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iBiomagazine and iBioseminars

December 15, 2011 By

Some video resources if you need to explain scientific topics to students (or need something explained to you!)

iBioMagazine.org features short (<15 min) talks that highlight the human side of research. iBioSeminars.org provides approximately hour-long seminars by high profile researchers.

Professor Emeritus of Biology Hugh Huxley discusses the sliding filament theory of muscle contraction in a November 2011 video from iBiomagazine.org

 

 

Professor of Biology Jim Haber discusses Mechanisms of DNA Repair in a 2009 video from iBioseminars.org

 

Filed Under: Biology, seminars Tagged With: , , , , , , ,

James P. Allison to deliver Gabbay Award Lecture

November 3, 2011 By

James Allison, PhD  from the Memorial Sloan-Kettering Cancer Center will receive the 2011 Jacob Heskel Gabbay Award in Biotechnology and Medicine “for his development of strategies for the treatment of autoimmune diseases and for immunotherapy of cancer”. The award, administered by the Rosenstiel Center at Brandeis, consists of a $15,000 cash prize and a medallion. Dr. Allison will deliver the award lecture on Mobilizing the immune system to treat cancer: Immune checkpoint blockade, on Monday, Nov 14, 2011 at 3:30 pm in Gerstenzang 121.

Allison and his lab are interested in the mechanisms that regulate T cell responses and using that understanding to improve clinical outcomes in areas ranging from autoimmunity, to allergy to vaccination to  tumor therapy.

Filed Under: awards, seminars Tagged With: , , , , , ,

Formins require assistance; not so different from other actin nucleators

September 29, 2011 By

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.

Filed Under: Biology, grad students, labs Tagged With: , , , , , ,

Cryo-electron tomography and the structure of doublet microtubules

September 22, 2011 By

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.

Filed Under: Biology, labs Tagged With: , , , , , , , , ,

3D electron microscopy reveals: twin spokes are not twins

May 31, 2011 By

Movement of cells has fascinated scientists for centuries. Improved handcrafted light microscopes in the late 17th century allowed observations of contracting muscle fibers, single-cell organisms gliding through water drops or cells crawling across surfaces. How cell motility is generated and regulated is an ongoing question researchers at Brandeis and many other institutions are trying to answer. The single-cell green algae Chlamydomonas reinhardtii has two eukaryotic flagella (Fig. A) and is a popular genetic model system for studying these motile organelles, which are also called cilia or undulipodia. Cilia and flagella are basically the same organelles that are highly similar from single-cell algae to humans, but when a cell has many relatively short and asymmetrically beating ones they are called cilia (e.g. on the multi-ciliated epithelial cells that line our airways and are important for mucus-clearance), while a few long ones with often symmetric waveforms are called (eukaryotic) flagella (e.g. the sperm flagellum). These should not be confused with bacterial and archaeal flagella, which are very different in structure and evolutionary origin. Eukaryotic cilia and flagella consist of a microtubule-based, cylindrical core with hundreds of similar building blocks that repeat along the length of the organelle (Fig. B-D). In a single flagellum the activity of thousands of motor proteins, dyneins, has to be coordinated to generate motility, and important regulatory complexes include the radial spokes, in Chlamydomonas two spokes per building block (RS1 and RS2) (Fig. D). Recently, Dr. Thomas Heuser, a postdoc in Dr. Daniela Nicastro’s lab at Brandeis, successfully used three-dimensional electron microscopy (electron tomography) to study the structure of rapidly frozen Chlamydomonas flagella in unprecedented detail (Heuser et al. 2009).

Erin Dymek from Dr. Elizabeth Smith’s laboratory at Dartmouth College found that the concentration of Calcium ions, a known regulatory signal modulating ciliary and flagellar motility, affects dynein activity through a conserved Calmodulin and Radial Spokes associated Complex (CSC) (Dymek and Smith, 2007). Erin Dymek and Elizabeth Smith have now teamed up with Tom Heuser and Daniela Nicastro to study the 3D location of this Calcium sensing complex in flagella. In a recent paper (Dymek et al. 2011 MBoC in press) they compared the wild type structure of Chlamydomonas flagella to several artificial microRNA-interference mutants lacking parts of the CSC. They found that in all amiRNAi mutants many of the flagellar building blocks were missing one specific radial spoke, RS2, while RS1 was always present (Fig. E-G), suggesting that the Calcium sensing CSC is located at or near RS2. Interestingly, RS1 and RS2 were previously assumed to be structurally identical, their different numbering simply referred to their proximal and distal location within the repeating building block. The current study not only indicates that the CSC is required for spoke assembly and wild type motility, but as one of the most surprising outcomes it also provides evidence for heterogeneity among the radial spokes, at least at the base where the spokes are anchored to the microtubules. The same team of biologists is now continuing to study the CSC location in the flagellar building block in greater detail by improving image processing strategies to increase resolution.

Filed Under: Biology, labs, postdocs Tagged With: , , , , , , , ,

High resolution virus structures from electron cryo-microscopy

February 24, 2011 By

Professor of Biochemistry Nikolaus Grigorieff discusses recent progress in obtaining virus structures at 4 Å or better resolution from electron microscopy in a new review “Near-atomic resolution reconstructions of icosahedral viruses from electron cryo-microscopy” in Current Opinon in Structural Biology.

Filed Under: Biochemistry Tagged With: , , , , ,

Allis, Grunstein to receive 2011 Rosenstiel Award

December 17, 2010 By

The 2011 recipients of the Lewis S. Rosenstiel Award for Distinguished Work in Basic Medical Science are C. David Allis (Rockefeller Univ.) and Michael Grunstein (UCLA) for their discovery that histones and histone acetylation directly regulate transcription.  There will be lectures and an award ceremony at Brandeis University on April 14, 2011 at 3:30 pm in the Carl J. Shapiro Theater, Shapiro Campus Center

C. David Allis
(Tri-Institutional Professor, Joy and Jack Fishman Professor, Laboratory of Chromatin Biology and Epigenetics, The Rockefeller University)
Beyond the Double Helix: Varying the ‘Histone Code’
Michael Grunstein
(Distinguished Professor , Department of Biological Chemistry , University of California, Los Angeles )
Towards histone function

A reception will follow in the Shapiro Science Center Atrium for all attendees of the talk, from roughly 5:30 until 7 pm.

For more information, see Brandeis NOW and the Rosenstiel Award website.

Filed Under: awards Tagged With: , , , , , , ,

Seeing key hinges in the lever arm of myosin at the atomic level

December 16, 2010 By

In this week’s on-line issue of the Proceedings of the National Academy of Sciences (PNAS), Brandeis researchers Jerry H. Brown, V. S. Senthil Kumar, Elizabeth O’Neall-Hennessey, Ludmila Reshetnikova, and Michelle Nguyen-McCarty ’10, together with Professors Andrew Szent-Györgyi and Carolyn Cohen, and Brookhaven National Laboratory researcher Howard Robinson, reveal the existence of a pair of major new hinges in the muscle protein myosin.

Muscle consists of myosin-containing thick filaments with projections, i.e. myosin heads, that exert force on actin-containing thin filaments during contraction. Previous crystal structures of the myosin head from bay scallop striated muscles and vertebrate muscles have already shown how this motion is produced by the amplification of small conformational changes about hinges in the motor domain (MD) by the so-called lever arm, which consists of the converter and elongated light chain binding domain (LCD).  Just like a baseball bat or other lever arms we are all familiar with in the “real world”, this LCD of myosin has appeared to be relatively rigid in these crystal structures, as it needs to be to transmit force effectively. But it has also long been expected that in muscle the myosin head, including its lever arm, is likely to contain elastic elements so that force can be produced under various strains.

(Left) Schematic of a myosin molecule and (right) the two conformations of the heavy chain portion of the LCD.

The Brandeis researchers originally set out to crystallize a myosin LCD corresponding to that from the catch muscle of sea scallop because it contains a specialized sequence whose structure was predicted to give insight into how muscle contraction of smooth muscles is turned on and off. Remarkably, however, as described in the PNAS article, this LCD forms two different conformations in the crystal, about mechanically linked hinges in the part of the lever arm distal from the motor. For the first time — and quite unexpectedly— a potential major elastic element in the lever arm has been visualized at atomic resolution, one that allows the length of the lever arm to change by about 10%. Sequence comparisons strongly suggest that these specific hinges are likely to be found in the lever arms of all muscle myosins. These comparisons also indicate differences in the degree of flexibility about these hinges in the different myosins, perhaps helping to account for the different properties (e.g., speed of contraction) of different types of muscle.

This result may also be important for mechanical engineers. In 2009, one of the authors (JHB) wrote an article in American Scientist that expands the concept of biomimicry by describing potentially novel joints, switches, and other mechanical designs that can be derived from the structures of various proteins. The current results in the PNAS seem to add one more. As described by Olena Pylypenko and former Brandeis researcher Anne Houdusse in a commentary scheduled to accompany the print version of the PNAS article, the motion about the hinge of the myosin LCD resembles the motion of a foot relative to a leg about an ankle. A lever “arm” that can extend or compress about an “ankle” may thus be one more novel mechanical design that nature can teach us about.

Filed Under: Biology, labs Tagged With: , , , , , ,

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