renal epithelia.

Reversible paralysis of Schistosoma mansoni by forchlorfenuron, a phenylurea cytokinin that affects septins.

Septins are guanosine-5'-triphosphate-binding proteins involved in wide-ranging cellular processes including cytokinesis, vesicle trafficking, membrane remodelling and scaffolds, and with diverse binding partners. Precise roles for these structural proteins in most processes often remain elusive. Identification of small molecules that inhibit septins could aid in elucidating the functions of septins and has become increasingly important, including the description of roles for septins in pathogenic phenomena such as tumorigenesis.

Identification of an actin binding surface on vinculin that mediates mechanical cell and focal adhesion properties.

Vinculin, a cytoskeletal scaffold protein essential for embryogenesis and cardiovascular function, localizes to focal adhesions and adherens junctions, connecting cell surface receptors to the actin cytoskeleton. While vinculin interacts with many adhesion proteins, its interaction with filamentous actin regulates cell morphology, motility, and mechanotransduction. Disruption of this interaction lowers cell traction forces and enhances actin flow rates.

The structure of the CS1 pilus of enterotoxigenic Escherichia coli reveals structural polymorphism.

Enterotoxigenic Escherichia coli (ETEC) is a bacterial pathogen that causes diarrhea in children and travelers in developing countries. ETEC adheres to host epithelial cells in the small intestine via a variety of different pili. The CS1 pilus is a prototype for a family of related pili, including the CFA/I pili, present on ETEC and other Gram-negative bacterial pathogens.

Are ParM filaments polar or bipolar?

A recent perspective [Erickson, H. (2012). Bacterial actin homolog ParM: arguments for an apolar, antiparallel double helix.