David A. Knecht - US grants

The long range goal of this project is the application of molecular genetic tools to understanding the cellular mechanisms and processes that are necessary for differentiation and morphogenesis. The objective is to identify, isolate and characterize genes that are essential for development of Dictoyostelium discoideum. This eucaryotic amoebal organism provides a unique model system for the study of cellular functions that are involved in development. During development, a uniform population of amoebal cells differentiates into a multicellular organism consisting of two cell types with a highly regulated spatial organization. Motility, signal transduction, adhesion, morphogenetic signalling and cell type specific gene regulation are among the fundamental processes utilized by this organism to accomplish its developmental program. The information gained about how these functions are accomplished in Dictyostelium will provide clues to similar functions in other organisms. An indirect genetic approach to understanding the cellular processes related to development will be taken. Mutants that affect processes that are essential for development will be identified, and then the genes that were mutated will be isolated. In this way, a direct handle on critical functional elements can be gained. Mutants that affect development are not new in Dictyostelium, but the means to isolate the mutated genes has never been available. The DNA mediated transformation system will provide the means for both mutating and isolating these genes. Two approaches to accomplishing this goal will be undertaken in parallel. Antisense RNA synthesized from a transformation vector will be used to inhibit expression of essential developmental genes, or a vector that inserts into the chromosome will be used to interrupt gene expression. In both cases, large numbers of transformants will be screened for defects in morphogenesis caused by the transformation vector. The vector sequence will then provide the means for isolating the inactivated gene. The purpose of this project is not simply to clone more genes to study their regulation. Rather it is to use functional criteria to isolate important genes as a means to eventually studying the function of the gene products.

microscopy; image processing; biomedical equipment resource; biomedical equipment purchase;

DESCRIPTION: The applicant will use molecular genetic, imaging and biophysical techniques to determine how the actin filament network is formed and shaped by actin cross-linking proteins. Mutant cell lines which contain alterations in genes coding for the actin binding proteins ABP-120, ABP-240 and a-actinin as well as myosin II will be studied. Several novel in vitro and in vivo assays will be used to assess the consequences of these alterations in the cytoskeleton.

The 1999 International Dictyostelium Meeting will be held August 14-19 in Bar Harbor, ME. This meeting brings together researchers from all over the world to discuss the latest advances in research on this important model system. Support is requested from the NSF to offset some of the cost of the meeting and to allow investigators with limited grant support to attend the meeting. The focus of these travel awards will be on new investigators, as well as women and minority post-doctoral fellows and graduate students in order to increase their representation among the attendees.

Cell shape, polarity and motility are controlled by the actin cytoskeleton. How F-actin filaments are assembled into functional arrays is still poorly understood. There are numerous F-actin binding proteins that are capable of cross-linking actin filaments and these proteins vary in the arrangement of filaments produced and the regulation of the binding activity. They can produce parallel, anti-parallel or orthogonal arrays of F-actin, but the complex interplay of these proteins is poorly understood. IN addition, a number of actin-binding proteins are regulated by factors such as Ca++ and the circumstance where this is important is not known. This proposal seeks to use molecular genetic and imaging approaches to continue to investigate the mechanisms used by cells to regulate the assembly of actin filament arrays. Myosin II has been found to be a major contributor to cytoskeletal integrity of Dictyostelium cells. The contribution of myosin II to forces applied to the surface will be investigated and compared to the contributions of other actin binding proteins using a flexible substratum assay. The contribution of myosin II to mammalian cell integrity will be investigated by using RNAi to inhibit the expression of myosin llB in tissue culture cells. Studies of ABP120 and alpha-actinin have shown that the actin binding domains of these proteins regulate their association with F-actin filaments. This has led to the hypothesis that not all actin filaments in cells are the same, since these proteins can distinguish between filaments in different parts of the cell. This important idea will be further investigated by studying the properties of the actin binding domains in vivo and in vitro. The role of Ca++ in regulating alpha-actinin localization will also be investigated in order to determine under what circumstances Ca++ regulation becomes important. Fimbrin is another member of this class of actin crosslinking proteins. There are two homologs of fimbrin in Dictyostelium cells, fimA and fimB. The fimA gene is similar to mammalian flmbrin having EF-hands and two actin-binding domains. FimB is a recently discovered homolog that lacks EF hands, but has PH domains and a talin homology domain. The function of each protein will be investigated by gene disruption, mutagenesis and localization techniques. The structure of the actin binding domains of each of these proteins will also be determined to investigate the mechanism by which they differentially recognize actin filaments.

An award has been made to the University of Connecticut under the direction of Dr. David A. Knecht to acquire a spinning disc confocal microscope to be used for observing living cells in real time. The instrument will allow real-time, fast imaging of cells to study a variety of cellular processes, including motility, auxin transport, vesicular transport, and auxin localization in plants, slime molds, and fish. The microscope will increase viability of cells and allow observations to be made over long periods of time. The institution has strong record in several programs for recruiting underrepresented groups to science, and the instrument will enhance the experiences of students in these programs. Most of the users of the confocal microscope will be graduate students, and undergraduate and high school students will be given demonstrations with the instrument.

Biological Sciences (61)
The advent of inexpensive digital video technology has revolutionized the ability to capture images of cell structure and behavior through the microscope. In parallel, the fluorescent protein revolution, initiated with the cloning of GFP, has given researchers dramatic insights into the dynamics of proteins in living cells. This project capitalizes on these advances to give students in biochemistry, cell biology and genetics courses the opportunity to engage in self-directed laboratory exploration of cellular processes by using green fluorescent probes to compare processes in live cells of mutant and wild strains of the social amoeba Dictyostelium. Equipment added to the laboratory includes several low-cost video fluorescent microscope workstations, a spinning disc confocal microscope system, and an electroporator (for the genetics and cell biology labs). The modules being developed are based on current research at the institution and experience gained within the institution's core microscopy facility.

The intellectual merits of this proposal stem from extensive use of live cell imaging, the emphasis on quantitative digital imaging, and the investigative nature of the course. Students learn what a phase contrast optical system does and how it differs from Differential Interference Contrast, how fluorescence filter sets work and the relationship of a CCD chip to an image on a computer screen. The microscope is used for a variety of activities including: checking cultures, titering cells on a hemocytometer, and time-lapse video movie acquisition. Another key aspect of the course is quantitative imaging. Students learn about digital image processing including quantification of parameters like cell size, cell speed, and persistence of chemotactic movement using the power of the video microscope as a quantitative measurement instrument, rather than a qualitative photographic device. In most experiments, students not only investigate a cellular process, but they compare wild-type cells with mutants. The students are given guidelines, but not protocols and are encouraged to devise their own experimental questions within the general parameters of the laboratory. Each laboratory report is written as a mini-research manuscript in which they describe the background, assumptions and hypotheses, as well as the results and discussion.

The implementation of this proposal is having a broad impact on undergraduate education. The equipment is being utilized not only to enhance the core microscopy course, but also by two other laboratory courses in the department, using course suitable inquiry based fluorescence microscopy experiments designed jointly by the PI and the faculty responsible for the course. In addition, modified versions of the laboratory modules are being used in undergraduate summer programs, high schools, and community colleges. To reach the wider audience of Cell Biology faculty, the modules are being distributed to the Dictyostelium community through Dictybase, ASCB Education, BiosciEdNet and other teaching resources.