University of Bonn, Bonn, Germany.Prostate apoptosis response-4 (Par-4) is a protein that has primarily been con-nectedwithapoptoticprocesses.Recently,wehaveshownthatPar-4alsoplaysa role in smooth muscle contractility. Here, we have tested the hypothesis thatPar-4 regulates the phosphorylation state of myosin regulatory light chain(LC20) by modulating the activity of myosin phosphatase. In the smooth mus-cle-derived cell line A7r5, Par-4 colocalizes with the targeting (MYPT1) andcatalytic(PP1cd) subunitsof myosinphosphatase onactinfilaments.Proximityligation assays demonstrate a close proximity of Par-4 and MYPT1 in vivo.Moreover, endogenous MYPT1 and PP1cd co-immunoprecipitate with endog-enous Par-4 from A7r5 lysates. Direct binding of Par-4 and MYPT1 is shownbysurfaceplasmonresonance,andtheleucinezipperofMYPT1isrequiredfordirect binding. The domain of Par-4 that mediates interaction with MYPT1 hasbeen mapped to the leucine zipper motif in co-immunoprecipitation experi-ments and in proximity ligation assays. LC20 phosphorylation assays usingthe proximity ligation assay revealed that overexpression of Par-4 and a phos-phorylation site mutant of Par-4 (T155A), but not a leucine-zipper defectivemutant(L3A), leadsto reduced phosphorylationlevels of LC20,suggestingac-tivationof myosin phosphatase by Par-4in a leucine-zipper dependentmanner.Moreover, the co-expression of either Par-4 mutant, but not wild type Par-4,with zipper-interacting protein kinase (ZIPK) interferes with ZIPK mediatedLC20 phosphorylation. Our results demonstrate that Par-4 interacts with sub-units of the myosin phosphatase in vivo, possibly as an accessory proteinthatsupportsthecatalyticactivityofmyosinphosphatasebyanasyetunknownmechanism. At the same time, Par-4 is required for efficient inhibition of my-osin phosphatase by ZIPK. Support: HL31704, HL80003, HL86655, AR41637from the NIH, AHA postdoctoral fellowship to SV.666-Pos Board B545Microinjection of Smooth Muscle Myosin in Cultured CellsKatherine McDonald, Renaud Leguillette.University of Calgary, Calgary, AB, Canada.Our current knowledge on the ultrastructure of smooth muscle (SM) cells doesnot satisfactorily account for the particular length-tension behavior of SM,whichseems to be able to adapt its contractile apparatus to generategreat forceover a wide range of lengths. A key question in the field remains: Are contrac-tile filaments in SM cells a fixed array or dynamic structures? There is insuffi-cient knowledge as to whether the myosin filaments can indeed reorganizethemselves in vivo as has been shown through proposed models. We are pro-posing here to develop a microinjection technique to address this question.Methods:Smoothmusclemyosinwaspurifiedfromchickengizzardusingserialprecipitation. As a control, monomeric myosin and self-assembled filaments ofthepurifiedmyosinweretestedforfunctionalityusinganinvitromotilityassay.The myosin was then labeled with a rhodamine-derivative fluorophore, tetra-methylrhodamine-5-iodoacetamidedihydroiodide(5-TMRIA).Thelabeledmy-osinwasthenself-assembledinvitrointofilamentsandimagesweretakenusinga spinning-disk confocal microscope. The labeled filaments, as well as mono-mericmyosinwerethenrespectivelymicroinjectedintoculturedA5R7vascularSMcells.Thevolumeofthecellswasestimatedbymeasuringthemusingacali-bratedmicroscopicruler.Thevolumeofmyosinsolutionthatwasmicroinjectedrepresented2to5%oftheestimatedvolumeofthecells.Thecells’viabilitywasestimated by observing their morphology after the microinjection.Results: The purified myosin was functional in the in vitro motility assay, bothin a monomeric and filamentous form. The microinjection technique could bedone with little modifications of the morphology of the injected cells in a rea-sonable number of cells.Conclusion: The microinjection technique of purified myosin can be used forfurther studies on the ultrastructure and dynamics of myosin filaments insmooth muscle cells.667-Pos Board B546Essential Features of a Non-processive Class V Myosin from BuddingYeast for ASH1 mRNA TransportCarol S. Bookwalter, Matthew Lord, Kathleen M. Trybus.University of Vermont, Burlington, VT, USA.A featureof mostclass V myosins istheir ability tomove processivelyonactin.ThebuddingyeastSaccharomycescerevisiaehasanon-processiveclassVmyo-sin,Myo4p,whichisasingle-headedbuthighdutycyclemotor.Itscellularroleistoasymmetricallytransportmorethan 20differentmRNAs,a widelyusedstrat-egytopolarizeaproteinwithinthecell.ThemoststudiedmRNAisASH1,whichismovedbyMyo4ptothebudtiptorepressmatingtypeswitchinginthedaughtercell. Here we determine the features of Myo4p that are necessary for correct lo-calizationofASH1mRNAtothedaughtercell.Thisprocessrequirestheadapterprotein She3p,andthemRNAbinding proteinShe2p,whichbindsASH1atspe-cific localization elements called zip codes. Based on a series of chimeric con-structs, we showed that the rod region of Myo4p, but not the globular tail, is es-sentialforcorrectlocalizationofASH1mRNA.Therodthuscontainstheprimarybinding site forShe3p,consistentwithourearlierinvitrostudies (Hodges etal.,2008). Totestif mRNA localizationis more efficient when twomotors arecou-pled together, we compared transport by a constitutive dimer of Myo4p/She3pwith a constitutive monomer. Correct ASH1 mRNA localization was achievedequally well with both constructs. This may reflect the fact that many mRNAsand thus many motors are part of the translocation complex. Our results showthatthemostimportantfeatureforcorrectlocalizationistheretentionofcouplingbetweenallthemembersofthecomplex(Myo4p- She3p-She2p-ASH1 mRNA),which is aided by She3p being a tightly bound subunit of Myo4p.668-Pos Board B547Towards Custom-topology Tracks For Probing Myosin Motor DynamicsHendrik Dietz
