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.

A lattice of interacting chemical oscillators

At Brandeis, there is a long tradition of interesting experiments on the Belousov-Zhabostinsky reaction system, with the legendary Zhabotinsky himself having been a part of the fraternity. This reaction system shows interesting oscillatory and stable patterns (see videos on Youtube). In the Fraden lab, an oil emulsion of micron-sized water droplets containing the BZ reactions, was shown to show interesting synchronization properties and complex spatial patterns [Toiya et al, J. Phys. Chem. Lett. 1, 1241 (2010)]. A coupling between the droplets due to preferential diffusion of an inhibitory reactant (bromine) in the oil medium was seen to be responsible for these collective phenomena.

In a new paper titled “Phase and frequency entrainment in locally coupled phase oscillators with repulsive interactions” in Phys. Rev. E, Physics Ph. D student Michael Giver, postdoc Zahera Jabeen and Prof. Bulbul Chakraborty show that neighboring oscillators can be modeled as Kuramoto phase oscillators, coupled nonlinearly to its nearest neighbors. The form of the coupling chosen is repulsive, which favors out of phase synchronization. They show using linear stability analysis as well as numerical study that the stable phase patterns depend on the geometry of the lattice. A linear chain of these repulsively coupled oscillators shows anti-phase synchronization, in which neighboring oscillators show a phase difference of π The phase difference between the neighboring oscillators when placed on a ring however depends on the number of oscillators. In such a case, the locally preferred phase difference of π is ruled out for an odd number of oscillators, as this may lead to frustration. When these oscillators are placed on a triangular lattice in two dimensions, the geometry of the lattice constrains the phase difference between two neighboring oscillators to 2 π /3. Interestingly, domains with different helicities form in the lattice. In each domain, the phases of any three neighboring oscillators can vary continuously in either clockwise or an anti-clockwise direction. Hence, phase difference between the nearest neighbors are seen to be ±2π /3 in the two domains (See figure). A phase difference of π is seen at the interfaces of these domains. These domains can grow in time, resembling domain coarsening in other statistical studies. At large coupling strengths, the domains freeze in size due to frequency synchronization of all the oscillators. Hence, an interplay between frequency synchronization and phase synchronization was seen in this system. Ongoing studies in the BZ experimental setup at the Fraden Lab, find correlations with the above results. Hence, insights into a complex system like the BZ oscillators could be gained using the phase oscillator formalism.

The research was supported by the ACS Petroleum Research Fund and the Brandeis MRSEC. Michael Giver is a trainee in the Brandeis NSF-sponsored IGERT program Time, Space & Structure: Physics and Chemistry of BIological Systems

A molecular function of Zillion Different Screens protein explained

In a recent paper in Journal of Cell Biology entitled “Spatial regulation of Cdc55-PP2A by Zds1/Zds2 controls mitotic entry and mitotic exit in budding yeast“, Brandeis postdoctoral fellow Valentina Rossio and Assistant Professor of Biology Satoshi Yoshida reveal a molecular function of a mysterious protein Zds1.

The Zds1 protein in yeast  was identified some years ago in “a zillion different screens” for cell cycle mutants, stress response mutants, RNA metabolism mutants, etc., but the molecular function of the protein remained a mystery for more than 15 years. Rossio revealed that Zds1’s key target is a protein phosphatase 2A (PP2A) complex. She showed that Zds1 controls nucleocytoplasmic distribution of PP2A complex, and that this regulation is critical for cells to know when to enter and to exit from mitosis (picture below; cells lacking Zds proteins adopt an abnormal shape because of problems in mitosis). Rossio thinks all the other complicated phenotypes associated with ZDS1 can also be explained by PP2A regulation and is currently studying mechanistic details about the Zds1-PP2A interaction.

See also the accompanying commentary “Proteins keep Cdc55 in its place

Yeast genetics and familial ALS

In a recent paper in PLoS Biology, “A Yeast Model of FUS/TLS-Dependent Cytotoxicity“, Brandeis postdoc Shulin Ju and coworkers applied yeast genetics to examine the function of the human protein FUS/TLS. The gene for FUS/TLS is mutated in 5-10$ of cases of Familial ALS. The yeast model expressing the mutant protein recapitulates many important features of the pathology.

A particular feature of interest is that  FUS/TLS form cytoplasmic inclusions of this protein which is normally localized to the nucleus. Over-expression of a number of yeast proteins rescues the cells from the toxic effect without removing the inclusions. The results are suggested to implicate RNA processing or RNA quality control in the mechanism of toxicity, which I find really interesting in light of the talk Susan Lindquist (an author on this paper) gave at Brandeis about yeast prions and regulatory proteins earlier this month.

Other authors on the paper include Brandeis professors Dagmar Ringe and Gregory Petsko, and Brandeis alumni Dan Tardiff (PhD, Mol. Cell. Biol.,  ’07), currently a postdoc in the Lindquist lab at the Whitehead Institute,  and Daryl Bosco (PhD, Bioorganic Chem, ’03), currently on the faculty at U. Mass. Medical School.

For more information, please see the paper itself or the longer article about the research on Brandeis NOW.

Mei Zeng Receives Genome Customization Award

Mei Zeng, a postdoc in Nelson Lau’s lab (Biology) has been selected to receive a postdoctoral fellowship award – the Genome Customization Award (TGCA) from Cellectis Bioresearch. The TGCA award was established by Cellectis Bioresearch in 2010 with the goal of spreading the use of meganucleases for genome customization throughout the life sciences

Meganucleases are endodeoxyribonucleases characterized by a large recognition site (12 to 40 base pairs) — so large that it  generally only occurs once in any given genome. The Lau group will apply the custom meganucleases to improve transgenesis of Xenopus tropicalis for RNA interference methodologies. The most widely used transgenesis method utilizes the yeast meganucleases I-SceI which cuts both the transgene vector and an unknown site in the genome into which the transgene gets integrated. This method has several limitations: it requires a large number of embryos for injection and screening,  the integration sites cut by I-Sce-I are unknown and likely stochastic, and it ultimately produces only 5-10% of germline transmission. The custom meganucleases engineered by Cellectis Bioresearch target a known single site (24bp) within the genome, allowing for increased specificity and efficiency of transgene intergration. Mei and colleagues hope to use the rational design to enforce the systemic constitutive expression of a short hairpin RNA cassette in a vertebrate model.

Marc Le Bozec, CEO of Cellectis Bioresearch, presented the award to Drs. Nelson Lau and Mei Zeng on March 16, 2011 at the grand opening of Cellectis Bioresearch Inc facilities in Cambridge, Massachusetts.

What are best friends for? Insights from 10 million friendships

Why do people have best friends? Why do we think of some individuals as “better” friends than other individuals? Why rank friends at all? And what is so special about the apex of the ranking, our “best” friend?

Peter DeScioli, a Kay Fellow at Brandeis University, and colleagues recently shed light on these questions by collecting a dataset of over 10 million people’s friendship decisions from the MySpace social network. The results support the “alliance hypothesis” which is based on the idea that people depend on their friends in conflicts. The findings were recently published in the journal Perspectives on Psychological Science.

MySpace has a feature that allows users to rank their “Top Friends,” providing a unique data source for testing how well different variables explain people’s rankings of friends. The alliance hypothesis predicts that people will feel closest to friends who rank them higher than others. Here’s why: If you need your friend to take your side in an argument, then they will have to side against someone else—which is unlikely if they are better friends with your adversary. The fewer people ranked above you, the more you can rely on your friend to take your side. According to the theory, people unconsciously track this strategic information and it shapes how we feel about our friends.

It turns out that the importance of friend rank was highly significant. Comparing first- and second-ranked friends, 69% chose for first-rank the individual who ranked them better. This was a considerably larger effect than the next best predictor, geographic proximity. The effects of sex, age, and popularity were small by comparison. Moreover, friend rank increased in strength when the analysis was extended to first- versus third- through eighth-ranked friends. In short, we now have 10 million more reasons to wonder if human friendship might be more strategic than it seems.

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