Myosin-like Proteins Research Articles

An actomyosin-like cytoskeleton in the cyanobiont (Nosctoc sp.) of Peltigera canina

Lichenized Nostoc cells isolated from the lichen Peltigera canina develop chemotaxis towards a lectin purified from the lichen thallus. Similar movements in unicellular eukaryotes require actin and myosin to generate contraction and relaxation along the chemotactic axis. We provide evidences for prokaryotic actin-like and myosin-like proteins in the cyanobiont Nostoc sp using cross-reacting antibodies against α- and β-actin and non-muscle myosin II light and heavy chains, and two-dimensional gel electrophoresis to determine the isoelectric point (IP) of the actin-like protein. Actin antibodies bound to a single reactive Nostoc polypeptide of an approximate molecular mass of 50kD with IP values between 4 and 7pH, similar to eukaryotic actin. The myosin light chain antibody reacted with a Nostoc protein with an apparent molecular weight of 20kDa and another of 48kDa. Immunoprecipitation of cell free extracts using anti-heavy chain myosin separately yielded only one signal corresponding to a protein of a molecular weight around 200kDa. A bioinformatics analysis indicated that (cyano)bacterial actins are rare but do exist. Our results are consistent with the possible existence of protein homologues of actins and myosins in cyanobionts of P. canina, suggesting the existence of an actin-like apparatus that supports chemotactic swimming.

Identification of 24 h Ixodes scapularis immunogenic tick saliva proteins

Ixodes scapularis is arguably the most medically important tick species in the United States. This tick transmits 5 of the 14 human tick-borne disease (TBD) agents in the USA: Borrelia burgdorferi, Anaplasma phagocytophilum, B. miyamotoi, Babesia microti, and Powassan virus disease. Except for the Powassan virus disease, I. scapularis-vectored TBD agents require more than 24h post attachment to be transmitted. This study describes identification of 24h immunogenic I. scapularis tick saliva proteins, which could provide opportunities to develop strategies to stop tick feeding before transmission of the majority of pathogens. A 24h fed female I. scapularis phage display cDNA expression library was biopanned using rabbit antibodies to 24h fed I. scapularis female tick saliva proteins, subjected to next generation sequencing, de novo assembly, and bioinformatic analyses. A total of 182 contigs were assembled, of which ∼19% (35/182) are novel and did not show identity to any known proteins in GenBank. The remaining ∼81% (147/182) of contigs were provisionally identified based on matches in GenBank including ∼18% (27/147) that matched protein sequences previously annotated as hypothetical and putative tick saliva proteins. Others include proteases and protease inhibitors (∼3%, 5/147), transporters and/or ligand binding proteins (∼6%, 9/147), immunogenic tick saliva housekeeping enzyme-like (17%, 25/147), ribosomal protein-like (∼31%, 46/147), and those classified as miscellaneous (∼24%, 35/147). Notable among the miscellaneous class include antimicrobial peptides (microplusin and ricinusin), myosin-like proteins that have been previously found in tick saliva, and heat shock tick saliva protein. Data in this study provides the foundation for in-depth analysis of I. scapularis feeding during the first 24h, before the majority of TBD agents can be transmitted.

The myosin ATPase inhibitor, 2,3-butanedione 2-monoxime, prevents protein secretion by the basidiomycete Coprinopsis cinerea

The plant-saprophytic basidiomycete, Coprinopsis cinerea, produces and secretes various cellulases during cellulose degradation as the main extracellular proteins. Although enzymatic characterization of such cellulases has been frequently reported, the mechanism of their secretion remains unclear. This study focused on myosins, actin-based motor proteins, involved in protein secretion in C. cinerea. During cultivation under cellulase-inducing condition, no cellulase activity was observed when the mycelia were treated with 2,3-butanedione 2-monoxime (BDM), a general inhibitor of myosin ATPase. Furthermore, BDM treatment disrupted the localization of the Golgi apparatus, but not that of the endoplasmic reticulum. Three genes encoding myosin-like proteins (CcMyo1, CcMyo2 and CcMyo5) were identified from the C. cinerea genome database. Transcription of these genes was promoted when the fungus was grown under cellulase-inducing condition.

Baculovirus Reveals a New pH-dependent Direct Cell-fusion Pathway For Cell Entry and Transgene Delivery

Evaluation of: Dong S, Wang M, Qiu Z et al.: AcMNPV efficiently infects Sf9 cells and transduces mammalian cells via direct fusion with the plasma membrane at low pH. J. Virol. 84(10), 5351–5359 (2010). The article evaluated unravels the existence of an alternative infection pathway of baculoviruses, such as Autographa californica multiple nuclear polyhedrosis virus, for infection of insect cells by altering pH conditions. The authors have demonstrated that lowering the pH within an insect cell can induce baculovirus cell entry through an alternative direct cell membrane fusion pathway independent of the normal endocytosis pathway. They explain that this mode of cell entry and the corridor to the nucleus is dependent on myosin-like proteins and not on microtubules. The article also reports, for the first time, that baculovirus can also efficiently transduce mammalian cells using a direct fusion pathway induced by a short pH trigger, which can be further enhanced by completely blocking the probable endocytosis pathway. These findings by Dong et al. provide us with a further step in understanding the different routes by which baculovirus can enter insect and mammalian cells. In fact, this study gives us an insight to the development of a novel pH-dependent baculovirus-mediated therapeutic strategy for efficient transgene delivery to mammalian cells and also for an advanced insect cell-based protein production system.

Autographa californica Multicapsid Nucleopolyhedrovirus Efficiently Infects Sf9 Cells and Transduces Mammalian Cells via Direct Fusion with the Plasma Membrane at Low pH

The budded virus (BV) of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) infects insect cells and transduces mammalian cells mainly through the endocytosis pathway. However, this study revealed that the treatment of the virus bound to Sf9 cells at low pH could efficiently rescue the infectivity of AcMNPV in the presence of endocytosis pathway inhibitors. A colocalization assay of the major capsid protein VP39 with the early endosome marker EEA1 showed that at low pH, AcMNPV entered Sf9 cells via an endosome-independent pathway. Using a fluorescent probe (R18), we showed that at low pH, the viral nucleocapsid entered Sf9 cells via direct fusion at the cell surface. By using the myosin-specific inhibitor 2,3-butanedione monoxime (BDM) and the microtubule inhibitor nocodazole, the low pH-triggered direct fusion was demonstrated to be dependent on myosin-like proteins and independent of microtubules. The reverse transcription-PCR of the IE1 gene as a marker for viral entry showed that the kinetics of AcMNPV in cells triggered by low pH was similar to that of the normal entry via endocytosis. The low pH-mediated infection assay and VP39 and EEA1 colocalization assay also demonstrated that AcMNPV could efficiently transduce mammalian cells via direct membrane fusion at the cell surface. More importantly, we found that a low-pH trigger could significantly improve the transduction efficiency of AcMNPV in mammalian cells, leading to the potential application of this method when using baculovirus as a vector for heterologous gene expression and for gene therapy.

Absence of yKu/Hdf1 but not myosin-like proteins alters chromosome dynamics during prophase I in yeast

Meiosis is central to the formation of haploid gametes or spores in that it segregates homologous chromosomes and halves the chromosome number. A prerequisite of this genome bisection is the pairing of homologous chromosomes during the first meiotic prophase. When budding yeast cells are induced to undergo meiosis, this has profound consequences for nuclear structure: after premeiotic DNA replication centromeres disperse, while telomeres move about the nuclear periphery and temporarily cluster during the leptotene/zygotene transition (bouquet stage) of the prophase to first meiotic division. In vegetative cells, Hdf1p (yKu) and the myosin-like proteins Mlp1p and Mlp2p have been suggested to contribute to the organization of silent chromatin, tethering of telomeres to the nuclear periphery, DNA repair, and telomere maintenance. Here, we investigated by molecular cytology whether yKu and Mlp proteins contribute to telomere and chromosome dynamics in meiosis. It was found that mlp1Δ mlp2Δ double-mutant cells undergo centromere dispersion, telomere clustering, homologue pairing, and sporulation like wild type. On the other hand, cells deficient for yKu underwent meiosis-specific chromosomal events with a delay, while they eventually sporulated like wild type. These results suggest that the absence of yKu not only affects vegetative nuclear architecture (Laroche et al., 1998) but also interferes with the ordered occurrence of chromosome dynamics during first meiotic prophase.

Mlp-dependent anchorage and stabilization of a desumoylating enzyme is required to prevent clonal lethality

Myosin-like proteins 1 and 2 (Mlp1 and Mlp2) form filaments attached to the nucleoplasmic side of the nuclear pore complexes via interaction with the nucleoporin Nup60. Here, we show that Mlps and Nup60, but not several other nucleoporins, are required to localize and stabilize a desumoylating enzyme Ulp1. Moreover, like Mlps, Ulp1 exhibits a unique asymmetric distribution on the nuclear envelope. Consistent with a role in regulating Ulp1, removal of either or both MLPs affects the SUMO conjugate pattern. We also show that deleting MLPs or the localization domains of Ulp1 results in DNA damage sensitivity and clonal lethality, the latter of which is caused by increased levels of 2-micron circle DNA. Epistatic and dosage suppression analyses further demonstrate that Mlps function upstream of Ulp1 in 2-micron circle maintenance and the damage response. Together, our results reveal that Mlps play important roles in regulating Ulp1 and subsequently affect sumoylation stasis, growth, and DNA repair.

Motor function of unconventional myosin.

Myosins are motor proteins that interact with actin filaments and convert energy from ATP hydrolysis into mechanical force. In addition to the well-characterized conventional, filament forming, two-headed myosin II of muscle and non-muscle cells, a number of myosin-like proteins have recently been discovered. Based upon their amino acid sequences, these newly found “myosins” do not seem to form myosin filaments, thus they are often called “unconventional” myosins. The discovery of these “myosin-like motor proteins” has fundamentally expanded the potential physiological importance of myosins in diverse biological processes such as chemotactic motility, endocytosis, exocytosis, phagocytosis, vesicular trafficking, secretion, etc. The myosins are classified based upon phylogenetie sequence comparisons of the motor domain (Cheney et al, 1993; Goodson and Spudich, 1993; Mooseker and Cheney, 1995; Cope et al, 1996; Titus, 1997; Hodge and Cope, 2000) and divided into at least 18 classes. In vertebrates, it has been shown that eleven classes of myosin, (including conventional filament forming myosin) are expressed. The N-terminal domains of these classes of unconventional myosins are relatively conserved and contain the primary force production machinery, whereas the C-terminal tail domains are highly divergent and are thought to function as targeting sites that bind to the cellular partner molecules. Between the motor and the diverse tail domains of myosin, there are neck regions that are composed of various numbers of light chain binding motifs (Mermall et al, 1998).

Live Imaging of Telomeres: yKu and Sir Proteins Define Redundant Telomere-Anchoring Pathways in Yeast

Background: The positioning of chromosomal domains within interphase nuclei is thought to facilitate transcriptional repression in yeast. Although this is particularly well characterized for telomeres, the molecular basis of their specific subnuclear organization is poorly understood. The use of live fluorescence imaging overcomes limitations of in situ staining on fixed cells and permits the analysis of chromatin dynamics in relation to stages of the cell cycle.Results: We have characterized the dynamics of yeast telomeres and their associated domains of silent chromatin by using rapid time-lapse microscopy. In interphase, native telomeres are highly dynamic but remain within a restricted volume adjacent to the nuclear envelope. This constraint is lost during mitosis. A quantitative analysis of selected mutants shows that the yKu complex is necessary for anchoring some telomeres at the nuclear envelope (NE), whereas the myosin-like proteins Mlp1 and Mlp2 are not. We are able to correlate increased telomeric repression with increased anchoring and show that silent chromatin is tethered to the NE in a Sir-dependent manner in the absence of the yKu complex. Sir-mediated anchoring is S phase specific, while the yKu-mediated pathway functions throughout interphase. Subtelomeric elements of yeast telomere structure influence the relative importance of the yKu- and Sir-dependent mechanisms.Conclusions: Interphase positioning of telomeres can be achieved through two partially redundant mechanisms. One requires the heterodimeric yKu complex, but not Mlp1 and Mlp2. The second requires Silent information regulators, correlates with transcriptional repression, and is specific to S phase.

Myosin-like proteins 1 and 2 are not required for silencing or telomere anchoring, but act in the Tel1 pathway of telomere length control

The positioning of chromosomal domains in interphase nuclei is thought to facilitate transcriptional repression in yeast. It has been reported that two large coiled-coil proteins of the nuclear envelope, myosin-like proteins 1 and 2, play direct roles in anchoring yeast telomeres to the nuclear periphery, thereby creating a subcompartment enriched for Sir proteins. We have created strains containing complete deletions of mlp1 and mlp2 genes, as well as the double null strain, and find no evidence for the disruption of telomere anchoring at the nuclear periphery in these cells. We also detect no disruption of telomere-associated gene silencing. We confirm, on the other hand, that mlp mutants are particularly sensitive to DNA-damaging agents, such as bleomycin. Moreover, we show that rather than having short telomeres as in yKu-deficient strains, the mlp1 mlp2 strains have extended telomeres, resembling phenotypes of mutations in rif1. Whereas the mlp1 mlp2 mutations act on a pathway of telomere length regulation different from that of yKu70, the effects of the tel1 deletion are epistatic to the mlp mutations, suggesting that the Mlp proteins restrict telomere length in wild-type cells by influencing the Rif–Tel1 pathway of telomerase regulation.

Protein kinase CK2 and regulation of Ca2+-ATPase activity in brain neuron chromatin in rats during aging.

Protein kinase CK2 from neuronal chromatin phosphorylates myosin-like proteins isolated from rat brain chromatin. The protein kinase CK2 activator 1-ethyl-4,5-di(N-methylcarbamoyl)imidazole in vitro stimulated phosphorylation of myosin-like proteins and increased myosin-type Ca(2+)-ATPase activity in neuronal chromatin. After systemic administration this agent normalized Ca(2+)-ATPase activity of brain chromatin in aged rats.

Rab-interacting lysosomal protein (RILP): the Rab7 effector required for transport to lysosomes.

Rab7 is a small GTPase that controls transport to endocytic degradative compartments. Here we report the identification of a novel 45 kDa protein that specifically binds Rab7GTP at its C-terminus. This protein contains a domain comprising two coiled-coil regions typical of myosin-like proteins and is found mainly in the cytosol. We named it RILP (Rab-interacting lysosomal protein) since it can be recruited efficiently on late endosomal and lysosomal membranes by Rab7GTP. RILP-C33 (a truncated form of the protein lacking the N-terminal half) strongly inhibits epidermal growth factor and low-density lipoprotein degradation, and causes dispersion of lysosomes similarly to Rab7 dominant-negative mutants. More importantly, expression of RILP reverses/prevents the effects of Rab7 dominant-negative mutants. All these data are consistent with a model in which RILP represents a downstream effector for Rab7 and both proteins act together in the regulation of late endocytic traffic.

Microfilaments in regenerating cells of rabbit cornea: immunological and ultrastructural observations.

The role of myosin-like protein in regenerating and proliferating corneal cells following a standard alkali-injury in the rabbit eye has been studied. Microfilaments were observed by conventional transmission electron microscopy (T.E.M.) in injured epithelium and in fibroblasts. Typical microfilament bundles with electron dense structures and with stress fibers were evident. The presence of myosin-like proteins was demonstrated by means of immunochemical and autoradiographical techniques. Both epithelial cells and fibroblasts bind antimyosin-like antibodies (AMA). The same cells studied with the E.M. showed bundles of microfilaments in the cortical areas of their cytoplasm in correspondence with the same side of fluorescent or labelled AMA. The immunochemical and ultrastructural results suggest that both cells are able to produce in vivo movements involved in morphogenetic phenomena. Therefore, these structures play a role in post-traumatic corneal regeneration.

Cse15, cse60, and csk22 are new members of mother-cell-specific sporulation regulons in Bacillus subtilis.

We report on the characterization of three new transcription units expressed during sporulation in Bacillus subtilis. Two of the units, cse15 and cse60, were mapped at about 123 degrees and 62 degrees on the genetic map, respectively. Their transcription commenced around h 2 of sporulation and showed an absolute requirement for sigmaE. Maximal expression of both cse15 and cse60 further depended on the DNA-binding protein SpoIIID. Primer extension results revealed -10 and -35 sequences upstream of the cse15 and cse60 coding sequences very similar to those utilized by sigmaE-containing RNA polymerase. Alignment of these and other regulatory regions led to a revised consensus sequence for sigmaE-dependent promoters. A third transcriptional unit, designated csk22, was localized at approximately 173 degrees on the chromosome. Transcription of csk22 was activated at h 4 of sporulation, required the late mother-cell regulator sigmaK, and was repressed by the GerE protein. Sequences in the csk22 promoter region were similar to those of other sigmaK-dependent promoters. The cse60 locus was deduced to encode an acidic product of only 60 residues. A 37.6-kDa protein apparently encoded by cse15 was weakly related to the heavy chain of myosins, as well as to other myosin-like proteins, and is predicted to contain a central, 100 residue-long coiled-coil domain. Finally, csk22 is inferred to encode a 18.2-kDa hydrophobic product with five possible membrane-spanning helices, which could function as a transporter.

Role of the ninaC proteins in photoreceptor cell structure: ultrastructure of ninaC deletion mutants and binding to actin filaments.

The ninaC proteins are found in Drosophila photoreceptor cells. Their primary sequences suggest they are kinase/myosin chimeras, but their myosin head-like domain is the most divergent amongst all the myosin-like proteins described to date. To investigate possible roles of the ninaC proteins in cell structure, we examined the ultrastructure of the photoreceptor cells in various ninaC mutants, and tested the ability of the proteins to interact with actin filaments in a myosin-like manner. In flies lacking the larger ninaC protein, p174, an ultrastructural phenotype was evident before eclosion. The axial actin cytoskeleton of the rhabdomeral microvilli appeared either fragmented or as an isolated structure, without linkage to the microvillar membrane. Deletion of the myosin head-like domain or the calmodulin-binding domain of p174 resulted in a similar abnormal cytoskeleton. Breakdown of the rhabdomeres followed, although at different rates depending on the deletion. Lack of the smaller protein, p132, per se did not result in photoreceptor degeneration, but in older flies there was an abnormal accumulation of multivesicular bodies. Moreover, the presence of p132 retarded the degeneration that occurs in the absence of p174, even though the p132 remained outside the rhabdomere. Biochemical studies showed that both ninaC proteins bind actin filaments and cosediment with actin filaments in an ATP-sensitive manner. These results outline structural roles for the ninaC proteins, and are consistent with the notion, suggested by their amino acid sequences, that the proteins are actin-based mechanoenzymes.

Myosin-like protein (M(r) 175,000) in Gregarina blaberae.

A myosin-like protein (M(r) 175,000) was detected in the parasitic protozoan Gregarina blaberae, by both immunofluorescence and immunoblotting of one- and two-dimensional electrophoresis gels using anti-myosin antibodies. This protein was present in the trophozoite ghost but not in the cytoplasmic extract, nor in extract from the sexual stage, suggesting a protein-stage-dependent expression. The protein tightly bound to the cortical membranes was insoluble at low ionic strength, or in detergent solutions, but could be extracted from Gregarina ghosts by 6 M urea in high ionic strength solution (0.5 M NaCl) and in the presence of reducing agents (20 mM DTT). The protein was localized by indirect immunofluorescence in the cortex of the epimerite, in the fibrillar disc (the so-called septum) separating the proto- and the deutomerite segments, in the contractile ring or sphincter at the top of the protomerite, and as longitudinal lines underlying the G. blaberae epicyte folds. The presence of both actin-like and myosin-like proteins would be consistent with a role in gliding and other cell motility processes of this parasite.

PCR-dependent amplification and sequence characterization of partial cDNAs encoding myosin-like proteins in Anemia phyllitidis (L.) Sw. and Arabidopsis thaliana (L.) Heynh.

Partial cDNAs encoding for myosin-like proteins from Anemia phyllitidis and Arabidopsis thaliana have been isolated using PCR technology. The deduced amino acid sequences show an average similarity up to 62% with known myosin heavy chain genes. From northern blot analysis we were able to estimate that transcripts of ca. 6.1 kb size are expressed in A. phyllitidis.

Immunolocalization of myosin in intact and wounded cells of the green alga Ernodesmis verticillata (Kützing) Borgesen

Myosin localization was examined in the coenocytic green alga E. verticillata using indirect immunofluorescence microscopy. A polyclonal antibody affinity-purified against the heavy chain of slime-mold myosin recognizes a 220000 to 230000 Mr protein that electrophoretically migrates slightly behind rabbit myosin. A second polypeptide of 85000 Mr is also consistently detected in immunoblots, indicating that two forms of myosin-like proteins may be present in these cells. In intact cells, myosin immunofluorescence is present on the chloroplast surfaces, in nuclei and in cytoplasmic strands between plastids. Myosin labeling also occurs in association with pyrenoids primarily in apical chloroplasts. During wound-induced cytoplasmic contractions, myosin is localized near the plasma membrane in longitudinal arrays superimposed over a reticulate pattern of fluorescence; both these patterns become apparent upon wounding. Double-label immunofluorescence of actin and myosin demonstrates that these arrays represent the longitudinal bundles of actin microfilaments and the actin-containing reticulum, the former being directly associated with contraction in these cells. These results indicate that both actin and myosin are associated with contractility in Ernodesmis, probably representing the apparatus and "molecular motor", respectively, which effect motility.

Evolutionarily Conserved Sequences of Striated Muscle Myosin Heavy Chain Isoforms: Epitope mapping by cDNA expression

A cDNA expression strategy was used to localize amino acid sequences which were specific for fast, as opposed to slow, isoforms of the chicken skeletal muscle myosin heavy chain (MHC) and which were conserved in vertebrate evolution. Five monoclonal antibodies (mAbs), termed F18, F27, F30, F47, and F59, were prepared that reacted with all of the known chicken fast MHC isoforms but did not react with any of the known chicken slow nor with smooth muscle MHC isoforms. The epitopes recognized by mAbs F18, F30, F47, and F59 were on the globular head fragment of the MHC, whereas the epitope recognized by mAb F27 was on the helical tail or rod fragment. Reactivity of all five mAbs also was confined to fast MHCs in the rat, with the exception of mAb F59, which also reacted with the beta-cardiac MHC, the single slow MHC isoform common to both the rat heart and skeletal muscle. None of the five epitopes was expressed on amphioxus, nematode, or Dictyostelium MHC. The F27 and F59 epitopes were found on shark, electric ray, goldfish, newt, frog, turtle, chicken, quail, rabbit, and rat MHCs. The epitopes recognized by these mAbs were conserved, therefore, to varying degrees through vertebrate evolution and differed in sequence from homologous regions of a number of invertebrate MHCs and myosin-like proteins. The sequence of those epitopes on the head were mapped using a two-part cDNA expression strategy. First, Bal31 exonuclease digestion was used to rapidly generate fragments of a chicken embryonic fast MHC cDNA that were progressively deleted from the 3' end. These cDNA fragments were expressed as beta-galactosidase/MHC fusion proteins using the pUR290 vector; the fusion proteins were tested by immunoblotting for reactivity with the mAbs; and the approximate locations of the epitopes were determined from the sizes of the cDNA fragments that encoded a particular epitope. The epitopes were then precisely mapped by expression of overlapping cDNA fragments of known sequence that covered the approximate location of the epitopes. With this method, the epitope recognized by mAb F59 was mapped to amino acids 211-231 of the chicken embryonic fast MHC and the three distinct epitopes recognized by mAbs F18, F30, and F47 were mapped to amino acids approximately 65-92. Each of these epitope sequences is at or near the ATPase active site.

Ultrastructural and biochemical changes in a prokaryote induced by cytoskeletal active drugs

A previous study has shown that the stress of freezing and thawing in saline can cause a condensation of otherwise dispersed bacterial nucleoids [1]. In other studies, the transition from active growth to cessation of growth either by attainment of stationary phase or addition of chloramphenicol was also accompanied by a condensation of the nucleoids in Escherichia coli [2-4]. All three treatments are bacteriostatic. It thus seems possible that structural alterations of the nucleoid might be important in regulating cell metabolism. Recently, actin-like and myosin-like proteins have been isolated from the cytoplasm of E. coli and Mycoplasma [5-9]. While the function of these and possibly other proteins is unclear, they could be involved in a structural function in the cell cytoplasm and nucleus [10-15]. Since specific inhibitors of microfilament (cytochalasin B) and microtubule (vinblastin and colchicine) assembly and stabilization in eukaryotes are available [16,17], we have determined their effects on the ultrastructure and on certain biochemical functions in a prokaryote, E. co#.