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The Structural Organization of the Cytoskeleton

Investigation of the 3D localization of various cytoskeletal proteins in motile cells. The image below is a rendering of a series of images obtained with a Biorad MRC600 confocal microscope. A single Dictyostelium amoeba was stained with anti-tubulin antibody (red) and fluorescein-phalloidin (green) to simultaneously visualize the microtubule and actin filament systems.

The cell below was stained with antibodies to myosin (red) and F-actin (green) and optical sections obtained with the confocal microscope. The yellow areas represent colocalization of the two proteins in the cell.     



Cell Motility During Development: 3D cell tracking (X-Y-time)

In order to investigate the mechanisms of motility in developing cells, cells were labeled with a fluorescent dye, mixed with unlabeled cells and then the mixture allowed to undergo development. Uncer these conditions, Dictyostelium amoebae aggregated together into multicellular mounds. The movement of the fluorescent cells was visualized during aggregation using the confocal microscope. The image below shows fluorescent cells (green) within the mass of unlabeled cells (red). The movie shows cells moving in a single focal plane over time.  For more information see:

Shelden, E. and Knecht, D.A. (1995) Mutants lacking myosin II cannot resist forces generated during multicellular morphogenesis. J. Cell Sci 108:1105-1115.

Knecht, D.A. and Shelden, E. (1995) Three Dimensional Localization of Wild-Type and Myosin II Mutant Cells during Morphogenesis of Dictyostelium. Developmental Biology, 170:434-444.

Xu, X.S., Kuspa, A., Fuller D., Loomis, W.F. and Knecht, D.A. (1996) Cell-cell adhesion prevents mutants lacking myosin II from penetrating aggregation streams of Dictyostelium. Dev Biol.175:218-226.


 

4D Cell Tracking (X-Y-Z-time)

Fluorescent cells mixed with unlabeled cells and then optical sections through an aggregation center were collected every few minutes.  The data was assembled into a stereo movie that can be viewed with red-blue glasses to see the 3D effect.



     

Later in development, the pseudoplasmodial (slug) stage is reached.  Z series were collected through the slug as it crawled through the field of view. Two different slugs are shown. Use red-blue glasses to see the stereo effect.



 
 

Curriculum Vitae

DAVID A. KNECHT, Ph.D.

Address: Department of Molecular and Cell Biology
University of Connecticut
Storrs, CT 06269
phone: 203-486-2200
fax: 203-486-4331
Email- KNECHT@UCONNVM.UCONN.EDU

Education and Positions:

1987-present Department of Molecular and Cell Biology, University of Connecticut

1982-1987 Postdoctoral Fellow University of California, San Diego

1975-1982 Ph.D. in Molecular Biology University of Wisconsin, Madison

1971-1975 B.A. in Chemistry, with honors Oberlin College
 

Recent Publications:

Knecht, D.A., Fuller, D.A., and Loomis, W.F. (1987) Surface glycoprotein, gp24, involved in early adhesion of Dictyostelium discoideum. Dev. Biol. 121:277-283.

Knecht, D. A. and Loomis, W.F. (1987) Antisense RNA inactivation of myosin heavy chain gene expression in Dictyostelium discoideum. Science 236:1081-1086.

Knecht, D. A. and Loomis, W. F. (1988) Developmental consequences of the lack of myosin heavy chain in Dictyostelium discoideum. Dev. Biol. 128: 178-184.

Wessels, D., Soll, D.R., Knecht, D.A., Loomis, W.F., De Lozanne, A. and Spudich, J.A. (1988) Cell motility and chemotaxis in Dictyostelium amebae lacking native myosin heavy chain. Dev. Biol. 128: 164-177.

Peters, D., Knecht, D.A., Loomis, W.F., De Lozanne, A., Spudich, J.A. and Van Haastert, P.J.M. (1988) Transmembrane signal transduction, chemotaxis and cell aggregation in Dictyostelium discoideum cells without myosin heavy chain. Dev. Biol. 128: 158-163.

Knecht, D.A. (1989) Application of antisense RNA to the study of the cytoskeleton: background, principles and a summary of results obtained with myosin heavy chain. Cell Motility and the Cytoskeleton 14:92-102.

Knecht, D.A., Jung, J. and Matthews, L.R. (1990) Quantification of transformation efficiency using a new method for clonal growth and selection of axenic Dictyostelium cells. Dev. Genet. 11:403-409.

Knecht, D.A. and Kessin, R.H. (1990) Recent Advances in the Molecular Genetics of Dictyostelium. Dev. Genet.11:388-390.

Reaume, A.G., Knecht, D.A., and Chovnick, A. (1991) The Rosy Locus in Drosophila melanogaster: Xanthine Dehydrogenase and Eye Pigments. Genetics, 129:1099-1109.

Cox, D., Condeelis, J., Wessels, D., Soll, D., Kern H., Knecht, D.A. (1992) Targeted disruption of the ABP-120 gene leads to cells with altered motility. J. Cell Biology, 116:943-956.

Dingermann, T. Werner, H., Schutz, A., Zundorf, I., Nerke, K., Knecht, D.A., and Marschalek, R. (1992) Establishment of a System for Conditional Gene Expression using an Inducible tRNA Suppressor Gene. Mol. Cell Biol. 12:4038-4042.

Scherczinger, C., Yates, A.A., and Knecht, D.A. (1992) Variables affecting antisense RNA inhibition of gene expression. Ann. N.Y. Acad. Sci. 660:45-56.

Knecht, D.A. (1993) Non-photographic (digital) preparation of data for publication. Biotechniques 14:1006-1008.

C. Scherczinger and Knecht, D.A. (1993) Systematic Analysis of Antisense RNA inhibition of Myosin heavy chain gene expression in Dictyostelium. Antisense Research and Development. 3:191-205.

C. Scherczinger and Knecht, D.A. (1993) Co-supression of Myosin heavy chain gene expression by a sense orientation transcript. Antisense Research and Development, 3:207-217.

Knecht, D.A., Kern, H.K. and Scherczinger, C., (1993) Bidirectional transcription from actin promoters in Dictyostelium. Biochem. Biophys. Acta 1216:105-109.

Shelden, E. and Knecht, D.A. (1995) Mutants lacking myosin II cannot resist forces generated during multicellular morphogenesis. J. Cell Sci 108:1105-1115.

Knecht, D.A. and Shelden, E. (1995) Three Dimensional Localization of Wild-Type and Myosin II Mutant Cells during Morphogenesis of Dictyostelium. Developmental Biology, 170:434-444.

Knecht, D.A. and Pang, K.M. (1995) Electroporation of Dictyostelium discoideum. in Methods in Molecular Biology vol. 47, Protocols for Electroporation and Electrofusion of Microorganisms, (J. Nickoloff ed.), Humana Press, Inc. Clifton, N.J.

Xu, X.S., Kuspa, A., Fuller D., Loomis, W.F. and Knecht, D.A. (1996) Cell-cell adhesion prevents mutants lacking myosin II from penetrating aggregation streams of Dictyostelium. Dev Biol.175:218-226.

Cohen, N.R., Knecht, D.A. and Lodish, H.F. (1996) Functional expression of rat Glut 1 glucose transporter in Dictyostleium discoideum. Biochem. J. 315:971-975.

Shelden, E. and Knecht, D.A. (1996) Dictyotelium cell shape generation requires myosin II. Cell Motil. and Cytoskel. 35:59-67.


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