Biology Keywords:

Circadian rhythms, fungal biology, Molecular Genetics, Genomics

Collaboration Keywords:

Network models, antifungal drugs, time series data analysis, proteomics

Unwinding the Neurospora Circadian Clock

Organisms from bacteria to humans use a circadian clock to control daily biochemical, physiological, and behavioral rhythms, and disruption of this clock in humans has severe consequences ranging from sleep disorders, to mental illness, to cancer. We are using the simple model organism Neurospora crassa to study the clock and its output pathways. 

Using molecular genetic techniques, we found that the Neurospora clock is complex and is composed of at least 3 overlapping oscillators that share components and regulate distinct outputs. A critical question that we are currently addressing and that is relevant to the organization of all clocks, including the human clock, is: how do multiple oscillators communicate with each other to coordinately control rhythmic processes. These analysis would benefit from computational modeling.

Our studies on the output pathways using microarrays suggest that about 20% of Neurospora genes are under control of the circadian clock system at the level of transcript accumulation, and that the bulk of the clock-controlled mRNAs have peak accumulation in the late night to early morning. These data suggest the existence of global mechanisms of rhythmic control of gene expression. Consistent with this idea, we found that the Neurospora OS pathway, a phosphorelay signal transduction pathway that responds to changes in osmotic stress, functions as an output pathway from the Neurospora clock system that regulates daily rhythms in expression of downstream genes. Hijacking conserved signaling pathways by the circadian clock provides a new paradigm for global rhythmic control of target genes of the pathway. We are now interested in persuing a global analysis of clock regulation of translation and post-translational regulation.

To understand how light and clock signaling pathways interact in cells, we used Chromatin immunoprecipitation followed by high throughput DNA sequencing (ChIP-Seq) to uncover direct targets of the Neurospora blue-light and circadian clock regulator WC-2. Surprisingly, we found that WC-2 binds to more than 100 different promoters regulating known clock and light-regulated genes, as well as previously unknown targets encoding transcription factors, chromatin modifying enzymes, kinases, phosphatases, cell cycle and DNA repair proteins. Our findings provide direct links between WC-2 and effector molecules in downstream regulatory pathways responsible for light-induced behavior and circadian rhythmicity. Our current goal is to develop a network model of the output pathways.

As an aside, we are also interested in developing new antifungal drugs.

This work is supported by the NIH (GM58529 and NS039546)

Biology Keywords:

microbiology, recombinant DNA, enzyme engineering, bioremediation, directed evolution

Collaboration Keywords:

synthetic biology, engineering microbes, designer enzymes

Biology Keywords:

parasite, microscopy, ecology, evolution, morphology

Collaboration Keywords:

3D-imaging, tomography, volume measurements

Biology Keywords:

neural networks, goal-oriented behavior, regulation of motivation, neural basis of instinct, muscle and neural excitability, ion channels, G-protein coupled neuro-modulatory receptors, sensory neurons, motor units, muscle contraction and relaxation, age-related degradation of neural networks

Collaboration Keywords:

mathematical/coputational modeling of biological circuits over the lifespan of an animal, remote stimulation of neural/muscular circuit components. conception and fabrication of non-invasive micro/nanoscale devices for measuring changes in neuro/muscular cell excitability

My lab is interested in understanding how networks of neuro-muscular circuits are assembled to produce and regulate goal-oriented behaviors. We use the nematode Caenorhabditis elegans to identify cellular and molecular substrates that: (1) compute when the animal should initiate a behavioral subroutine; (2) monitor if the motor outcome was successful; (3) reinitiate the sub-behavior if the intended outcome failed; and (4) activate the next behavioral subroutine if the preceding behavioral outcome is successful. C. elegans is 1 mm in length, transparent and has approximately 1000 somatic cells. The nematode contains two sexes, self-fertilizing hermaphrodites and males. The hermaphrodite contains 302 neurons total, and the male has 387 neurons; the extra male neurons are used to regulate male mating behavior. The gross neural muscular connectivity that controls male-specific mating behavior has been solved. My lab uses C. elegans male mating behavior as a model to determine how different biological molecules such as potassium channels, calcium channels and receptors for neurotransmitters such as acetylcholine, dopamine and serotonin shape the activity of circuit components, so that the male can initiate and regulate the proper execution of the instinctive mating behavior. With C. elegans, one has the experimental advantages of manipulating a compact nervous system with lasers, doing classical Mendellian genetics using mutations in behavioral genes and studying the behavior of transgenic animals. However, there are some limitations to this model organism. The small size of C. elegans, which makes it so convenient to grow, store and feed in the laboratory, requires novel devices that allows monitoring of the ever-changing electrophysiological states of neurons and muscles before, during and after behavior. The size of C. elegans neuronal cell bodies is ~5 micrometers in diameter. The cell size is large enough for current electrodes to impale the cell body, but this classical electrophysiological technique, requiring the animal to be immobilized and cut opened, is much too invasive to obtain data on how circuits function during natural conditions. My aim for collaborations with engineers is to brainstorm new ideas on conceptualizing and fabricating micro or nano scale devices that allow monitoring and eventually manipulating, in noninvasive ways, the electrical activity of discrete neuronal and muscle cells.

Biology Keywords:

Plant cell biology, Cell polarity, Cell development, Cytoskeleton, Membrane Traffic, Nuclear Envelope-ER interface, Golgi-ER inteface, Endosomes-Golgi interface

Collaboration Keywords:

Molecular motors, 3D reconstruction, morphometric image processing, modeling cellular dynamics, modeling cellular development

Biology Keywords:

Embryonic development, brain development, gene expression, patterning, signal transduction, cis-regulatory elements

Collaboration Keywords:

visualization methods, NLOM

The vertebrate central nervous system (CNS) develops from three gross subdivisions, the prosencephalon, mesencephalon and rhombencephalon, organized along the anterior-posterior axis of the embryo. Each of these initial divisions becomes further refined into smaller domains through tissue interactions or through the function of signaling centers in specific positions. How the initial subdivisions are specified during development has been a long-standing question in the field of developmental biology. It is clear that certain cell-signaling pathways are responsible for providing spatial information in the embryo that is interpreted by the neural plate to establish these subdivisions, which are reflected in altered patterns of gene expression. The downstream mechanism by which cells interpret positional information into patterns of gene expression that are crucial for early brain development is not understood.

One hurdle to overcome in the effort to understand brain development has been the difficulty in visualizing dynamic gene expression patterns in living, developing embryos.
To approach the question of how different gene expression domains are established in the vertebrate brain, the Yeh and Lekven laboratories are collaborating to develop a new approach for visualizing fluorescent protein reporter genes in living embryos. We are exploiting the optical properties and genetic capabilities of the zebrafish to generate lines that express multiple fluorescent proteins in specific regions of the developing brain, then we will visualize these fluorescent proteins with broadband, ultra-short pulse multispectral non-linear optical microscopy. The goals of this project are to develop the founding technologies on which dynamic gene expression profiles and subsequent morphogenetic events can be visualized for every cell in a live, developing embryo.

Biology Keywords:

cell culture, neuron cells

Collaboration Keywords:

biomaterials, cell-material interfaces, cell imaging and characterization, nanoparticles

Biology Keywords:

Systems biology, modeling, sensitivity analysis, experimental design, inverse problems, optimization

Collaboration Keywords:

Systems biology, modeling, optimization

Biology Keywords:

neurobiology, single cell analysis, co-culture

Collaboration Keywords:

microfluidics, bioMEMS, micro contact printing of protein/cell, cell/microbe culture microsystems, lab on a chip, high throughput screening platform, energy harvesting microsystems

Research activities in the NanoBio Systems Lab. include the development of miniaturized systems for cellular and molecular analysis using micro and nano fabrication technologies. Micro and nanofluidic based systems with integrated analysis techniques enable fast and accurate analysis at low cost and portable setting. Micro and nanofabrication of silicon, glass, and polymer are being used in my lab to realize such system. I have interest in developing systems that can analyze and control various physiological properties of individual cells in an array format. Cancer cells and neuronal cells are of particular interest. I also have interest in utilizing the unique properties of micro and nano scale fluidic phenomena to develop novel systems and interface those systems with biological systems. More information can be found at http://biomems.tamu.edu.

Biology Keywords:

neuroscience, learning, memory, motor production, behavior, electrophysiology, voltage-sensitive fluorescent proteins

Collaboration Keywords:

microelectromechanical systems, microfluidics, nanomaterials, photonics, video telemetry

My lab studies the behavioral, molecular and neurophysiological mechanisms of vocal learning, a sensorimotor integration process that is central to human speech and language, and upon which the whole edifice of human culture is built. We study this process in songbirds as these animals are the best non-human models for understanding vocal imitation and other learning phenomena that are shaped by ongoing cognitive development. Behavioral and neuroanatomical features of song production make it an ideal model for understanding the motor coding of learned social behaviors.
In the context of BECSI, we are particularly interested in developing gene manipulation and neural recording techniques in the song system to delve into basic mechanisms controlling moment-to-moment timing in the production of complex behavioral outputs. Recently, important progress has been made in understanding single-neuron firing properties that code for song production (esp. work by Michale Fee, an engineer/physicist by training, now at MIT). However, we still do not know how the activity patterns of large ensembles of neurons are sequentially orchestrated to produce behavior, either at the molecular or electrophysiological level. This is a fundamental problem in neuroscience with significant ramifications for understanding brain function in health and disease. We will sketch a vague and fanciful outline of a novel approach to substantially increase the number of single unit recordings simultaneously obtained from an unrestrained awake behaving animal. The proposed setup could be utilized in the song system to study neuronal ensemble dynamics during adult vocal communication, the developmental ontogeny of imitative learning, and perturbation to ensemble function using (relatively) high-throughput neuropharmacology.
The architecture of this airborne castle is based on a combination of miniaturized wireless videotelemetry (based on commercial capsule endoscopy products), nanomaterial photonics, dual-color genetically encoded voltage-sensitive fluorophores, and a microelectromechanical drug delivery device, (e.g. Langer, Cima et al). We conceive of applying our approach to zebra finches (~14g; the standard laboratory songbird) and/or mockingbirds (~50g; vocal mimics with large song repertoires), depending on realized device characteristics (ideally <10% of bodyweight).
Looking beyond this particular notion, we hope to illustrate that the songbird represents a superb platform for exploring many potential interfaces between MEMS technology, nanomaterials and neuroscience.

Biology Keywords:

vocal learning, neural activity, immediate early gene expression, sleep

Collaboration Keywords:

volumetric data, 3D-reconstruction, microvascular filament tracing, statistical parametric mapping, Brain image visualization

Song imitation by juvenile male songbirds provides an interesting analogy to human infant speech and language development. Vocal development in songbirds and humans occurs over a protracted period of juvenile life and follows surprising non-linear trajectories based on central and peripheral constraints imposed by the brain and vocal organ, respectively. We have developed operant training methods that enable us to time lock the onset of song learning. Using this training paradigm, we are attempting to describe how global neural activity unfolds, at the genomic level, during the first 48hr of imitative vocal development. During this brief window, young males display two very interesting behaviors. Initial exposure to adult male song triggers a rapid descent into a sleep state and, over the next few hours (or on the following day), juvenile male subsong undergoes rapid vocal changes.
The function of this experience-dependent sleep state is still unknown, although a mnemonic role is an attractive idea. Conceivably, this process may facilitate encoding of the song template (i.e. memory of the tutor song), a holy grail of songbird biology. Vocal learning is also strongly facilitated by visual input to songbirds and human infants, and other brain structures involved in motor learning, such as the cerebellum, could also be involved. We are therefore taking a whole-brain 3D mapping approach to assess the spatiotemporal dynamics of immediate-early gene (IEG) activation patterns, as markers for neural activity. This approach, based on radioactive in situ hybridization, may reveal transient engagement of brain regions outside the song system at the onset of the vocal learning process.
Our BECIS interest for this project is that we wish to significantly increase the multimodality and spatial resolution of these whole brain volumes by switching to quantum dot based multispectral in situ hybridization. This would also enable whole-brain catFISH approaches (e.g. Guzowski, JF) to identify distributed populations of neurons that are differentially activated by specific stimuli, for example playback of the tutor song compared with the birds’ own song. High accuracy 3D-image registration across successive brain sections, querying many genes, would be achieved by including on each slide a qdot-labeled probe to vascular/capillary specific markers. This provides a spectral channel dedicated to brain microvasculature as input data for filament network tracing algorithms (e.g. Mayerich & Keyser, Computer Science, TAMU). Our combination of songbird behavioral analysis and these mapping approaches would represent an unprecedented description of a developmental learning process essential to social communication.

Primary: U.J. McMahan and Mark Harlow
Authors: U.J. McMahan, Mark Harlow, Joseph Szule and Robert Marshall
Office: Butler 100/ BSBE 314b
Phone Number: 650-575-5599/ 979-845-9823
E-mail: grantser@tamu.edu / mharlow@tamu.edu

Biology Keywords:

synaptic transmission, organization and behavior of macromolecules, structural biology, electron microscope tomography

Collaboration Keywords:

software for 3D imaging at nanometer scale

Improvement and Extension of EM3D, a unified software package for electron microscope tomography

We are currently using the nascent technology of high-resolution electron microscope tomography (also called electron tomography) to study at 2-3 nm spatial resolution in 3-dimensions the organization and behavior of macromolecules in tissue sections of synapses. The information that is obtained provides unique insights about the molecular mechanisms involved in synaptic impulse transmission and in synapse formation. To aid in theses studies we have developed a software package called EM3D (see http://em3d.stanford.edu/). EM3D is a unified application designed specifically for structural cell biologists that allows a user to start with a raw set of EM images and proceed through the functions of alignment, reconstruction, segmentation, and volume analysis - ultimately producing 3D surface models of structures within the specimen. EM3D also includes computational tools that quantify spatial characteristics on a vertex-by-vertex basis upon the surface models. These technologies can be used to examine how macromolecular organization regulates cell function in any tissue. We are interested in collaborating with computer scientists to improve and extend EM3D’s capabilities.

Harlow , M.L., Ress, D., Stoschek, A., Marshall , R.M. and McMahan, U.J. The architecture of active zone material at the frog's neuromuscular junction. Nature 409: 479-484, 2001.

Ress, D., Harlow, M.L., Marshall, R.M. and U. J. McMahan. Optimization method for isodensity surface models obtained with electron microscope tomography data. Engineering in Medicine and Biology Society, 2003. Proceedings of the 25th Annual Conference of the IEEE 1: 774-77, 2003.

Ress, D.B., Harlow, M.L., Marshall, R.M. and McMahan, U.J. Methods for generating high-resolution structural models from electron microscope tomography data. &#8232;Structure : 12 (10):1763-1774, 2004

Nagwaney, S., Harlow, M.L., Jung, J.H., Szule, J.A., Ress, D., Xu, J., Marshall, R.M. and U.J. McMahan. Macromolecular connections of active zone material to docked synaptic vesicles and presynaptic membrane at neuromuscular junctions of mouse. J. Comp. Neurol. 513: 457-468, 2009.

Primary: U.J. McMahan and Mark Harlow
Authors: U.J. McMahan, Mark Harlow, Joseph Szule and Robert Marshall
Office: Butler 100/ BSBE 314b
Phone Number: 650-575-5599/ 979-845-9823
E-mail: grantser@tamu.edu / mharlow@tamu.edu

Biology Keywords:

synaptic transmission, organization and behavior of macromolecules, structural biology, electron microscope tomography

Collaboration Keywords:

engineering improved metallic or organometallic tissue stains for high resolution electron microscopy

Developing fine-grain, high-contrast metallic stains for electron tomography

A useful method for high-resolution imaging of sub-cellular structures in tissue samples by electron microscope tomography is to enhance their contrast by staining them with heavy metals, which deflect electrons. The most commonly used stain is osmium tetroxide. First applied more than 50 years ago for low-resolution imaging, it stains primarily lipids and/or proteins that compose sub-cellular structures. We use electron microscope tomography on osmium tetroxide stained tissues to study subcellular components at the nervous system’s synapses, particularly macromolecules, at 2-3 nm resolution in 3D. Macromolecules are small groups of proteins that perform specific functions and we have found that certain of those at synapses regulate impulse transmission in a stereotypic way. A disadvantage of osmium tetroxide for high-resolution studies is its large grain size and its discontinuous spatial pattern along continuous structures. We are interested in collaborating with chemical engineers to develop metallic or organometallic stains providing finer grain and more uniform distribution, which would significantly improve electron tomographic spatial resolution. Such stains attached to antibody fragments or toxins might also be useful for localizing biochemically identified proteins of interest to specific macromolecules.

Ress, D.B., Harlow, M.L., Marshall, R.M. and McMahan, U.J. Methods for generating high-resolution structural models from electron microscope tomography data. &#8232;Structure : 12 (10):1763-1774, 2004

Biology Keywords:

photosynthesis, mass culture of microbes, energy harvesting, biofuels

Collaboration Keywords:

bioreactors, biomass growth and harvesting techniques, fiber optics, light sources

Recent rises in energy costs have triggered widespread industrial interest in technologies leading to biofuel production. Photosynthetic microbes offer many attractive features for biofuel feedstock production. Among these attractive features are ease of cultivation, rapid growth rates, existing techniques for genetic manipulation, etc. Photosynthetic microbes have been cultivated in lab-scale bioreactors since the 1940s, but surprisingly little work has been done on scaling-up such bioreactors to industrial volumes. Similarly, open-pond cultures have been used for some specific applications in photosynthetic microbial cultivation. Open ponds have certain disadvantages, including contamination of the cultures by competing species or grazers, difficulty of maintaining constant growth conditions, and permitting/licensing/monitoring restrictions on genetically-modified organisms for open cultivation.

I have initiated an on-going conversation with a large industrial concern that produces extremely large quantities of carbon dioxide as a by-product of their plant operations. They are interested in photosynthetic microbes for 2 reasons: to capture and sequester some fraction (the more the better) of their CO2 output, and to generate high-energy biomass that can be fed into their processing stream. They are not at all interested in open-pond cultivation, but have expressed interest in closed bioreactor designs. I would be very interested to talk with anyone familiar with scale-up problems in bioreactors.

An example of a specific problem that needs to be addressed: delivery of optimal intensities of light throughout a dense suspension of photosynthesizing cells. To maximize production rates, cell suspensions should be as concentrated as possible. But if light is delivered unidirectionally from a source outside the culture vessel, the cells nearest the surface of the container absorb or reflect virtually all photons: most of the cells are in the shade. Rapid mixing minimizes this problem, but introduces new problems of physical damage to the cells if mixing is too violent. Sunlight appears to be the cheapest light source. For efficient use of space, it appears that a vertical cylindrical bioreactor, up to 30 - 40 meters tall and 40 - 60 cm in diameter would be attractive in its production capacity. A "forest" of such bioreactors would yield the production capacity needed for my industrial partner's application. But such an array presents many difficulties in delivering sufficient intensities of light to the cells deep within a dense suspension. I have tried to dream up an arrangement of Fresnel lenses linked to optical fibers, but I don't know enough about those technologies to do anything useful.

I would be very interested to talk to anyone with interests/expertise in large-capacity bioreactors or light-distribution techniques.

Biology Keywords:

zebrafish, retina, photoreceptor, macular degeneration, retinal pigment epithelium

Collaboration Keywords:

hypoxia, nanoparticles,

Biology Keywords:

zebrafish, inner ear, hair cells, support cells, hearing, regeneration, stem cells, Atoh1, Sox2

Collaboration Keywords:

We study how sensory hair cells in the inner ear are regulated during development and regeneration. Hair cells are modified neurons that form in discrete patches on the inner surface of the inner ear. Once formed, hair cell can no longer divide. In most vertebrates, damaged hair cells can be replaced by trans-differentiation of support cells, an auxiliary cell type interspersed amongst hair cells within each patch. In humans, however, the capacity for hair cell regeneration has been lost, leading to progressive and irreversible loss of hearing as we age. Our lab has identified genes that regulate hair cell development and regeneration in zebrafish, a commonly studied model organism. Our studies show that both hair cells and support cells arise from a pool of identical progenitor cells, each poised to choose between these alternate cell fates. Initially, all progenitors express two transcription factors, Atoh1 and Sox2. Atoh1 stimulates differentiation of hair cells whereas Sox2 generally represses differentiation and maintains the ability of cells to divide. The mutually antagonistic activities of Atoh1 and Sox2 help orchestrate formation of “salt-and-pepper” patterns of hair cells and support cells. Formation of sensory patches is also associated with two extracellular signaling molecules, which regulate, and are regulated by, expression levels of Atoh1 and Sox2. Moreover, these signaling molecules appear to facilitate post-embryonic growth of sensory patches, which occurs by ongoing recruitment of surrounding cells into the sensory patch as the animal ages. This is in contrast to humans in which the number of hair cells and support cells is fixed at birth. We are working to develop a model in which dynamic feedback between the genes encoding Atoh1, Sox2 and the extracellular signaling molecules coordinates formation, growth, maintenance and repair of sensory patches. It is hoped that modifying the expression levels or feedback relationships between the human orthologs of these genes can restore the regenerative capacity of the human inner ear.

Biology Keywords:

biosensing, biological agents, biomolecular self-assembly, molecule targeting, molecular recognition, miRNA, neural microtubules, enzymes

Collaboration Keywords:

biosensing, nanoencapsulation of molecules, drug delivery, biomolecule-functionalizated nanodevices, RNA-based nanostructures, protein-based nanostructures, enzymes for chemical sensing

Biology Keywords:

Sensory systems. Motor control. Bats. Sonar. Neurobiology. Psychopharmacology.

Collaboration Keywords:

Active Sensing. Sonar. Acoustics. Computational Neuroscience. Neural Networks.

Biology Keywords:

Gene regulation, Transcription factors, DNA/protein interactions

Collaboration Keywords:

Gene regulation, Transcription factors, DNA/protein interactions, Automation, Robotics, Microfluidics

A Biology-Engineering Interface (BEI) Initiative: High-Throughput Analysis of Transcription Factor Interactions with DNA Regulatory Elements

Specific interaction of transcription factors (TFs) with DNA regulatory elements is the primary gene regulatory mechanism in all organisms. We have developed a prototype high-throughput chemiluminescent DNA binding assay to assess the specificity of TF interactions with DNA regulatory elements. TFs with a C-terminal hemaglutinin (HA) epitope tag are expressed in vitro in a coupled transcription/translation system. HA-tagged TFs are then reacted in solution with biotinylated double-stranded oligonucleotides containing the TF binding site. Following the binding reaction, the mixture is added to microtiter plate wells coated with streptavidin; subsequently, the bound HA-tagged/DNA protein complexes are detected by a chemiluminescence reaction via the HA-tag. The objective of this BEI initiative is to automate the screening process using robotics and possibly microfluidics to develop a scalable system to analyze the dynamics of DNA/TF interactions. One application of such an automated system is to identify small molecules that interact with TFs as an initial step in identifying new drug targets.

Biology Keywords:

Twilight reefs, collecting, remote operated vehicle (ROV), biodiversity, northern Gulf of Mexico

Collaboration Keywords:

remote operating vehicle, sampling arm, collecting baskets

The Deep Fish Habitat (DFH) program of the National Oceanographic and Atmospheric Administration, Flower Gardens Banks National Marine Sanctuary, seeks to characterize habitat for fishes on the deeper (50 m and more) reefs of the northern Gulf of Mexico. The fishes usually can be identified from photographs. However, the corals, sponges, crabs and many other invertebrate animals cannot be identified until after a biologist has compared a specimen with existing descriptions. Many of these descriptions are based on preserved and often broken material, much of it taken prior to 1920. The "Phantom" ROV (picture) is equipped with cameras only. We' seek assistance in equipping the ROV with an arm and basket.The new set-up must not interfere with the camera or be in danger of "hanging up" on the sea floor. It must be durable. The basket must be able to be opened and closed securely. Controlling cables can be bundled into the guidance cables that guide the propellers and camera angles.