Motor properties of Myosin 5c are modulated by tropomyosin isoforms and inhibited by pentabromopseudilin.

Myosin 5c (Myo5c) is a motor protein that is produced in epithelial and glandular tissues, where it plays an important role in secretory processes. Myo5c is composed of two heavy chains, each containing a generic motor domain, an elongated neck domain consisting of a single α-helix with six IQ motifs, each of which binds to a calmodulin (CaM) or a myosin light chain from the EF-hand protein family, a coiled-coil dimer-forming region and a carboxyl-terminal globular tail domain. Although Myo5c is a low duty cycle motor, when two or more Myo5c-heavy meromyosin (HMM) molecules are linked together, they move processively along actin filaments. We describe the purification and functional characterization of human Myo5c-HMM co-produced either with CaM alone or with CaM and the essential and regulatory light chains Myl6 and Myl12b. We describe the extent to which cofilaments of actin and Tpm1.6, Tpm1.8 or Tpm3.1 alter the maximum actin-activated ATPase and motile activity of the recombinant Myo5c constructs. The small allosteric effector pentabromopseudilin (PBP), which is predicted to bind in a groove close to the actin and nucleotide binding site with a calculated ΔG of -18.44 kcal/mol, inhibits the motor function of Myo5c with a half-maximal concentration of 280 nM. Using immunohistochemical staining, we determined the distribution and exact localization of Myo5c in endothelial and endocrine cells from rat and human tissue. Particular high levels of Myo5c were observed in insulin-producing ß-cells located within the pancreatic islets of Langerhans.

Molecular regulatory mechanism of human myosin-7a.

Myosin-7a is an actin-based motor protein essential for vision and hearing. Mutations of myosin-7a cause type 1 Usher syndrome, the most common and severe form of deafblindness in humans. The molecular mechanisms that govern its mechanochemistry remain poorly understood, primarily because of the difficulty of purifying stable intact protein. Here, we recombinantly produce the complete human myosin-7a holoenzyme in insect cells and characterize its biochemical and motile properties. Unlike the Drosophila ortholog that primarily associates with calmodulin (CaM), we found that human myosin-7a utilizes a unique combination of light chains including regulatory light chain, CaM, and CaM-like protein 4. Our results further reveal that CaM-like protein 4 does not function as a Ca2+ sensor but plays a crucial role in maintaining the lever arm's structural-functional integrity. Using our recombinant protein system, we purified two myosin-7a splicing isoforms that have been shown to be differentially expressed along the cochlear tonotopic axis. We show that they possess distinct mechanoenzymatic properties despite differing by only 11 amino acids at their N termini. Using single-molecule in vitro motility assays, we demonstrate that human myosin-7a exists as an autoinhibited monomer and can move processively along actin when artificially dimerized or bound to cargo adaptor proteins. These results suggest that myosin-7a can serve multiple roles in sensory systems such as acting as a transporter or an anchor/force sensor. Furthermore, our research highlights that human myosin-7a has evolved unique regulatory elements that enable precise tuning of its mechanical properties suitable for mammalian auditory functions.

The C-terminal tail extension of myosin 16 acts as a molten globule, including intrinsically disordered regions, and interacts with the N-terminal ankyrin.

The lesser-known unconventional myosin 16 protein is essential in proper neuronal functioning and has been implicated in cell cycle regulation. Its longer Myo16b isoform contains a C-terminal tail extension (Myo16Tail), which has been shown to play a role in the neuronal phosphoinositide 3-kinase signaling pathway. Myo16Tail mediates the actin cytoskeleton remodeling, downregulates the actin dynamics at the postsynaptic site of dendritic spines, and is involved in the organization of the presynaptic axon terminals. However, the functional and structural features of this C-terminal tail extension are not well known. Here, we report the purification and biophysical characterization of the Myo16Tail by bioinformatics, fluorescence spectroscopy, and CD. Our results revealed that the Myo16Tail is functionally active and interacts with the N-terminal ankyrin domain of myosin 16, suggesting an intramolecular binding between the C and N termini of Myo16 as an autoregulatory mechanism involving backfolding of the motor domain. In addition, the Myo16Tail possesses high structural flexibility and a solvent-exposed hydrophobic core, indicating the largely unstructured, intrinsically disordered nature of this protein region. Some secondary structure elements were also observed, indicating that the Myo16Tail likely adopts a molten globule-like structure. These structural features imply that the Myo16Tail may function as a flexible display site particularly relevant in post-translational modifications, regulatory functions such as backfolding, and phosphoinositide 3-kinase signaling.

Myosin XVI in the Nervous System.

The myosin family is a large inventory of actin-associated motor proteins that participate in a diverse array of cellular functions. Several myosin classes are expressed in neural cells and play important roles in neural functioning. A recently discovered member of the myosin superfamily, the vertebrate-specific myosin XVI (Myo16) class is expressed predominantly in neural tissues and appears to be involved in the development and proper functioning of the nervous system. Accordingly, the alterations of MYO16 has been linked to neurological disorders. Although the role of Myo16 as a generic actin-associated motor is still enigmatic, the N-, and C-terminal extensions that flank the motor domain seem to confer unique structural features and versatile interactions to the protein. Recent biochemical and physiological examinations portray Myo16 as a signal transduction element that integrates cell signaling pathways to actin cytoskeleton reorganization. This review discusses the current knowledge of the structure-function relation of Myo16. In light of its prevalent localization, the emphasis is laid on the neural aspects.

Myosin XVI.

Myosin XVI (Myo16), a vertebrate-specific motor protein, is a recently discovered member of the myosin superfamily. The detailed functionality regarding myosin XVI requires elucidating or clarification; however, it appears to portray an important role in neural development and in the proper functioning of the nervous system. It is expressed in the largest amount in neural tissues in the late embryonic-early postnatal period, specifically the time in which neuronal cell migration and dendritic elaboration coincide. The impaired expression of myosin XVI has been found lurking in the background of several neuropsychiatric disorders including autism, schizophrenia and/or bipolar disorders.Two principal isoforms of class XVI myosins have been thus far described: Myo16a, the tailless cytoplasmic isoform and Myo16b, the full-length molecule featuring both cytoplasmic and nuclear localization. Both isoforms contain a class-specific N-terminal ankyrin repeat domain that binds to the protein phosphatase catalytic subunit. Myo16b, the predominant isoform, exhibits a diverse function. In the cytoplasm, it participates in the reorganization of the actin cytoskeleton through activation of the PI3K pathway and the WAVE-complex, while in the nucleus it may possess a role in cell cycle regulation. Based on the sequence, myosin XVI may have a compromised ATPase activity, implying a potential stationary role.

Activation of mitochondrial fusion provides a new treatment for mitochondria-related diseases.

Mitochondria fragmentation destabilizes mitochondrial membranes, promotes oxidative stress and facilitates cell death, thereby contributing to the development and the progression of several mitochondria-related diseases. Accordingly, compounds that reverse mitochondrial fragmentation could have therapeutic potential in treating such diseases. BGP-15, a hydroxylamine derivative, prevents insulin resistance in humans and protects against several oxidative stress-related diseases in animal models. Here we show that BGP-15 promotes mitochondrial fusion by activating optic atrophy 1 (OPA1), a GTPase dynamin protein that assist fusion of the inner mitochondrial membranes. Suppression of Mfn1, Mfn2 or OPA1 prevents BGP-15-induced mitochondrial fusion. BGP-15 activates Akt, S6K, mTOR, ERK1/2 and AS160, and reduces JNK phosphorylation which can contribute to its protective effects. Furthermore, BGP-15 protects lung structure, activates mitochondrial fusion, and stabilizes cristae membranes in vivo determined by electron microscopy in a model of pulmonary arterial hypertension. These data provide the first evidence that a drug promoting mitochondrial fusion in in vitro and in vivo systems can reduce or prevent the progression of mitochondria-related disorders.

Ankyrin domain of myosin 16 influences motor function and decreases protein phosphatase catalytic activity.

The unconventional myosin 16 (Myo16), which may have a role in regulation of cell cycle and cell proliferation, can be found in both the nucleus and the cytoplasm. It has a unique, eight ankyrin repeat containing pre-motor domain, the so-called ankyrin domain (My16Ank). Ankyrin repeats are present in several other proteins, e.g., in the regulatory subunit (MYPT1) of the myosin phosphatase holoenzyme, which binds to the protein phosphatase-1 catalytic subunit (PP1c). My16Ank shows sequence similarity to MYPT1. In this work, the interactions of recombinant and isolated My16Ank were examined in vitro. To test the effects of My16Ank on myosin motor function, we used skeletal muscle myosin or nonmuscle myosin 2B. The results showed that My16Ank bound to skeletal muscle myosin (K D ≈ 2.4 µM) and the actin-activated ATPase activity of heavy meromyosin (HMM) was increased in the presence of My16Ank, suggesting that the ankyrin domain can modulate myosin motor activity. My16Ank showed no direct interaction with either globular or filamentous actin. We found, using a surface plasmon resonance-based binding technique, that My16Ank bound to PP1cα (K D ≈ 540 nM) and also to PP1cδ (K D ≈ 600 nM) and decreased its phosphatase activity towards the phosphorylated myosin regulatory light chain. Our results suggest that one function of the ankyrin domain is probably to regulate the function of Myo16. It may influence the motor activity, and in complex with the PP1c isoforms, it can play an important role in the targeted dephosphorylation of certain, as yet unidentified, intracellular proteins.

Nonmuscle myosin IIA with a GFP fused to the N-terminus of the regulatory light chain is regulated normally.

Nonmuscle myosin II plays a crucial role in a variety of cellular processes (e.g., polarity formation, cell motility, and cytokinesis). It is composed of two heavy chains, two regulatory light chains and two essential light chains. The ATPase activity of the myosin II motor domain is regulated through phosphorylation of the regulatory light chain (RLC) by myosin light chain kinase. To study myosin function and localization in cellular processes, GFP-fused RLCs are widely used; however, the exact kinetic properties of myosins with bound GFP-RLC are poorly described. More importantly, it has not been shown that a regulatory light chain fused at its N-terminus with GFP can maintain the normal phosphorylation-dependent regulation of nonmuscle myosin or serve as a substrate for myosin light chain kinase. We coexpressed N-terminal GFP-RLC with a heavy meromyosin (HMM)-like fragment of nonmuscle myosin IIA and essential light chain to characterize the phosphorylation dynamics and in vitro kinetic properties of the resulting HMM. Myosin light chain kinase phosphorylates the GFP-RLC bound to HMM IIA with the same V(max) as it does the wild type RLC bound to HMM IIA, but the K(m) is about two fold higher for the GFP fusion protein, meaning that it is a somewhat poorer substrate. The steady-state actin-activated MgATPase activity of the GFP-RLC HMM is very low in the absence of phosphorylation demonstrating that the GFP moiety does not prevent formation of the off state. The actin-activated MgATPase activity of phosphorylated GFP-RLC-HMM and is about half that of wild type phosphorylated HMM. The ability of phosphorylated GFP-RLC-HMM to move actin filaments in the actin gliding assay is also slightly compromised. These data indicate that despite some kinetic differences the N-terminal GFP fusion to the regulatory light chain is a reasonable model system for studying myosin function in vivo.

Interaction forces between F-actin and titin PEVK domain measured with optical tweezers.

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.

Spatially and temporally synchronized atomic force and total internal reflection fluorescence microscopy for imaging and manipulating cells and biomolecules.

The atomic force microscope is a high-resolution scanning-probe instrument which has become an important tool for cellular and molecular biophysics in recent years but lacks the time resolution and functional specificities offered by fluorescence microscopic techniques. To exploit the advantages of both methods, here we developed a spatially and temporally synchronized total internal reflection fluorescence and atomic force microscope system. The instrument, which we hereby call STIRF-AFM, is a stage-scanning device in which the mechanical and optical axes are coaligned to achieve spatial synchrony. At each point of the scan the sample topography (atomic force microscope) and fluorescence (photon count or intensity) information are simultaneously recorded. The tool was tested and validated on various cellular (monolayer cells in which actin filaments and intermediate filaments were fluorescently labeled) and biomolecular (actin filaments and titin molecules) systems. We demonstrate that with the technique, correlated sample topography and fluorescence images can be recorded, soft biomolecular systems can be mechanically manipulated in a targeted fashion, and the fluorescence of mechanically stretched titin can be followed with high temporal resolution.

Differential actin binding along the PEVK domain of skeletal muscle titin.

Parts of the PEVK (Pro-Glu-Val-Lys) domain of the skeletal muscle isoform of the giant intrasarcomeric protein titin have been shown to bind F-actin. However, the mechanisms and physiological function of this are poorly understood. To test for actin binding along PEVK, we expressed contiguous N-terminal (PEVKI), middle (PEVKII), and C-terminal (PEVKIII) PEVK segments of the human soleus muscle isoform. We found a differential actin binding along PEVK in solid-state binding, cross-linking and in vitro motility assays. The order of apparent affinity is PEVKII>PEVKI>PEVKIII. To explore which sequence motifs convey the actin-binding property, we cloned and expressed PEVK fragments with different motif structure: PPAK, polyE-rich and pure polyE fragments. The polyE-containing fragments had a stronger apparent actin binding, suggesting that a local preponderance of polyE motifs conveys an enhanced local actin-binding property to PEVK. The actin binding of PEVK may serve as a viscous bumper mechanism that limits the velocity of unloaded muscle shortening towards short sarcomere lengths. Variations in the motif structure of PEVK might be a method of regulating the magnitude of the viscous drag.