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    Roger W Craig PhD

    TitleProfessor
    InstitutionUniversity of Massachusetts Medical School
    DepartmentCell and Developmental Biology
    AddressUniversity of Massachusetts Medical School
    55 Lake Avenue North
    Worcester MA 01655
    Phone508-856-2474
      Other Positions
      InstitutionUMMS - Graduate School of Biomedical Sciences
      DepartmentCell Biology

      InstitutionUMMS - Programs, Centers and Institutes
      DepartmentProgram in Cell Dynamics

        Overview 
        Narrative

        CDB Department Website

        Craig Lab Website

        Academic Background

        Ph.D., London University, King's College, 1975

         

        Molecular Structure, Dynamics, and Contractile Mechanism of Muscle

         

        We use state-of-the-art electron microscopic techniques to understand how muscles contract. By studying the molecular structures of the actin and myosin filaments, whose interaction is responsible for contraction, we can elucidate the molecular mechanism of force generation and the processes responsible for regulating contraction. We are investigating systems as diverse as the rapidly contracting striated muscles of the skeleton and heart, and the smooth muscles of the internal organs (e.g. blood vessels), which are specialized to contract slowly and to maintain tension over long periods of time. These studies are adding to our basic understanding of muscle function, and also providing a structural basis for understanding muscle diseases caused by malfunction in the actin or myosin filaments.

        Techniques: high resolution electron microscopy, 3D reconstruction, and atomic fitting

        To decipher these filament structures in three dimensions at the molecular level, we use high resolution electron microscopy combined with computer image reconstruction. Specimens are observed by negative staining or cryo-electron microscopy, and 3D reconstructions of filaments are computed using helical or single particle methods. Atomic level detail is achieved by computationally 'fitting' atomic structures of filament subunits into the reconstruction. To study dynamic changes in filament structure that occur in active muscle, we have developed methods for capturing transient structural intermediates on the millisecond time scale for observation by EM.

        Myosin filaments

        Using these approaches we have recently achieved a major breakthrough in defining the 3D configuration of the key energy-transducing molecules, the myosin heads, on the surface of striated muscle myosin filaments (Woodhead et al., 2005). These results show for the first time, and in atomic detail, how myosin molecules are switched 'off', bringing about relaxation of muscle. The results suggest that the structure we observe is common to muscles of animals throughout most of the animal kingdom, and they provide a basis for understanding how these filaments are activated in contracting muscle. Our results also reveal for the first time how the tails of the myosin molecules are packed into the backbone of the thick filament, forming small 'subfilaments' that themselves assemble to form the thick filament core. This provides key background information for understanding how myosin filaments assemble in the cell.

        Actin filaments

        We have also made the first direct observations of how the protein tropomyosin, on the actin filament, regulates contraction by sterically blocking sites of myosin head attachment on actin filaments (Lehman et al., 1994; Xu et al., 1999; Pirani et al., 2005). We are currently determining the organization of the Ca2+-sensitive regulatory complex, troponin, on the thin filament, and how this changes on Ca2+ activation. These studies are revealing in atomic detail the molecular dynamics regulating muscle contraction.

        Smooth muscle

        In addition to our work on striated muscle, we have also shown that the myosin filaments of smooth muscle have a unique 'side-polar' structure, different from the helical organization in striated muscle. This structure helps to explain the characteristic ability of smooth muscles to undergo high degrees of shortening (Xu et al., 1996). Actin filaments from smooth muscle also differ from those in striated muscle, and we have gained new insights into their functioning in terms of the organization of their associated regulatory proteins (Hodgkinson et al., 1997; Lehman et al., 1997).

        Current studies

        We are currently determining the head organization in striated muscle myosin filaments from several key organisms, to test the generality of our model of the off state, and to determine whether subfilaments are a common feature of different species. We are imaging filaments at higher resolution to determine further details of their structure, and are carrying out tomographic studies of smooth muscle filaments to determine the three-dimensional details of their side-polar structure. In our studies of thin filaments, we are developing new methods of 3D reconstruction to reveal further details of the organization of troponin on actin, and we are combining the reconstructions with crystallographic structures of the thin filament components to produce a 3D thin filament model at the atomic level.

        Myosin figure
         
        Figure 1. 3D reconstruction and atomic fitting of (thick) myosin filament (from Woodhead et al., 2005). Left: 3D reconstruction showing arrangement of myosin heads on filament surface, and subfilaments running parallel to axis in filament backbone. Right: fitting of atomic structure of myosin heads (space-filling colored balls) into reconstruction of one pair of heads. The fitting reveals that the two heads interact with each other, preventing interaction with actin and thereby switching contraction off.
         

        Actin figure

        Figure 2. 3D reconstruction and atomic fitting of thin filament. Left: 3D reconstruction based on cryo images of thin filaments (from Xu et al., 1999). Actin in gold, tropomyosin in red (myosin blocking position), and green (non-blocking position). Right: fitting of actin atomic structure (yellow, α-carbon chain) into reconstruction of one actin subunit (blue wire). Highlighted in orange are amino acid clusters on actin that are blocked by tropomyosin in blocking position (white arrow). From Vibert et al., 1997.



        Rotation Projects

        Potential Rotation Projects

        Project #1: Lipid-Layer Protein Crystallization for Electron Microscopy.While individual protein molecules can be readily visualized by EM, structural information is greatly enhanced if the molecules can by crystallized into 2-dimensional ordered arrays. Methods for achieving this are well established, making use of lipid monolayers at an air-water interface.

        1. Follow literature methods to crystallize "standard" proteins using the lipid-layer method.
        2. Observe results by electron microscopy following negative staining.
        3. If time permits, carry out preliminary image processing of micrographs and/or crystallization of unknown proteins. Techniques to be learned: lipid layer crystallization methods; grid preparation; use of electron microscope; image processing.


        Post Docs

        A postdoctoral position is available to study in this laboratory. Contact Dr. Craig for additional details.

        Bibliographic 
        selected publications
        List All   |   Timeline
        1. Lin B, Govindan S, Lee K, Zhao P, Han R, Runte KE, Craig R, Palmer BM, Sadayappan S. Cardiac Myosin binding protein-C plays no regulatory role in skeletal muscle structure and function. PLoS One. 2013; 8(7):e69671.
          View in: PubMed
        2. Woodhead JL, Zhao FQ, Craig R. Structural basis of the relaxed state of a Ca2+-regulated myosin filament and its evolutionary implications. Proc Natl Acad Sci U S A. 2013 May 21; 110(21):8561-6.
          View in: PubMed
        3. Craig R. Isolation, electron microscopy and 3D reconstruction of invertebrate muscle myofilaments. Methods. 2012 Jan; 56(1):33-43.
          View in: PubMed
        4. Brito R, Alamo L, Lundberg U, Guerrero JR, Pinto A, Sulbarán G, Gawinowicz MA, Craig R, Padrón R. A molecular model of phosphorylation-based activation and potentiation of tarantula muscle thick filaments. J Mol Biol. 2011 Nov 18; 414(1):44-61.
          View in: PubMed
        5. Luther PK, Winkler H, Taylor K, Zoghbi ME, Craig R, Padrón R, Squire JM, Liu J. Direct visualization of myosin-binding protein C bridging myosin and actin filaments in intact muscle. Proc Natl Acad Sci U S A. 2011 Jul 12; 108(28):11423-8.
          View in: PubMed
        6. Mun JY, Gulick J, Robbins J, Woodhead J, Lehman W, Craig R. Electron Microscopy and 3D Reconstruction of F-Actin Decorated with Cardiac Myosin-Binding Protein C (cMyBP-C). J Mol Biol. 2011 Jul 8; 410(2):214-25.
          View in: PubMed
        7. Li XE, Tobacman LS, Mun JY, Craig R, Fischer S, Lehman W. Tropomyosin position on f-actin revealed by em reconstruction and computational chemistry. Biophys J. 2011 Feb 16; 100(4):1005-13.
          View in: PubMed
        8. Badyal SK, Basran J, Bhanji N, Kim JH, Chavda AP, Jung HS, Craig R, Elliott PR, Irvine AF, Barsukov IL, Kriajevska M, Bagshaw CR. Mechanism of the Ca(2+)-Dependent Interaction between S100A4 and Tail Fragments of Nonmuscle Myosin Heavy Chain IIA. J Mol Biol. 2011 Jan 28; 405(4):1004-26.
          View in: PubMed
        9. Galinska A, Hatch V, Craig R, Murphy AM, Van Eyk JE, Wang CL, Lehman W, Foster DB. The C terminus of cardiac troponin I stabilizes the Ca2+-activated state of tropomyosin on actin filaments. Circ Res. 2010 Mar 5; 106(4):705-11.
          View in: PubMed
        10. Cammarato A, Craig R, Lehman W. Electron microscopy and three-dimensional reconstruction of native thin filaments reveal species-specific differences in regulatory strand densities. Biochem Biophys Res Commun. 2010 Jan 1; 391(1):193-7.
          View in: PubMed
        11. Umeki N, Jung HS, Watanabe S, Sakai T, Li XD, Ikebe R, Craig R, Ikebe M. The tail binds to the head-neck domain, inhibiting ATPase activity of myosin VIIA. Proc Natl Acad Sci U S A. 2009 May 26; 106(21):8483-8.
          View in: PubMed
        12. Lehman W, Galinska-Rakoczy A, Hatch V, Tobacman LS, Craig R. Structural basis for the activation of muscle contraction by troponin and tropomyosin. J Mol Biol. 2009 May 15; 388(4):673-81.
          View in: PubMed
        13. Zhu J, Sun Y, Zhao FQ, Yu J, Craig R, Hu S. Analysis of tarantula skeletal muscle protein sequences and identification of transcriptional isoforms. BMC Genomics. 2009; 10:117.
          View in: PubMed
        14. Zhao FQ, Craig R, Woodhead JL. Head-head interaction characterizes the relaxed state of Limulus muscle myosin filaments. J Mol Biol. 2009 Jan 16; 385(2):423-31.
          View in: PubMed
        15. Alamo L, Wriggers W, Pinto A, Bártoli F, Salazar L, Zhao FQ, Craig R, Padrón R. Three-dimensional reconstruction of tarantula myosin filaments suggests how phosphorylation may regulate myosin activity. J Mol Biol. 2008 Dec 26; 384(4):780-97.
          View in: PubMed
        16. Luther PK, Bennett PM, Knupp C, Craig R, Padrón R, Harris SP, Patel J, Moss RL. Understanding the organisation and role of myosin binding protein C in normal striated muscle by comparison with MyBP-C knockout cardiac muscle. J Mol Biol. 2008 Dec 5; 384(1):60-72.
          View in: PubMed
        17. Jung HS, Craig R. Ca2+ -induced tropomyosin movement in scallop striated muscle thin filaments. J Mol Biol. 2008 Nov 14; 383(3):512-9.
          View in: PubMed
        18. Zhao FQ, Padrón R, Craig R. Blebbistatin stabilizes the helical order of myosin filaments by promoting the switch 2 closed state. Biophys J. 2008 Oct; 95(7):3322-9.
          View in: PubMed
        19. Zhao FQ, Craig R. Millisecond time-resolved changes occurring in Ca2+-regulated myosin filaments upon relaxation. J Mol Biol. 2008 Aug 29; 381(2):256-60.
          View in: PubMed
        20. Jung HS, Komatsu S, Ikebe M, Craig R. Head-head and head-tail interaction: a general mechanism for switching off myosin II activity in cells. Mol Biol Cell. 2008 Aug; 19(8):3234-42.
          View in: PubMed
        21. Galinska-Rakoczy A, Engel P, Xu C, Jung H, Craig R, Tobacman LS, Lehman W. Structural basis for the regulation of muscle contraction by troponin and tropomyosin. J Mol Biol. 2008 Jun 20; 379(5):929-35.
          View in: PubMed
        22. Zoghbi ME, Woodhead JL, Moss RL, Craig R. Three-dimensional structure of vertebrate cardiac muscle myosin filaments. Proc Natl Acad Sci U S A. 2008 Feb 19; 105(7):2386-90.
          View in: PubMed
        23. Li XD, Jung HS, Wang Q, Ikebe R, Craig R, Ikebe M. The globular tail domain puts on the brake to stop the ATPase cycle of myosin Va. Proc Natl Acad Sci U S A. 2008 Jan 29; 105(4):1140-5.
          View in: PubMed
        24. Lehman W, Craig R. Tropomyosin and the steric mechanism of muscle regulation. Adv Exp Med Biol. 2008; 644:95-109.
          View in: PubMed
        25. Li XD, Jung HS, Mabuchi K, Craig R, Ikebe M. The globular tail domain of myosin Va functions as an inhibitor of the myosin Va motor. J Biol Chem. 2006 Aug 4; 281(31):21789-98.
          View in: PubMed
        26. Poole KJ, Lorenz M, Evans G, Rosenbaum G, Pirani A, Craig R, Tobacman LS, Lehman W, Holmes KC. A comparison of muscle thin filament models obtained from electron microscopy reconstructions and low-angle X-ray fibre diagrams from non-overlap muscle. J Struct Biol. 2006 Aug; 155(2):273-84.
          View in: PubMed
        27. Craig R, Woodhead JL. Structure and function of myosin filaments. Curr Opin Struct Biol. 2006 Apr; 16(2):204-12.
          View in: PubMed
        28. Pirani A, Vinogradova MV, Curmi PM, King WA, Fletterick RJ, Craig R, Tobacman LS, Xu C, Hatch V, Lehman W. An atomic model of the thin filament in the relaxed and Ca2+-activated states. J Mol Biol. 2006 Mar 31; 357(3):707-17.
          View in: PubMed
        29. Woodhead JL, Zhao FQ, Craig R, Egelman EH, Alamo L, Padrón R. Atomic model of a myosin filament in the relaxed state. Nature. 2005 Aug 25; 436(7054):1195-9.
          View in: PubMed
        30. Cammarato A, Craig R, Sparrow JC, Lehman W. E93K charge reversal on actin perturbs steric regulation of thin filaments. J Mol Biol. 2005 Apr 15; 347(5):889-94.
          View in: PubMed
        31. Pirani A, Xu C, Hatch V, Craig R, Tobacman LS, Lehman W. Single particle analysis of relaxed and activated muscle thin filaments. J Mol Biol. 2005 Feb 25; 346(3):761-72.
          View in: PubMed
        32. Gong H, Hatch V, Ali L, Lehman W, Craig R, Tobacman LS. Mini-thin filaments regulated by troponin-tropomyosin. Proc Natl Acad Sci U S A. 2005 Jan 18; 102(3):656-61.
          View in: PubMed
        33. Foster DB, Huang R, Hatch V, Craig R, Graceffa P, Lehman W, Wang CL. Modes of caldesmon binding to actin: sites of caldesmon contact and modulation of interactions by phosphorylation. J Biol Chem. 2004 Dec 17; 279(51):53387-94.
          View in: PubMed
        34. Cammarato A, Hatch V, Saide J, Craig R, Sparrow JC, Tobacman LS, Lehman W. Drosophila muscle regulation characterized by electron microscopy and three-dimensional reconstruction of thin filament mutants. Biophys J. 2004 Mar; 86(3):1618-24.
          View in: PubMed
        35. Lehman W, Craig R. The structure of the vertebrate striated muscle thin filament: a tribute to the contributions of Jean Hanson. J Muscle Res Cell Motil. 2004; 25(6):455-66.
          View in: PubMed
        36. Lehman W, Craig R, Kendrick-Jones J, Sutherland-Smith AJ. An open or closed case for the conformation of calponin homology domains on F-actin? J Muscle Res Cell Motil. 2004; 25(4-5):351-8.
          View in: PubMed
        37. Luther PK, Padrón R, Ritter S, Craig R, Squire JM. Heterogeneity of Z-band structure within a single muscle sarcomere: implications for sarcomere assembly. J Mol Biol. 2003 Sep 5; 332(1):161-9.
          View in: PubMed
        38. Sutherland-Smith AJ, Moores CA, Norwood FL, Hatch V, Craig R, Kendrick-Jones J, Lehman W. An atomic model for actin binding by the CH domains and spectrin-repeat modules of utrophin and dystrophin. J Mol Biol. 2003 May 23; 329(1):15-33.
          View in: PubMed
        39. Zhao FQ, Craig R. Ca2+ causes release of myosin heads from the thick filament surface on the milliseconds time scale. J Mol Biol. 2003 Mar 14; 327(1):145-58.
          View in: PubMed
        40. Zhao FQ, Craig R. Capturing time-resolved changes in molecular structure by negative staining. J Struct Biol. 2003 Jan; 141(1):43-52.
          View in: PubMed
        41. Tobacman LS, Nihli M, Butters C, Heller M, Hatch V, Craig R, Lehman W, Homsher E. The troponin tail domain promotes a conformational state of the thin filament that suppresses myosin activity. J Biol Chem. 2002 Aug 2; 277(31):27636-42.
          View in: PubMed
        42. Tonino P, Simon M, Craig R. Mass determination of native smooth muscle myosin filaments by scanning transmission electron microscopy. J Mol Biol. 2002 May 10; 318(4):999-1007.
          View in: PubMed
        43. Craig R, Lehman W. The ultrastructural basis of actin filament regulation. Results Probl Cell Differ. 2002; 36:149-69.
          View in: PubMed
        44. Craig R, Lehman W. Crossbridge and tropomyosin positions observed in native, interacting thick and thin filaments. J Mol Biol. 2001 Aug 31; 311(5):1027-36.
          View in: PubMed
        45. Wang L, Zhou P, Craig RW, Lu L. Protection from cell death by mcl-1 is mediated by membrane hyperpolarization induced by K(+) channel activation. J Membr Biol. 1999 Nov 15; 172(2):113-20.
          View in: PubMed
        46. Lu L, Yang T, Markakis D, Guggino WB, Craig RW. Alterations in a voltage-gated K+ current during the differentiation of ML-1 human myeloblastic leukemia cells. J Membr Biol. 1993 Mar; 132(3):267-74.
          View in: PubMed
        47. Cohen C, Vibert PJ, Craig RW, Phillips GN. Protein switches in muscle contraction. Prog Clin Biol Res. 1980; 40:209-31.
          View in: PubMed
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