Myosin S2 is not required for effects of myosin binding protein-C on motility

The unique myosin binding protein-c "motif" near the N-terminus of myosin binding protein-C (MyBP-C) binds myosin S2. Previous studies demonstrated that recombinant proteins containing the motif and flanking regions (e.g., C1C2) affect thin filament movement in motility assays using heavy meromyosin (S1 plus S2) as the molecular motor. To determine if S2 is required for these effects we investigated whether C1C2 affects motility in assays using only myosin S1 as the motor protein. Results demonstrate that effects of C1C2 are comparable in both systems and suggest that the MyBP-C motif affects motility through direct interactions with actin and/or myosin S1.     

Interaction of pollen F-actin with rabbit muscle myosin and its subfragments (HMM,S1)

Summary Actin purified from maize pollen grains can be polymerized into F-actin which increased the ATPase activities of proteolytic fragments (HMM, S₁) of rabbit muscle myosin. The values of K_app is 232 M for HMM and 290 M for S₁, which are six- and seven-fold higher than those of rabbit muscle F-actin under the same conditions. Pollen actin and rabbit muscle myosin form hybrid actomyosin showing increase in viscosity and turbidity of solution. Viscosity and turbidity of the actomyosin dropped and then increased again with addition of ATP. Polymerized pollen actin can be decorated in vitro with both rabbit muscle HMM and S₁ to form an arrowhead-shaped structure like that observed in living plant cells. The results show that pollen actin is similar to muscle actin at a qualitative level. But there are differences between them at a quantitative level.Abbreviations HMM heavy meromyosin - S₁ myosin subfragment 1 - ATP adenosine-5-triphosphate     

Mutation of a conserved glycine in the SH1-SH2 helix affects the load-dependent kinetics of myosin

The ATP hydrolysis rate and shortening velocity of muscle are load-dependent. At the molecular level, myosin generates force and motion by coupling ATP hydrolysis to lever arm rotation. When a laser trap was used to apply load to single heads of expressed smooth muscle myosin (S1), the ADP release kinetics accelerated with an assistive load and slowed with a resistive load; however, ATP binding was mostly unaffected. To investigate how load is communicated within the motor, a glycine located at the putative fulcrum of the lever arm was mutated to valine (G709V). In the absence of load, stopped-flow and laser trap studies showed that the mutation significantly slowed the rates of ADP release and ATP binding, accounting for the ~270-fold decrease in actin sliding velocity. The load dependence of the mutant's ADP release rate was the same as that of wild-type S1 (WT) despite the slower rate. In contrast, load accelerated ATP binding by ~20-fold, irrespective of loading direction. Imparting mechanical energy to the mutant motor partially reversed the slowed ATP binding by overcoming the elevated activation energy barrier. These results imply that conformational changes near the conserved G709 are critical for the transmission of mechanochemical information between myosin's active site and lever arm.     

A new member of the NY-ESO-1 gene family is ubiquitously expressed in somatic tissues and evolutionarily conserved

Three single copy ATP-binding cassette (ABC) transporter encoding genes, designated MgAtr3, MgAtr4, and MgAtr5, were cloned and sequenced from the plant pathogenic fungus Mycosphaerella graminicola. The encoded ABC proteins all exhibit the [NBD-TMS(6)](2) configuration and can be classified as novel members of the pleiotropic drug resistance (PDR) class of ABC transporters. The three proteins are highly homologous to other fungal and yeast, ABC proteins involved in multidrug resistance or plant pathogenesis. MgAtr4 and MgAtr5 possess a conserved ABC motif at both the N- and C-terminal domain of the protein. In contrast, the Walker A motif in the N-terminal and the ABC signature in the C-terminal domain of MgAtr3, deviate significantly from the consensus sequence found in other members of the PDR class of ABC transporters. Expression of MgAtr3 could not be detected under any of the conditions tested. However, MgAtr4 and MgAtr5 displayed distinct expression profiles when treated with a range of compounds known to be either substrates or inducers of ABC transporters. These included synthetic fungitoxic compounds, such as imazalil and cyproconazole, natural toxic compounds, such as the plant defence compounds eugenol and psoralen, and the antibiotics cycloheximide and neomycin. The expression pattern of the genes was also dependent on the morphological state of the fungus. The findings suggest a role for MgAtr4 and MgAtr5 during plant pathogenesis and in protection against toxic compounds.     

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.     

Nuclear ultrastructure: Condensed chromatin in plants is species-specific (karyotypical), but not tissue-specific (functional)

Summary In contrast to mammalian cell nuclei those of plants display nearly an identical ultrastructure in all developmental stages and tissues. This indicates that the gross organization of chromatin is species-specific, but not tissue-specific and function-dependent. The species-specific nuclear ultrastructure is determined by the basic nuclear DNA content (2 C value). The higher the DNA content, the more the euchromatin remains in the condensed state during interphase, but to a lower coiling order than the heterochromatin.Some difficulties in the interpretation of electron micrographs of cell nuclei, and the possible role of repetitive DNA sequences in the karyotypical condensation of euchromatin in plants are discussed.     

Myo1c is designed for the adaptation response in the inner ear

The molecular motor, Myo1c, a member of the myosin family, is widely expressed in vertebrate tissues. Its presence at strategic places in the stereocilia of the hair cells in the inner ear and studies using transgenic mice expressing a mutant Myo1c that can be selectively inhibited implicate it as the mediator of slow adaptation of mechanoelectrical transduction, which is required for balance. Here, we have studied the structural, mechanical and biochemical properties of Myo1c to gain an insight into how this molecular motor works. Our results support a model in which Myo1c possesses a strain-sensing ADP-release mechanism, which allows it to adapt to mechanical load.     

Crosslinking of tropomyosin to myosin subfragment-1 in reconstituted rabbit skeletal thin filaments

Rabbit skeletal tropomyosin was labeled with the bifunctional photoactivatable crosslinker N-succinimidyl-6- (4'-azido-2'-nitrophenylamino)hexanoate. After irradiating the rigor complex composed of myosin subfragment-1, crosslinker-labeled tropomyosin, and F-actin, a crosslinked product was formed. This product was identified as a 1:1 adduct of tropomyosin and subfragment-1. This finding is in support of recent structural studies which suggest that tropomyosin and subfragment-1 are in close proximity to each other, and may be relevant to the mechanism of thin filament regulation.     

Involvement of RhoA, ROCK I and myosin II in inverted orientation of epithelial polarity

In multicellular epithelial tissues, the orientation of polarity of each cell must be coordinated. Previously, we reported that for Madin-Darby canine kidney cells in three-dimensional collagen gel culture, blockade of beta1-integrin by the AIIB2 antibody or expression of dominant-negative Rac1N17 led to an inversion of polarity, such that the apical surfaces of the cells were misorientated towards the extracellular matrix. Here, we show that this process results from the activation of RhoA. Knockdown of RhoA by short hairpin RNA reverses the inverted orientation of polarity, resulting in normal cysts. Inhibition of RhoA downstream effectors, Rho kinase (ROCK I) and myosin II, has similar effects. We conclude that the RhoA-ROCK I-myosin II pathway controls the inversion of orientation of epithelial polarity caused by AIIB2 or Rac1N17. These results might be relevant to the hyperactivation of RhoA and disruption of normal polarity frequently observed in human epithelial cancers.