3D electron microscopy reveals: twin spokes are not twins

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.

BIOL 99 AND NEUR 99 Senior Honors Talks

Senior honors presentations and defenses for Biology and Neuroscience are this week and next Monday.

Name      Faculty Sponsor & Committee  Time & Location of Talk

Biol 99

Alicia Bach Dagmar Ringe, Neil Simister, Liz Hedstrom May 10   3 pm    Bassine 251
Kristin Little Bruce Goode, Joan Press, Satoshi Yoshida May 6    10 am    Bassine 251
Spencer Rittner KC Hayes, Carolyn Cohen, Larry Wangh May 6    3 pm      Bassine 251
Danielle Saly Michael Rosbash, Mike Marr, Nelson Lau May 10   11 am   Bassine 251
Sue Yen Tay Jim Haber, Sue Lovett, Joan Press  May 7    11am     Bassine 251
Alan Tso Daniela Nicastro, Liz Hedstrom, Greg Petsko May 10   2 pm    Bassine 251
Hannah Worchel Jim Morris, Ruibao Ren, Paul Garrity   May 6    2 pm     Bassine 251

Neur 99
Sarah Pease Sue Paradis, Gina Turrigiano, Paul Miller  May 10   11 am   Volen 201
Solon Schur John Lisman, Eve Marder, Paul Miller May 6    10 am   Volen 201
Alexander Trott Leslie Griffith, Piali Sengupa, Melissa Kosinski-Collins May 6    11 am   Volen 201
Dylan Wolman Sue Paradis, Sacha Nelson, Piali Sengupta May 10   1 pm    Volen 201

Faculty research mentor (emphasized) is chair of the committee.

Postdoctoral position: functional organization of cilia and flagella using molecular genetic approaches

A postdoctoral position is immediately available in the laboratory of Dr. Nicastro at Brandeis University to study the functional organization of cilia and flagella using molecular genetic approaches.

Our lab has in the past mainly been focused on high-resolution structural studies of these highly conserved organelles and defects in mutants, as well as the cytoskeleton and molecular motor in general. One of our long-term goals is to better understand ciliary diseases and identify therapeutic targets. Recently we have expanded our expertise in biochemistry and we are now seeking to complement our highly interactive team with an expert in genetics.

Applicants should have a PhD degree, a strong background in molecular biology/genetic techniques, and an edge for technology development. Responsibilities will include the establishment of a new model organism optimized for reverse genetics to target complexes in cilia and flagella. Familiarity with RNAi and one of the following model organisms is a plus, but not required: Chlamydomonas or Tetrahymena or Planaria. The candidate should be team-oriented and have excellent oral and written communication skills.
The position is available April 1st for up to three years with the possibility of extension. Interested candidates should send an application, including a CV, areas of expertise and interest, publications list, and names and contact information for 3 references to:

Dr. Daniela Nicastro
MS 029
Rosenstiel Center
Brandeis University
415 South Street
Waltham, MA 02454, USA.

The Nicastro Lab is located in the well-equipped and vibrant Biology Department of Brandeis University in Waltham, Massachusetts (eight miles west of Boston). Brandeis has a state-of-the-art electron microscopy facility, a newly implemented facility for Correlative Light and Electron Microscopy and an extensive computational facility. Life Science Research is highly collaborative and interdisciplinary at Brandeis, and offers excellent opportunities for scientific interaction on campus and other scientific institutions in the Boston area. Brandeis University is committed to diversity and equality in education and employment.

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