CIMBio - CIMBio Groups

Principal Investigator:

Ron Milligan

Research:

Structure and Action of Molecular Machines R.A. Milligan, J.M. Al-Bassami, T. Dang, * S. Farah, ** J.H. Holtmann, C.M. Birdsell, C. Arthur, B.O. Carragher, A.W. Lin, C.A. Moores, C.S. Potter, I. Rouiller, B. Sheehan, E.M. Wilson-Kubalek, K. Littlefield, F. Mouche, A.B. Ward
* Stanford University, Stanford, CA
** Freie Universitat, Berlin, Germany

Macromolecular assemblies may be composed of only a few or perhaps scores of proteins and are the functional units - the molecular machines - of the cell. We use cryo-electron microscopy and image analysis to study the structure and mechanism of action of several of these molecular machines. The three-dimensional maps we calculate from electron images of the assemblies are used together with the x-ray structures of the individual components to build models of the working machines.

In our work on myosin and kinesin motors we have used this approach to visualize the various stages in the chemo-mechanical cycle of the track-motor complexes. Our findings have been combined with the wealth of biochemical and biophysical data from other laboratories to provide models for the action of the most well studied motors. Movies showing the motions of brain kinesin and conventional myosin can be viewed at www.scripps.edu/milligan/projects.html.

Although the mechanism of plus-end directed, processive motion by the conventional KinN kinesins is now well understood, the mechanism by which members of the KinC kinesins (e.g. Ncd) move towards the minus ends of microtubules is not. Likewise in the myosin superfamily, it is not known how nucleotide-mediated conformational changes in the motor domain of Class VI myosins result in "backwards" motility. We are now expending considerable effort elucidating the molecular mechanisms of these more unusual members of the myosin and kinesin superfamilies.

Whereas KinN and KinC kinesins move along intact microtubules, members of the KinI kinesins are unusual in that they depolymerize microtubules and do not appear to have motile properties. We have found that a KinI fragment consisting of only the conserved motor core is necessary and sufficient for ATP-dependent depolymerization. The motor core binds along microtubules in all nucleotide states, but in the presence of a non-hydrolysable ATP analogue depolymerization also occurs. Structural characterization of the analogue-induced depolymerization products revealed a snapshot of the disassembly machine as it precisely deformed tubulin dimers at the microtubule ends (figure 1). Our data indicate that while conventional kinesins use the energy of ATP binding to execute a powerstroke that results in unidirectional motion along the microtubule surface, KinIs use it to bend the underlying protofilament, thereby destabilizing the microtubule lattice and leading to microtubule depolymerization.

We have recently investigated the interaction of Map2c and tau with microtubules. These microtubule stabilizing proteins are unstructured in solution but appear to become folded when they interact with the tubulin C-terminus and bind to microtubules. We have shown that they bind longitudinally along the outer crest of tubulin protofilaments, very close to the primary binding site for microtubule motors. The longitudinal interaction geometry suggests that Map2c and tau exert their microtubule stabilizing activity by bridging tubulin interfaces along the protofilament and preventing the straight-to-curled transition that results in depolymerization.

We have extended our studies on VCP/p97, a member of the AAA ATPase family of proteins. This protein is involved in a wide variety of cellular processes including organelle assembly, homotypic membrane fusion and protein degradation. We have examined VCP/p97 in various nucleotide states by cryo-electron microscopy and single particle image analysis. The resulting three-dimensional maps of the hexameric protein assembly show that it undergoes substantial conformational changes during the ATPase cycle. Nucleotide-dependent rearrangements of the subunits are accompanied by constriction of the central channel opening and changes in the interaction geometry of the N-terminal domain of the protein.

Having developed a general method for helical crystallization of proteins on lipid tubules, we are using it to study the virulence factor (PFO) from Clostridium Perfringens. PFO is a cytolysin, an important class of proteins that oligomerize and embed within membranes as part of their lytic function. We have obtained helical crystals of wild type and several mutant forms of PFO on nickel-lipid tubules. 3-D maps of these proteins derived from images of the helical crystals, will be used to compliment our studies of PFO pore formation on lipid layers. These studies will allow us to obtain a better understanding of the pathogenic function of cytolysins. Additional studies involving tubular crystallization of membrane proteins and other bacterial toxins are opening up promising new areas for future research. Finally, collaborations with the Automated Imaging Group led by Bridget Carragher and Clint Potter are focused on the development and implementation of automatic grid searching, image acquisition and image analysis protocols for molecular microscopy.

Figure 1

KinI motor cores caught in the act of deforming a microtubule protofilament. When the KinI motor core binds to microtubules in the presence of a non-hydrolysable ATP analogue it induces curvature in the protofilaments. Locked at the pre-hydrolysis stage in the ATPase cycle, the KinI-tubulin complexes remain as rings consisting of 13 tubulin heterodimers (26 outer densities) and 13 KinI motors (13 inner densities). This projection map was obtained by averaging 250 individual images of the ring complex.

Representative Publications:

Rouiller, I., Pulokas, J., Butel, V.M., Milligan, R.A., Wilson-Kubalek, E.M., Potter, C.S., Carragher, B.O. Automated image acquisition for single-particle reconstruction using p97 as the biological sample. J. Struct. Biol. 133:102, 2001.

Zhu, Y, Carragher, B., Kriegman, D.J., Milligan, R.A., Potter, C.S. Automated identification of filaments in cryo-electron microscopy images. J. Struct. Biol. 135:302, 2001.

Gillespie P.G., Albanesi, J.P., Bahler, M,, Bement, W.M., Berg, J.S., Burgess, D.R., Burnside, B., Cheney, R.E., Corey, D.P., Coudrier, E, de Lanerolle, P., Hammer, J.A., Hasson, T., Holt, J.R., Hudspeth, A.J, Ikebe, M., Kendrick-Jones, J., Korn, E.D., Li, R., Mercer, J.A., Milligan, R.A, Mooseker, M S, Ostap, E.M, Petit, C, Pollard, T.D., Sellers, J,R,, Soldati, T,, Titus, M.A. Myosin-I nomenclature. J. Cell Biol. 155:703, 2001.

Moores, C.A., Yu, M., Guo, J., Beraud, C., Sakowicz, R., Milligan, R.A. A mechanism for microtubule depolymerizatin by KinI kinesins. Molecular Cell , 9:903, 2002.

Fellmann, D., Pulokas, J., Milligan, R., Carragher, B., Potter, C. A relational database for cryoEM: Experience at one year and 50,000 images. J. Struct. Biol., in press.

Al-Bassam, J., Ozer, R., Safer, D., Halpain, S., Milligan, R.A. MAP2 and tau bind longitudinally along the outer ridges of microtubule protofilaments J. Cell Biol., submitted.

Jontes, J.D and R.A. Milligan. 1997. Brush border myosin-I structure and ADP-dependent conformational changes by cryo-electron microscopy and image analysis. J. Cell Biol. 139:683-693.

Wells, A.L., A.W. Lin, L.-Q. Chen, D. Safer, S.M. Cain, T. Hasson, B.O. Carragher, R.A. Milligan and H.L. Sweeney. 1999. Myosin VI is an actin-based motor that moves backwards. Nature 401:505-508.

Rice, S., A.W. Lin, D. Safer, C.L. Hart, N.Naber, B.O. Carragher, S.M. Cain, E. Pechatnikova, E.M. Wilson-Kubalek, M. Whittaker, E. Pate, R. Cooke, E.W. Taylor, R.A. Milligan and R.D. Vale. 1999. A structural change in the kinesin motor protein that drives motility. Nature 402:778-784.

Vale, R.D. and R.A. Milligan. 2000. The way things move: Looking under the hood of molecular motor proteins. Science 288:88-95

Moores, C.A., M. Yu, J. Guo, C. Beraud, R. Sakowicz and R.A. Milligan. 2002. A mechanism for microtubule depolymerization by KinI kinesins. Mol. Cell 9:903-909.

Al-Bassam, J., R. Ozer, D. Safer, S. Halpain and R.A. Milligan. 2002. MAP2 and tau bind longitudinally along the outer ridges of microtubule protofilaments. J. Cell Biol. 157:1187-1196.

Hinshaw, J.E. and R.A. Milligan. Nuclear pore complexes exceeding eight-fold rotational symmetry. 2003. J. Struct. Biol., in press.

D.P. Dias A.L. Wells, M. Whittaker, A. Lin, E. De La Cruz, H.L. Sweeney, R.A. Milligan. Structure of myosin V bound to actin filaments. Submitted.

Contact Information:

milligan@scripps.edu

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Ron Milligan's Research Group

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	Homepage	Ron Milligan's Research Group
	Groups AMI Asturias Danuser Dawson Finn Fowler Gchang Gekakis/Joazeiro Hahn Johnson Kubalek Kuhn Manchester Milligan Otomo Paulson Schneemann Sullivan Unwin Waterman Facilities Microscopes Ancillary Equipment Scanners Resources NRAMM Glycomics Documentation/Help Scheduler People Media Miscellaneous Building Curiosities Image Gallery	Principal Investigator: Ron Milligan Research: Structure and Action of Molecular Machines R.A. Milligan, J.M. Al-Bassami, T. Dang, * S. Farah, ** J.H. Holtmann, C.M. Birdsell, C. Arthur, B.O. Carragher, A.W. Lin, C.A. Moores, C.S. Potter, I. Rouiller, B. Sheehan, E.M. Wilson-Kubalek, K. Littlefield, F. Mouche, A.B. Ward * Stanford University, Stanford, CA Freie Universitat, Berlin, Germany Macromolecular assemblies may be composed of only a few or perhaps scores of proteins and are the functional units - the molecular machines - of the cell. We use cryo-electron microscopy and image analysis to study the structure and mechanism of action of several of these molecular machines. The three-dimensional maps we calculate from electron images of the assemblies are used together with the x-ray structures of the individual components to build models of the working machines. In our work on myosin and kinesin motors we have used this approach to visualize the various stages in the chemo-mechanical cycle of the track-motor complexes. Our findings have been combined with the wealth of biochemical and biophysical data from other laboratories to provide models for the action of the most well studied motors. Movies showing the motions of brain kinesin and conventional myosin can be viewed at www.scripps.edu/milligan/projects.html. Although the mechanism of plus-end directed, processive motion by the conventional KinN kinesins is now well understood, the mechanism by which members of the KinC kinesins (e.g. Ncd) move towards the minus ends of microtubules is not. Likewise in the myosin superfamily, it is not known how nucleotide-mediated conformational changes in the motor domain of Class VI myosins result in "backwards" motility. We are now expending considerable effort elucidating the molecular mechanisms of these more unusual members of the myosin and kinesin superfamilies. Whereas KinN and KinC kinesins move along intact microtubules, members of the KinI kinesins are unusual in that they depolymerize microtubules and do not appear to have motile properties. We have found that a KinI fragment consisting of only the conserved motor core is necessary and sufficient for ATP-dependent depolymerization. The motor core binds along microtubules in all nucleotide states, but in the presence of a non-hydrolysable ATP analogue depolymerization also occurs. Structural characterization of the analogue-induced depolymerization products revealed a snapshot of the disassembly machine as it precisely deformed tubulin dimers at the microtubule ends (figure 1). Our data indicate that while conventional kinesins use the energy of ATP binding to execute a powerstroke that results in unidirectional motion along the microtubule surface, KinIs use it to bend the underlying protofilament, thereby destabilizing the microtubule lattice and leading to microtubule depolymerization. We have recently investigated the interaction of Map2c and tau with microtubules. These microtubule stabilizing proteins are unstructured in solution but appear to become folded when they interact with the tubulin C-terminus and bind to microtubules. We have shown that they bind longitudinally along the outer crest of tubulin protofilaments, very close to the primary binding site for microtubule motors. The longitudinal interaction geometry suggests that Map2c and tau exert their microtubule stabilizing activity by bridging tubulin interfaces along the protofilament and preventing the straight-to-curled transition that results in depolymerization. We have extended our studies on VCP/p97, a member of the AAA ATPase family of proteins. This protein is involved in a wide variety of cellular processes including organelle assembly, homotypic membrane fusion and protein degradation. We have examined VCP/p97 in various nucleotide states by cryo-electron microscopy and single particle image analysis. The resulting three-dimensional maps of the hexameric protein assembly show that it undergoes substantial conformational changes during the ATPase cycle. Nucleotide-dependent rearrangements of the subunits are accompanied by constriction of the central channel opening and changes in the interaction geometry of the N-terminal domain of the protein. Having developed a general method for helical crystallization of proteins on lipid tubules, we are using it to study the virulence factor (PFO) from Clostridium Perfringens. PFO is a cytolysin, an important class of proteins that oligomerize and embed within membranes as part of their lytic function. We have obtained helical crystals of wild type and several mutant forms of PFO on nickel-lipid tubules. 3-D maps of these proteins derived from images of the helical crystals, will be used to compliment our studies of PFO pore formation on lipid layers. These studies will allow us to obtain a better understanding of the pathogenic function of cytolysins. Additional studies involving tubular crystallization of membrane proteins and other bacterial toxins are opening up promising new areas for future research. Finally, collaborations with the Automated Imaging Group led by Bridget Carragher and Clint Potter are focused on the development and implementation of automatic grid searching, image acquisition and image analysis protocols for molecular microscopy. Figure 1** KinI motor cores caught in the act of deforming a microtubule protofilament. When the KinI motor core binds to microtubules in the presence of a non-hydrolysable ATP analogue it induces curvature in the protofilaments. Locked at the pre-hydrolysis stage in the ATPase cycle, the KinI-tubulin complexes remain as rings consisting of 13 tubulin heterodimers (26 outer densities) and 13 KinI motors (13 inner densities). This projection map was obtained by averaging 250 individual images of the ring complex. Representative Publications: Rouiller, I., Pulokas, J., Butel, V.M., Milligan, R.A., Wilson-Kubalek, E.M., Potter, C.S., Carragher, B.O. Automated image acquisition for single-particle reconstruction using p97 as the biological sample. J. Struct. Biol. 133:102, 2001. Zhu, Y, Carragher, B., Kriegman, D.J., Milligan, R.A., Potter, C.S. Automated identification of filaments in cryo-electron microscopy images. J. Struct. Biol. 135:302, 2001. Gillespie P.G., Albanesi, J.P., Bahler, M,, Bement, W.M., Berg, J.S., Burgess, D.R., Burnside, B., Cheney, R.E., Corey, D.P., Coudrier, E, de Lanerolle, P., Hammer, J.A., Hasson, T., Holt, J.R., Hudspeth, A.J, Ikebe, M., Kendrick-Jones, J., Korn, E.D., Li, R., Mercer, J.A., Milligan, R.A, Mooseker, M S, Ostap, E.M, Petit, C, Pollard, T.D., Sellers, J,R,, Soldati, T,, Titus, M.A. Myosin-I nomenclature. J. Cell Biol. 155:703, 2001. Moores, C.A., Yu, M., Guo, J., Beraud, C., Sakowicz, R., Milligan, R.A. A mechanism for microtubule depolymerizatin by KinI kinesins. Molecular Cell , 9:903, 2002. Fellmann, D., Pulokas, J., Milligan, R., Carragher, B., Potter, C. A relational database for cryoEM: Experience at one year and 50,000 images. J. Struct. Biol., in press. Al-Bassam, J., Ozer, R., Safer, D., Halpain, S., Milligan, R.A. MAP2 and tau bind longitudinally along the outer ridges of microtubule protofilaments J. Cell Biol., submitted. Jontes, J.D and R.A. Milligan. 1997. Brush border myosin-I structure and ADP-dependent conformational changes by cryo-electron microscopy and image analysis. J. Cell Biol. 139:683-693. Wells, A.L., A.W. Lin, L.-Q. Chen, D. Safer, S.M. Cain, T. Hasson, B.O. Carragher, R.A. Milligan and H.L. Sweeney. 1999. Myosin VI is an actin-based motor that moves backwards. Nature 401:505-508. Rice, S., A.W. Lin, D. Safer, C.L. Hart, N.Naber, B.O. Carragher, S.M. Cain, E. Pechatnikova, E.M. Wilson-Kubalek, M. Whittaker, E. Pate, R. Cooke, E.W. Taylor, R.A. Milligan and R.D. Vale. 1999. A structural change in the kinesin motor protein that drives motility. Nature 402:778-784. Vale, R.D. and R.A. Milligan. 2000. The way things move: Looking under the hood of molecular motor proteins. Science 288:88-95 Moores, C.A., M. Yu, J. Guo, C. Beraud, R. Sakowicz and R.A. Milligan. 2002. A mechanism for microtubule depolymerization by KinI kinesins. Mol. Cell 9:903-909. Al-Bassam, J., R. Ozer, D. Safer, S. Halpain and R.A. Milligan. 2002. MAP2 and tau bind longitudinally along the outer ridges of microtubule protofilaments. J. Cell Biol. 157:1187-1196. Hinshaw, J.E. and R.A. Milligan. Nuclear pore complexes exceeding eight-fold rotational symmetry. 2003. J. Struct. Biol., in press. D.P. Dias A.L. Wells, M. Whittaker, A. Lin, E. De La Cruz, H.L. Sweeney, R.A. Milligan. Structure of myosin V bound to actin filaments. Submitted. Contact Information: Ron Milligan The Scripps Research Institute Department of Cell Biology 10550 North Torrey Pines Road La Jolla, CA 92037 Office: (858)784-9827 Fax: (858)784-2749 milligan@scripps.edu Group Homepage Ron Milligan's Research Group
T.S.R.I		Center for Integrative Molecular Biosciences To contact us: e-mail: phone: (858) 784-9714 fax: (858) 784-9763