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Ronald A Milligan

Publications and source records attributed to Ronald A Milligan.

At least 19 recordsLinked to original sources
PubMed2006

Lucky 13-microtubule depolymerisation by kinesin-13 motors.

The kinesin-13 class of motors catalyses microtubule depolymerisation by bending tubulins at microtubule ends. Depolymerisation activity is intrinsic to the kinesin-13 motor core but the activity of the core alone is very low compared with that of constructs that also contain a conserved neck sequence. The full-length dimeric motor is an efficient depolymeriser and also diffuses along the microtubule lattice, which helps it to find microtubule ends. Current evidence supports the idea of a generic mechanism for kinesin-13-catalysed depolymerisation. However, the activity of kinesin-13 motors is precisely localised and regulated in vivo to enable a wide range of cellular roles. The proteins are involved in global control of microtubule dynamics. They also localise to mitotic and meiotic spindles, where they contribute to formation and maintenance of spindle bipolarity, chromosomal congression, attachment correction and chromatid separation. In interphase cells, intricate and subtle mechanisms appear to allow kinesin-13 motors to act on specific populations of microtubules. Such carefully controlled localisation and regulation makes these kinesins efficient, multi-tasking molecular motors.

Amino Acid Sequence↗
PubMed2006

The role of the kinesin-13 neck in microtubule depolymerization.

To ensure genetic integrity, replicated chromosomes must be accurately distributed to daughter cells-a process that is accomplished on the microtubule spindle. Kinesin-13 motors play an essential role in this process by performing regulated microtubule depolymerization. We set out to dissect the depolymerization mechanism of these kinesins, and in particular, the role of their conserved neck sequence. We used a monomeric kinesin-13 MCAK, consisting of the neck and motor core, which has strong depolymerizing activity. In the presence of a non-hydrolysable ATP analogue, this construct induced formation of rings around microtubules. The rings are built from tubulin protofilaments that are bent by the kinesin-13 motor engaged at the ATP-binding step of its ATPase cycle. Our data suggest that the ring-microtubule interaction is mediated by the neck and support the idea of a role for the kinesin-13 neck in depolymerization efficiency, acting by optimizing release of tubulin from microtubule ends.

Adenosine Triphosphate↗
PubMed2006

Supramolecular architecture of severe acute respiratory syndrome coronavirus revealed by electron cryomicroscopy.

Coronavirus particles are enveloped and pleomorphic and are thus refractory to crystallization and symmetry-assisted reconstruction. A novel methodology of single-particle image analysis was applied to selected virus features to obtain a detailed model of the oligomeric state and spatial relationships among viral structural proteins. Two-dimensional images of the S, M, and N structural proteins of severe acute respiratory syndrome coronavirus and two other coronaviruses were refined to a resolution of approximately 4 nm. Proteins near the viral membrane were arranged in overlapping lattices surrounding a disordered core. Trimeric glycoprotein spikes were in register with four underlying ribonucleoprotein densities. However, the spikes were dispensable for ribonucleoprotein lattice formation. The ribonucleoprotein particles displayed coiled shapes when released from the viral membrane. Our results contribute to the understanding of the assembly pathway used by coronaviruses and other pleomorphic viruses and provide the first detailed view of coronavirus ultrastructure.

Cryoelectron Microscopy↗
PubMed2005

A lever-arm rotation drives motility of the minus-end-directed kinesin Ncd.

Kinesins are microtubule-based motor proteins that power intracellular transport. Most kinesin motors, exemplified by Kinesin-1, move towards the microtubule plus end, and the structural changes that govern this directional preference have been described. By contrast, the nature and timing of the structural changes underlying the minus-end-directed motility of Kinesin-14 motors (such as Drosophila Ncd) are less well understood. Using cryo-electron microscopy, here we demonstrate that a coiled-coil mechanical element of microtubule-bound Ncd rotates approximately 70 degrees towards the minus end upon ATP binding. Extending or shortening this coiled coil increases or decreases velocity, respectively, without affecting ATPase activity. An unusual Ncd mutant that lacks directional preference shows unstable nucleotide-dependent conformations of its coiled coil, underscoring the role of this mechanical element in motility. These results show that the force-producing conformational change in Ncd occurs on ATP binding, as in other kinesins, but involves the swing of a lever-arm mechanical element similar to that described for myosins.

Adenosine Triphosphate↗
PubMed2005

Helical crystallization on nickel-lipid nanotubes: perfringolysin O as a model protein.

To facilitate purification and subsequent structural studies of recombinant proteins the most widely used genetically encoded tag is the histidine tag (His-tag) which specifically binds to N-nitrilotriacetic-acid-chelated nickel ions. Lipids derivatized with a nickel-chelating head group can be mixed with galactosylceramide glycolipids to prepare lipid nanotubes that bind His-tagged proteins. In this study, we use His-tagged perfringolysin O (PFO), a soluble toxin secreted by the bacterial pathogen Clostridium perfringens, as a model protein to test the utility of nickel-lipid nanotubes as a tool for structural studies of His-tagged proteins. PFO is a member of the cholesterol dependent cytolysin family (CDC) of oligomerizing, pore-forming toxins found in a variety of Gram-positive bacterial pathogens. CDC pores have been difficult to study by X-ray crystallography because they are membrane associated and vary in size. We demonstrate that both a wild-type and a mutant form of PFO form helical arrays on nickel-lipid containing nanotubes. Cryo-electron microscopy and image analysis of the helical arrays were used to reconstruct a 3D density map of wild-type PFO. This study suggests that the use of nickel-lipid nanotubes may offer a general approach for structural studies of recombinant proteins and may provide insights into the molecular interactions of proteins that have a natural affinity for a membrane surface.

Bacterial Toxins↗
PubMed2005

Composition and structure of the 80S ribosome from the green alga Chlamydomonas reinhardtii: 80S ribosomes are conserved in plants and animals.

We have conducted a proteomic analysis of the 80S cytosolic ribosome from the eukaryotic green alga Chlamydomonas reinhardtii, and accompany this with a cryo-electron microscopy structure of the ribosome. Proteins homologous to all but one rat 40S subunit protein, including a homolog of RACK1, and all but three rat 60S subunit proteins were identified as components of the C. reinhardtii ribosome. Expressed Sequence Tag (EST) evidence and annotation of the completed C. reinhardtii genome identified genes for each of the four proteins not identified by proteomic analysis, showing that algae potentially have a complete set of orthologs to mammalian 80S ribosomal proteins. Presented at 25A, the algal 80S ribosome is very similar in structure to the yeast 80S ribosome, with only minor distinguishable differences. These data show that, although separated by billions of years of evolution, cytosolic ribosomes from photosynthetic organisms are highly conserved with their yeast and animal counterparts.

Algal Proteins↗
PubMed2005

Complementarity in the supramolecular design of arenaviruses and retroviruses revealed by electron cryomicroscopy and image analysis.

Arenaviruses are rodent-borne agents of diseases, including potentially lethal human hemorrhagic fevers. These enveloped viruses encapsidate a bisegmented ambisense single-stranded RNA genome that can be packaged in variable copy number. Electron cryomicroscopy and image analysis of New World Pichinde and Tacaribe arenaviruses and Old World lymphocytic choriomeningitis virus revealed pleomorphic enveloped particles ranging in diameter from approximately 400 to approximately 2,000 A. The surface spikes were spaced approximately 100 A apart and extended approximately 90 A from the maximum phospholipid headgroup density of the outer bilayer leaflet. Distinctive stalk and head regions extended radially approximately 30 and approximately 60 A from the outer bilayer leaflet, respectively. Two interior layers of density apposed to the inner leaflet of the viral lipid bilayer were assigned as protein Z and nucleoprotein (NP) molecules on the basis of their appearance, spacing, and projected volume. Analysis of en face views of virions lacking the GP-C spikes showed reflections consistent with paracrystalline packing of the NP molecules in a lattice with edges of approximately 57 and approximately 74 A. The structural proteins of retroviruses and arenaviruses assemble with similar radial density distributions, using common cellular components.

Animals↗
PubMed2004

Mechanism of microtubule stabilization by doublecortin.

Neurons undertake an amazing journey from the center of the developing mammalian brain to the outer layers of the cerebral cortex. Doublecortin, a component of the microtubule cytoskeleton, is essential in postmitotic neurons and was identified because its mutation disrupts human brain development. Doublecortin stabilizes microtubules and stimulates their polymerization but has no homology with other MAPs. We used electron microscopy to characterize microtubule binding by doublecortin and visualize its binding site. Doublecortin binds selectively to 13 protofilament microtubules, its in vivo substrate, and also causes preferential assembly of 13 protofilament microtubules. This specificity was explained when we found that doublecortin binds between the protofilaments from which microtubules are built, a previously uncharacterized binding site that is ideal for microtubule stabilization. These data reveal the structural basis for doublecortin's binding selectivity and provide insight into its role in maintaining microtubule architecture in maturing neurons.

Binding Sites↗
PubMed2004

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.

Adaptation, Biological↗
PubMed2004

MAP2c, but not tau, binds and bundles F-actin via its microtubule binding domain.

BACKGROUND: MAP2 and tau are abundant microtubule-associated proteins (MAPs) in neurons. The development of neuronal dendrites and axons requires a dynamic interaction between microtubules and actin filaments. MAPs represent good candidates to mediate such interactions. Although MAP2c and tau have similar, well-characterized microtubule binding activities, their actin interaction is poorly understood. RESULTS: Here, we show by using a cosedimentation assay that MAP2c binds F-actin. Upon actin binding, MAP2c organizes F-actin into closely packed actin bundles. Moreover, we show by using a deletion approach that MAP2c's microtubule binding domain (MTBD) is both necessary and sufficient for both F-actin binding and bundling activities. Surprisingly, even though the MAP2 and tau MTBDs share high sequence homology and possess similar microtubule binding activities, tau is unable to bind or bundle F-actin. Furthermore, experiments with chimeric proteins demonstrate that the actin binding activity fully correlates with the ability to promote neurite initiation in neuroblastoma cells. CONCLUSIONS: These results provide the first demonstration that the MAP2c and tau MTBD domains exhibit distinct properties, diverging in actin binding and neurite initiation activities. These results implicate a novel actin function for MAP2c in neuronal morphogenesis and furthermore suggest that actin interactions could contribute to functional differences between MAP2 and tau in neurons.

Actins↗
PubMed2003

Regulation of KinI kinesin ATPase activity by binding to the microtubule lattice.

KinI kinesins are important in regulating the complex dynamics of the microtubule cytoskeleton. They are unusual in that they depolymerize, rather than move along microtubules. To determine the attributes of KinIs that distinguish them from translocating kinesins, we examined the ATPase activity, microtubule affinity, and three-dimensional microtubule-bound structure of a minimal KinI motor domain. Together, the kinetic, affinity, and structural data lead to the conclusion that on binding to the microtubule lattice, KinIs release ADP and enter a stable, low-affinity, regulated state, from which they do not readily progress through the ATPase cycle. This state may favor detachment, or diffusion of the KinI to its site of action, the microtubule ends. Unlike conventional translocating kinesins, which are microtubule lattice-stimulated ATPases, it seems that with KinIs, nucleotide-mediated modulation of tubulin affinity is only possible when it is coupled to protofilament deformation. This provides an elegant mechanistic basis for their unique depolymerizing activity.

Adenosine Triphosphatases↗
PubMed2003

Rapid routine structure determination of macromolecular assemblies using electron microscopy: current progress and further challenges.

Although the methodology of molecular microscopy has enormous potential, it is time consuming and labor intensive. The techniques required to produce a three-dimensional (3D) electron density map of a macromolecular structure normally require manual operation of an electron microscope by a skilled operator and manual supervision of the sometimes complex software needed for analysis and calculation of 3D maps. Systems to automate the process of data acquisition from an electron microscope are being developing and these systems are being integrated with specimen handling operations and post acquisition data processing. Here, the current performance of our existing systems and the future challenges involved in substantially improving both the sustained throughput and the yield of automated data collection and analysis are reported.

Algorithms↗
PubMed2003

Distinct conformations of the kinesin Unc104 neck regulate a monomer to dimer motor transition.

Caenhorhabditis elegans Unc104 kinesin transports synaptic vesicles at rapid velocities. Unc104 is primarily monomeric in solution, but recent motility studies suggest that it may dimerize when concentrated on membranes. Using cryo-electron microscopy, we observe two conformations of microtubule-bound Unc104: a monomeric state in which the two neck helices form an intramolecular, parallel coiled coil; and a dimeric state in which the neck helices form an intermolecular coiled coil. The intramolecular folded conformation is abolished by deletion of a flexible hinge separating the neck helices, indicating that it acts as a spacer to accommodate the parallel coiled-coil configuration. The neck hinge deletion mutation does not alter motor velocity in vitro but produces a severe uncoordinated phenotype in transgenic C. elegans, suggesting that the folded conformation plays an important role in motor regulation. We suggest that the Unc104 neck regulates motility by switching from a self-folded, repressed state to a dimerized conformation that can support fast processive movement.

Adenosine Diphosphate↗
PubMed2003

Nuclear pore complexes exceeding eightfold rotational symmetry.

Nuclear pore complexes are rotationally symmetric structures that span the nuclear envelope and provide channels for nucleocytoplasmic traffic. These large complexes normally consist of eight spokes arranged around a central channel, although, occasionally, 9- and 10-fold nuclear pore complexes are found in preparations of Xenopus oocyte macronuclei. Here we examine these unusual nuclear pore complexes by negative stain electron microscopy and image analysis and compare the results with data previously obtained from 8-fold structures. The details in two-dimensional and three-dimensional maps indicate that the substructure of the spoke is the same in 8-, 9- and 10-fold nuclear pore complexes: therefore, the spoke is likely an immutable structural component. In all three variant forms, the spacing between adjacent annular subunits, which surround the central channel, is identical. Distances between spokes at higher radius decrease in the 9- and 10-fold nuclear pore complexes. These data imply that the most important connections holding the nuclear pore complex together are those between adjacent annular subunits and that these interactions may play a predominant role in nuclear pore complex assembly. Circumferential connections mediated by ring subunits and radial arms presumably further stabilize the structure and are flexible enough to accommodate additional spokes.

Animals↗
PubMed2002

MAP2 and tau bind longitudinally along the outer ridges of microtubule protofilaments.

MAP2 and tau exhibit microtubule-stabilizing activities that are implicated in the development and maintenance of neuronal axons and dendrites. The proteins share a homologous COOH-terminal domain, composed of three or four microtubule binding repeats separated by inter-repeats (IRs). To investigate how MAP2 and tau stabilize microtubules, we calculated 3D maps of microtubules fully decorated with MAP2c or tau using cryo-EM and helical image analysis. Comparing these maps with an undecorated microtubule map revealed additional densities along protofilament ridges on the microtubule exterior, indicating that MAP2c and tau form an ordered structure when they bind microtubules. Localization of undecagold attached to the second IR of MAP2c showed that IRs also lie along the ridges, not between protofilaments. The densities attributable to the microtubule-associated proteins lie in close proximity to helices 11 and 12 and the COOH terminus of tubulin. Our data further suggest that the evolutionarily maintained differences observed in the repeat domain may be important for the specific targeting of different repeats to either alpha or beta tubulin. These results provide strong evidence suggesting that MAP2c and tau stabilize microtubules by binding along individual protofilaments, possibly by bridging the tubulin interfaces.

Animals↗
PubMed2002

A relational database for cryoEM: experience at one year and 50 000 images.

For the past year we have been using a relational database as part of an automated data collection system for cryoEM. The database is vital for keeping track of the very large number of images collected and analyzed by the automated system and essential for quantitatively evaluating the utility of methods and algorithms used in the data collection. The database can be accessed using a variety of tools including specially developed Web-based interfaces that enable a user to annotate and categorize images using a Web-based form.

Algorithms↗
PubMed2002

A mechanism for microtubule depolymerization by KinI kinesins.

Whereas most kinesins motor along microtubules, KinI kinesins are microtubule depolymerizing machines. Surprisingly, we found that a KinI fragment consisting of only the motor core is capable of ATP-dependent depolymerization. The motor binds along microtubules in all nucleotide states, but in the presence of AMPPNP, microtubule depolymerization also occurs. Structural characterization of the products of AMPPNP-induced destabilization revealed a snapshot of the disassembly machine in action as it precisely deformed a tubulin dimer. While conventional kinesins use the energy of ATP binding to execute a "powerstroke," KinIs use it to bend the underlying protofilament. Thus, the relatively small class-specific differences within the KinI motor core modulate a fundamentally conserved mode of interaction with microtubules to produce a unique depolymerizing activity.

Adenosine Triphosphate↗