CYTOSKELETON, Issue 2 2003
Rosalind V. Silverman-Gavrila
Abstract We showed previously that in crane-fly spermatocytes myosin is required for tubulin flux [Silverman-Gavrila and Forer, 2000a: J Cell Sci 113:597,609], and for normal anaphase chromosome movement and contractile ring contraction [Silverman-Gavrila and Forer, 2001: Cell Motil Cytoskeleton 50:180,197]. Neither the identity nor the distribution of myosin(s) were known. In the present work, we used immunofluorescence and confocal microscopy to study myosin during meiosis-I of crane-fly spermatocytes compared to tubulin, actin, and skeletor, a spindle matrix protein, in order to further understand how myosin might function during cell division. Antibodies to myosin II regulatory light chain and myosin II heavy chain gave similar staining patterns, both dependent on stage: myosin is associated with nuclei, asters, centrosomes, chromosomes, spindle microtubules, midbody microtubules, and contractile rings. Myosin and actin colocalization along kinetochore fibers from prometaphase to anaphase are consistent with suggestions that acto-myosin forces in these stages propel kinetochore fibres poleward and trigger tubulin flux in kinetochore fibres, contributing in this way to poleward chromosome movement. Myosin and actin colocalization at the cell equator in cytokinesis, similar to studies in other cells [e.g., Fujiwara and Pollard, 1978: J Cell Biol 77:182,195], supports a role of actin-myosin interactions in contractile ring function. Myosin and skeletor colocalization in prometaphase spindles is consistent with a role of these proteins in spindle formation. After microtubules or actin were disrupted, myosin remained in spindles and contractile rings, suggesting that the presence of myosin in these structures does not require the continued presence of microtubules or actin. BDM (2,3 butanedione, 2 monoxime) treatment that inhibits chromosome movement and cytokinesis also altered myosin distributions in anaphase spindles and contractile rings, consistent with the physiological effects, suggesting also that myosin needs to be active in order to be properly distributed. Cell Motil. Cytoskeleton 55:97,113, 2003. © 2003 Wiley-Liss, Inc. [source]

Antihypertensive Activities of Peptides Derived from Porcine Skeletal Muscle Myosin in Spontaneously Hypertensive Rats

JOURNAL OF FOOD SCIENCE, Issue 1 2002
Y. Nakashima
ABSTRACT: Antihypertensive activities derived from porcine skeletal muscle proteins were investigated. Thermolysin hydrolysates of porcine muscle water-insoluble proteins demonstrated antihypertensive activities in spontaneously hypertensive rats when administrated in single oral doses. Hydrolysates of porcine myosin and peptides (Met-Asn-Pro-Pro-Lys, Ile-Thr-Thr-Asn-Pro, Met-Asn-Pro, Pro-Pro-Lys) with parts of the sequence of myosin showed antihypertensive activities. This is the first report of antihypertensive activities of peptides derived from muscle proteins of domestic animals. The hydrolysates of porcine muscle protein and their corresponding bioactive peptides might be utilized for physiologically functional foods. [source]

Actin and myosin regulate cytoplasm stiffness in plant cells: a study using optical tweezers

NEW PHYTOLOGIST, Issue 1 2010
Hannie S. Van Der Honing
Summary ,,Here, we produced cytoplasmic protrusions with optical tweezers in mature BY-2 suspension cultured cells to study the parameters involved in the movement of actin filaments during changes in cytoplasmic organization and to determine whether stiffness is an actin-related property of plant cytoplasm. ,,Optical tweezers were used to create cytoplasmic protrusions resembling cytoplasmic strands. Simultaneously, the behavior of the actin cytoskeleton was imaged. ,,After actin filament depolymerization, less force was needed to create cytoplasmic protrusions. During treatment with the myosin ATPase inhibitor 2,3-butanedione monoxime, more trapping force was needed to create and maintain cytoplasmic protrusions. Thus, the presence of actin filaments and, even more so, the deactivation of a 2,3-butanedione monoxime-sensitive factor, probably myosin, stiffens the cytoplasm. During 2,3-butanedione monoxime treatment, none of the tweezer-formed protrusions contained filamentous actin, showing that a 2,3-butanedione monoxime-sensitive factor, probably myosin, is responsible for the movement of actin filaments, and implying that myosin serves as a static cross-linker of actin filaments when its motor function is inhibited. The presence of actin filaments does not delay the collapse of cytoplasmic protrusions after tweezer release. ,,Myosin-based reorganization of the existing actin cytoskeleton could be the basis for new cytoplasmic strand formation, and thus the production of an organized cytoarchitecture. [source]

Actin-Based Motility in the Net Slime Mould Labyrinthula: Evidence for the Role of Myosin in Gliding Movement

THE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 6 2005
TERENCE M. PRESTON
Abstract. In contrast to crawling movement (e.g. in amoebae and tissue cells) the other major class of substratum-associated motility in eukaryotes, gliding, has received relatively little attention. The net slime mold Labyrinthula provides a useful laboratory model for studying this process since it exhibits a particular kind of gliding in its plasmodial stage. Here nucleated spindle cells glide along self-established cytoplasmic trackways in a predominantly unidirectional manner, at 1,2 ,m/s. These trackways, upon which gliding is dependent, are held by filopodial tethers some distance off the well-developed reticulopodial mesh anchoring the plasmodium onto the substratum. Reflection interference microscopy resolves this matrix in live plasmodia. The axially disposed cytoskeletal elements of the trackways are revealed by rhodamine-labelled phalloidin to be rich in F-actin. A weft of peripheral, rapidly extending filopodia (50 ,m/min) typifies the expanding regions of the plasmodium. Here spindle cells are recruited before emigrating into newly differentiated trackways. Immunoblotting whole plasmodia or a sucrose-soluble cytoplasmic extract reveals a single actin-positive band of Mr 48 kDa. Polyclonal antibodies to two distinct myosin peptide sequences identify a single myosin HC (Mr 96 kDa) in immunoblots. Gliding was reversibly blocked by 10 mM 2,3-butanedione-2-monoxime, a myosin ATPase inhibitor, but it was insensitive to the actin-binding drugs cytochalasin D and phalloidin. We suggest that the force (>50 pN) for gliding motility results from interaction of myosin molecules, associated with the spindle cells, with trackway F-actin via the bothrosomes. [source]

Adhesion Effects of a Guanidinium Ion Appended Dendritic "Molecular Glue" on the ATP-Driven Sliding Motion of Actomyosin,

ANGEWANDTE CHEMIE, Issue 17 2010
Kou Okuro
Festgeklebt: Ein heterotropes Konjugat aus Actin und Myosin , Actomysin , wird durch einen wasserlöslichen ,molekularen Klebstoff", ein Dendrimer mit neun Guanidinium-Resten, stabilisiert (siehe Bild; ATP=Adenosintriphosphat, ADP=Adenosindiphosphat). Während das Dendrimer erster Generation die ATP-getriebene Gleitbewegung von Actinfilamenten auf dem Myosin-funktionalisierten Deckglas bremst oder zum Stillstand bringt, ist ein verwandtes Dendrimer nullter Generation dazu nicht in der Lage. [source]

Myosins of Babesia bovis: Molecular characterisation, erythrocyte invasion, and phylogeny

CYTOSKELETON, Issue 4 2002
A.E. Lew
Abstract Using degenerate primers, three putative myosin sequences were amplified from Australian isolates of Babesa bovis and confirmed as myosins (termed Bbmyo-A, Bbmyo-B, and Bbmyo-C) from in vitro cultures of the W strain of B. bovis. Comprehensive analysis of 15 apicomplexan myosins suggests that members of Class XIV be defined as those with greater than 35% myosin head sequence identity and that these be further subclassed into groups bearing above 50,60% identity. Bbmyo-A protein bears a strong similarity with other apicomplexan myosin-A type proteins (subclass XIVa), the Bbmyo-B myosin head protein sequence exhibits low identity (35,39%) with all members of Class XIV, and 5,-sequence of Bbmyo-C shows strong identity (60%) with P. falciparum myosin-C protein. Domain analysis revealed five divergent IQ domains within the neck of Pfmyo-C, and a myosin-N terminal domain as well as a classical IQ sequence unusually located within the head converter domain of Bbmyo-B. A cross-reacting antibody directed against P. falciparum myosin-A (Pfmyo-A) revealed a zone of approximately 85 kDa in immunoblots prepared with B. bovis total protein, and immunofluorescence inferred stage-specific myosin-A expression since only 25% of infected erythrocytes with mostly paired B. bovis were immuno-positive. Multiplication of B. bovis in in vitro culture was inhibited by myosin- and actin-binding drugs at concentrations lower than those that inhibit P. falciparum. This study identifies and classifies three myosin genes and an actin gene in B. bovis, and provides the first evidence for the participation of an actomyosin-based motor in erythrocyte invasion in this species of apicomplexan parasite. Cell Motil. Cytoskeleton 52:202,220, 2002. © 2002 Wiley-Liss, Inc. [source]

Thermal Effects on Fast Skeletal Myosins from Alaska Pollock, White Croaker, and Rabbit in Relation to Gel Formation

JOURNAL OF FOOD SCIENCE, Issue 5 2003
H. Fukushima
ABSTRACT Thermodynamic properties in differential scanning calorimetry (DSC) and changes in viscoelasticity upon heating of myosins from white croaker, Alaska pollock, and rabbit fast muscles were investigated in relation to their thermal gel formation abilities. Alaska pollock myosin unfolded in a wide temperature range of 19 to 69°C as revealed by DSC, whereas rabbit myosin unfolded in very narrow range of 32 to 56°C. Thermal unfolding of white croaker myosin occurred in an intermediate temperature range of 30 to 60°C. Viscoelastic properties determined as storage modulus, G,, and loss modulus, G,, reflected differences observed in DSC for the 3 myosins. [source]

Differential activation of stress-responsive signalling proteins associated with altered loading in a rat skeletal muscle

JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2005
Inho Choi
Abstract Skeletal muscle undergoes a significant reduction in tension upon unloading. To explore intracellular signalling mechanisms underlying this phenomenon, we investigated twitch tension, the ratio of actin/myosin filaments, and activities of key signalling molecules in rat soleus muscle during a 3-week hindlimb suspension and 2-week reloading. Twitch tension and myofilament ratio (actin/myosin) gradually decreased during unloading but progressively recovered to initial levels during reloading. To study the involvement of stress-responsive signalling proteins during these changes, the activities of protein kinase C alpha (PKC,) and three mitogen-activated protein kinases (MAPKs),c-Jun NH2 -terminal kinase (JNK), extracellular signal-regulated protein kinase (ERK), and p38 MAPK,were examined using immunoblotting and immune complex kinase assays. PKC, phosphorylation correlated positively with the tension (Pearson's r,=,0.97, P,<,0.001) and the myofilament ratio (r,=,0.83, P,<,0.01) over the entire unloading and reloading period. Treatment of the soleus muscle with a PKC activator resulted in a similar paralleled increment in both PKC, phosphorylation and the ,-sarcomeric actin expression. The three MAPKs differed in the pattern of activation in that JNK activity peaked only for the first hours of reloading, whereas ERK and p38 MAPK activities remained elevated during reloading. These results suggest that PKC, may play a pivotal role in converting loading stress to intracellular changes in contractile proteins that determine muscle tension. Differential activation of MAPKs may also help alleviate muscle damage, modulate energy transport and/or regulate the expression of contractile proteins upon altered loading. J. Cell. Biochem. © 2005 Wiley-Liss, Inc. [source]

Molecular characterization of regenerated cardiomyocytes derived from adult mesenchymal stem cells

CONGENITAL ANOMALIES, Issue 1 2002
Keiichi Fukuda
ABSTRACT, We recently isolated a cardiomyogenic (CMG) cell line from murine bone marrow stroma, and in this paper characterize regenerated cardiomyocytes derived from adult mesenchymal stem cells at the molecular level. Stromal cells were immortalized, exposed to 5-azacytidine, and repeatedly screened for spontaneously beating cells. CMG cells began to beat spontaneously after 2 weeks, and beat synchronously after 3 weeks. They exhibited sinus-node-like or ventricular-cell-like action potentials. Analysis of the isoforms of contractile protein genes, such as of myosin and ,-actin, indicated that their phenotype was similar to that of fetal ventricular cardiomyocytes. The cells expressed Nkx2.5, GATA4, TEF-1, and MEF2-C mRNA before 5-azacytidine exposure, and MEF2-A and MEF2-D after exposure. CMG cells expressed ,1A, ,1B, and ,1D -adrenergic receptor mRNA prior to differentiation, and ,1, ,2 -adrenergic and M1, M2 -muscarinic receptors after acquiring the cardiomyocyte phenotype. Phenylephrine induced phosphorylation of ERK1/ 2, and the phosphorylation was inhibited by prazosin. Isoproterenol increased the cAMP level 38-fold and beating rate, cell motion, % shortening, and contractile velocity by 48%, 38%, 27%, and 51%, respectively, and the increases were blocked by CGP20712A (,1 -selective blocker). Car-bachol increased IP3 32-fold, and the increase was inhibited by AFDX116 (M2 -selective blocker). These findings demonstrated that the regenerated cardiomyocytes were capable of responding to adrenergic and muscarinic stimulation. This new cell line provides a model for the study of cardiomyocyte transplantation. [source]

Myosin diversity in the diatom Phaeodactylum tricornutum,

CYTOSKELETON, Issue 3 2010
Matthew B. Heintzelman
Abstract This report describes the domain architecture of ten myosins cloned from the pennate diatom Phaeodactylum tricornutum. Several of the P. tricornutum myosins show similarity to myosins from the centric diatom Thalassiosira pseudonana as well as to one myosin from the oomycete Phytophthora ramorum. The P. tricornutum myosins, ranging in size from 126 kDa to over 250 kDa, all possess the canonical head, neck and tail domains common to most myosins, though variations in each of these domains is evident. Among the features distinguishing several of the diatom myosin head domains are N-terminal SH3-like domains, variations in or near the P-loop and Loop 1 regions close to the nucleotide binding pocket, and extended converter domains. Variations in the length of the neck domain or lever arm, defined by the light chain-binding IQ motifs, are apparent with the different diatom myosins predicted to contain from one to nine IQ motifs. Protein domains found within the P. tricornutum myosin tails include regions of coiled-coil structure, ankyrin repeats, CBS domain pairs, a PB1 domain, a kinase domain and a FYVE-finger motif. As many of these features have never before been characterized in myosins of any type, it is likely that these new diatom myosins will expand the repertoire of known myosin behaviors. © 2010 Wiley-Liss, Inc. [source]

Susceptibility of isolated myofibrils to in vitro glutathionylation: Potential relevance to muscle functions,

CYTOSKELETON, Issue 2 2010
Chiara Passarelli
Abstract In this study we investigated the molecular mechanism of glutathionylation on isolated human cardiac myofibrils using several pro-glutathionylating agents. Total glutathionylated proteins appeared significantly enhanced with all the pro-oxidants used. The increase was completely reversed by the addition of a reducing agent, demonstrating that glutathione binding occurs by a disulfide and that the process is reversible. A sensitive target of glutathionylation was ,-actin, showing a different reactivity to the several pro-glutathionylating agents by ELISA. Noteworthy, myosin although highly sensitive to the in vitro glutathionylation does not represent the primary glutathionylation target in isolated myofibrils. Light scattering measurements of the glutathionylated ,-actin showed a slower polymerisation compared to the non-glutathionylated protein and force development was depressed after glutathionylation, when the myofibrils were mounted in a force recording apparatus. Interestingly, confocal laser scanning microscopy of cardiac cryosections indicated, for the first time, the constitutive glutathionylation of ,-cardiac actin in human heart. Due to the critical location of ,-actin in the contractile machinery and to its susceptibility to the oxidative modifications, glutathionylation may represent a mechanism for modulating sarcomere assembly and muscle functionality under patho-physiological conditions in vivo. © 2009 Wiley-Liss, Inc. [source]

A FERM domain in a class XIV myosin interacts with actin and tubulin and localizes to the cytoskeleton, phagosomes, and nucleus in Tetrahymena thermophila,

CYTOSKELETON, Issue 2 2010
Michael Gotesman
Abstract Previous studies have shown that Myo1(myosin class XIV) localizes to the cytoskeleton and is involved in amitosis of the macronucleus and trafficking of phagosomes. Myo1 contains a FERM domain that could be a site for interaction between Myo1 and the cytoskeleton. Here, we explore the function of FERM by investigating its cytoskeleton binding partners and involvement in localization of Myo1. Alignment of Myo1 FERM with a talin actin-binding sequence, a MAP-2 tubulin-binding sequence, the radixin FERM dimerization motif, and the SV40 nuclear localization sequence (NLS) revealed putative actin- and tubulin-binding sequences, a putative FERM dimerization motif, and NLS-like sequences in both the N-terminal and C-terminal regions of Myo1 FERM. Alignment of Myo1 with an ERM C-terminal motif revealed a similar sequence in the Myo1 motor domain. GFP-FERM and two truncated FERM domains were separately expressed in Tetrahymena. GFP-FERM contained the entire Myo1 FERM. Truncated Myo1 FERM domains contained either the N-terminal or the C-terminal region of FERM and one putative sequence for actin-binding, one for tubulin-binding, a putative dimerization motif, and a NLS-like sequence. Actin antibody coprecipitated GFP-fusion polypeptides and tubulin from lysate of cells expressing GFP-fusions. Cosedimentation assays performed with either whole cell extracts or anti-actin immunoprecipitation pellets revealed that F-actin (independent of ATP) and microtubules cosedimented with GFP-fusion polypeptides. GFP-FERM localized to the cytoskeleton, phagosomes, and nucleus. Truncated GFP-FERM domains localized to phagosomes but not to the cytoskeleton or nucleus. © 2009 Wiley-Liss, Inc. [source]

Isolation and partial purification of the Saccharomyces cerevisiae cytokinetic apparatus,

CYTOSKELETON, Issue 1 2010
Brian A. Young
Abstract Cytokinesis is the process by which a cell physically divides in two at the conclusion of a cell cycle. In animal and fungal cells, this process is mediated by a conserved set of proteins including actin, type II myosin, IQGAP proteins, F-BAR proteins, and the septins. To facilitate biochemical and ultrastructural analysis of cytokinesis, we have isolated and partially purified the Saccharomyces cerevisiae cytokinetic apparatus. The isolated apparatus contains all components of the actomyosin ring for which we tested,actin, myosin heavy and light chain, and IQGAP,as well as septins and the cytokinetic F-BAR protein, Hof1p. We also present evidence indicating that the actomyosin rings associated with isolated cytokinetic apparati may be contractile in vitro, and show preliminary electron microscopic imaging of the cytokinetic apparatus. This first successful isolation of the cytokinetic apparatus from a genetically tractable organism promises to make possible a deeper understanding of cytokinesis. © 2009 Wiley-Liss, Inc. [source]

Phosphorylation of tropomyosin extends cooperative binding of myosin beyond a single regulatory unit

CYTOSKELETON, Issue 1 2009
Vijay S. Rao
Abstract Tropomyosin (Tm) is one of the major phosphoproteins comprising the thin filament of muscle. However, the specific role of Tm phosphorylation in modulating the mechanics of actomyosin interaction has not been determined. Here we show that Tm phosphorylation is necessary for long-range cooperative activation of myosin binding. We used a novel optical trapping assay to measure the isometric stall force of an ensemble of myosin molecules moving actin filaments reconstituted with either natively phosphorylated or dephosphorylated Tm. The data show that the thin filament is cooperatively activated by myosin across regulatory units when Tm is phosphorylated. When Tm is dephosphorylated, this "long-range" cooperative activation is lost and the filament behaves identically to bare actin filaments. However, these effects are not due to dissociation of dephosphorylated Tm from the reconstituted thin filament. The data suggest that end-to-end interactions of adjacent Tm molecules are strengthened when Tm is phosphorylated, and that phosphorylation is thus essential for long range cooperative activation along the thin filament. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source]

In vivo phosphorylation of regulatory light chain of myosin II in sea urchin eggs and its role in controlling myosin localization and function during cytokinesis

CYTOSKELETON, Issue 2 2008
Ryota Uehara
Abstract Phosphorylation of myosin regulatory light chain (RLC) at Ser19 (mono-phosphorylation) promotes filament assembly and enhances actin-activated ATPase activity of non-muscle myosin, while phosphorylation at both Ser19 and Thr18 (di-phosphorylation) further enhances the ATPase activity. However, it has not well been addressed which type of phosphorylation is important in regulating myosin during cytokinesis. Here, we investigated subcellular localization in sea urchin eggs of mono-phosphorylated and di-phosphorylated RLC by both quantitative biochemical and spatiotemporal cytological approaches. Mono-phosphorylated RLC was dominant in the equatorial cortex throughout the whole process of cytokinesis. Inhibition of myosin light chain kinase (MLCK) decreased mono-phosphorylated RLC both in the cortex and in the cleavage furrow, and blocked both formation and contraction of the contractile ring. Two different types of ROCK inhibitor gave inconsistent results: H1152 blocked both RLC mono-phosphorylation in the cleavage furrow and contraction of the contractile ring, while Y27632 affected neither the mono-phosphorylation nor cell division. These results suggest that there may be other targets of H1152 than ROCK, which is involved in the RLC phosphorylation in the cleavage furrow. Furthermore, it was revealed that localization of myosin heavy chain in the cleavage furrow, but not in the cortex, was perturbed by inhibition of RLC mono-phosphorylation. These results suggested that RLC mono-phosphorylation by more than two RLC kinases play a main role in regulation and localization of myosin in the dividing sea urchin eggs. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source]

Myosin16b: The COOH-tail region directs localization to the nucleus and overexpression delays S-phase progression

CYTOSKELETON, Issue 1 2007
Richard S. Cameron
Abstract Rat Myo16a and Myo16b comprise the founding members of class XVI myosin and are characterized by an N-terminal ankyrin repeat domain thought to mediate an association with protein phosphatase 1 catalytic subunits 1, and 1,. Myo16b is the principal isoform and reveals predominant expression in developing neural tissue. Here, we use COS-7 cells as a model system to develop an understanding of Myo16b function. We find that Myo16b displays predominant localization in the nucleus of cells transitioning through interphase, but is not associated with processes of mitosis. Using a panel of EGFP-Myo16b-expression plasmids in transient transfection studies, we identified the COOH-terminal residues 1616,1912 as necessary and solely sufficient to target Myo16b to the nucleus. We show that the Myo16b-tail region directs localization to a nuclear compartment containing profilin and polymerized actin, which appears to form a three-dimensional meshwork through the depth of the nucleus. Further, we demonstrate that this compartment localizes within euchromatic regions of the genome and contains proliferating cell nuclear antigen (PCNA) and cyclin A, both markers of S-phase of the cell cycle. Cells transiently expressing Myo16b or Myo16b-tail region show limited incorporation of BrdU, delayed progression through S-phase of the cell cycle, and curtailed cellular proliferation. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source]

Actin and myosin in Gregarina polymorpha

CYTOSKELETON, Issue 2 2004
Matthew B. Heintzelman
Abstract Actin and two class XIV unconventional myosins have been cloned from Gregarina polymorpha, a large protozoan parasite inhabiting the gut of the mealworm Tenebrio molitor. These proteins were most similar to their homologues expressed in the coccidian and haemosporidian Apicomplexa such as Toxoplasma and Plasmodium despite the significant morphological differences among these parasites. Both actin and G. polymorpha myosin A (GpMyoA), a 92.6-kDa protein characterized by a canonical myosin head domain and short, highly basic tail, localized to both the longitudinally-disposed surface membrane folds (epicytic folds) of the parasite as well as to the subjacent rib-like myonemes that gird the parasite cortex. G. polymorpha myosin B (GpMyoB), a 96.3-kDa myosin, localized exclusively to the epicytic folds of the parasite. Both myosins were tightly associated with the cortical cytoskeleton and were solubilized only with a combination of high salt and detergent. Both GpMyoA and GpMyoB could bind to actin in an ATP-sensitive fashion. The distribution of actin and the unconventional myosins in G. polymorpha was consistent with their proposed participation in both the rapid (1,10 ,m/sec) gliding motility exhibited by the gregarines as well as the myoneme-mediated bending motions that have been observed in these parasites. Cell Motil. Cytoskeleton 58:83,95, 2004. © 2004 Wiley-Liss, Inc. [source]

A subclass of myosin XI is associated with mitochondria, plastids, and the molecular chaperone subunit TCP-1, in maize

CYTOSKELETON, Issue 4 2004
Zhengyuan Wang
Abstract The role and regulation of specific plant myosins in cyclosis is not well understood. In the present report, an affinity-purified antibody generated against a conserved tail region of some class XI plant myosin isoforms was used for biochemical and immunofluorescence studies of Zea mays. Myosin XI co-localized with plastids and mitochondria but not with nuclei, the Golgi apparatus, endoplasmic reticulum, or peroxisomes. This suggests that myosin XI is involved in the motility of specific organelles. Myosin XI was more than 50% co-localized with tailless complex polypeptide-1, (TCP-1,) in tissue sections of mature tissues located more than 1.0 mm from the apex, and the two proteins co-eluted from gel filtration and ion exchange columns. On Western blots, TCP-1, isoforms showed a developmental shift from the youngest 5.0 mm of the root to more mature regions that were more than 10.0 mm from the apex. This developmental shift coincided with a higher percentage of myosin XI /TCP-1, co-localization, and faster degradation of myosin XI by serine protease. Our results suggest that class XI plant myosin requires TCP-1, for regulating folding or providing protection against denaturation. Cell Motil. Cytoskeleton 57:218,232, 2004. © 2004 Wiley-Liss, Inc. [source]

Myosin localization during meiosis I of crane-fly spermatocytes gives indications about its role in division

Acute effects of desmin mutations on cytoskeletal and cellular integrity in cardiac myocytes

CYTOSKELETON, Issue 2 2003
Kurt Haubold
Mutations in desmin have been associated with a subset of human myopathies. Symptoms typically appear in the second to third decades of life, but in the most severe cases can manifest themselves earlier. How desmin mutations lead to aberrant muscle function, however, remains poorly defined. We created a series of four mutations in rat desmin and tested their in vitro filament assembly properties. RDM-G, a chimera between desmin and green fluorescent protein, formed protofilament-like structures in vitro. RDM-1 and RDM-2 blocked in vitro assembly at the unit-length filament stage, while RDM-3 had more subtle effects on assembly. When expressed in cultured rat neonatal cardiac myocytes via adenovirus infection, these mutant proteins disrupted the endogenous desmin filament to an extent that correlated with their defects in in vitro assembly properties. Disruption of the desmin network by RDM-1 was also associated with disruption of plectin, myosin, and ,-actinin organization in a significant percentage of infected cells. In contrast, expression of RDM-2, which is similar to previously characterized human mutant desmins, took longer to disrupt desmin and plectin organization and had no significant effect on myosin or ,-actinin organization over the 5-day time course of our studies. RDM-3 had the mildest effect on in vitro assembly and no discernable effect on either desmin, plectin, myosin, or ,-actinin organization in vivo. These results indicate that mutations in desmin have both direct and indirect effects on the cytoarchitecture of cardiac myocytes. Cell Motil. Cytoskeleton 54:105,121, 2003. © 2003 Wiley-Liss, Inc. [source]

Myosins of Babesia bovis: Molecular characterisation, erythrocyte invasion, and phylogeny

An effective skeletal muscle prefractionation method to remove abundant structural proteins for optimized two-dimensional gel electrophoresis

ELECTROPHORESIS, Issue 11 2005
Bradley Jarrold
Abstract Proteomic analysis of biological samples in disease models or therapeutic intervention studies requires the ability to detect and identify biologically relevant proteins present in relatively low concentrations. The detection and analysis of these low-level proteins is hindered by the presence of a few proteins that are expressed in relatively high concentrations. In the case of muscle tissue, highly abundant structural proteins, such as actin, myosin, and tropomyosin, compromise the detection and analysis of more biologically relevant proteins. We have developed a practical protocol which exploits high-pH extraction to reduce or remove abundant structural proteins from skeletal muscle crude membrane preparations in a manner suitable for two dimensional gel electrophoresis. An initial whole-cell muscle lysate is generated by homogenization of powdered tissue in Tris-base. This lysate is subsequently partitioned into a supernatant and pellet containing the majority of structural proteins. Treatment of the pellet with high-pH conditions effectively releases structural proteins from membrane compartments which are then removed through ultracentrifugation. Mass spectrometric identification shows that the majority of protein spots reduced or removed by high-pH treatment were contractile proteins or contractile-related proteins. Removal of these proteins enabled successful detection and identification of minor proteins. Structural protein removal also results in significant improvement of gel quality and the ability to load higher amounts of total protein for the detection of lower abundant protein classes. [source]

Electrophoretic variants of cardiac myosin heavy chain-, in Sprague Dawley rats

ELECTROPHORESIS, Issue 3 2004
Peter J. Reiser
Abstract Analysis of cardiac myosin revealed differences in gel electrophoretic migration patterns of the ,-isoform of myosin heavy chain, but not the ,-isoform, in Sprague Dawley rats. No differences in the migration patterns of the ,-or ,-isoforms were observed in other rat strains. Three electrophoretic migration patterns of the ,-isoforms were observed in individual rats: a slower migrating isoform alone (4% of all rats tested), a faster migrating isoform alone (55%), and both isoforms (41%). The isoform expression pattern was identical in all myocardial regions in each rat. Frequency of expression patterns suggests multiple gene sequences for ,-cardiac myosin heavy chain in Sprague Dawley rats. Sequence analysis of amplified regions of the Sprague Dawley and Brown Norway rat ,-myosin genes, specifically the 5'-untranslated region, exons 1,3, and associated introns, showed numerous single nucleotide polymorphisms in coding and noncoding regions, including putative regulatory sites in Sprague Dawley rats, but not in Brown Norway rats. All Sprague Dawley rats varied from Brown Norway rats and no heterogeneity was observed in Brown Norway rats. Several deletions and dimorphic positions were also observed. Dimorphic positions were evident on automated sequencing comparisons. The data indicate that at least two ,-myosin heavy chain isoforms exist in Sprague Dawley rats and these rats exhibit sequence diversity within that portion of the ,-myosin heavy chain gene reported in this study. [source]

Molecular mechanisms activating muscle protein degradation in chronic kidney disease and other catabolic conditions

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 3 2005
J. Du
Abstract Muscle atrophy is a prominent feature of chronic kidney disease (CKD) and is frequent in other catabolic conditions. Results from animal models of these conditions as well as patients indicate that atrophy is mainly owing to accelerated muscle proteolysis in the ubiquitin-proteasome (Ub-P'some) proteolytic system. The Ub-P'some system, however, rapidly degrades actin or myosin but cannot breakdown actomyosin or myofibrils. Consequently, another protease must initially cleave the complex structure of muscle. We identified caspase-3 as an initial and potentially rate-limiting proteolytic step that cleaves actomyosin/myofibrils to produce substrates degraded by the Ub-P'some system. In rodent models of CKD and other catabolic conditions, we find that caspase-3 is activated and cleaves actomyosin to actin, myosin and their fragments. This initial proteolytic step in muscle leaves a characteristic footprint, a 14-kDa actin band, providing a potential diagnostic tool to detect muscle catabolism. We also found that stimulation of caspase-3 activity depends on inhibition of IRS-1-associated phosphatidylinositol 3-kinase (PI3K) activity; inhibiting PI3K in muscle cells also leads to expression of a critical E3-ubiquitin-conjugating enzyme involved in muscle protein breakdown: atrogin-1/MAFbx. Thus, protein breakdown by caspase-3 and the ubiquitin-proteasome system in muscle are stimulated by the same signal: a low PI3K activity. These responses could yield therapeutic strategies to block muscle atrophy. [source]

Neutralization of IL-17 by active vaccination inhibits IL-23-dependent autoimmune myocarditis

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2006
Ivo Sonderegger
Abstract The most common reason for heart failure in young adults is dilated cardiomyopathy often resulting from myocarditis. Clinical studies and animal models provide evidence that an autoimmune response against heart myosin is the underlying reason for the disease. IL-12 has been suggested to play a key role in development of experimental autoimmune myocarditis (EAM), as IL-12p40 and IL-12R,1 knockouts are protected from disease. In this study, we have compared IL-12p40,/, mice, IL-12p35,/, mice and mice treated with a neutralizing IL-23 antibody in EAM and found that in fact IL-23, not IL-12, is responsible for inflammatory heart disease. However, these cytokines appear to have redundant activity for priming and expansion of autoreactive CD4 T cells, as specific T cell proliferation was only defective in the absence of both cytokines. IL-23 has been suggested to promote a pathogenic IL-17-producing T cell population. We targeted IL-17 by capitalizing on an active vaccination approach that effectively breaks B cell tolerance. Neutralization of IL-17 reduced myocarditis and heart autoantibody responses, suggesting that IL-17 is the critical effector cytokine responsible for EAM. Thus, targeting of IL-23 and IL-17 by passive and active vaccination strategies holds promise as a therapeutic approach to treat patients at risk for development of dilated cardiomyopathy. See accompanying commentary: http://dx.doi.org/10.1002/eji.200636760 [source]

Functional regions in the essential light chain of smooth muscle myosin as revealed by the mutagenesis approach

FEBS JOURNAL, Issue 20 2000
Sophie Quevillon-Chéruel
The endogenous essential light chain (LC17) of myosin from intestine smooth muscle was replaced with mutated essential light chains prepared using recombinant techniques. Complete exchange was observed with histidine-tagged derivatives of LC17a, LC17b and E122A-LC17a (LC17a and LC17b are the usual constituants of smooth muscle myosin), with small changes in the ATPase activity of reconstituted myosins. Much less exchange was observed with the light-chain derivative lacking the last 12 amino acid residues, demonstrating the importance of this segment, which may act as one arm of a pair of pincers to bind the myosin heavy chain. [source]

Deficiency of Myo18B in mice results in embryonic lethality with cardiac myofibrillar aberrations

GENES TO CELLS, Issue 10 2008
Rieko Ajima
Myo18B is an unconventional myosin family protein expressed predominantly in muscle cells. Although conventional myosins are known to be localized on the A-bands and function as a molecular motor for muscle contraction, Myo18B protein was localized on the Z-lines of myofibrils in striated muscles. Like Myo18A, another 18th class of myosin, the N-terminal unique domain of the protein and not the motor domain and the coiled-coil tail is critical for its localization to F-actin in myocytes. Myo18B expression was induced by myogenic differentiation through the binding of myocyte-specific enhancer factor-2 to its promoter. Deficiency of Myo18B caused an embryonic lethality in mice accompanied by disruption of myofibrillar structures in cardiac myocytes at embryonic day 10.5. Thus, Myo18B is a unique unconventional myosin that is predominantly expressed in myocytes and whose expression is essential for the development and/or maintenance of myofibrillar structure. [source]

Calcineurin phosphatase in signal transduction: lessons from fission yeast

GENES TO CELLS, Issue 7 2002
Reiko Sugiura
Calcineurin (protein phosphatase 2B), the only serine/threonine phosphatase under the control of Ca2+/calmodulin, is an important mediator in signal transmission, connecting the Ca2+ -dependent signalling to a wide variety of cellular responses. Furthermore, calcineurin is specifically inhibited by the immunosuppressant drugs cyclosporin A and tacrolimus (FK506), and these drugs have been a powerful tool for identifying many of the roles of calcineurin. Calcineurin is enriched in the neural tissues, and also distributes broadly in other tissues. The structure of the protein is highly conserved from yeast to man. The combined use of powerful genetics and of specific calcineurin inhibitors in fission yeast Schizosaccharomyces pombe (S. pombe) identified new components of the calcineurin pathway, and defined new roles of calcineurin in the regulation of the many cellular processes. Recent data has revealed functional interactions in which calcineurin phosphatase is involved, such as the cross-talk between the Pmk1 MAP kinase signalling, or the PI signalling. Calcineurin also participates in membrane traffic and cytokinesis of fission yeast through its functional connection with members of the small GTPase Rab/Ypt family, and Type II myosin, respectively. These findings highlight the potential of fission yeast genetic studies to elucidate conserved elements of signal transduction cascades. [source]

Identification and functional analysis of the gene for type I myosin in fission yeast

GENES TO CELLS, Issue 3 2001
Mika Toya
Background Type I myosin is highly conserved among eukaryotes, and apparently plays important roles in a number of cellular processes. In the budding yeast, two myosin I species have been identified and their role in F-actin assembly has been inferred. Results We cloned the fission yeast myo1 gene, which apparently encoded a myosin I protein. Disruption of myo1 was not lethal, but it caused growth retardation at high and low temperatures, sensitivity to a high concentration of KCl, and aberrance in cell morphology associated with an abnormal distribution of F-actin patches. An abnormal deposition of cell wall materials was also seen. Homothallic myo1, cells could mate, but heterothallic myo1, cells were poor in conjugation. Myo1p was necessary for the encapsulation of spores. The tail domain of Myo1p was pivotal for its function. Calmodulin could bind to Myo1p through the IQ domain at the neck. Conclusions Myo1p appears to control the redistribution of F-actin patches during the cell cycle. Loss of Myo1p function is likely to slow down the actin assembly/disassembly process, which results in a failure of the actin cycle to catch up with other events in both the mitotic and meiotic cell cycles, including extension of the conjugation tubes. [source]

A phylogeny of Vetigastropoda and other "archaeogastropods": re-organizing old gastropod clades

INVERTEBRATE BIOLOGY, Issue 3 2010
Stephanie W. Aktipis
Abstract. The phylogenetic relationships among the "archaeogastropod" clades Patellogastropoda, Vetigastropoda, Neritimorpha, and Neomphalina are uncertain; the phylogenetic placement of these clades varies across different analyses, and particularly among those using morphological characteristics and those relying on molecular data. This study explores the relationships among these groups using a combined analysis with seven molecular loci (18S rRNA, 28S rRNA, histone H3, 16S rRNA, cytochrome c oxidase subunit I [COI], myosin heavy-chain type II, and elongation factor-1, [EF-1,]) sequenced for 31 ingroup taxa and eight outgroup taxa. The deep evolutionary splits among these groups have made resolution of stable relationships difficult, and so EF-1, and myosin are used in an attempt to re-examine these ancient radiation events. Three phylogenetic analyses were performed utilizing all seven genes: a single-step direct optimization analysis using parsimony, and two-step approaches using parsimony and maximum likelihood. A single-step direct optimization parsimony analysis was also performed using only five molecular loci (18S rRNA, 28S rRNA, histone H3, 16S rRNA, and COI) in order to determine the utility of EF-1, and myosin in resolving deep relationships. In the likelihood and POY optimal phylogenetic analyses, Gastropoda, Caenogastropoda, Neritimorpha, Neomphalina, and Patellogastropoda were monophyletic. Additionally, Neomphalina and Pleurotomariidae fell outside the remaining vetigastropods, indicating the need for further investigation into the relationship of these groups with other gastropods. [source]