Katsuhide Mabuchi

Hsp72 and Stress Kinase c-jun N-Terminal Kinase Regulate the Bid-Dependent Pathway in Tumor Necrosis Factor-Induced Apoptosis

ABSTRACT The major inducible heat shock protein Hsp72 has been shown to protect cells from certain apoptotic stimuli. Here we investigated the mechanism of Hsp72-mediated protection from tumor necrosis factor (TNF)-induced apoptosis of primary culture of IMR90 human fibroblasts. Hsp72 temporarily blocked apoptosis in response to TNF and permanently protected cells from heat shock. An Hsp72 mutant (Hsp72ΔEEVD) with a deletion of the four C-terminal amino acids, which are essential for the chaperone function, blocked TNF-induced apoptosis in a manner similar to that of normal Hsp72 but did not inhibit heat shock-induced death. Therefore, the chaperone activity of Hsp72 is dispensable for suppression of TNF-induced apoptosis but is required for protection from heat shock. In fibroblasts derived from Bid knockout mice, similar temporal inhibition of TNF-induced apoptosis was seen. In these cells neither normal Hsp72 nor Hsp72ΔEEVD conferred additional protection from apoptosis, suggesting that Hsp72 specifically affects Bid-dependent but not Bid-independent apoptotic pathways. Furthermore, both normal Hsp72 and ΔHsp72EEVD inhibited Bid activation and downstream events, including release of cytochrome c, activation of caspase 3, and cleavage of poly-ADP-ribose polymerase. Both Hsp72 and ΔHsp72EEVD blocked activation of the stress kinase c-jun N-terminal kinase (JNK) by TNF, and specific inhibition of JNK similarly temporarily blocked Bid activation and the downstream apoptotic events. These data strongly suggest that in TNF-induced apoptosis, Hsp72 specifically interferes with the Bid-dependent apoptotic pathway via inhibition of JNK.

The motor activity of myosin-X promotes actin fiber convergence at the cell periphery to initiate filopodia formation.

Filopodia are actin-rich fingerlike protrusions found at the leading edge of migrating cells and are believed to play a role in directional sensing. Previous studies have shown that myosin-X (myoX) promotes filopodia formation and that this is mediated through its ability to deliver specific cargoes to the cell periphery (Tokuo, H., and M. Ikebe. 2004. Biochem Biophys. Commun. 319:214–220; Zhang, H., J.S. Berg, Z. Li, Y. Wang, P. Lang, A.D. Sousa, A. Bhaskar, R.E. Cheney, and S. Stromblad. 2004. Nat. Cell Biol. 6:523–531; Bohil, A.B., B.W. Robertson, and R.E. Cheney. 2006. Proc. Natl. Acad. Sci. USA. 103:12411–12416; Zhu, X.J., C.Z. Wang, P.G. Dai, Y. Xie, N.N. Song, Y. Liu, Q.S. Du, L. Mei, Y.Q. Ding, and W.C. Xiong. 2007. Nat. Cell Biol. 9:184–192). In this study, we show that the motor function of myoX and not the cargo function is critical for initiating filopodia formation. Using a dimer-inducing technique, we find that myoX lacking its cargo-binding tail moves laterally at the leading edge of lamellipodia and induces filopodia in living cells. We conclude that the motor function of the two-headed form of myoX is critical for actin reorganization at the leading edge, leading to filopodia formation.

IMMUNOCYTOCHEMICAL LOCALIZATION OF CALDESMON AND CALPONIN IN CHICKEN GIZZARD SMOOTH MUSCLE

SummaryThe distribution of caldesmon and calponin in chicken gizzard smooth muscle was investigated with immunofluorescence and immunogold electron microscopy. Immunofluorescence microscopy showed that in verapamil treated (relaxed) muscles the distributions of caldesmon and myosin appeared to be uniform throughout the cytoplasm, but clearly more textured than that of actin filaments as revealed by the distribution of tropomyosin. In shortened muscles both caldesmon and myosin became segregated, in contrast to the distribution of actin, which remained uniform. The distribution of calponin was even more textured, with no similarity to those of caldesmon or myosin. Instead, considerable overlap was observed between calponin and the cytoskeletal protein desmin and, to a lesser extent, β-actin. By immunogold electron microscopy caldesmon appeared mostly near and around myosin filaments in both relaxed and shortened muscle. Calponin, on the other hand, was found primarily at the periphery of cytoskeletal structures in the same general region as desmin, and very often adjacent to β-actin, which is mainly in the core. These observations indicated that caldesmon and calponin are associated with different subsets of actin filaments, caldesmon with contractile actin, while calponin with cytoskeletal actin. Thus the in situ localization of caldesmon is consistent with its proposed regulatory function. Calponin, on the other hand, is unlikely to directly regulate actomyosin interactions in these cells; instead, it may function as a bridging protein between the actin and the intermediate filament networks.

The Globular Tail Domain of Myosin Va Functions as an Inhibitor of the Myosin Va Motor

The actin-activated ATPase activity of full-length mammalian myosin Va is well regulated by Ca2+, whereas that of truncated myosin Va without the C-terminal globular tail domain (GTD) is not. Here, we have found that exogenous GTD is capable of inhibiting the actin-activated ATPase activity of GTD-deleted myosin Va. A series of truncated constructs of myosin Va further showed that the entire length of the first coiled-coil (coil-1) of the tail domain is critical for GTD-dependent regulation of myosin Va and that deletion of 58 residues from the C-terminal end of coil-1 markedly hampered regulation. Negative staining electron microscopy revealed that GTD-deleted myosin Va formed a “Y”-shaped structure, which was converted to a triangular shape, similar to the structure of full-length myosin Va in the inhibited state, by addition of exogenous GTD. In contrast, the triangular shape was not observed when the C-terminal 58 residues of coil-1 were deleted, even in the presence of exogenous GTD. Based on these results, we propose a model for the formation of the inhibited state of myosin Va. GTD binds to the C-terminal end of coil-1. The neck-tail junction of myosin Va is flexible, and the long neck enables the head domain to reach the GTD associated with the end of coil-1. Once the head interacts with the GTD, the triangular inhibited conformation is stabilized. Consistent with this model, we found that shortening of the neck of myosin Va by two IQ motifs abolished the regulation by GTD, whereas regulation was partially restored by shortening of coil-1 by an amount comparable to that of the two IQ motifs.

Electron microscopic studies of chicken gizzard caldesmon and its complex with calmodulin

SummaryCaldesmon samples mounted on a stage rotating about a horizontal axis were shadowed keeping the shadow angle at about 3°. This technique minimizes background metal deposits compared with the conventional method. The identity of caldesmon was confirmed by comparing the images of caldesmon alone with those of the caldesmon-calmodulin complex. In these samples the caldesmon molecules appeared to be elongated; most were between 30 and 80 nm in length. The maximum length was in good agreement with the earlier estimate of 74 nm based on hydrodynamic studies. Our observations also suggested the presence of a rather rigid 30–40 nm stretch in the middle of the caldesmon molecule, which was always visible under rotary shadowing, and a flexible structure of about 20 nm in length at each end of the molecule, which may or may not be visible depending on their orientation on the mica surface. In the samples of caldesmon crosslinked with calmodulin, we noticed the existence of complexes containing two calmodulin molecules per caldesmon molecule, separated by a distance of 60 nm, consistent with the suggestion that each end of caldesmon can interact with calmodulin.

Heavy-meromyosin-decorated actin filaments: A simple method to preserve actin filaments for rotary shadowing

It has become accepted that deep-freeze-drying at or below -90 degrees C is necessary to preserve the structure of supramolecular assemblies such as actin filaments (AFs) for metal shadowing. This has kept the metal shadowing technique from widespread use in the study of proteins complexed with AFs because of the limited availability of the apparatus for deep-freeze-drying. I report here that adsorption to freshly cleaved mica, treatment with buffered uranyl acetate in glycerol solution, rinsing, and removal of liquid eliminate the need of freeze-drying to preserve the structure of AFs. This technique, in combination with metal shadowing, was applied to the study of AFs decorated with heavy meromyosin (HMM). It was observed that (1) when HMM molecules are associated with single AFs in the majority of cases only one head of each HMM molecule makes contact at the point furthest from the neck region; (2) binding of HMM causes bundling of AFs, probably by the two heads of each molecule binding different filaments; and (3) the binding of HMM to the bundled AFs appears to be more stable than that to a single AF. This method of specimen preparation requires no freeze-drying and is therefore easily applicable to other large protein complexes.

Melting of myosin and tropomyosin: Electron microscopic observations

A method was devised to maintain a very low angle (2-3 degrees) during the metal casting of specimens for electron microscopy. With this modified rotary shadowing procedure the melting of myosin and tropomyosin (TM) was investigated. When protein solutions were sprayed on mica sheets and then heated to melt alpha-helices, myosin molecules did not show any sign of chain separation but appeared to have collapsed into loose clumps. A few molecules showed separation of the two chains at the light meromyosin-heavy meromyosin hinge region. Heating myosin in bulk solution at 65 degrees C before spraying caused extensive fusing of the myosin heads. In contrast, in the case of TM, separation of the chains appeared to occur at temperatures at which the unfolding of alpha-helices had been shown by circular dichroism. Dissolution of TM and myosin in 0.5% SDS followed by 150-fold dilution led to single chain species. This method capable of detecting single chain peptides of melting TM whose thickness is of the order of 1 nm may be applicable to the study of the structure of proteins previously not considered possible.

Electron microscopic images suggest both ends of caldesmon interact with actin filaments

SummaryAn improved rotary shadowing technique enabled us to visualize chicken gizzard caldesmon (CaD) and its complexes with one or two covalently linked calmodulin (CaM) molecules by electron microscopy. Using a monoclonal antibody against an epitope in the N-terminal region of CaD (anti-N), we can now identify the end of the molecule that is involved in binding to another protein molecule. Thus in the 1:1 complex of CaD and CaM, the CaM molecule was almost always associated with the C-terminus of CaD, indicating preferential CaM-binding to the C-terminal region. We have also studied binding of CaD to filamentous actin (F-actin), using an EM technique that avoids spraying or freeze drying and thereby preserves the structure of F-actin. Only one end of CaD appeared to bind to F-actin, leaving the rest of the molecule projecting away from the filament. While the majority of anti-N bound at the free end of CaD, some antibody molecules were found on F-actin. These findings suggest that either end of CaD can bind to F-actin. Experiments using a monoclonal antibody against the C-terminus of CaD (anti-C) supported this idea. When the native thin filaments that contain endogenous CaD were incubated with anti-N, almost all the bound antibodies were found on the filaments, indicating that the N-terminal regions of CaD interact with actin, and that the binding affinity of the N-terminal region of CaD for actin is higher in vivo than that in vitro, either because the properties of CaD have been altered during purification, or because of the presence of some other component(s) associated with the native filaments.

Modular structure of smooth muscle Myosin light chain kinase: hydrodynamic modeling and functional implications.

Smooth muscle myosin light chain kinase (smMLCK) is a calcium-calmodulin complex-dependent enzyme that activates contraction of smooth muscle. The polypeptide chain of rabbit uterine smMLCK (Swiss-Prot entry P29294) contains the catalytic/regulatory domain, three immunoglobulin-related motifs (Ig), one fibronectin-related motif (Fn3), a repetitive, proline-rich segment (PEVK), and, at the N-terminus, a unique F-actin-binding domain. We have evaluated the spatial arrangement of these domains in a recombinant 125 kDa full-length smMLCK and its two catalytically active C-terminal fragments (77 kDa, residues 461-1147, and 61 kDa, residues 461-1002). Electron microscopic images of smMLCK cross-linked to F-actin show particles at variable distances (11-55 nm) from the filament, suggesting that a well-structured C-terminal segment of smMLCK is connected to the actin-binding domain by a long, flexible tether. We have used structural homology and molecular dynamics methods to construct various all-atom representation models of smMLCK and its two fragments. The theoretical sedimentation coefficients computed with HYDROPRO were compared with those determined by sedimentation velocity. We found agreement between the predicted and observed sedimentation coefficients for models in which the independently folded catalytic domain, Fn3, and Ig domains are aligned consecutively on the long axis of the molecule. The PEVK segment is modeled as an extensible linker that enables smMLCK to remain bound to F-actin and simultaneously activate the myosin heads of adjacent myosin filaments at a distance of >or=40 nm. The structural properties of smMLCK may contribute to the elasticity of smooth muscle cells.

Heat Treatment Could Affect the Biochemical Properties of Caldesmon

Smooth muscle caldesmon (CaD) exhibits apparent heat stability. A widely used purification procedure of CaD involves extensive heat treatment (Bretscher, A. (1984) J. Biol. Chem. 259, 12873-12880). CaD thus purified co-sediments with actin, inhibits actomyosin ATPase activity, and interacts with Ca2+/calmodulin, similarly to the unheated protein. On the other hand, heat-treated CaD binds to actin filaments in a tether-like fashion, whereas lengthwise binding dominates in vivo (Mabuchi, K., Lin, J. J.-C., and Wang, C.-L. A. (1993) J. Muscle Res. Cell Motil. 14, 54-64), suggesting that differences do exist between heat-purified CaD and the native protein. We have isolated, without heat treatment, full-length recombinant chicken gizzard CaD overexpressed in insect cells (High-Five™) using a baculovirus expression system. We found that such unheated CaD interacts with calmodulin 10 times stronger than does the heated CaD; its inhibitory action on actomyosin ATPase is reversed by a much lesser amount of calmodulin. Moreover, electron microscopic examination indicated that actin binding at the N-terminal region is more frequent in the unheated CaD, resulting in more lengthwise binding. These findings point to the fact that CaD is not entirely heat-stable; the C-terminal CaM-binding regions and the N-terminal actin-binding region are possibly affected by heat treatment.