Research interests

1. Pollen Tube Growth:

Nicotiana pollen tube growing in a polar fashion

Calcium gradient at the tip of pollen tube

a. Ionic control of growth:

Pollen tubes of flowering plants grow quickly, and exclusively at their apex. Of special note the rate of growth oscillates; in lily pollen tubes the rate changes from 100 to 500 nm/sec with a frequency of 15-50 sec. Many underlying physiological processes also oscillate with the same frequency, but with varying phase relationships to growth rate. For example, the intracellular Ca²⁺ gradient oscillates in phase or slightly behind the growth peak, while the extracellular Ca²⁺ influx exhibits a 10-15 sec delay. H⁺ also oscillate. Current studies are aimed at experimentally modifying these phase relationships in an effort to identify the central control elements.

b. Cytoskeletal control of growth:

Recent studies indicate that actin polymerization is necessary for pollen tube growth. Using different actin binding proteins, e.g., profilin, cofilin/ADF, and villin, we are attempting to define the locus of actin filament assembly, and the underlying controlling processes. Here we also give attention to the response of these proteins to the Ca²⁺ and H⁺ ion gradients. We are also examining myosin, which together with actin, causes cytoplasmic streaming.

Cytokinesis in a Tradescantia stamen hair cell

2. Cell Division:

The formation of the new cross wall during cytokinesis is brought about by the phragmoplast, a complex structure consisting of microtubules (MTs), actin microfilaments (MFs), and membrane elements. Previous work has identified two phases in phragmolast dynamics as follows: 1. initiation and early expansion, followed by 2. late lateral expansion. Phase 2 involves attachment of the expanding cell plate to the parent wall together with reorientation to the correct plane. We have examined the role of MT and MF motor proteins on cell division. For example studies with myosin inhibitors indicate that phase 2 is highly dependent upon the acto-myosin system. We are attempting therefore to define the organization and activity of this system more completely, with particular attention to the possible role of small "G" proteins.

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3. Methods/Techniques:

Emphasis is placed on the study of living cells using various types of light microscopy. For normal visualization we use Nomarski differential interference contrast optics. To examine particular structures such as actin MFs or MTs we use either wide field fluorescence microscopy or confocal microscopy of cells that have been microinjected with fluorescent analogs (e.g., fluorescent profilin for profilin, or tubulin for MTs), or specific dyes (e.g., fluorescent phalloidin, for MFs). More recently we have expressed GFP fusion proteins to vitally label specific structures (e.g., GFP-talin for MFs). To study intracellular ion activity we microinject fura-2 dextran for Ca²⁺ and BCECF-dextran for H⁺, and perform ratiometric ion imaging with a computer controlled (MetaMorph/Meta/Fluor) wide-field fluorescence microscope. Extracellular ion fluxes are examined using a self referencing vibrating electrode system.

While emphasis is placed on analyzing the living cell, we also perform electron microscopy on fixed cells to achieve high resolution of structural components. Here freeze fix cells in order to preserve structures in their life-like state. Immunogold studies, for localizing molecules, also employ freeze fixation methods.

Biochemical and molecular biological methods are being increasingly used. For example, we have recently identified an actin binding protein, ABP-135, as villin. Using a specific antibody to probe an expression cDNA library, we isolated a full-length clone, which on sequence analysis indicated that the protein was plant villin. We are also using the actin binding domain of mouse talin, which is expressed as a GFP-fusion protein for the localization of MFs in living cells.

4. Collaborators:

We have very active collaborations at the University of Massachusetts with Joseph Kunkel, for the study of ion currents associated with pollen tube growth, with Alice Cheung for molecular biological studies on both pollen tubes and dividing cells, and with Patricia Wadsworth for studies on cytoskeletal structural dynamics.

We have several collaborations in other laboratories as follows:

Jonathan Chernoff at the Fox Chase Cancer Center, Philadelphia, PA for studies on small "G" proteins in plants;

Peter John and Brian Gunning, at the Australian National University, Canberra, Australia for studies on cyclin and other cell division regulators;

A.S.N. Reddy, Colorado State University, Fort Collin, CO, for studies on kinesins in plant cell division;

Jose Feijo, Gulbenkian Foundation, Lisbon, Portugal for studies on proton regulation of pollen tube growth;

Mauro Cresti, University of Siena, Siena, Italy, for studies on pollen tube ultrastructure;

Betty Lord, University of Calif., Riverside, for studies on pollen arabinogalactan proteins;

Luis Cardenas, Carmen Quinto, and Federico Sanchez, National University of Mexico, Cuernavaca, MX for studies on the effects of Nod factors on legume root hairs;

Etsuo Yokota and Teruo Shimmen, Himeji Technical Institute, Hyogo, Japan, for studies on villin and myosin in pollen tubes.