Department of Cell Biology & Molecular Genetics
University of Maryland
College Park, Maryland, 20742
Ph.D. Botany, University of California,
M.S. Botany, University of Illinois, Urbana, 1974
B.A. Biology, State University of New York, Oswego, 1972
- Regulatory Cascades that Control Entry into Anaphase -
The centerpiece of my research program is a project centered on the role of ions and other factors in the regulation of progression through mitosis, with a particular focus on the signalling pathway that is responsible for controlling sister chromatid separation at the onset of anaphase. The majority of our experiments have been performed on living stamen hair cells of the spiderwort plant, Tradescantia virginiana, selected because the cells exhibit an extrordinarily predictable rate of progression through mitosis. Click here to see a mature Tradescantia flower. This rate of mitotic progression has served as a bioassay for the detection of regulatory factors and events that control entry into anaphase. Our early experiments were primarily pharmacological, and they have led to the conclucion that a multifaceted phosphorylation/dephosphorylation cascade controls entry into anaphase. The precise timing of these events during prometaphase and metaphase has been tested in a series of microinjection experiments, where small quantites of protein kinase or protein phosphatase substrates were microinjected into cells at known times after nuclear envelope breakdown. The presence of these substrates at specfic times during metaphase has enabled us to define inflection points in the cascade, where kinase and phosphatase activities are changing in rapid succession. We are currently focusing on the identification of particular protein kinases or phosphatases and the substrates they act upon. We have generated a cDNA library from stamen hair cells, and have isolated characterized several cloned cDNAs, one of which resembles CDPK, a likely component in the mitotic regulatory cascade. From these efforts, we are designing peptides that are identical to phosphorylation domains of genuine stamen hair cell proteins for microinjection experiments, with the aim of using these peptides as specific competitors or inhibitors for the enzymatic activities that underlie mitotic progression.
We have tested the hypothesis that a transient rise in the concentration of free cytosolic calcium is necessary for sister chromatid separation at the onset of anaphase. Our work has shown that an influx of the cation across the plasma membrane from the wall space into the cytosolic compartment appears to be a necessary prerequisite for anaphase onset. However, the influx of calcium appears not to be sufficient for entry into anaphase and the late-metapahse influx of calcium may only be a portion of the total calcium mobilized for anaphase chromosome movement. We determined that calcium fluxes themselves may be under the control of polyphosphoinositide cycling that occurs at specific intervals during metaphase and that changes in calcium activity may be linked to changes in the activities of specific protein kinases and protein phosphatases at different points during metaphase. Treatments of cells at specific times during metaphase with protein kinase activators or with protein phosphatase inhibitors indicate that the cells undergo a pronounced shift from protein phosphorylation to dephosphorylation during mid-metaphase. Later in metaphase, there is an apparent reactivation of protein kinase(s) that is necessary for entry into anaphase. We have shown that the mid-metaphase interval of protein dephosphorylation is an essential facet of the regulatory cascade responsible for the synchronous separation of sister chromatids. We found that asynchronous entry into anaphase can be induced by the brief treatment of cells in metaphase with protein phosphatase inhibitors, but is not caused either by a disruption of spindle microtubule organization, or by the dissociation of kinetochores from kinetochore fiber microtubules. In recent experiments, we have determined the timing of protein kinase activation/inactivation in this regulatory cascade. I have found that the microinjection of a small quantity of a phosphorylatable substrate peptide (that apparently acts as a competitive inhibitor to one or more protein kinases in the cell) alters the rate of progression into anaphase, but only when it is introduced into the cytosolic compartment at four specific points during prometaphase and metaphase. These intervals appear to be inflection points, when kinase activation or inactivation is occurring. Conversely, the microinjection of a phospho-peptide appears to act as an inhibitor of protein phosphatase activity in stamen hair cells. These microinjections reveal there are at least two peaks of phosphatase activity that occur during prometaphase and metaphase.
- Control of Cell Cycle Activity in the Maize Root Tip Meristem -
This project is focused on mitotic regulation in plant cells. The first part of this project is directed toward assessing the changes in the relative expression and abundance of several cell cycle control proteins in the well-ordered root meristem of maize. Specifically, Wen-ling Hsieh focused on a group of cell cycle control protein known collectively as the cyclins. In a variety of organisms, the A- and B-type cyclins bind to cyclin dependent kinase (cdk) and activate its enzymatic activity for entry into and progression through early parts of mitosis. cdk is known to expressed in higher plants, and, in concert with its regulatory cyclins, is a likely regulator of cell cycle progression in all eukaryotes. The significance of this approach is that it will provide new information about regulatory mechanisms that control cell division in the complex, but nevertheless, well-defined root tip of corn, and thereby contribute to our overall understanding of cell cycle regulation in a meristem where adjacent cells exhibit strikingly different rate of cell cycle activity. By using heterologous cloning, she isolated an A-type cyclin cDNA from the root tips of maize, and in addition to the standard sequencing characterizations, she successfully performed functional complementation with this cDNA in a conditionally-deficient mutant of yeast (S. cerevisiae). Alpha-factor resistance assays indicate that this A-type cyclin appears to function during the G2/M transition in transformed yeast cells.
Cell cycle activity in the maize root tip can be altered in predictable ways via surgical manipulation of the meristem. Wen-ling took advantage of this feature in maize root tips, with the aim of detecting changes in the patterns of expression and abundance of the cyclins in cells entering and exiting the proliferation pathway. She found by RNase protection assays that the pattern of our A-type cyclin changes with patterns of proliferation in the surgically-altered root. We predict that changes in cyclin abundance should be manifested by corresponding shifts in the activity of the enzymatic activity of cdk. Shane Hardin joined my lab as Wen Ling was finishing her dissertation, and picked up on the maize project. He has been testing whether root cap removal is followed by the activation of the MAP kinase pathway. He isolated and cloned a member of this pathway, a cDNA that encodes an enzyme known as MEK. He expressed the protein in E. coli, and has performed in vitro assays for its enzymatic activity. We intend to determine whether changes in cyclin activity are the consequence of activites of a signalling cascade involving MEK and MAP kinase that is activated after root cap removal. Results obtained from this work should provide new information about regulatory mechanisms that control cell division in higher plant cells and thereby contribute to our overall understanding of mitotic control in eukaryotes.
- Formation of the Cytoskeleton and Motile Apparatus in Spermatids of Marsilea -
In the process of spermatogenesis, lower plants provide us with cells that exhibit a feat of development that is remarkable and unique in the eukaryotic realm. In a fairly short period of time, the spermatids develop from something resembling standard plant parenchyma cells into free-swimming gametes that lack walls and possess as few as two to as many as thirty thousand cilia. Click kere to see light micrographs of a mature Marsilea spermatozoid. The cilia are situated at the anterior end of the cell body, and are attached by their basal bodies to an elaborate cytoskeleton, consisting of heavily cross-linked microtubules. The developmental process leading to spermatozoid formation is unique among eukaryotes because the generation of ciliary axonemes occurs in spermatids that previously lacked basal bodies, or centrioles. De novo synthesis and assembly of basal bodies in these spermatids is initiated approximately 5 h after the time of imbibition, and occurs in a structure known as the blepharoplast. In animals, ciliogenesis occurs in a variety of cells, but always, these cells had centrioles prior to axonemal formation. Our initial approach to this problem has been to characterize the temporal patterns of gene expression during the 11.5 h period required for spermatozoid formation and maturation in Marsilea vestita. Several years ago, Peter Hart joined my lab and began to perform a series of electrophoretic and western blot analyses on cells fractionated at different points in the developmental process, both under controlled conditions, and after treatments at known times with a -amanitin or with cycloheximide. (A key question is whether the the rapid process of spermatogenesis involves significant levels of transcription and translation, and if so, when.) We have found that the incubation of spores at the time of imbibition with transcriptional and translational inhibitors blocks spermatozoid maturation, and that the inclusion of 35S-mentionine in the imbibition medium results in the labelling of a relatively low numbers of polypeptide bands on electrophoretic gels, a result indicating that few proteins must be made for the process to reach completion.
Pete performed a series of immunoblotting assays and found that the dry spores contain large quantities of alpha-, beta-, and gamma- tubulin. Tubulin was an obvious protein to study, because it is the dominant protein in the cytoskeleton and motile apparatus. He found that none of the tubulin isoform levels appears to change significantly during the 11 h period of development. In contrast, we have found that centrin, a protein found in association with centrioles, basal bodies, and microtubule organizing centers, increases significantly in abundance approximately 5 h after imbibition. Northern blot analyses demonstrate that centrin mRNA is present in spores from the time of imbibition; we believe that stored centrin mRNA underlies its rapid translation in the spermatids, 5 h after imbibition. A series of in vitro translation experiments indicate that considerable quantities of centrin mRNA are available for translation soon after the time of imbibition. The in vitro translation of isolated mRNA from cells in early phases of development reveals that centrin is one of the major products that is made. From these results, we hypothesize that centrin translation is limiting for the assembly of basal bodies and the formation of the microtubule-based cytoskeleton of the spirally-shaped gamete. We believe that in this developmental program, the cells possess the bulk of the structural proteins necessary to build a motile apparatus, but must synthesize a select population of regulatory proteins in order for the self-assembly process to occur. In a real way, the cells possess the blocks to build an arch, but must make a few keystone proteins in order for the arch to form.
Pete has generated a cDNA library from male gametophytes of Marsilea vestita at all stages of spermatogenesis. He has cloned a centrin cDNA from the library, determined that it appears to be a single gene in the fern gametophyte, and that the encoded protein possesses all of the important motifs common to centrin, including several EF-hands and a PKA phosphorylation site. Our future aims center on determining the identities of other proteins that serve as regulators for rapid spermiogenesis in this organism.
Hart, P.E. and S.M. Wolniak. 1999. Molecular
cloning of a centrin homolog from Marsilea vestita and
evidence for its translational control during spermiogenesis.
Biochem. Cell Biol. (in press).
Hsieh, W.-L. and S.M. Wolniak. 1998. Isolation and characterization of a functional A-type cyclin from maize. Pl. Molec. Biol.37: 121-129.
Hardin, S.and S.M. Wolniak. 1998. Low-voltage separation of phosphoamino acids by silica gel thin-layer electrophoresis in a DNA electrophoresis cell. Biotechniques 24: 344-346.
Hardin , S. and S.M. Wolniak. 1998. Molecular cloning and characterization of maize ZmMEK1, a protein kinase with a catalytic domain homologous to mitogen- and stress- activated protein kinase kinases. Planta 206: 577-584.
Hart, P.E. and S.M. Wolniak. 1998. Spermiogenesis in Marsilea vestita: a temporal correlation between centrin expression and blepharoplast differentiation. Cell Motil. Cytoskelet. 41: 39-48.
Wolniak, S.M. and P.M. Larsen. 1995. The timing of protein kinase activation events in the cascade that regulates mitotic progression in Tradescantia stamen hair cells. Plant Cell 7: 431-445.
Larsen, P.M. and S.M. Wolniak. 1993. Asynchronous entry into anaphase induced by okadaic acid: the organization of the mitotic spindle and microtubule/kinetochore attachments. Protoplasma 177: 53-65.
Wolniak, S.M. and P.M. Larsen. 1992. Changes in the metaphase transit times and the pattern of sister chromatid separation in stamen hair cells of Tradescantia after treatment with protein phosphatase inhibitors. J. Cell Sci. 102: 691-715.
Larsen, P.M., T.-L.L. Chen and S.M. Wolniak. 1991. Neomycin reversibly disrupts mitotic progression in stamen hair cells of Tradescantia. J. Cell Sci. 98: 159-168.
Larsen, P.M. and S.M. Wolniak. 1990. 1,2-dioctanoylglycerol accelerates or retards mitotic progression in Tradescantia stamen hair cells as a function of the time of its addition. Cell Motility Cytoskelet. 16: 190-203.
Please see my other pages for updated publications.
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