Cryo-electron tomography and the structure of doublet microtubules

In a new paper in PNAS entitled “Cryo-electron tomography reveals conserved features of doublet microtubules“, Assistant Professor of Biology Daniela Nicastro and coworkers describe in striking new detail the structure and organization of the doublet microtubules (DMTs), the most conserved feature of eukaryotic cilia and flagella.

Cilia and flagella are thin, hair-like appendages on the surface of most animal and lower plant cells, which use these organelles to move, and to sense the environment. Defects in cilia and flagella are known to cause disease and developmental disorders, including polycystic kidney disease, respiratory disease, and neurological disorders. An essential feature of these organelles is the presence of nine outer DMTs (hollow protein tubes) that form the cylindrical core of the structure known as the axoneme. The doublet microtubule is formed by tubulin protofilaments and other structural proteins, which provide a scaffold for the attachment of dynein motors (that drive ciliary and flagellar motility) and regulatory components in a highly specific and ordered manner.

To address long-standing questions and controversies about the assembly, stability, and detailed structure of DMTs , the Nicastro lab used a high-resolution imaging technique, cryo-electron microscope tomography (cryo-ET), to probe the structure of DMTs from Chlamydomonas (single-celled algae) and sea urchin sperm flagella. Cryo-ET involves:

  1. rapid freezing of the sample to cryo-immobilize the molecules without forming ice crystals,
  2. tilting the specimen in the electron microscope to collect ~70 different views from +65° to –65°,
  3. computational alignment of the views to calculate a tomogram (a three-dimensional reconstruction of the imaged sample), and
  4. computational averaging of repeating structures in the tomogram to reduce noise and increase resolution.

Cryo-ET provided the necessary resolution to show that the B-tubules of DMTs are composed of 10 protofilaments, not 11, and that the inner and outer junctions between the A- and B-tubules are fundamentally different (see figure). The outer junction, crucial for the initial formation of the DMT, appears to be formed by interactions between the tubulin subunits of three protofilaments with unusual tubulin interfaces, but one of these protofilaments does not fit with the conventionally accepted orientation for tubulin protofilaments. This outer junction is important physiologically, as shown by mutations affecting the usual pattern of posttranslational modifications of tubulin. In contrast, the inner junction is not formed by direct interactions between tubulin protofilaments. Instead, a ladder-like structure that is clearly thinner than tubulin connects protofilaments of the A- and B-tubules.

The level of detail also allowed the Nicastro lab to show that the recently discovered microtubule inner proteins (MIPs) located within the A- and B-tubules are more complex than previously thought. MIPs 1 and 2 are both composed of alternating small and large subunits recurring every 16 and/or 48 nm along the inner A-tubule wall. MIP 3 forms small protein arches connecting the two B-tubule protofilaments closest to the inner junction, but does not form the inner junction itself. MIP 4 is associated with the inner surface of the A-tubule along the partition protofilaments, i.e., the five protofilaments of the A-tubule bounded by the two junctions with the B-tubule.

The Nicastro lab plans to build on this foundation in future work on the molecular assembly and stability of the doublet microtubule and axoneme, and hope to use it to elucidate molecular mechanisms of ciliary and flagellar motility and signal transduction in normal and disease states.

Other authors on the paper include Brandeis postdocs Xiaofeng Fu and Thomas Heuser, Brandeis undergrad Alan Tso (’10), and collaborators Mary Porter and Richard Linck from the University of Minnesota.

Complex Fluids Workshop on Sep 23

On Friday, Sep 23 2011, Brandeis will play host to the 48th New England Complex Fluids Meeting, run by the New England Complex Fluids Workgroup, of which the Brandeis Complex Fluids group is a charter participant. These quarterly meetings foster collaboration among researchers from industry and academia in the New England area studying Soft Condensed Matter, offer the opportunity to exchange ideas, and help introduce students and post-docs to the local academic and industrial research community.

The workshop, to be held in the Shapiro Campus Center, will have four talks by invited speakers, each about 30 minutes long with ample time for questions. In addition, everyone who attends is encouraged to give a five minute update (soundbite) of their current work.

Schedule

9:30 AM – Krystyn Van Vliet (Materials Science and Engineering, MIT), Chemomechanics of responsive gels
10:15 AM – Jeremy England (Physics, MIT), Shape Shifting: the statistical physics of protein conformational change

Soundbites: 11:30 – 12:30 PM Five minute updates of current research

1:30 PM – Francis Starr (Physics, Wesleyan), DNA-linked Nanoparticle Assemblies
2:15 PM – Jennifer Ross (Physics, UMass Amherst), Controlling Microtubules Through Severing

More Soundbites: 3:30 PM – 4:30 PM

GC student wins fellowship to study Cystic Fibrosis carrier screening

Erica Wellington (MS ’12), a student in the Genetic Counseling Program, has been awarded a Jane Engelberg Memorial Fellowship Student Research Award by the National Society of Genetic Counselors. The purpose of the JEMF Student Research Award is to foster research and grant writing skills in genetic counseling students, skills which can continue to be used throughout their careers. Erica’s masters research project is entitled Cystic fibrosis carrier screening: Current practices and challenges in genetic counseling.

Cystic fibrosis (CF) is one of the most common recessive genetic conditions in the Caucasian population, making it a frequent counseling topic in the prenatal clinic. However, because CF displays allelic and phenotypic heterogeneity, genetic counseling of carrier couples is often a challenging task. Depending on the mutations found, carrier couples are at risk of having a child anywhere along a phenotypic spectrum that includes classic CF, nonclassic CF, congenital bilateral absence of the vas deferens (CBAVD) and subclinical manifestations. Genotype-phenotype correlations are highly variable, and complex alleles, the effects of which are mediated by chromosomal background, further complicate counseling. Although there is a large body of literature describing the prognostic ambiguities associated with CF, the CF carrier screening and counseling practices of genetic counselors have not yet been described. This study will use an online survey and telephone interviews to evaluate prenatal genetic counselors’ knowledge of CF carrier screening guidelines, describe current practices, and identify the genetic counseling challenges presented by complex screening scenarios. Establishing this baseline description of practices and challenges is one of the first steps toward helping genetic counselors provide accurate, consistent and useful CF counseling as part of quality preconception and prenatal patient care.

Sleepy and seasick

Associate Professor of Psychology Paul DiZio is interviewed about recent research in the Ashton Graybiel Spatial Orientation Laboratory on the interactions of sleep deprivation and body motion in a new article at BrandeisNOW, “How much sleep’s enough? Navy wants to know

citations everywhere but not a drop to drink

more papers, not otherwised discussed on this blog. Brandeis authors in boldface.

  • Strahler J, Kirschbaum C, Rohleder N. Association of blood pressure and antihypertensive drugs with diurnal alpha-amylase activity. Int J Psychophysiol. 2011;81(1):31-7.
  • Carr M, Devadoss SL, Forcey S. Pseudograph associahedra. J Comb Theory A. 2011;118(7):2035-55.
  • Naculich SG, Schnitzer HJ. Eikonal methods applied to gravitational scattering amplitudes. J High Energy Phys. 2011(5).
  • Vizcarra CL, Kreutz B, Rodal AA, Toms AV, Lu J, Zheng W, Quinlan ME, Eck MJ. Structure and function of the interacting domains of Spire and Fmn-family formins. Proc Natl Acad Sci U S A. 2011;108(29):11884-9.
  • Aaltonen et al. (CDF Collaboration). Search for a Very Light CP-Odd Higgs Boson in Top Quark Decays from p(p)over-bar Collisions at root s=1.96 TeV. Physical Review Letters. 2011;107(3).
  • Wu Y, Singh RP, Deng L. Asymmetric Olefin Isomerization of Butenolides via Proton Transfer Catalysis by an Organic Molecule. J Am Chem Soc. 2011.
  • Kim YS, Ryu YB, Curtis-Long MJ, Yuk HJ, Cho JK, Kim JY, Kim KD, Lee WS, Park KH. Flavanones and rotenoids from the roots of Amorpha fruticosa L. that inhibit bacterial neuraminidase. Food Chem Toxicol. 2011;49(8):1849-56.
  • Broderick R, Fishman L, Kleinbock D. Schmidt’s game, fractals, and orbits of toral endomorphisms. Ergod Theor Dyn Syst. 2011;31:1095-107.
  • Lisman, J., Grace, A.A., and Duzel, E. (2011). A neoHebbian framework for episodic memory; role of dopamine-dependent late LTP. Trends Neurosci. (online)
  • Reeves, D., Cheveralls, K., and Kondev, J. (2011). Regulation of biochemical reaction rates by flexible tethers. Phys Rev E 84.
  • Bell, M.R., Roberts, D.H., and Wardle, J.F.C. (2011). Structure and Magnetic Fields in the Precessing Jet System Ss 433. III. Evolution of the Intrinsic Brightness of the Jets from a Deep Multi-Epoch Very Large Array Campaign. Astrophys J 736.
  • Krogman, J.P., Foxman, B.M., and Thomas, C.M. (2011). Activation of CO(2) by a Heterobimetallic Zr/Co Complex. J Am Chem Soc.
  • Friedman, E.J., Wang, H.X., Jiang, K., Perovic, I., Deshpande, A., Pochapsky, T.C., Temple, B.R., Hicks, S.N., Harden, T.K., and Jones, A.M. (2011). Acireductone Dioxygenase 1 (ARD1) Is an Effector of the Heterotrimeric G Protein β Subunit in Arabidopsis. J Biol Chem 286, 30107-30118.
  • Lahiri, S., Shen, K., Klein, M., Tang, A., Kane, E., Gershow, M., Garrity, P., and Samuel, A.D. (2011). Two alternating motor programs drive navigation in Drosophila larva. PLoS One 6, e23180.
  • Fritz-Laylin, L.K., Ginger, M.L., Walsh, C., Dawson, S.C., and Fulton, C. (2011). The Naegleria genome: a free-living microbial eukaryote lends unique insights into core eukaryotic cell biology. Research in microbiology 162, 607-618.
  • Yan, Q., Xin, Y., Zhou, R., Yin, Y., and Yuan, J. (2011). Light-controlled smart nanotubes based on the orthogonal assembly of two homopolymers. Chem Commun (Camb) 47, 9594-9596.
  • Peelle, J.E., Troiani, V., Grossman, M., and Wingfield, A. (2011). Hearing loss in older adults affects neural systems supporting speech comprehension. J Neurosci 31, 12638-12643.
  • Graziano, B.R., Dupage, A.G., Michelot, A., Breitsprecher, D., Moseley, J.B., Sagot, I., Blanchoin, L., and Goode, B.L. (2011). Mechanism and cellular function of Bud6 as an actin nucleation-promoting factor. Mol Biol Cell.
  • Park, D., Hadzic, T., Yin, P., Rusch, J., Abruzzi, K., Rosbash, M., Skeath, J.B., Panda, S., Sweedler, J.V., and Taghert, P.H. (2011). Molecular Organization of Drosophila Neuroendocrine Cells by Dimmed. Curr Biol.
  • Helminck, A.G., and Schwarz, G.W. (2011). On generalized Cartan subspaces. Transform Groups 16, 783-805.

New Computational Neuroscience Training Program

The National Institute on Drug Abuse has recently awarded Brandeis a pair of linked training grants to support student training in computational neuroscience. The program is unusual for NIH training grants in supporting both undergraduate and graduate student research. Funding for the program is approximately $1.8 million over the next five years.

Modeling a biconditional discrimination task, see Bourjaily & Miller, 2011

The program, directed by Professor Eve Marder, will support six Ph.D. students and six undergraduates (juniors or seniors) each year. Students must be working to fulfill an appropriate degree in the Division of Science at Brandeis, and must engaged in research in computational neuroscience. Said Marder,

We are extremely pleased to have received this grant, as it continues a long Brandeis tradition of integrating theory and experimental work in the neurosciences.  We are especially pleased to have the undergraduate component, as we know there are students who are interested in learning how to employ rigorous quantitative methods to study the brain.

Eligibility and program requirements to participate in the program will soon be available at the training grant website.

Some recent publications:

Bourjaily, M.A., and Miller, P. (2011). Synaptic plasticity and connectivity requirements to produce stimulus-pair specific responses in recurrent networks of spiking neurons. Plos Comput Biol 7, e1001091.

Piquado, T., Cousins, K.A., Wingfield, A., and Miller, P. (2010). Effects of degraded sensory input on memory for speech: Behavioral data and a test of biologically constrained computational models. Brain Res 1365, 48-65.

Berkes, P., Orban, G., Lengyel, M., and Fiser, J. (2011). Spontaneous cortical activity reveals hallmarks of an optimal internal model of the environment. Science 331, 83-87.

Grashow, R., Brookings, T., and Marder, E. (2010). Compensation for variable intrinsic neuronal excitability by circuit-synaptic interactions. J Neurosci 30, 9145-9156.

Geometry and Dynamics IGERT Awarded

Brandeis has just been awarded an NSF Integrative Graduate Education and Research Traineeship (IGERT) grant in the mathematical sciences.  The grant, titled Geometry and Dynamics: integrated education in the mathematical sciences, is designed to foster interdisciplinary research and education by and for graduate students across the mathematical and theoretical sciences, including chemistry, economics, mathematics, neuroscience, and physics.  It is structured around a number of themes common to these disciplines: complex dynamical systems, stochastic processes, quantum and statistical field theory; and geometry and topology. We believe that it is the first IGERT awarded for the theoretical (as opposed to laboratory) sciences, and are very excited about what we believe to be a highly novel program which will cement existing interdepartmental relationships and encourage exciting new collaborations in the mathematical sciences, including collaborations between the natural sciences and the International Business School (IBS).

The resolution of a singularity that develops along Ricci flow, understood mathematically by Grigori Perelman.  If the red manifold represents the target space of a string, it is conjectured that the corresponding two-dimensonal field theory describing the string undergoes confinement and develops a mass gap for the degrees of freedom corresponding to the singular regime.

The award, for $2,867,668 spread out over five years, provides funds for graduate student stipends, travel, seminar speakers, and interdisciplinary course development.  It contains activities and research opportunities in partnership with the New England Complex Systems Institute (NECSI) in Cambridge, MA.  It also provides opportunities for research internships at the International Center for the Theoretical Sciences in Bangalore.

The PIs on the grant are: Bulbul Chakraborty (Physics); Albion Lawrence (Physics: lead PI); Blake LeBaron (IBS); Paul Miller (Neuroscience); and Daniel Ruberman (Mathematics).  There are 11 additional affiliated Brandeis faculty across biology, chemistry, mathematics, neuroscience, physics, and psychology.  Contact Albion Lawrence (albion@brandeis.edu) for more information about the program.

Arrays of repulsively coupled Kuramoto oscillators on a triangular lattice organize into domains with opposite helicities in which phases of any three neighboring oscillators either increase or decrease in a given direction. Fig. (a) illustrates these two helicities in which cyan, ma- genta and blue vary in opposite directions. In Fig. (b), white and green regions represent domains of opposite helicities. The red regions indicate the frequency entrained oscillators, which are predominantly seen in the interior of the domains.

Admission to the program is handled through the Ph.D programs in the various disciplines:

Who pulls the strings in actin cable assembly?

When large structures are built inside of cells, how are their dimensions determined? Are cues received that tell the structure to keep growing, or to slow down, or to stop growing altogether? A recent study published in Developmental Cell by a team led by Molecular and Cell Biology PhD student Melissa Chesarone-Cataldo and Professor of Biology Bruce Goode begins to address these questions by focusing on cytoskeletal structures called yeast actin cables.

Actin cables serve as essential railways for myosin-dependent transport of vesicles, organelles and other cargo, required for yeast cells to grow asymmetrically and produce a daughter cell. Cables are assembled at one end of the mother cell and run the length of the entire cell, but no longer, or else they would hit the back of the cell, buckle and misdirect transport. So how does an actin cable know how long to grow? How are other properties of the cable, such as its thickness and mechanical rigidity determined, and how important are these properties for cable function in vivo?

Actin cables are assembled at the bud neck by the formin protein Bnr1, and rapidly extend into the mother cell at a rate of 0.5-1 µm/s. At this speed, the tip of the actin cable reaches the back end of the cell in about 5-10 seconds. Each cable consists of many shorter overlapping pieces (individual actin filaments) that are stitched or cross-linked together to form a single cable, and cables continuously stream out of the bud neck due to the robust actin assembly activity of Bnr1. Chesarone-Cataldo et al. asked the question, “what mechanism prevents the cables from colliding with the back of the cell and overgrowing?” In doing so, they identified a novel actin cable ‘length sensing’ feedback loop, dependent on the myosin-passenger protein Smy1.

Using live-cell imaging, they showed that Smy1 molecules are transported by myosin from the mother cell to the bud neck, where they pause to interact with the formin Bnr1. Purified Smy1 attenuated Bnr1 activity by slowing down the rate of actin filament elongation. When the SMY1 gene was deleted, cables grew too long, hit the rear of the cell and buckled (see image, right). In addition, the mutant cables abnormally fluctuated in thickness and were kinked, impairing transport of myosin and its cargoes.

The authors propose that a negative feedback loop controls actin cable length. In their model, the cargo (Smy1 in this case) communicates with the machinery that is making the cable (the formin Bnr1), as a means of sensing ‘railway’ length. The longer the railway grows, the more passengers it picks up, and the more transient inhibitory pulses the formin receives. As such, longer cables are selectively attenuated, while shorter cables are allowed to grow rapidly. This negative feedback loop allows yeast cells to tailor actin cable length to the dimensions of the cell and to the needs of its myosin-based transport system.

Current work in the Goode lab is aimed at testing many of the mechanistic predictions of the model above and understanding how Smy1 functions in coordination with other known regulators of Bnr1, all simultaneously present in a cell, to produce actin cables with proper architecture and function. In addition, experiments are underway to find out whether related mechanisms are used to control formins in mammalian cells and to understand the physiological consequences of disrupting those mechanisms.

Chesarone-Cataldo M, Guérin C, Yu JH, Wedlich-Söldner R, Blanchoin L, Goode BL. The Myosin passenger protein Smy1 controls actin cable structure and dynamics by acting as a formin damper. Dev Cell. 2011 Aug 16;21(2):217-30.

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