Characterization of p80, a novel nuclear and cytoplasmic protein in dinoflagellates.

The presence of myosin in dinoflagellates was tested using an anti-Acanthamoeba castellanii myosin II polyclonal antibody on the heterotrophic dinoflagellate Crypthecodinium cohnii Seligo. Western blots revealed the presence of a unique band of 80 kDa in total protein extracts and after immunoprecipitation. Expression of this 80 kDa protein appeared constant during the different phases of the cell cycle. In protein extracts from various other dinoflagellates, this 80 kDa protein was detected only in the autotrophic species Prorocentrum micans Ehr. Screening of a C. cohnii cDNA expression library with this antibody revealed a cDNA coding for an amino acid sequence without homology in the databases. However, particular regions were detected: - a polyglutamine repeat domain in the N-terminal part of the protein, - four peptide sequences associated with GTP-binding sites, - a sequence with slight homology to the rod tail of Caenorhabditis elegans myosin II, -a sequence with homology to a human kinesin motor domain. Immunocytolocalization performed on C. cohnii thin sections with a polyclonal antibody raised against the recombinant protein showed p80 to be present both within the nucleus and in the cytoplasm. Labelling was widespread in the nucleoplasm and more concentrated at the periphery of the permanently condensed chromosomes. In the cytoplasm, labelling appeared in a punctate region close to the nucleus and in the flagellum. Potential functions of this novel protein are discussed.     

Developmental expression of Xenopus myosin 1d and identification of a myo1d tail homology that overlaps TH1

Xenopus laevis myosin 1d (XlMyo1d) is a member of the myosin I class, subclass 4. Members of this class are single headed, bind calmodulin light chains and have lipid binding domains in their tails. The rat myo1d homologue has been implicated in endosome vesicle recycling in epithelial cells. Mutations in the Drosophila myosin 1d homologue cause situs inversus in the abdomen. The XlMyo1d cDNA has been cloned and the derived amino acid sequence is 80% identical to the rat and human homologues. Sequence comparison revealed a novel isoform‐specific tail homology embedded in the Tail Homology 1 (TH1) domain characteristic of myosin I isoforms. Western blot analysis using a polyclonal antibody raised against an isoform‐specific peptide showed that the protein is present in eggs and levels increase at early neurula through tadpole stages. Whole mount in situ hybridization using a probe containing the 5′UTR (untranslated region) showed that XlMyo1d mRNA is expressed in neural tube, pre‐somitic mesoderm, somites and all three segments of cranial neural crest cells during their migration. Sections of the in situ hybridizations revealed that during somitogenesis, XlMyo1d mRNA was localized to a stripe overlapping the nuclear region of somites during early tadpole stages.     

Immunolocalization of myosin in rhizoids ofChara globularis Thuill

Summary Myosin-related proteins have been localized immunocytochemically in gravity-sensing rhizoids of the green algaChara globularis using a monoclonal antibody against the heavy chain of myosin from mouse 3T3 cells and a polyclonal antibody to bovine skeletal and smooth muscle myosin. In the basal zone of the rhizoids which contain a large vacuole, streaming endoplasm and stationary cortical cytoplasm, the monoclonal antibody stained myosin-related proteins as diffusely fluorescing endoplasmic strands. This pattern is similar to the arrangement of subcortical actin filament bundles. In the apical zone which contains an aggregation of ER membranes and secretory vesicles for tip growth, diffuse immunofluorescence was detected; the intensity of the signal increasing towards the apical cell wall. The most prominent myosin-staining was associated with the surface of statoliths in the apical zone. The polyclonal antibody produced a punctate staining pattern in the basal zone, caused by myosin-related proteins associated with the surface of drganelles in the streaming endoplasm and the periphery of the nucleus. In the apical zone, this antibody revealed myosin-immunofluorescence on the surface of statoliths in methacrylate-embedded rhizoids. Neither antibody revealed myosin-immunofluorescence on the surface of organelles and vesicles in the relatively stationary cytoplasm of the subapical zone. These results indicate (i) that different classes of myosin are involved in the various transport processes inChara rhizoids; (ii) that cytoplasmic streaming in rhizoids is driven by actomyosin, corresponding to the findings onChara internodal cells; (iii) that actindependent control of statolith position and active movement is mediated by myosin-related proteins associated with the statolith surfaces; and (iv) that myosin-related proteins are involved in the process of tip growth.     

Class VI myosin moves processively along actin filaments backward with large steps.

Among a superfamily of myosin, class VI myosin moves actin filaments backwards. Here we show that myosin VI moves processively on actin filaments backwards with large ( approximately 36 nm) steps, nevertheless it has an extremely short neck domain. Myosin V also moves processively with large ( approximately 36 nm) steps and it is believed that myosin V strides along the actin helical repeat with its elongated neck domain that is critical for its processive movement with large steps. Myosin VI having a short neck cannot take this scenario. We found by electron microscopy that myosin VI cooperatively binds to an actin filament at approximately 36 nm intervals in the presence of ATP, raising a hypothesis that the binding of myosin VI evokes "hot spots" on actin filaments that attract myosin heads. Myosin VI may step on these "hot spots" on actin filaments in every helical pitch, thus producing processive movement with 36 nm steps.     

Immunogold method evidences that kinesin and myosin bind to and couple microtubules and actin filaments in lipotubuloids of Ornithogalum umbellatum ovary epidermis

Lipotubuloids in ovary epidermis of Ornithogalum umbellatum which are a domain of cytoplasm containing a lot of lipid bodies, microtubules and actin filaments, ribosomes, endoplasmic reticulum as well as scarce mitochondria, microbodies, dictyosomes, autolytic vacuoles, exhibit progressive-rotary motion. The immunogold method demonstrated that microtubules and actin filaments of lipotubuloids might be connected with one another by myosin and kinesin. It was supposed that collaboration of motor proteins with actin filaments and microtubules makes autonomic high peripheral speed rotary motion of lipotubuloids in epidermis cells possible. Moreover, myosin was also detected in Golgi bodies in lipotubuloid. In lipotubuloids, the immunogold method demonstrated immunosignals after the use of an antibody to dynein light chains but spectroscopy mass analysis showed that in O. umbellatum epidermis lacked dynein heavy chains.     

Myosins from angiosperms, ferns, and algae amplification of gene fragments with versatile PCR primers and detection of protein products with a monoclonal antibody to a conserved head epitope

Summary Myosins providing the motors for the actin-based motility that occurs in diverse plants have proved difficult to study. To facilitate those studies, we describe polymerase chain reaction primers that reliably amplify part of the myosin head from diverse plants, consensus sequences that characterise the amplified product as encoding a class V or class VIII myosin, and a monoclonal antibody that recognises an epitope conserved in the head of most plant, fungal, and animal myosins. A pair of stringent oligonucleotide primers was designed that, when used in the polymerase chain reaction, amplified at least eleven different myosins from five species of angiosperms and one sequence from each of the fernAzolla and the algaeNitella andPhaeodactylum. The amplified products, comprising 126 to 135 nucleotides encoding part of the myosin head domain, can be used as myosin-specific probes to screen genomic and cDNA libraries. To identify the products of plant myosin genes, we raised a monoclonal antibody (anti-CHE) to a nine amino acid peptide matching a conserved head epitope showing not more than single amino acid substitutions in most published myosin genes. This antibody recognises rabbit skeletal myosin and multiple polypeptides of >100 kDa in four angiosperms and in the algaNitella. Relating the M_r values of immunoreactive bands inArabidopsis extracts to the predicted M_r values of the products of five myosin genes supports the view that the antibody recognises both myosins V and VIII together with the products of some as yet unsequenced genes. The previously described MB170 antibodies may, in contrast, be specific for one or more type V myosins. Together, the polymerase chain reaction primers and the antibody represent versatile tools for identifying and categorising myosins in diverse plants.     

The changing epidemiology of dengue in Delhi, India

The distribution of deformed wing virus infection within the honey bee reproductive castes (queens, drones) was investigated by in situ hybridization and immunohistology from paraffin embedded sections. Digoxygenin or CY5.5 fluorochrome end-labelled nucleotide probes hybridizing to the 3' portion of the DWV genome were used to identify DWV RNA, while a monospecific antibody to the DWV-VP1 structural protein was used to identify viral proteins and particles. The histological data were confirmed by quantitative RT-PCR of dissected organs. Results showed that DWV infection is not restricted to the digestive tract of the bee but spread in the whole body, including queen ovaries, queen fat body and drone seminal vesicles.     

Interactions and regulation of molecular motors in Xenopus melanophores

Many cellular components are transported using a combination of the actin- and microtubule-based transport systems. However, how these two systems work together to allow well-regulated transport is not clearly understood. We investigate this question in the Xenopus melanophore model system, where three motors, kinesin II, cytoplasmic dynein, and myosin V, drive aggregation or dispersion of pigment organelles called melanosomes. During dispersion, myosin V functions as a "molecular ratchet" to increase outward transport by selectively terminating dynein-driven minus end runs. We show that there is a continual tug-of-war between the actin and microtubule transport systems, but the microtubule motors kinesin II and dynein are likely coordinated. Finally, we find that the transition from dispersion to aggregation increases dynein-mediated motion, decreases myosin V--mediated motion, and does not change kinesin II--dependent motion. Down-regulation of myosin V contributes to aggregation by impairing its ability to effectively compete with movement along microtubules.     

The temporal and spatial expression pattern of myosin Va, Vb and VI in the mouse ovary

There are 16 classes of unconventional myosins. Class V myosins have been shown to be involved in transporting cargo to and from the cell periphery. Class VI myosins have also been shown to transport cargo from the cell periphery, although it seems that these proteins have many roles which include the mediation of cell migration and stereocillia stabilisation. With the requirement of myosin VI for Drosophila oogenesis, the localised expression of Myosin V in the developing egg chamber and recent mounting evidence which links myosin VI to the migration of human ovarian cancer cell lines, we wanted to investigate the expression pattern of these two myosin classes in the normal mouse ovary. Here we show that these myosins are expressed, localised and regulated within the oocyte and granulosa cells of the developing mouse follicle.     

PARTIAL CHARACTERIZATION OF A PUTATIVE 110kDa MYOSIN FROM THE GREEN ALGACHARA CORALLINABYIN VITROBINDING OF FLUORESCENT F-ACTIN

Using the binding of heterologous, rhodamine phalloidin-labelled F-actinin vitro, two F-actin binding proteins were identified in protein extracts from the green algaChara corallinaafter fractionation by anion exchange chromatography. The first protein, a putative myosin, released laterally bound F-actin at ATP-concentrations as low as 1μm ; equivalent concentrations of ADP were not effective. Binding of F-actin was inhibited by the sulfhydryl-alkylating agent N-ethylmaleimide (NEM). Binding of F-actin was also abolished by a monoclonal anti-myosin (J14) previously used for immunodetection and immunolocalization in internodal cells (Groliget al., 1988,Eur J Cell Biol 47 : 22–31). Immunoblotting with J14 detected a 110kDa polypeptide only in those protein fractions that had revealed ATP-sensitive binding of F-actin. The putative myosin bound with mediocre affinity to immobilized calmodulin and free Ca²⁺-concentration made no difference to this binding affinity. In contrast to the putative myosin, the second, less abundant protein revealed ATP-insensitive and end-wise binding to the microfilament and was not recognized by the anti-myosin antibody.     

Myosin VI and Optineurin Are Required for Polarized EGFR Delivery and Directed Migration

The polarized trafficking of membrane proteins into the leading edge of the cell is an integral requirement for cell migration. Myosin VI and its interacting protein optineurin have previously been shown to operate in anterograde trafficking pathways, especially for the polarized delivery of cargo to the basolateral domain in epithelial cells. Here we show that in migratory cells ablation of myosin VI or optineurin inhibits the polarized delivery of the epidermal growth factor receptor (EGFR) into the leading edge and leads to profound defects in lamellipodia formation. Depletion of either myosin VI or optineurin, however, does not impair the overall ability of cells to migrate in a random migration assay, but it dramatically reduces directed migration towards a growth factor stimulus. In summary, we identified a specific role for myosin VI and optineurin in directionally persistent cell migration, which involves the polarized delivery of vesicles containing EGFR into the leading edge of the cell.     

Evidence suggesting the presence of common antigenic determinant between dynein and tubulin.

The present experiments showed that the guinea pig antiserum prepared against the main polypeptides of 14 S dynein from Tetrahymena cilia reacted with sea urchin sperm flagellar dynein and with bovine brain high molecular weight protein to give rise to a precipitin line confluent with that formed between the antiserum and Tetrahymena dynein. Furthermore, it was found that this antiserum also reacted with tubulins from Tetrahymena cilia, sea urchin sperm flagella and bovine brain to give rise to the confluent precipitin line. Among muscle proteins, only actin preparation from rabbit skeletal muscle reacted with the anti-Tetrahymena dynein serum, whereas neither rabbit skeletal muscle myosin, chicken skeletal muscle tropomyosin nor chicken skeletal muscle troponin reacted with the antiserum. These results suggest that dynein and tubulin and probably actin share an antigenic determinant regardless of different protein species and of different animal species. The common antigenic determinant was detected only when the proteins denatured with urea/sodium dodecyl sulfate/beta-mercaptoethanol/N-ethylmaleimide were used, but it was not detected at all when the native proteins were used. This implies that a certain common antigenic determinant which is involved in the precipitin line formation exists in the primary structures of dyneins and tubulins and probably actin, and is hidden inside the tertiary structures of the native protein molecules.     

Myosin VI binds to and localises with Dab2, potentially linking receptor-mediated endocytosis and the actin cytoskeleton

Myosin VI, an actin-based motor protein, and Disabled 2 (Dab2), a molecule involved in endocytosis and cell signalling, have been found to bind together using yeast and mammalian two-hybrid screens. In polarised epithelial cells, myosin VI is known to be associated with apical clathrin-coated vesicles and is believed to move them towards the minus end of actin filaments, away from the plasma membrane and into the cell. Dab2 belongs to a group of signal transduction proteins that bind in vitro to the FXNPXY sequence found in the cytosolic tails of members of the low-density lipoprotein receptor family. The central region of Dab2, containing two DPF motifs, binds to the clathrin adaptor protein AP-2, whereas a C-terminal region contains the binding site for myosin VI. This site is conserved in Dab1, the neuronal counterpart of Dab2. The interaction between Dab2 and myosin VI was confirmed by in vitro binding assays and coimmunoprecipitation and by their colocalisation in clathrin-coated pits/vesicles concentrated at the apical domain of polarised cells. These results suggest that the myosin VI–Dab2 interaction may be one link between the actin cytoskeleton and receptors undergoing endocytosis.     

Dissociation of double-headed cytoplasmic dynein into single-headed species and its motile properties

Cytoplasmic dynein is a minus-end directed microtubule motor and plays important roles in the transport of various intracellular cargoes. Cytoplasmic dynein comprises two identical heavy chains and forms a dimer (double-headed dynein); the total molecular weight of the cytoplasmic dynein complex is about 1.5 million. The dynein motor domain is structurally very different from those of kinesin and myosin, and our understanding of the mechanisms of dynein energy transduction is limited mainly because of the difficulty in obtaining a sufficient quantity of purified and active cytoplasmic dynein. We purified cytoplasmic dynein, which was free from dynactin and other dynein-associated proteins. The purified cytoplasmic dynein was active in an in vitro motility assay. The controlled dialysis of the purified dynein against 4 M urea resulted in its complete dissociation into monomeric species (single-headed dynein). The separation of the dynein heads by the treatment was reversible. The MgATPase activities of the single-headed and reconstituted double-headed dynein were comparable to that of intact dynein. The double-headed dynein bundled microtubules in the absence of ATP; the single-headed dynein did not. The single-headed dynein produced in vitro microtubule-gliding motility at velocities very similar to those of double-headed dynein at various ATP concentrations. These results indicate that a single cytoplasmic dynein heavy chain is sufficient to produce robust microtubule motility. Application of the double- and single-headed dynein molecules in various assay systems will elucidate the mechanism of action of the cytoplasmic dynein.     

Properties of a monoclonal antibody directed to the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase

A synthetic peptide representing the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase (K-R-R-W-K-K-N-F-I-A-V-S-A-A-N-R-F-K-K-I-S-S-S-G-A-L) was used as an antigen to produce a monoclonal antibody. The antibody (designated MAb RSkCBP1, of the IgM class) reacted with similar affinity (KD approximately 20 nM) by competitive enzyme-linked immunoassay (ELISA) with the antigen peptide and intact rabbit skeletal muscle myosin light chain kinase. MAb RSkCBP1 inhibited rabbit skeletal muscle myosin light chain kinase activity competitively with respect to calmodulin (Ki = 20 nM). The antibody also inhibited myosin light chain kinase activity in extracts of skeletal muscle from several mammalian species (rabbit, sheep, and bovine) and an avian species (chicken). The concentration of MAb RSKCBP1 required for 50% inhibition of enzyme activity was similar for the mammalian species (80 nM) but was significantly higher for the avian species (1.2 microM). A competitive ELISA protocol was used to analyze weak cross-reactivity to other calmodulin-binding peptides and proteins. This assay demonstrated no cross-reactivity with the venom peptides melittin or mastoparan; smooth muscle myosin light chain kinases from hog carotid, bovine trachea, or chicken gizzard; bovine brain calmodulin-dependent calcineurin; or rabbit skeletal muscle troponin I. These data support the contention that the synthetic peptide used as the antigen represents the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase and that the calmodulin-binding domains of different calmodulin-regulated proteins may have distinct primary and/or higher order structures.     

Multimodal regulation of myosin VI ensemble transport by cargo adaptor protein GIPC

A range of cargo adaptor proteins are known to recruit cytoskeletal motors to distinct subcellular compartments. However, the structural impact of cargo recruitment on motor function is poorly understood. Here, we dissect the multimodal regulation of myosin VI activity through the cargo adaptor GAIP-interacting protein, C terminus (GIPC), whose overexpression with this motor in cancer enhances cell migration. Using a range of biophysical techniques, including motility assays, FRET-based conformational sensors, optical trapping, and DNA origami–based cargo scaffolds to probe the individual and ensemble properties of GIPC–myosin VI motility, we report that the GIPC myosin-interacting region (MIR) releases an autoinhibitory interaction within myosin VI. We show that the resulting conformational changes in the myosin lever arm, including the proximal tail domain, increase the flexibility of the adaptor–motor linkage, and that increased flexibility correlates with faster actomyosin association and dissociation rates. Taken together, the GIPC MIR–myosin VI interaction stimulates a twofold to threefold increase in ensemble cargo speed. Furthermore, the GIPC MIR–myosin VI ensembles yield similar cargo run lengths as forced processive myosin VI dimers. We conclude that the emergent behavior from these individual aspects of myosin regulation is the fast, processive, and smooth cargo transport on cellular actin networks. Our study delineates the multimodal regulation of myosin VI by the cargo adaptor GIPC, while highlighting linkage flexibility as a novel biophysical mechanism for modulating cellular cargo motility.     

Conformationally altered aortic myosin light chains

Aorta smooth myosin contains two types of light chain, LC20 and LC17, which fold together with the N-terminal region of each heavy chain to form the globular head region of myosin. We demonstrate an altered conformation of LC20 after its separation from heavy chain by high concentrations of urea, on the basis of the following evidende: 1) A polyclonal antibody against LC20 was not able to recognize this conformationally altered form; 2) Myosin reconstituted from heavy chains and urea-dissociated light chains exhibited extremely low ATPase activity. Circular dichroism unfolding profiles showed that light chains dissociated from heavy chains by SDS appeared to be more stable than those generated by urea dissociation.     

Amino acid composition of dynein and comparison with myosin.

A comparison is made between dynein [flagellar ATPase; EC 3.6.1.3], purified from sea urchin sperm flagella, and muscle myosin. The amino acid composition of dynein was found to be statistically different from that of myosin. The same was true of their tryptic fragments retaining ATPase activity, i.e., Fragment A of dynein and heavy meromyosin. At low ionic strength, no superprecipitation took place when ATP was added to a mixture of dynein and actin, and stimulation of the Mg2+-ATPase activity of dynein remained below 50% even when a one-hundred-fold excess of actin was present. No viscosity drop was caused by adding ATP to a solution containing dynein and actin. Anti-myosin antiserum did not react with dynein, while anti-Fragment A antiserum formed no precipit-n line against myosin. Furthermore, the amount of dynein that combined with F-actin was less than one-fifth of the amount of dynein that fully combined with microtubules. These results are consistent with the dissimilarity in enzymatic and other physiocochemical properties of these two proteins.