U.S.-Australian Cooperative Research: Pollination Ecology and Selection in
Plants that Mimic Insects
Prgm Manager Charles Wallace
INT DIVISION OF INTERNATIONAL PROGRAMS
SBE DIRECT FOR SOCIAL, BEHAV & ECONOMIC SCIE
Expected Total Amt.
$29967 (Estimated)
Investigator Steven N. Handel (Principal Investigator current)
Sponsor Rutgers Univ New Brunswick
Administrative Services Building
New Brunswick, NJ 08903 201/932-1766
Abstract
This award will support a three-year collaborative research project between Professor
Steven N. Handel, Department of Biological Sciences, Rutgers University, and Professor
Andrew J. Beattie, School of Biological Sciences, Macquarie University, Sydney,
Australia. The activity takes place under the U.S.-Australia Cooperative Science
Program. The scientists will study the ecology and evolutionary biology of so-called
sexually deceptive plants (plants with flowers that mimic female insects, thus attracting
male insects to mate and thereby effecting pollen transport). The mimicry is known to
occur only within the orchid family, and though unusual, it is observed among hundreds
of species in Australia, and to a much lesser extent elsewhere. The investigators wish to
explore the hypothesis that the genetic structure of these plant populations is significantly
different from those in which pollination is the result of an insect foraging strategy based
on food rewards (nectar or pollen). Specific species of the orchid genus Chiloglottis,
pollinated by male thynnine wasps, will be studied. Field observations of the wasp flight
movements among the flowers, as well as pollen transfer dynamics, will be carried out.
The effects of varying average inter-flower distance and flower height on pollinator
behavior will be investigated. In addition, plant samples will be taken from representative
populations in the field and their genetic profiles determined, using the technique of gel
electrophoresis.
Correlations between the observed pollination patterns and gene flow in these species
will be sought. This research in evolutionary ecology will provide an independent test of
current theoretical generalizations that insect movement patterns determine the genetic
structure of plant populations. In addition, the work has implications for the conservation
of these orchids, some of which are endangered species. The cooperating scientists in this
project bring complementary research strengths to the study. Professor Handel's expertise
is in plant ecology, especially pollination biology and gene flow dynamics. Professor
Beattie has considerable experience in population genetics and the biochemical analysis
of plant populations. The field studies will be carried out at selected Australian locations,
and the laboratory analyses performed at Macquarie University, in Sydney.
Mechanism of Protein Retention in the Golgi Apparatus
Expected Total Amt. $300000 (Estimated)
Investigator M. Gerard Waters (Principal Investigator current)
Sponsor Princeton University
Princeton, NJ 08544 609/452-3000
Abstract
The secretory pathway of eukaryotic cells consists of a set of membrane-bounded
compartments that are involved in delivery of newly synthesized proteins to their final
destinations. The Golgi apparatus is involved in the post-translational modification and
sorting of proteins that pass through it. The Golgi consists of several topologically
distinct cisternae, each contains a unique set of resident proteins. These proteins resist
dispersion by the large flux of vesicular traffic to and from the Golgi apparatus. The
maintenance of the Golgi integrity is an essential process, yet it is poorly understood. Do
newly synthesized Golgi proteins simply cease their forward movement upon reaching
the correct cisternae, or do they localized by a more dynamic mechanism involving
retrieval from distal compartments? The goal of this proposal is to gain insights into
Golgi retention mechanisms. The budding yeast Saccharomyces cerevisiae is used as a
model system since it is amenable to biochemical, cell biological, and genetic analyses.
The Golgi mannosyltransferase, Och1p, will serve as the model protein because the
inability to retain Och1p in the Golgi is readily detectable by both biochemical and
genetic techniques. Fusion proteins of Och1p fused to the reporter protein invertase have
been used in initial studies. Inclusion of a cleavage site specific for a late Golgi
endoprotease, Kex2p, in some of the fusion proteins, allows monitoring of the release of
the invertase moiety if the fusion protein reaches the distal compartments of the Golgi.
Initial results from these studies imply that newly synthesized Och1p is not retained
efficiently on its first passage through the Golgi apparatus, that is, Och1p moves to distal
compartments and is retrieved. Retention in the Golgi apparatus is not a static process,
but is dynamic, involving, apparently, both anterograde and retrograde vesicular
transport. The mechanism of this process will be further investigated by identifying
components o f the cellular machinery that retains Och1p and mediates its retrieval from
distal compartments; by determining if Och1p recycles through the ER and the
endosomes, as well as the Golgi, by using mutants that can "trap" recycling Och1p in the
ER or divert it from the endosomes to the plasma membrane; and by biochemically
characterizing the statically-retained form of Och1p. Known mutants of the retrograde
vesicular traffic will be examined to study their impact on the recycling of Och1p through
the Golgi. New components of the retrograde transport apparatus will be identified by
selecting mutants defective in recovering Och1p from distal compartments. These
mutants will be phenotypically characterized and the gene will be examined at the
molecular level. Studying protein retention in the Golgi of S. cerevisiae will be relevant
to Golgi retention processes in mammalian cells as well as to the maintenance of other
organelles. Since there exist numerous examples of evolutionary conservation of
biological processes among various species, these studies will help
reveal how human cells maintain compartmentalization that is so critical to cell viability.
%%% The secretory pathway of eukaryotic cells consists of a set of membrane-bounded
compartments that are involved in delivery of newly synthesized proteins to their final
destinations. The Golgi apparatus is involved in the post-translational modification and
sorting of proteins that pass through it. The Golgi consists of several topologically
distinct cisternae, each contains a unique set of resident proteins. These proteins resist
dispersion by the large flux of vesicular traffic to and from the Golgi apparatus. The
maintenance of the Golgi integrity is an essential process, yet it is poorly understood. Do
newly synthesized Golgi proteins simply cease their forward movement upon reaching
the correct cisternae, or do they localized by a more dynamic mechanism involving
retrieval from distal compartments? The goal of this proposal is to gain insi ghts into
Golgi retention mechanisms. Studying protein retention in the Golgi of S. cerevisiae will
be relevant to Golgi retention processes in mammalian cells as well as to the maintenance
of other organelles. Since there exist numerous examples of evolutionary conservation of
biological processes among various species, these studies will help reveal how human
cells maintain compartmentalization that is so critical to cell viability. ***
Cloning of the TuMV Genome and Analysis of Its Movement Protein
Expected Total Amt.
$12000 (Estimated)
Investigator
Jerry L. Bryant (Principal Investigator current)
Sponsor
U of Missouri Saint Louis
8001 Natural Bridge Road
Saint Louis, MO 631214401 314/553-0111
Abstract
The proposed planning activity will enable the applicant to prepare a cDNA library
of the Turnip mosaic virus and develop viral mutants and probes as well as other reagents
required for a longer term research plan directed toward establishing the gene sequence,
the protein structure and the function of the turnip mosaic virus movement protein.
Turnip mosaic virus is a member of an agriculturally important and widespread group of
plant viruses. These viruses occur worldwide and infect all vegetable and ornamental
plants of the mustard and cress family. These viruses are transmitted by many species of
aphid. When viruses are inoculated onto host plants, either by a feeding insect or by an
abrasion, the initial infection is limited to a single or few cells of the plant. In order for
the virus to successfully infect the entire plant, movement from cell to cell is necessary.
This movement involves the plasmodesmata, which are membrane-lined pores extending
through the cell walls and connecting adjacent plant cells. A virus-encoded factor termed
the "movement protein" is also required. The movement protein of another plant virus has
been shown to increase the effective size of the channels between cells, and this is
thought to facilitate movement of virus from cell to cell. The movement protein of turnip
mosaic virus probably has a similar function. The wide- ranging turnip mosaic virus and
its movement protein are excellent candidates for genetic manipulation for crop
protection, but the molecular biology of this system also has the potential for providing
new information on the biology of plant-virus interactions, the structure and function of
the plasmodesmata and the molecular biology of plant virus replication. The goal of the
proposed planning activity is to lay the experimental groundwork for determining the
structure of the gene encoding the turnip mosaic virus movement protein and ultimately
studying its function.
Biogenesis and Function of Cortical Granule Contents
Expected Total Amt.
$300000 (Estimated)
Investigator
Gary M. Wessel (Principal Investigator current)
Sponsor
Brown University
164 Angell Street
Providence, RI 02912 401/863-1000
Abstract
Cortical granules are secretory vesicles unique to eggs and oocytes. At fertilization
they secrete their contents to form both a permanent block to polyspermy and to provide
protection for early embryonic development. In this application we will address three
questions: What is in the cortical granule?, What does it do?, and How does it get in
there? We will use sea urchin eggs and oocytes to answer these questions because in this
animal we can obtain approximately 106 eggs and 1 ~ oocytes per female, and because
the approximately 15,000 cortical granules in each oocyte are synchronous in biogenesis,
in translocation to the surface, in docking to the plasma membrane, and in secretion in
response to sperm or parthenogenic activation. This system offers a unique opportunity to
examine the molecular mechanism of cortical granule biogenesis and flinction. The sea
urchin oocyte is also the only oocyte in which 1) cDNA clones have been isolated that
encode content and membrane proteins specific to the cortical granules; 2) the cortical
granules can be isolated in a fimctional form; and 3) in vitro culture and maturation of
oocytes and direct visualization of cortical granules is possible. In addition to the
synchrony of vesicle biogenic steps, cortical granules are different from most other
secretory vesicles in that they are nonrecycling, and contain over a dozen different
proteins that are specific to cortical granules and are subcompartmentalized within the
vesicle. Three specific aims are proposed:
1. Identify the contents of the cortical granules. We will focus on the cDNA cloning
of the three fertilization envelope proteins: proteohaisin, p90 and p63. These are major
envelope proteins that must quickly interact with each other and with the vitelline layer to
form an impenetrable layer within seconds of fertilization.
2. Characterize the function and regulation of the cortical granule proteins. We will
determine the mechanism of fertilization envelope construction, a specialized
extracellular matrix, by identiiying domains of cortical granule protein interactions that
are responsible for envelope construction. We will then use this information to examine
the hierarchy, and identity of interactions with the vitelline layer to understand the
molecular mechanism for the rapid condensation of the fertilization envelope.
3. Determine how the contents are packaged selectively into cortical granules. We
will make use of the newly identified cDNA clones to determine the mechanism of
cortical granule biogenesis. Selective protein targeting into cortical granules will be
studied using recombinant, tagged cortical granule proteins. The tags will include the
myc epitope and the green fluorescent protein, and we will follow the fates of wild-type
and modified cortical granule protein sequences during cortical granule biogenesis. We
will also use these tagged proteins, in conjunction with biotinylated amino acid markers,
to assay cortical granule biogenesis. Because of our recent success with in vitro
maturation of sea urchin oocytes, we will also be able to study cortical granule protein
function in vivo.
Mechanisms and Integration of Signal Pathways: A Role for Calpains?
Expected Total Amt.
$300000 (Estimated)
Investigator
Dorothy E. Croall (Principal Investigator current)
Sponsor
University of Maine
5717 Corbett Hall
Orono, ME 044695717 207/581-1110
Abstract
Cells receive environmental signals which determine their development, growth and
differentiation. How external signals transmit information to the nucleus to regulate these
processes is a fundamental question of cell biology. The long range goal of this research
is to understand the biochemical mechanisms that mediate cellular responses to these
signals. This project focuses on the two calcium-dependent proteolytic enzymes, microand milli- calpain (EC 3.4.22.17), and a protein inhibitor that specifically inhibits them,
calpastatin. These enzymes may play a direct role in calcium-linked signal pathways and
also may mediate integration (cross-talk) between signalling pathways. The proteins
susceptible to calpain represent each phase of signal transduction including receptors,
kinases, phosphatases and transcription factors. It is not known whether these signalling
components share common features that are recognized by calpain. These studies will
characterize the substrate recognition determinants for calpains by 1) identifying the
calpastatin binding site within calpain; 2) testing the hypothesis that either calmodulinbinding domains, PEST- regions, or both, mediate substrate recognition; and 3) analyzing
the amino acid sequences surrounding calpain-cleavage sites within potentially,
physiologically significant protein targets. Results of these studies will provide new
approaches for experimental manipulation of calcium-linked signal pathways. Another
objective is to discover how calcium regulates calpain activity. It is hypothesized that the
regulation of calpain by calcium is analogous to calmodulin regulation of its target
enzymes. A first step toward testing this hypothesis will be to examine the effects of
calmodulin antagonists on calpain. Further tests of the hypothesis are envisioned, but are
beyond the scope of this project. The results of these experiments should provide
important insights towards a general understanding of how calcium regulates the
functions of the proteins and enzymes that can bind calcium ion directly.
%%% Cells receive environmental signals which determine their development, growth
and differentiation. How external signals transmit information to the cell nucleus to
regulate these processes is a fundamental question of cell biology. One major internal
messenger in signaling is calcium. Intracellular calcium levels have been found to go up
in response to a variety of signals in many types of cells, but the mechanisms by which
calcium acts in various signaling pathways are not yet understood despite much research.
This project focuses on two calcium-dependent enzymes that break down specific
proteins, and an inhibitor that specifically inhibits them. These enzymes may play direct
roles in calcium-linked signal pathways and also may mediate cross-talk between
different signalling pathways.
A primary goal of this research is to characterize the mechanisms by which the
calcium- dependent enzymes recognize and act on other proteins in the signaling
pathways. The results will be an important contribution to our understanding of the role
of calcium in cellular signal transduction mechanisms.***