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Brandeis grad students guest edit The Biochemist

December 16, 2011 By

Brandeis graduate students Kene Piasta (Biochemistry, PhD ’11), Dharia McGrew (Mol. Cell. Biology, PhD ’11) and Lena Webb (Mol. Cell. Biology, MS ’11) were recently rewarded with the chance to be guest editors for The Biochemist, the bi-monthly magazine for members of the Biochemical Society. The issue they produced, with articles covering “The Science of Sensation“, is now available. The issue covers the biology of the five senses people have (and one that people don’t have but fish do). All three students have moved on from Brandeis: Lena now edits scripts for the Journal of Visualized Experiments, Kene is a postdoc in Joseph Falke’s lab at U. Colorado, Boulder, studying bacterial two-component chemoreceptors, and Dharia is a science and technology policy fellow with the state of California.

Filed Under: Biochemistry, Biology, grad students 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: , , , , , ,

Who pulls the strings in actin cable assembly?

August 26, 2011 By

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.

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

How many neurons does it take to stay cool?

August 17, 2011 By

The worm (nematode) C. elegans is a nice model system for studying neuroscience, combining

  • genetic tools that allow genes to be turned on or off, often on a per cell basis, in the whole organism,
  • tools like laser or genetic ablation that allow individual, identified cells to be selectively eliminated,
  • robust behaviors that can be readily measured, and
  • a well defined nervous system consisting of 302 neurons, each of which can be identified, and whoseanatomical connectivity has been established.

In a paper appearing this month in Journal of Neuroscience, Molecular and Cell Biology grad student Matthew Beverly, undergrad Sriram Anbil, and Professor of Biology Piali Sengupta examined the contribution of sensory neurons to controlling thermotaxis in C. elegans. Worms develop a memory of the temperature at which they have been cultivated, and display a robust behavior in which worms placed on a temperature gradient at temperatures higher than their cultivation temperature will crawl back towards colder temperature (negative thermotaxis – see movie). The behavior depends on TAX-4, a channel protein expressed in a subset of the sensory neurons. In this study, the Brandeis researchers asked the question “how many and which of the sensory neurons are required for the worms to perform negative thermotaxis, and are the required sensory neurons the same regardless of the temperature range examined?” (or, in my paraphrase, “how many and which neurons does it take to stay cool?”)

Worm head, showing expression of the calcim indicator GCaMP in ASI and AWA neurons (used in calcium imaging experiments)

As it turns out, the answer is complicated (and readers are encouraged to read the paper). The researchers found that in addition to the previously known thermosensory neurons AFD and AWC, the ASI neurons previously known to be involved in chemosensation play a significant role in regulating negative thermotaxis. Interestingly, the circuits used seem to be degenerate; under one condition, for example, a particular combination of AFD, AWC or ASI is necessary to generate the behavior, although at other conditions, a different combination is required to generate the same behavior.. And only a couple of degrees Celsius makes a difference — the circuit required for negative thermotaxis on a gradient centered at 8oC above the cultivation temperature is different from a gradient centered at 6oC above.

These and other results taken together suggest that even in the worm, a complex circuit has evolved to control crawling behaviors to cope with temperature changes, and that having degeneracy in the underlying circuits may be a common feature that ensures that behaviors crucial to survival are maintained in a variety of environmental conditions..

Beverly M, Anbil S, Sengupta P. Degeneracy and Neuromodulation among Thermosensory Neurons Contribute to Robust Thermosensory Behaviors in Caenorhabditis elegans. J Neurosci. 2011;31(32):11718-27.

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

Genetics Training Grant Symposium to be held Sep 2

August 10, 2011 By

The Genetics Training Grant at Brandeis (GTG) is an important part of the graduate programs in Molecular & Cell Biology and Biochemistry & Biophysics, teaching students to critically evaluate both their own research and the scientific literature, while also developing their communication skills. The annual symposium, organized and hosted by the GTG students, is central to this mission. This year’s GTG Symposium is entitled “Signal Transduction: Insights gained from diverse species”, and will take place on September 2.  Four distinguished scientists will be presenting their recent work:

  • Gary Ruvkun (Harvard Medical School), our Keynote Speaker, will speak about neuroendocrine control of C. elegans development, metabolism and longevity;
  • Marcia Haigis (Harvard Medical School) will present her work on mitochondrial sirtuins and aging;
  • Morris White (Children’s Hospital Boston) will talk about the molecular basis of mammalian insulin-like signaling in the pathophysiology of metabolic disease;
  • Cynthia Bradham (Boston University) will present work on secondary axis specification and patterning in the sea urchin.

These talks will be followed by a Poster Session and Reception (see schedule). Current and former GTG trainees will be presenting posters from 3:40 to 5:00 PM in the Shapiro Science Center Atrium, All life sciences graduate students are encouraged to present posters.

The entire event is free and open to the public.  For planning purposes, we ask anyone attending the symposium and/or presenting a poster to pre-register by August 24th, 2011. Poster titles will be available after registration is complete.

Please join us for this exciting symposium showcasing genetics at Brandeis.

Filed Under: Biochemistry, Biology, events, grad students 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: , , , , , , , ,

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