Carla B. Green - US grants

The vertebrate retina changes many aspects of its physiology with respect to time of day, which allows the retina to adapt efficiently to varying light intensities of many orders of magnitude. These changes include retinomotor movements, visual sensitivity, synaptic function, synthesis and release of neuromodulators and gene expression. Although it is known that vertebrate retinas contain endogenous circadian clocks that control these physiologies, little is known about how these clocks transmit this temporal information to the retinal cells. A powerful model system for studies of retinal and clock function is Xenopus laevis. The eyes from these frogs are very robust in culture for many days and maintain most aspects of normal physiology, including rhythmic physiologies of many types. Furthermore, new technology now allows the production of transgenic frogs providing the opportunity to perform precise mechanistic studies of these cellular functions in vivo. Previously, my lab isolated a novel gene named nocturnin that is expressed for only a few hours in early night in the photoreceptor cells of the retina. This gene encodes a protein that resembles a transcriptional coactivator and our hypothesis is that this gene product is involved in regulating night-time events in the retina. The first aim of this proposal continues the characterization of the nocturnin protein, investigating its intracellular location, its phosphorylation status and its turnover rates. The second aim will focus on identifying proteins that interact with nocturnin, through biochemical means and yeast two-hybrid screens. The third aim will address the role of nocturnin within the photoreceptors by producing transgenic frogs with various gain or loss-of-function mutations of the nocturnin gene. Finally, the fourth aim will continue our studies on the nocturnin promoter, by identifying the elements and proteins involved in its precise temporal regulation. Our overall goal is to provide data about nocturnin that links the central circadian timekeeping mechanism to the cellular rhythms that are so important for the normal homeostasis of the retina.

[unreadable] DESCRIPTION (provided by applicant): Circadian clocks control many processes important for normal functioning of living organisms, including behavior, physiology and biochemistry. These clocks are endogenous timekeeping devices and have been shown to be present in organisms ranging from bacteria to humans. In humans, disruptions of these clocks occur during jet lag, shift work and in some sleep disorders. Recent work has resulted in the identification of a number of genes involved in the central circadian clock and it has become clear that many of these genes are conserved within the animal kingdom. Although a general molecular clock model has been proposed, many of the steps within this model are still not well understood. In this proposal, experiments are described to study the molecular mechanism of the vertebrate circadian clock within the retina of Xenopus laevis. The Xenopus retina contains many well-described cellular and biochemical rhythms that can be manipulated in vitro. Furthermore, new methods for generating transgenic Xenopus embryos allow precise manipulation of gene expression within the intact retina, making this an extremely tractable system for studies of clock mechanism in vivo. The first and second aims of this proposal will focus on in vitro studies of two aspects of cryptochrome function. These proteins are critical components of the negative feedback loop of the clock and we will analyze how the cryptochromes move from the cytoplasm to the nucleus (aim 1) and how they cause repression of the transcriptional apparatus once they are in the nucleus (aim 2). The third aim will test the function of the cryptochromes in vivo by introduction of mutant versions and/or by altering expression levels of these genes in transgenic Xenopus embryos. In the fourth aim, we will make specific "molecular lesions" that disrupt the clock in specific cell types within the retina. This will be done by overexpressing mutant clock genes under the control of several different cell-specific promoters in order to address how individual clocks in the different cell types orchestrate tissue-level rhythmicity. These experiments take advantage of the strengths of the Xenopus system which allow mechanistic studies to be done that are difficult to do in other vertebrate systems. Because these clocks are conserved, information gained from these studies will provide insight into vertebrate clocks in general, including those in humans. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The Pineal Cell Biology Gordon Conference will be held in Ventura, California from February 10-15, 2002. The chair of this conference is Carla B. Green from the Department of Biology and the NSF Center for Biological Timing at the University of Virginia. The co-chair is Elizabeth Maywood from the Department of Anatomy, Cambridge University, U.K. This conference will explore topics related to pineal cell biology on a broad scale. Although the major focus of this meeting is on cellular and molecular aspects of pineal function, these topics will be augmented by sessions that investigate systems-level studies of pineal function and melatonin action in both animals and humans. This broad range of topics is indicative of this field, which is by its nature very multi-disciplinary. The program includes the following sessions: regulation of melatonin synthesis, signal transduction in the pineal and retina, non-visual photoreceptive mechanisms, regulation of gene expression in the pineal, molecular mechanisms of circadian clocks, regulation of photoperiodic responses, melatonin pharmacology, and human responses to melatonin including clinical applications. The speakers and discussion leaders for this conference have been chosen so that many areas will be well represented, including academic researchers (including those from minority institutions), government scientists, and industrial researchers. In addition, we have a large representation of women (including the chair and vice-chair) and have made an effort to invite scientists from Europe and Asia, in addition to those from the US. This meeting is held every two years and this next meeting will be the fifth time it has been held. The previous meetings have been quite successful, and attendance of this conference has steadily increased such that the last meeting was well over-subscribed.

Circadian clocks are intracellular timekeeping mechanisms that allow cells to coordinate various aspects of their physiology with time of day. These clocks are self-sustained oscillators with periods of about 24 hours and they are known to drive many rhythmic processes ranging from gene expression to complex behaviors. The central feature of this circadian clock is a core transcriptional/translational negative feedback loop in which a set of "clock genes" are transcribed rhythmically and then their protein products feed back to inhibit their own synthesis. This feedback loop takes one circadian cycle (24 hours) to complete. The many rhythmic "outputs" of the clock are thought to be controlled by coordinate regulation of many other genes by the clock genes themselves. The majority of studies on this mechanism have focused on transcriptional and post-translational modifications that are important for proper clock function. However, although there is significant evidence that post-transcriptional mechanisms must also be involved in generating stable circadian rhythmicity (both at the level of the central clock and in control of various output rhythms), almost nothing is known about this level of regulation. The best candidate for mediating circadian posttranscriptional control in vertebrates is the protein nocturnin, a deadenylase (an enzyme that removes the polyA tail from mRNAs) that is expressed in clock-containing cells with high amplitude rhythms. Our central hypothesis is that nocturnin is an important regulatory mechanism of the circadian clock through its control of the half-life (or translatability) of specific rhythmic mRNAs. In this proposal, we examine the biochemical function of nocturnin in detail in four Specific Aims: (1) Characterization of the complex of proteins in which nocturnin resides;(2) Examine nocturnin's intracellular localization;(3) Identify nocturnin's target mRNAs;and (4) Determine how these target mRNAs are recognized. Circadian clocks control many critical aspects of physiology and disruptions of clocks have recently been implicated in many human diseases. The studies proposed here will contribute to the understanding of the molecular underpinnings of these clocks as well as to the general area of post-transcriptional control of gene expression.

[unreadable] DESCRIPTION (provided by applicant): The Society for Research on Biological Rhythms (SRBR) will be held at Sandestin Resort in Destin, Florida from May 21-25, 2006. The Program Chair of this conference is Carla B. Green from the Department of Biology and the Center for Biological Timing at the University of Virginia. This conference will explore topics related to circadian biology on a broad scale ranging from biochemical/molecular and genetic analyses to whole organism behavior and physiology, including human clinical studies. This broad range of topics is indicative of this field, which is by its nature very multi-disciplinary. The program includes presentations in a variety of formats, including large symposia with invited speakers, small slide sessions and poster sessions programmed from the submitted abstracts and a number of workshops exploring specific hot areas of the field. The speakers and discussion leaders for this conference have been chosen so that many areas will be well represented, including academic researchers (including those from minority institutions), government scientists, and industrial researchers. In addition, we have a large representation of women (including the Program Chair, and members of the program committee) and have made an effort to include scientists from Europe, Latin America and Asia in addition to those from the US. Although this meeting is held every two years, the field is moving so rapidly that each meeting is a significant departure from previous meetings. A goal of the 2006 meeting is to bring in a number of excellent scientists from outside fields to increase the inter-disciplinary aspects of clock studies. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Timing plays a critical role in biology. From precisely sequenced "once-in-a-lifetime" developmental events to neural, endocrine and motor oscillations, biological systems have evolved a capacity to internally time myriad physiological processes and behaviors. Taking advantage of recent technical advances in Internet-based real-time video communications, the proposed Temporal Biology Training Program will join the investigative talent of three research institutions, the University of Virginia, Northwestern University and the Morehouse School of Medicine, in a multi-disciplinary group drawn from biology, endocrinology, biomathematics, chemistry, physics and engineering to provide novel graduate and undergraduate training in the temporal aspects of biological organization and function. Within the proposed training program, students will develop an appreciation for the role of temporal organization within biological systems. At the core of the proposed training program is the goal of increasing diversity within biomedical academic disciplines by providing an exceptional multi-institutional training opportunity focused on an increasingly important area of contemporary neuroscience. The two-stage program consisting of undergraduate summer research experience and graduate study will assist in developing the pipeline that will ultimately result in increased participation of minority populations at all levels of biomedical academic or industrial careers.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

With this award to the Biology department at the University of Virginia a bioluminescence/biofluorescence imaging system will be acquired to study molecular signals associated with daily time keeping mechanisms. The instrument will be used for experiments aimed at gaining a better understanding of the internal circadian clocks that allow animals to anticipate daily changes in their environment and to organize a multitude of bodily functions in an optimized daily schedule. The new imaging system optimally visualizes bioluminescent signals produced by clock-controlled expression of luciferase reporter genes in combination with fluorescent signals produced by additional reporter genes that act as markers for specific cell types or biological activities. With the new technology it will be possible to image cell-type specific circadian gene expression at single cell resolution. The proposed experiments will focus on the cellular and network properties of circadian clock function in fruit fly and rodent model systems. These research activities will strongly stimulate the research programs of the (Co-)PIs as well as help in the recruitment and training of researchers and students to the University of Virginia. The instrument and planned research activities will be prominently featured in several undergraduate courses. In addition, existing affiliations will be used to also incorporate training and outreach at the K-12 level. Participating researchers and students will not only be involved in projects at the cutting edge of chronobiological research and imaging technology, but also receive invaluable training in microscopy and data analysis techniques that will be an asset to them throughout their scientific or professional careers.

DESCRIPTION (provided by applicant): We have discovered a new spontaneous circadian mutation in Syrian hamsters, which confers a robust phenotype on the animals' locomotor rhythmicity. The free running period of animals homozygous for the mutation is approximately 28 hours, 4 hours per daily cycle longer than that of wild type animals. Such mutations, most produced in mice by chemical mutagenesis, have been critical in working out what we know thus far about the molecular mechanism that generates cell autonomous circadian rhythmicity in mammals, but our knowledge is far from complete and it is clear that additional unidentified genes must contribute to its function. Furthermore, because hamsters provide many advantages over mice for behavioral and physiological studies, this new mutation provides a unique opportunity for studies of the circadian clock mechanism in mammals. We propose to analyze this new mutation at 3 levels: genetic, behavioral and molecular. Genetic analysis will test our assumption, based on preliminary data, that the mutation is a single, autosomal co-dominant allele. The work in the Menaker laboratory will be focused on the behavioral phenotype and will explore the responses of wild type, heterozygous and homozygous littermates of both sexes to constant darkness, constant light, single light pulses, photoperiods with different light/dark ratis and entraining cycles with different periods, These data will address the role of the mutant gene in the circadian molecular mechanism as well as its impact on the organism's physiology. The Green lab will perform molecular and biochemical analysis of these mutants to determine whether this mutation affects the core intracellular oscillator mechanism or some system-level aspect, such as intercellular coupling. The nature of the mutation will be addressed by sequencing of the known circadian genes, and by analysis of their expression levels. This will also lay important groundwork for the eventual identification of the mutation by whole-genome sequencing. Circadian rhythms modulate much of normal physiology and behavior and circadian disruptions increase vulnerability to a variety of environmental insults. Knowledge of the underlying mechanism is essential to controlling these deleterious effects.