1985 — 1990 |
Wolniak, Stephen |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Ionic Regulation in the Mitotic Apparatus @ University of Maryland College Park |
0.988 |
1986 — 1987 |
Wolniak, Stephen Millay, Michael |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Establishment of a Scanning Electron Microscope Facility in the Botany Department At the University of Maryland @ University of Maryland College Park |
0.915 |
1994 — 1999 |
Wolniak, Stephen |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
A Regulatory Cascade For Entry Into Anaphase @ University of Maryland College Park
9403689 Wolniak The goal of this project is to investigate the involvement of three protein kinases in a signaling cascade that triggers sister chromatid separation at the onset of anaphase during mitosis. The proposed experiments will be performed on stamen hair cells of the spider plant ,Tradescantia virginiana because they exhibit a rate of progression from nuclear envelope breakdown to anaphase onset that is remarkably predictable and useful as a temporal assay for mitotic progression. The proposed experiments are based on a body of published work from the PI's laboratory that shows the regulatory cascade to consist of two or three successive waves of protein kinase activity, punctuated by an interval in mid-metaphase when protein phosphatase activity is elevated. Protein phosphatase activation is a necessary requirement for synchronous entry into anaphase. In this proposal a series of microinjections in living cells with a series of peptides that serve as activators, inhibitors and substrates of specific protein kinases will provide an assessment of the timing of involvement of p34cdc2 protein kinase, calcium calmodulin dependent protein kinase II, and calcium dependent but calmodulin-independent protein kinase (cdpk) in the regulation of progression through metaphase. All three of these protein kinases are present in plant cells, and the experiments include the utilization of the best available oligopeptide probes for the detection, inhibition or activation. The microinjection protocol developed by the PI permits the introduction of a known quantity of charged or uncharged molecule (MW < 6,000, though probably much larger) into the cytosolic compartment of a living stamen hair cell at a known time point during prometaphase or metaphase. The injection protocol, by itself, has no significant event on the metaphase transit time of these cells. These experiments will contribute to our understanding of the regulatory mechanisms that mediate progr ession through metaphase and trigger entry into anaphase. %%% Mitosis is the process that facilitates the equal partitioning of the replicated chromosomes to the incipient daughter cells that will separate from each other as one cell becomes two. In this proposal experiments are presented to test for the involvement of a set of protein phosphorylating enzymes in the regulation of progression through a specific cell division stage that is called mitosis, with a particular focus on sister chromatid separation, the morphological marker for entry into the anaphase stage of mitosis. The model system utilized in these experiments is the stamen hair cell from the spiderworth plant, Tradescantia virginiana. These plant cells progress through mitosis at a highly predictable rate, and they have been proven to be uniquely suited for studies of the timing of regulatory events involved in mitotic progression. In the current experiments living cells will be injected with known quantities of synthesized peptides that will affect the activities of specific protein kinases in known ways. Some of these peptides are kinase activators, while others are inhibitors or substrates for these enzymes. Because the cell can be injected at known time points during metaphase, their effect on the protein kinases at different times can provide clues about the environment of these enzymes in the regulatory cascade that controls the progression through metaphase and entry into anaphase. Results from this project will contribute to our overall understanding of the regulatory mechanisms thatcontrol mitotic progression in intact cells. ***
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0.915 |
1995 — 1997 |
Wolniak, Stephen Sze, Heven [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
A Phosphorimager For Research in Plant Biology @ University of Maryland College Park
A multi-user phosphorimager is requested by a group of investigators in the Department of Plant Biology and the Center for Agricultural Biotechnology. This instrument is often referred to as a fast, accurate, filmless autoradiography system. It facilitates the rapid and sensitive quantitation of radioactivity on gels or blots without scintillation counting or autoradiography. Highly sensitive storage phosphor screens capture the image of the experimental sample ten times faster than film. The broad linear dynamic range of the phosphoimager permits quantitative detection of both strong and weak signals in a way that film simply cannot. Digital images are compiled and analyzed with a microcomputer. We propose to use this powerful instrumentation in a wide range of applications, including the quantitative analyses of DNA-protein interactions by gel retardation, measurement of transcript levels by RNA gel blots or RNase protection assays, and kinetic measurements of enzyme activity and protein phosphorylation. The acquisition of a phosphorimager will permit us to obtain quantitative information and answers to research questions that would otherwise be impossible to obtain using conventional X-ray film and autoradiography. Six of the seven investigators are currently supported by competitive grants from federal agencies. In all cases, the proposed experiments requiring analyses by the phosphorimager are essential to the long-range goals of understanding the molecular basis for plant pathogenesis, signal transduction, membrane transport and mitosis.
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0.915 |
1999 — 2001 |
Wolniak, Stephen |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Rate-Limiting Steps in the Assembly of a Motile Apparatus During Spermiogenesis in Marsilea Vestita @ University of Maryland College Park
Microtubules (MTs) are long, slender polymers made up of the protein, tubulin, that play critical roles in the life of the cell. They are the key functional components of two extremely important subcellular structures that are involved in cellular motility phenomena: mitotic spindles, which are responsible for the orderly movement of chromosomes to the opposite ends of a cell just before the cell divides into two daughter cells; and cell surface appendages known as cilia or flagella, which beat in an orderly wave-like fashion and thereby move the watery environment of the cell across the cell surface, resulting in either whole cell movement (swimming) or water turbulence around the surface of the cell. These MT-containing structures are complex, and are generally associated with complex MT-containing subcellular structures, termed either centrioles, in the case of the spindles, or basal bodies, in the case of cilia or flagella.
This project is focused on increasing our understanding of basal body formation. Basal bodies are cylindrical organelles that reside at the base of a cilium or flagellum, and are comprised of 9 triplet microtubules held together by a series of proteinaceous spokes, links and plates. They are structurally similar to centrioles and they usually form in close proximity to centrioles in an elaborate assembly process. The details of this basal body assembly and the components present in these organelles have been under investigation at various times through the last century, but the process is still not well understood. In this project, the unique biological properties of an unconventional model organism, the water fern Marsilea vestita, will be exploited to expand our understanding of basal body formation. Specifically, Dr. Wolniak will study the role played by two proteins, centrin and Xgrip109, in forming the basal body precursor structure, the blepharoplast, in spermatocytes of M. vestita. In lower plant spermatocytes, basal bodies arise in the absence of any preexisting centrioles, so the synthesis, assembly and maturation processes occur without the background presence of related centriolar structures. The synchronous activation of male gametophytes of M. vestita permits time course studies on blepharoplast formation in large populations of developing spermatocytes or spermatids. In preliminary work, Dr. Wolniak has found that the rate and extent of spermiogenesis appears to be regulated post-transcriptionally, that is, it is dependent on the synthesis of certain new proteins that are translated from mRNAs that were stored in the dry microspore. Centrin is one of the proteins translated from stored mRNA midway through the developmental process, and its abundance increases just as the blepharoplast forms. Immunolocalizations with anticentrin antibody show an intense localization in fixed cells at a discrete spot at the time that the blepharoplasts assemble. Xgrip109 abundance on immunoblots increases in a fashion similar to those observed for centrin. In contrast to centrin and Xgrip109, the tubulins are abundant as stored protein in the dry spore and there is no change in tubulin abundance until late in development. From these preliminary observations, it is hypothesized that some of the newly translated proteins appear to control the rate, organization and extent of assembly of the blepharoplast. Newly assembled structures containing regulatory proteins like centrin and Xgrip109 then serve as cores or templates for the addition of the tubulins and other stored proteins that are already present in the gametophyte. Thus, the newly-made proteins appear to be rate-limiting for spermiogenesis.
A series of antisense experiments will be performed that are aimed at determining whether centrin translation is necessary for blepharoplast formation. Preliminary experiments show the efficacy of an antisense approach, but reveal that dsRNA and certain sense centrin constructs also slow or block development. An important part of the work during the period of funding will be directed toward understanding the differences in development that result from each of these treatments. In addition, Dr. Wolniak will study the linkage between cell divisions in the gametophyte, the activation of translation of stored mRNAs and the formation of the blepharoplast. Three ways to block mitosis in this organism have been found, and these will be used to study centrin and Xgrip109 protein accumulation in the one-celled gametophyte over time after imbibition. Extended time course experiments will be performed to determine whether the accumulated centrin and Xgrip109 have the capacity to aggregate into blepharoplast-like particles in the absence of cytoplasmic partitioning between spermatogenous and sterile cells of the gametophyte. This work is expected to establish the feasibility of using Marsilea vestita as a model for basal body development.
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0.915 |
2000 — 2002 |
Song, Wenxia (co-PI) [⬀] Mount, Stephen (co-PI) [⬀] Wolniak, Stephen Delwiche, Charles Baehrecke, Eric (co-PI) [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
A Fluorescence Deconvolution Microscope At the University of Maryland @ University of Maryland College Park
A Fluorescence Image Deconvolution-Reconstruction Microscope will be used for a variety of different applications with living cells. Use will be restricted to observations of living cells that are present singly, or in thin specimens. This microscope generates a stack of fluorescent images at different focal planes, and then employs a set of sophisticated algorithms to determine point-spread functions for sources of fluorescence in the specimen. Out-of focus noise is subtracted from the signals in the image slices, and the slices are restacked to generate a three-dimensional reconstruction of the object at high resolution. Since small, bright objects placed against a dark background are detected as spots, it is possible to image fluorescence sources that are smaller than the theoretical limit of resolution for the microscope.
During the last twenty years, developments in the design of novel indicator fluorescent probes have enabled biologists to attack formerly intractable problems in cell biology and cell physiology. The cellular processes that have been amenable to this kind of analysis include photoreception, neuronal transmission, animal development, hormonal signaling, triggered gene expression, mitotic regulation, and chemotaxis. The developments in fluorescent dye design have moved in parallel with improvements in our ability to visualize and measure low light intensity signals from small numbers of molecules in living cells, with newly-designed optical microscopes and large pixel array CCD camera detectors. It is reasonable to expect that a significant expansion of analysis of processes in living cells will result from combined developments of reporter molecules and digital imaging technologies. It seems clear that the convergence of developments in photochemistry, biochemistry, cell biology, physiology and microscopy are all about to intersect within the living cell, and when accurate assessments of changing abundance and activity of a variety of molecules can be made in vivo and through time.
This microscope will be placed in an imaging facility that provides faculty, postdocs, graduate and undergraduate students with access to several high performance microscopes equipped with the capacity to view small quantities of fluorescent reporter molecules in living cells.
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0.915 |
2001 — 2015 |
Wolniak, Stephen |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Translational Patterns During Spermiogenesis in Marsilea @ University of Maryland College Park
Marsilea vestita is a water fern that makes motile spermatozoids. Each of these gametes possesses ~140 cilia and a complex cytoskeleton. The gametes are formed in a rapid process that is initiated by placing dry microspores (meiotic products) into water, in a process known as imbibition. Spermiogenesis is synchronous in populations of microspores, and a distinctive feature of gamete formation in this organism is the de novo formation of basal bodies in cells that lack preexisting centrioles. In each spermatocyte, a discrete particle known as a blepharoplast forms approximately 4 h after imbibition, and during the next two hours the blepharoplast splits and functions as a centrosome for the last mitotic division in the gametophyte, and then it matures. Each blepharoplast produces approximately 140 basal bodies. Dry microspores contain large quantities of stored protein and stored mRNA. The translation of specific mRNAs at particular stages of spermiogenesis is necessary for gamete maturation, including formation of the blepharoplast and basal bodies. The translation of centrin is essential for blepharoplast formation in these gametophytes, and centrin and b-tubulin can be detected immunocytochemically in blepharoplasts (where they serve as marker proteins for the organelle), which form 4 h after imbibition. This project will focus on aspects of spermiogenesis in M. vestita that extend ongoing work and utilize probes and procedures this lab has developed in the past several years. Studies on essential proteins that participate in the de novo formation of basal bodies will be expanded. Reverse-genetics with RNAi will be used to perform a series of translational knockouts to see how Xgrip109, RanBPM and other members of the g-tubulin ring complex participate in the basal body formation. Then, ongoing studies with mago nashi (a polarity gene in embryogenesis in a variety of organisms) will be extended to examine its role in the early segregation of cytoplasm in the gametophyte into spermatogenous and sterile domains as the single-cell gametophyte develops and forms its two distinct types of progeny - sterile somatic jacket cells and spermatogenous cells. The movements of stored mRNA and proteins will be monitored, and actomyosin as a driving force for some of these events will be examined. This project will potentially provide new insights into the roles played by various proteins as cells assemble new basal bodies, the site in all eukaryotic cells where the growth of cilia and flagella initiates. Also, new perspectives will be provided into how cells differentiate their cytoplasm into functionally distinct regions, a process of central importance in the growth and development of all cells.
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0.915 |
2007 — 2009 |
Thompson, Matthew Wolniak, Stephen |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Defining Whole-Plant Phloem Function With Molecular Studies of Anatomy @ University of Maryland College Park
To link the long-distance fluid mechanical behavior of sugar transport in the phloem vasculature of plants with the short-distance regulation of sugar exchange between the phloem and surrounding tissue requires much deeper knowledge of the phloem's quantitative anatomy than is currently available. Accurate measurements of phloem anatomy are now possible with live-cell imaging of transgenic Arabidopsis plants that express mCitrine (monomeric yellow fluorescent protein, YFP)-labeled plasma membrane proteins in phloem sieve elements. Some of these transgenics (developed by the PI) provide excellent illumination of sieve elements for confocal laser scanning microscopy (even through live tissue). Sieve plates and pores show thorough YFP labeling, holding out the promise for immunohistochemical fluorescence imaging of fixed and sectioned tissues. The PI is currently exporting successful constructs to other species, including Nicotiana benthamiana, N. tabacum, and Populus, for cross-species comparisons of phloem anatomy. The results will help the PI to predict the gross-scale physicochemical behavior of the phloem across a set of species and plant organs, which will provide the context for other experiments, such as "black-box" manipulations of whole-plant source and sink strength using local and chemically inducible silencing of phloem-specific transporters. Analysis of the constructs will also aid in understanding how proteins expressed in nucleate companion cells are trafficked to enucleate sieve elements.
The broader impacts of this study include a "re-invention" of American phloem biology to cut across disciplines, drawing from applied mathematics, systems biology, and molecular genetics, as well as a truly interdisciplinary opportunity for undergraduate training. The work could be very important for our understanding of the movement of phloem-borne plant diseases, plant resource allocation, and the role of trees in the global carbon cycle.
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0.915 |
2008 — 2011 |
Wolniak, Stephen Mather, Ian (co-PI) [⬀] Delwiche, Charles Pick, Leslie (co-PI) [⬀] Kwak, June |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Mri: Acquisition of a Leica Confocal Microscope to Develop a Visual Imaging Center @ University of Maryland College Park
In this project, a group of five principal investigators (Drs. S. Wolniak, I. Mather, J. Kwak, L. Pick and C. Delwiche) describe a plan to use a new-generation laser scan confocal microscope for a variety of experiments on living cells and tissues. Experiments focus on the trafficking of proteins and other molecules in cells, on the development of a ciliary apparatus in developing sperm cells, on the regulation of uptake of carbon dioxide by plant leaves, on mechanisms leading to the specific distribution patterns of growth factors in developing fly embryos, and on algal parasite development. The microscope permits live cell imaging using both modern confocal scanning and multi-photon excitation for penetration into thick specimens. The specific instrument to be purchased with these funds is a Leica SP-5, which will be equipped with four lasers and dual detectors for prism-based spectroscopic detection (to distinguish among overlapping signals) and resonance detection (for rapid imaging of sensitive cells). These features will greatly enhance the imaging capabilities of faculty and students who use the instrument. The microscope will be installed in a Shared Core Microscopy Imaging laboratory, where several other heavily used instruments are already housed. The core lab is overseen by Ms. Amy Beaven, a research support technician, who is most capable of providing training and overseeing the use of the microscope. Beyond the five major users of this microscope, there is an additional group of seven individuals who list themselves as occasional users of this instrument. Their research interests center on the development of the visual system in mammals, the dynamics of antigen presentation in immune cells, the structure and function of phloem in plants, the formation, development and maintenance of neurons in flies, and on programmed cell death in flies and mammals.
This core laboratory provides access to state-or-the-art microscopes to over eighty faculty and students who are currently working at the University of Maryland and to approximately twenty collaborators and colleagues at nearby institutions. These microscopes are far too expensive to purchase and maintain in individual research laboratories, but they are essential for critical imaging of cells and tissues in ongoing research projects. A critical mission of the institution is training students in modern techniques with state-of the-art instrumentation. Thus, both research and training are conducted at the Visual Imaging Center. A formal training course in light microscopy is taught by S. Wolniak and A. Beaven each fall semester to approximately a dozen graduate students and undergraduates. These students receive hands-on training with the instrumentation currently housed in the laboratory, and they generate images of their samples with the microscopes. Students will be trained on the SP-5 through this course. In addition, practical training on the microscopes is conducted on an as-needed basis. Over 120 individuals have been trained in the Visual Imaging Center during the last six years through formal and informal mechanisms.
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0.915 |