Intellectual Merit: Actin microfilaments are critical cytoskeletal structures in all eukaryotes. The rate-limiting step in the formation of actin microfilaments is the polymerization of the first two to three monomeric actin protein molecules, known as actin nucleation. Formins are one of two known families of actin-nucleating proteins. Plant formins share similarity with their counterparts from other organisms in their actin-nucleating activity domain, but a large number of them (referred to as Group I plant formins) have the plant-unique feature of having a potentially glycosylated extracellular domain and a transmembrane domain that anchor the cytosolic actin-nucleating domain to the cell surface. This project aims to determine the functional roles of 3 group I formins from Arabidopsis, the pollen-specific AFH3, the pollen-, endosperm- and vegetative cell-expressed AFH5, and the root meristem-expressed AFH6, in plant growth and development. As group I formins, these AFHs serve as perfect candidates for mediating extracellular stimuli directly to the actin cytoskeleton, in a manner analogous to the function of integrins of animal cells.
Drs. Cheung, Wu and Kieliszewski plan to elucidate the functional roles for these Group I AFHs in plant growth and development through a combination of genetic and cell biological analyses. The glycosylation properties of selected group I extracellular domains and the functional significance of the extracellular domains for these three AFHs (and that of AFH1, a prototypical and thus far the best-characterized plant formin) will be examined. Dr. Cheung and her colleagues will also begin efforts to identify proteins that interact with AFH3 and AFH5, which should lead to elucidation of the protein networks that underlie the regulation and functions of these plant formins.
Broader Impacts: For resource development, Dr. Cheung and her colleagues will provide constructs, seeds and antibodies to the community. The PIs routinely host and train postdoctoral, graduate and undergraduate students in their laboratories, and plan to continue to do so in the context of this project. Since 1997, Dr. Cheung has mentored approximately 40 undergraduates, including several from unrepresented ethnic groups, and there are currently undergraduates participating in on-going efforts related to the project. Dr. Kieliszewski also consistently supervises undergraduates in her laboratory. Many of these undergraduates have gone on to graduate school in the sciences. Besides the PIs themselves, postdocs and senior graduate students in the Cheung and Kieliszewski laboratories all contribute to the direct training of undergraduates, therefore providing them with mentoring and teaching experience in preparation for their own future career development. Overall, this research project will provide a broad spectrum of training areas on different levels of sophistication suitable for students at the undergraduate, graduate and postdoctoral level.
The goal of this project is to understand how a pollen tube, a specialized plant cell whose function is to transport sperm cells to fertilize the female and is therefore essential for seed production. In addition to its importance as the conduit for fertilization in plants, pollen tube is also an excellent cell system to explore the mechanisms underlying cell growth in general and in particular "polarized" cell growth, which establishes the head to tail body axis in animals and the shoot-root axis in plants. In the case of the pollen tube growth, polarized growth, also referred to as tip-growth, underlies the production of a long tubular structure thousands times its width and transport the sperm cells over long distances to the egg cells. The long term goal of this project is to understand how a pollen tube achieves this polarity. The hallmark cellular feature of a pollen tube is an elaborate intracellular organization (image 1) that is also remarkably dynamic and is crucial for maintaining growth exclusively at the apical region to give rise to its long tubular morphology. The actin cytoskeleton, the basic cellular structure that supports cellular and intracellular motility, is crucial for pollen tube growth. Actin exists in several configuration, as monomers, long filamentous structures that arise from head-to-tail polymerization of the monomers, and more complex higher order structures such as bundles, meshes or patches, each with its specialized functions in different kinds of cells. Elongating pollen tubes have a prominent actin mesh at the subapical region forming a basket/cone shaped structure, this is trailed by long actin cables that run back and forth along the length of the tube. There is few organized actin structures between the subapical actin mesh and the extreme apex of the tube. This apcial region is occupied almost exclusively by vesicles and they deliver, as well as retrieve, membrane and secreted materials to support the apical growth process. This project addresses the question of how the formation of actin polymers is related to the overall actin cytoskeleton and thus the tube growth process. We specifically examine the function of a family of proteins, call formins, which nucleate actin filament polymerization, i.e. stimulate the production of filamentous actin from monomers. When we started this project, the family of formins is plants is only known through bioinfomatics analysis based on homology with yeast and animal formins whose function as actin nucleators was just revealed. Besides their fundamental importance to cell growth all cellular processes that are actin dependent, some plant formins (which are referred to as group I) have a unique feature that compels attention. They are unique as transmembraneous proteins, each with an extracellular domain. This configuration renders them ideal as receptors for extracellular cues and signal transducers to the cytoplasm to regulate actin polymerization and therefore many cellular processes. Our first goal is to establish that group 1 plant formins indeed function as predicted by their primary structure and we focus our study on three of them, FH1, FH3 and FH5 from the model plant Arabidopsis. We chose these three because they are the most prominently represented in pollen, among 21 family members. Our work clearly established that these group 1 formins indeed are transmembraneous and stimulate actin polymerization from the cell membrane. In particular, FH5 is located around the apical dome and is important for the assembly and maintenance of the subapical actin structure (image 2). FH3 is located around the entire pollen tube cell surface, and regulates the production of long actin cables along the length of the tube. FH1 is similarly located as FH3, and they stimulate actin bundles emanating from the cell membrane. In contributing to the production and maintenance of the actin cytoskeleton in pollen tubes, these formins provide the basis for how intracellular trafficking and apical growth are orchestrated. Our results show that these formin-produced actin support cell expansion most prominently at the apical flank and subapical region, pushing the tip forward. The subapical actin structure forms a partition between the apcial and distal cytoplasm and its radial organization relative to the apex controls the tube growth direction. When the subapical actin structure is radially symmetrical, tube growth is straight; when it is radially assymmetrical, a pollen tube changes its direction. Our results suggest that the activity of formins around the apical/subapical circumference of the tube determines the orientation of the subapical actin structure and thus pollen tube growth orientation. With these results, we provide important new insight of how pollen tubes grow and control growth direction, the latter is controlled by the female target cells the tubes aim to enter. As we conclude this project, our existing results are leading us to understand how female cues act to attract pollen tubes.
