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Aurora B (AurB) is a mitotic kinase responsible for multiple aspects of mitotic progression, including assembly of the outer kinetochore. Cytoplasmic dynein is an abundant kinetochore protein whose recruitment to kinetochores requires phosphorylation. To assess whether AurB regulates recruitment of dynein to kinetochores, we inhibited AurB using ZM447439 or a kinase-dead AurB construct. Inhibition of AurB reduced accumulation of dynein at kinetochores substantially; however, this reflected a loss of dynein-associated proteins rather than a defect in dynein phosphorylation. We determined that AurB inhibition affected recruitment of the ROD, ZW10, zwilch (RZZ) complex to kinetochores but not zwint-1 or more-proximal kinetochore proteins. AurB phosphorylated zwint-1 but not ZW10 in vitro, and three novel phosphorylation sites were identified by tandem mass spectrometry analysis. Expression of a triple-Ala zwint-1 mutant blocked kinetochore assembly of RZZ-dependent proteins and induced defects in chromosome movement during prometaphase. Expression of a triple-Glu zwint-1 mutant rendered cells resistant to AurB inhibition during prometaphase. However, cells expressing the triple-Glu mutant failed to satisfy the spindle assembly checkpoint (SAC) at metaphase because poleward streaming of dynein/dynactin/RZZ was inhibited. These studies identify zwint-1 as a novel AurB substrate required for kinetochore assembly and for proper SAC silencing at metaphase.

A polyclonal antibody (C4), raised against the head domain of chicken myosin Va, reacted strongly towards a 65 kDa polypeptide (p65) on Western blots of extracts from squid optic lobes but did not recognize the heavy chain of squid myosin V. This peptide was not recognized by other myosin Va antibodies, nor by an antibody specific for squid myosin V. In an attempt to identify it, p65 was purified from optic lobes of Loligo plei by cationic exchange and reverse phase chromatography. Several peptide sequences were obtained by mass spectroscopy from p65 cut from sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels. BLAST analysis and partial matching with expressed sequence tags (ESTs) from a Loligo pealei data bank indicated that p65 contains consensus signatures for the heterogeneous nuclear ribonucleoprotein (hnRNP) A/B family of RNA-binding proteins. Centrifugation of post mitochondrial extracts from optic lobes on sucrose gradients after treatment with RNase gave biochemical evidence that p65 associates with cytoplasmic RNP complexes in an RNA-dependent manner. Immunohistochemistry and immunofluorescence studies using the C4 antibody showed partial co-labeling with an antibody against squid synaptotagmin in bands within the outer plexiform layer of the optic lobes and at the presynaptic zone of the stellate ganglion. Also, punctate labeling by the C4 antibody was observed within isolated optic lobe synaptosomes. The data indicate that p65 is a novel RNA-binding protein located to the presynaptic terminal within squid neurons and may have a role in synaptic localization of RNA and its translation or processing., (Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Cytoplasmic dynein contributes to the localization and transport of multiple membranous organelles, including late endosomes, lysosomes, and the Golgi complex. It remains unclear which subunits of dynein are directly responsible for linking the dynein complex to these organelles, however the intermediate chain (IC), light intermediate chain (LIC) and light chain (LC) subunits are each thought to be important. Based on previous mapping of a dynein IC phosphorylation site (S84), we measured the impact of transfected ICs on dynein-driven organelle transport (Vaughan et al.,2001). Wild-type and S84A constructs disrupted organelle transport, whereas the S84D construct induced no defects. In this study we investigated the mechanisms of transfection-induced disruption of organelle transport. Transfected ICs did not: (1) disrupt the dynein holoenzyme, (2) incorporate into the native dynein complex, (3) dimerize with native dynein ICs or (4) sequester dynein LCs in a phosphorylation-sensitive manner. Consistent with saturation of dynactin as an inhibitory mechanism, truncated ICs containing only the dynactin-binding domain were as effective as full-length IC constructs in disrupting organelle transport, and this effect was influenced by phosphorylation-state. Competition analysis demonstrated that S84D ICs were less capable than dephosphorylated ICs in disrupting the dynein-dynactin interaction. Finally, two-dimensional gel analysis revealed phosphorylation of the wild-type but not S84D ICs, providing an explanation for the incomplete effects of the wild-type ICs. Together these findings suggest that transfected ICs disrupt organelle transport by competing with native dynein for dynactin binding in a phosphorylation-sensitive manner.

Cytoplasmic dynein functions at several sites during mitosis; however, the basis of targeting to each site remains unclear. Tandem mass spectrometry analysis of mitotic dynein revealed a phosphorylation site in the dynein intermediate chains (ICs) that mediates binding to kinetochores. IC phosphorylation directs binding to zw10 rather than dynactin, and this interaction is needed for kinetochore dynein localization. Phosphodynein associates with kinetochores from nuclear envelope breakdown to metaphase, but bioriented microtubule (MT) attachment and chromosome alignment induce IC dephosphorylation. IC dephosphorylation stimulates binding to dynactin and poleward streaming. MT depolymerization, release of kinetochore tension, and a PP1-gamma mutant each inhibited IC dephosphorylation, leading to the retention of phosphodynein at kinetochores and reduced poleward streaming. The depletion of kinetochore dynactin by moderate levels of p50(dynamitin) expression disrupted the ability of dynein to remove checkpoint proteins by streaming at metaphase but not other aspects of kinetochore dynein activity. Together, these results suggest a new model for localization of kinetochore dynein and the contribution of kinetochore dynactin.

Myosin-Va is a molecular motor that may participate in synaptic vesicle cycling. Calpain cleaves myosin-Va in vitro at methionine 1141 in the tail domain. We show that intracellular proteolysis of myosin-Va occurs in rat cortical synaptosomes depolarized in the presence of calcium, evidenced by the formation of an 80 k polypeptide that co-migrates in SDS-PAGE with the 80 k fragment produced by the in vitro proteolysis of myosin-Va by calpain. Anti-myosin-Va antibody recognized this polypeptide in Western blots and immunoprecipitated it from synaptosome extracts. Calpastatin, a calpain-specific inhibitor, or leupeptin, a general cysteine protease inhibitor, suppressed or blocked formation of the 80 k polypeptide depending on membrane permeability. We conclude that myosin-Va undergoes intracellular proteolysis by endogenous calpain, when synaptosomes are depolarized in the presence of calcium, at the same cleavage site previously identified in vitro, thus, making it a target for calcium signaling during synaptic activation.

Brain myosin-Va consists of two heavy chains, each containing a neck domain with six tandem IQ motifs that bind four to five calmodulins and one to two essential light chains. Previous studies demonstrated that myosin-Va exhibits an unusually high affinity for F-actin in the presence of ATP and that its MgATPase activity is stimulated by micromolar Ca(2+) in a highly cooperative manner. We demonstrate here that Ca(2+) also induces myosin-Va binding to and cosedimentation with F-actin in the presence of ATP in a similar cooperative manner and calcium concentration range as that observed for the ATPase activity. Neither hydrolysis of ATP nor buildup of ADP was required for Ca(2+)-induced cosedimentation. The Ca(2+)-induced binding was inhibited by low temperature or by 0.6 m NaCl, but not by 1% Triton X-100. Tight binding between myosin-Va and pyrene-labeled F-actin in the presence of ATP and Ca(2+) was also detected by quenching of the pyrene fluorescence. Negatively stained preparations of actomyosin-Va under Ca(2+)-induced binding conditions showed tightly packed F-actin bundles cross-linked by myosin-Va. Our data demonstrate that high affinity binding of myosin-Va and F-actin in the presence of ATP or 5'-O-(thiotriphosphate) is induced by micromolar concentrations of Ca(2+). Since Ca(2+) regulates both the actin binding properties and actin-activated ATPase of myosin-Va over the same concentration range, we suggest that the calcium signal may regulate the mechanism of processivity of myosin Va.

Myosin V isolated from chick brain (BM V) is a multimeric protein of about 640 kDa consisting of two intertwined heavy chains of 212 kDa and multiple light chains of 10 to 20 kDa. A distinctive feature of the heavy chain is an extended neck region with six consensus IQ sites for the binding of calmodulin (CaM) and myosin light chains. The actin-activated MgATPase has been shown to require >/=1 microM Ca2+ for full activity, and evidence points to a myosin-linked regulatory system where the CaM light chains participate as modulators for the Ca2+ signal. Still, the precise mechanism of Ca2+ regulation remains unknown. In the present study we have used the intrinsic tryptophan fluorescence of native BM V to monitor conformational changes of BM V induced by Ca2+, and we relate these changes to CaM dissociation from the BM V molecule. The fluorescence intensity decreases approximately 17% upon addition of sub-micromolar concentrations of Ca2+ (K0.5 = 0.038 microM). This decrease in fluorescence, which is dominated by a conformational change in the heavy chain, can be reversed by addition of 1, 2-di(2-aminoethoxy)ethane-N,N,N',N'tetraacetic acid (EGTA) followed by an excess of CaM, but not by addition of EGTA alone. Gel filtration of native BM V using HPLC shows that CaM is partially dissociated from the heavy chain in EGTA and dissociates further upon addition of sub-micromolar concentrations of Ca2+. These observations suggest that the affinity of CaM for at least one of the IQ sites on the BM V heavy chain decreases with Ca2+ and that the Ca2+ concentration required for this effect is lower than that needed to activate acto-BM V. Using a cosedimentation assay in the presence of actin, we also observe partial dissociation of CaM when Ca2+ is absent, but now the addition of Ca2+ has a biphasic effect: sub-micromolar Ca2+ concentrations lead to reassociation of CaM with the heavy chain, followed by dissociation when Ca2+ exceeds 5-10 microM. Thus, the binding of CaM to BM V is affected by both actin and Ca2+., (Copyright 1998 Academic Press.)

The actin binding and ATPase properties, as well as the functional domain structure of chick brain myosin-V, a two-headed, unconventional myosin, is reported here. Compared to conventional myosin from skeletal muscle, brain myosin-V exhibits low K-EDTA- and Ca-ATPase activities (1.8 and 0.8 ATP/s per head). The physiologically relevant Mg-ATPase is also low (approximately 0.3 ATP/s), unless activated by the presence of both F-actin and Ca2+ (Vmax of 27 ATP/s). Ca2+ stimulates the actin-activated Mg-ATPase over a narrow concentration range between 1 and 3 microM. In the presence of saturating Ca2+ and 75 mM KCl, surprisingly low concentrations of F-actin activate the Mg-ATPase in a hyperbolic manner (KATPase of 1.3 microM). Brain myosin-V also binds with relatively high affinity (compared to other known myosins) to F-actin in the presence of ATP, as assayed by cosedimentation. Digestion of brain myosin-V with calpain yielded a 65-kDa head domain fragment that cosediments with actin in an ATP-sensitive manner and a 80-kDa tail fragment that does not interact with F-actin. The 80-kDa fragment results from cleavage one residue beyond the proline-, glutamate-, serine-, threonine-rich region. Our data indicate that the Mg-ATPase cycle of brain myosin-V is tightly regulated by Ca2+, probably via direct binding to the calmodulin light chains in the neck domain, which like brush border myosin-I, results in partial (approximately 30%) dissociation of the calmodulin associated with brain myosin-V. The effect of Ca2+ binding, which appears to relieve suppression by the neck domain, can be mimicked by calpain cleavage near the head/neck junction.