Formins require assistance; not so different from other actin nucleators

Formins are a family of proteins conserved across a wide range of eukaryotes and constitute a major class of actin nucleators. In a paper recently published in Molecular Biology of the Cell, a team led by Ph.D. student Brian Graziano in the laboratory of Professor Bruce Goode made the surprising finding that formins depend on co-factors to efficiently nucleate actin assembly both in vitro and in vivo. This discovery was unanticipated because earlier studies had shown that purified formins are sufficient to catalyze actin polymerization in vitro. Graziano, working in collaboration with the labs of Laurent Blanchoin and Isabelle Sagot, investigated the mechanism and function of a formin-binding protein called Bud6 and found that it elevates formin nucleation activity by 5-10 fold. Further, they showed that this activity of Bud6 is critical in vivo for maintaining normal levels of actin cable assembly and polarized cell growth (see figure).

Earlier work from the Goode lab had shown that Bud6 enhances formin-mediated actin assembly in vitro (Moseley et al., 2004), but had left open the question of whether Bud6 stimulates the nucleation or elongation phase of filament growth (an important mechanistic distinction), and whether the activities of Bud6 are important in vivo. Graziano and collaborators dissected Bud6 mechanism by: (a) generating mutations in Bud6 that separately disrupt its interactions with formins (bu6-35) and actin monomers (bud6-8), (b) using TIRF (total internal reflection fluorescence) microscopy to visualize the effects of Bud6 and formins on individual actin filaments polymerizing in real time, and (c) performing a genetic analysis of bud6 alleles. They made three important observations. First, Bud6 enhances the nucleation rather than elongation phase of actin assembly, in sharp contrast to another formin ligand, profilin, that enhances elongation. Second, this activity of Bud6 requires its direct interactions with both the formin and actin monomers, suggesting that Bud6 recruits monomers to the formin to help assemble an actin ‘seed’. Third, genetic perturbation of these activities of Bud6 results in reduced levels of actin cable formation in vivo, in turn causing defects in polarized secretion and cell growth.

Until now, formins were thought to nucleate actin assembly by themselves, which is mechanistically distinct from the Arp2/3 complex (another major actin nucleator). Efficient nucleation by Arp2/3 requires the addition of a nucleation-promoting factor (NPF) such as WASp or WAVE, which recruits actin monomers. Graziano et al. reveal that some formins are similar to Arp2/3 in that they too require an NPF for robust nucleation. Their findings also uncover unanticipated mechanistic parallels between the two systems, since in each case nucleation requires both an actin filament end-capping component (formin or Arp2/3) and an actin monomer-recruiting factor (Bud6 or WASp).

How well is this formin-NPF mechanism conserved? Clues to this question have recently emerged from other studies. A paper published last year in The Journal of Cell Biology by the Goode lab, working in collaboration with the labs of Niko Grigorieff (Brandeis) and Gregg Gundersen (Columbia), implicates the human tumor suppressor protein Adenomatous polyposis coli (APC) in functioning as a formin NPF (Okada et al., 2010). Another study published in The Proceedings of the National Academy of Sciences by the labs of Mike Eck (Dana Farber Cancer Institute), Margot Quinlan (UCLA), and Avital Rodal (Brandeis), suggests that Spire, which is conserved in mammals and flies, may serve as a formin NPF (Vizcarra et al., 2011). Bud6, Spire, and APC all bind multiple actin monomers and interact with the C-terminus of formins to enhance actin assembly, suggesting that they may have related mechanisms and perform functionally analogous roles.

Although the requirement of NPFs increases the complexity of the formin mechanism, it offers an explanation for how cells simultaneously overcome two prominent barriers to actin assembly found in vivo – actin monomer binding proteins (e.g. profilin) that suppress formation of an actin nucleus and capping proteins that terminate growth by associating with the growing end of the filament. NPFs can facilitate nucleation by recruiting actin monomers in the presence of profilin, and formins protect growing ends of filaments from capping proteins. Future work will focus on identifying new formin-NFP pairs, defining the cellular processes with which they are associated, and distinguishing the underlying mechanistic differences among each set.

XJ Wang, Ranjan Sen return to Brandeis for lectures in October

Former Brandeis faculty members Xiao-Jing Wang (Physics, Neuroscience) and Ranjan Sen (Biology, Rosenstiel Center) will be back on campus next week to give seminars. Wang, now in the Department of Neurobiology and Kavli Institute for Neuroscience at the Yale University School of Medicine, will speak twiceon Monday, Oct 3. At 11:00 am in Volen 201, he will present “Context-dependent decision making” at Computational/Systems Neuroscience Journal Club, followed by a talk at 3:45 pm in Gzang 121, discussing “A ‘cognitive-type’ cortical microcircuit: decision-making and short-term memory” as first lecturer in this year’s IGERT Computational Neuroscience Seminar series. Sen, now Chief, Laboratory of Molecular Biology and Immunology, National Institute on Aging, will speak on Wednesday, Oct 5 at 4:00 pm in Gzang 121, on the subject of how “Loops within Loops Generate the Chromosome Conformation of the Immunoglobulin Heavy Chain Gene Locus”.

Ranjan Sen (left) and Xiao-Jing Wang (right), shown as they appeared in old Brandeis faculty webpages from days past.

For background on the subject of Ranjan’s talk, see The origins of NF-κB, published recently in Nature Immunology.

KC Hayes, obesity, and the Asian Food Network

BrandeisNOW has a new story about Professor KC Hayes, the Asian Food Network, the worldwide trend to greater obesity, and what should and shouldn’t be in your diet.

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.

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