Light buffers the wake‐promoting effect of dopamine

Sleep is driven and regulated by the integration of diverse internal and external (environmental) cues. Light is known to be a potent inhibitor of sleep in diurnal animals (awake during daylight hours and sleep at night), including both humans and fruit flies. Yet wakefulness does not scale linearly with light intensity and a lack of light does not automatically result in sleep. (Evolution seems unlikely to favor animals who become hyperactive in dangerously hot midday sunlight and fall asleep in an uncontrollable narcoleptic fashion when the sun goes down, unable to wake until the next morning.) The sleep regulatory system must be plastic — capable of weighing the relative importance of incoming sleep and wake‐promoting cues, and buffering the effects of those cues on sleep drive accordingly. In a recent Nature Neuroscience paper from a team led by postdoc Yuhua Shang (Rosbash lab), with collaborators from the Griffth, Pollack, and Hong labs at Brandeis, we determined at the cell and molecular level how the fruit fly, Drosophila melanogaster, is able to buffer the wake‐promoting effects of the neurotransmitters dopamine and octopamine in the presence of light in order to maintain a proper sleep:wake balance.

It is known that dopamine and octopamine both promote wakefulness in flies. Previous work in the Rosbash and Griffith labs has shown that 10 neurons in the Drosophila brain that release the neuropeptide pigment‐dispersing factor (PDF), known as the l‐LNvs, are critical for transducing the wake‐promoting effects of light. Quantifying mRNAs from all 18 PDF-expressing neurons revealed an enrichment of octopamine and dopamine receptors specifically in the ten wake‐promoting l‐LNvs. We wondered if the l‐LNvs were also able to respond to and transduce the wake‐promoting effects of dopamine and octopamine, and if so, how these effects were integrated with the wake‐promoting effects of light by these cells.

Figure: The l-LNvs use two parallel intracellular pathways to regulate the stimulating effects of DA and OA. Both DA and OA increase the cAMP levels in the l-LNvs. Light in the housing environment suppresses the effects of both DA and OA, but in different ways. In the case of dopamine, light induces increased expression of an inhibitory D2R receptor and in the case of octopamine, the effect is dependent on the circadian clock (Per.)

Using a fluorescence resonance energy transfer (FRET)‐based cyclic AMP reporter expressed in all 18 Pdf neurons, we were able to see robust responses to both octopamine and dopamine in only the t0 l‐LNvs, confirming the mRNA result. To verify that the l‐LNvs are in fact in close apposition to presynaptic octopaminergic and dopaminergic neurons, we looked for reconstitution of a split GFP protein between pre- and post‐synaptic cells. With different GFP fragments expressed at the membrane of the l‐LNvs and presynaptic dopaminergic or octopaminergic neurons, reconstituted GFP would only be visible if these cell populations were in close contact. Reconstituted GFP was seen in both cases around l‐LNv cell bodies and dendritic areas.

To determine the behavioral effect of increased dopaminergic neuron activity on sleep, we transiently hyper‐excited the dopaminergic neurons in flies using the Garrity lab’s heat‐activated dTrpA1 channel. When the housing temperature of flies expressing dTrpA1 in dopaminergic neurons was increased, activating dTrpA1 activity, flies exhibited increased wakefulness. Interestingly, this increased wakefulness was much greater in flies housed in constant darkness as compared to those housed in light:dark cycling conditions. This suggested that the l‐LNvs are a convergence point for the wakepromoting effects of dopamine and light. FRET analysis confirmed this, showing that the l‐LNv response to both dopamine and octopamine is much weaker in flies kept in light:dark conditions as compared to those kept in constant darkness. We then determined that light causes increased expression of an inhibitory dopamine receptor, resulting in a weaker excitatory response to dopamine by the l‐LNvs. In the case of octopamine, the circadian clock was found to regulate the effects of light. Such plasticity allows flies to maintain similar amounts of total sleep in varying environmental conditions, decreasing the relevance of internally generated wake‐promoting cues, in the presence of stronger environmental cues (light). It will be interesting to see how these results generalize to mammals, since light and dopamine also both promote wakefulness in mammals.

Collective behaviors in active matter

Active matter is describes systems whose constituent elements consume energy and are thus out-of-equilibrium. Examples include flocks or herds of animals, collections of cells, and components of the cellular cytoskeleton. When these objects interact with each other, collective behavior can emerge that is unlike anything possible with an equilibrium system. The types of behaviors and the factors that control them however, remain incompletely understood. In a recent paper in Physical Review Letters, “Excitable patterns in active nematics“, Giomi and coworkers develop a continuum theoretical description motivated by recent experiments from the Dogic group at Brandeis in which microtubules (filamentous cytoskeletal molecules) and clusters of kinesin (a molecular motor) exhibit dramatic spatiotemporal fluctuations in density and alignment. Specifically, they consider a hydrodynamic description for density, flow, and nematic alignment. In contrast to previous theories of this type, the degree of nematic alignment is allowed to vary in space and time.  Remarkably, the theory predicts that the interplay between non-uniform nematic order, activity and flow results in spatially modulated relaxation oscillations, similar to those seen in excitable media and biological examples such as the cardiac cycle. At even higher activity the dynamics is chaotic and leads to large-scale swirling patterns which resemble those seen in recent experiments. An example of the flow pattern is shown below left, and the nematic order parameter, which describes the degree of alignment of the filaments, as shown for the same configuration below right. These predictions can be tested in future experiments on systems of microtubules and motor proteins.

The system behavior for an active nematic at high activity. (left) The velocity field (arrows) is superimposed on a plot of the concentration of active nematogens (green=large concentration, red=small concentration). (right) A plot of the nematic order parameter, S,  (blue=large S, brown=small S) is superimposed on a plot of the nematic director (arrows). The flow under high activity is characterized by large vortices that span lengths of the order of the system size and the director field is organized in grains.


Prolonging assembly through dissociation

Microtubules are semiflexible polymers that serve as structural components inside the eukaryotic cell and are involved in many cellular processes such as mitosis, cytokinesis, and vesicular transport. In order to perform these functions, microtubules continually rearrange through a process known as dynamic instability, in which they switch from a phase of slow elongation to rapid shortening (catastrophe), and from rapid shortening to growth (rescue). The basic self-assembly mechanism underlying this process, assembly mediated by nucleotide phosphate activity, is omnipresent in biological systems.  A recent paper, Prolonging assembly through dissociation: A self-assembly paradigm in microtubules ,  published in the May 3 issue of Physical Review E,  presents a new paradigm for such self-assembly in which increasing depolymerization rate can enhance assembly.  Such a scenario can occur only out of equilibrium. Brandeis Physics postdoc Sumedha, working with Chakraborty and Hagan, carried out theoretical analysis of a stochastic hydrolysis model to demonstrate the effect and predict features of growth fluctuations, which should be measurable in experiments that probe microtubule dynamics at the nanoscale.

Model for microtuble dynamics. All activity is assumed to occur at the right end of the microtubule (denoted as ">")

The essential features of the model that leads to the counterintuitive result of depolymerization helping assembly are (a) stochastic hydrolysis that allows GTP to transform into GDP  in any part of the microtubule, and (b) a much higher rate of GTP attachment if the end of the microtubule has a GTP-bound tubulin dimer, compared to a GDP-bound tubulin dimer.    Process (a) leads to islands of GTP-bound tubulins to be buried deep in the microtubule.   Depolymerization from the end reveals these islands and enhances assembly because of the biased attachment rate (b).  The simplicity of the model lent itself to analytical results for various aspects of the growth statistics in particular parameter regimes.   Simulations of the model supported these analytical results, and extended them to regimes where it was not possible to solve the model analytically.  The statistics of the growth fluctuations in this stochastic hydrolysis model are very different from “cap models” which do not have GTP remnants buried inside a growing microtubule.   Testing the predictions in experiments could, therefore, lead to a better understanding of the processes underlying dynamical instability in-vivo and in-vitro.   An interesting question to explore is whether the bias in the attachment rates is different under different conditions of microtubule growth.

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.

Sprout Grant Winners 2011

Entrepreneurship is alive and well at Brandeis.

Last week, fourteen teams of Brandeis scientists presented their research to a panel of industry experts to compete for funding from the Brandeis University Virtual Incubator Sprout Grant Program.  The Virtual Incubator seeks to nurture and support entrepreneurial scientists at Brandeis by providing education, mentoring, networking and seed grants to help move their discoveries from the laboratory to the market.

Judges were impressed by the team presentations. The teams ranged from biologists who have projects that could be ready for licensing as early as next year, to computer science / IT entrepreneurship students with a web application that already has 1200 users.

“We were overwhelmed by the phenomenal proposals we received” says Irene Abrams, Associate Provost for Innovation.  “The response was incredible – with only a few weeks notice, 23 teams applied for Sprout Grants and 14 presented their proposals to the panel of judges.  I was impressed by the level of creativity among the applicants, and by the hard work the teams put into the presentations.  We only had $50,000, so we had to turn down many excellent applications, which we would have funded if we had more money.”

The 2011 winning projects are:

  • Generation Of A Rapid And Efficient Protein Knockout System, Lead Scientist:  Erin Jonasson (with Satoshi Yoshida)
  • Identification Of Molecules For Stabilizing DJ-1, A Protein Involved In Parkinson And Alzheimer Diseases. Lead Scientist: Joey Salisbury (with Brian Williams, Ala Nassar, Jeff Agar and Greg Petsko)
  • Targeting Oncogenic Ras For Protein Degradation, A Novel Approach To Therapy. Lead Scientist: Rory Coffey (with Marcus Long, Ruibao Ren, and Liz Hedstrom)
  • Identifying Pharmacological Chaperones that Promote Survival in Mouse Models of ALS, Lead Scientist: Jared Auclair (with Joey Salisbury, Dagmar Ringe, Greg Petsko, and Jeff Agar)
  • A Novel, Low Cost, Highly Sensitive Form Of Suppression PCR, Lead Scientist: Ken Sugino (with Sean O’Toole and Sacha Nelson)
  • Zen.Do, Team: Bill DeRusha, Joshua Silverman, Jason Urton (Computer Science)

see also: Brandeis NOW

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.

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