Location: Bowdoin / Bruce D. Kohorn

Biology

Bruce D. Kohorn

Professor of Biology and Biochemistry, Chair of Biology Department

Spring 2008

Biochemistry and Cell Biology (BIO 224)

Intermediate Independent Study in Biology (BIO 291)

Advanced Independent Study and Honors in Biology (BIO 402)

Advanced Independent Study and Honors in Biochemistry (BIOC 401)

Advanced Independent Study and Honors in Biochemistry (BIOC 402)

Phone	(207) 798-7068
Title	Professor
Department	BIOLOGY
2nd Title	Professor
2nd Department	BIOCHEMISTRY
3rd Title	Chair
3rd Department	BIOLOGY
Work Location	Druckenmiller Hall
E-Mail	bkohorn@bowdoin.edu

Bio 224 Cell Biology

Dr. Bruce D. Kohorn

Professor of Biology

Biology Department
Bowdoin College
Brunswick ME 04011

Ph.D., Yale University, 1983

Tel: (207) 798-7068
email: bkohorn@bowdoin.edu

fim vac

Images taken of plant leaf cells on Confocal Microscope.
Left: Actin in green, red is chloroplasts.
Right: vacuolemembrane in green with red chloroplasts.
Confocal microscope cross section

Left:View of Arabidopsis leaf; nuclei in green, chloroplasts in red.
Right: Confocal Image of Arabidopsis protoplast expressing WAK in green, Vacuole marker in red, chlorophyll in blue

Research Interests

My studies now concentrate on two types of signaling pathways. One involves communication between the extracellular matrix (ECM) and the cytoplasm of angiosperms. The other project involves a redox controlled, membrane bound organelle protein kinase that regulates light energy transduction and the perception of light. .

WAKs; WALL ASSOCIATED PROTEIN KINASES

Background

The cell wall, or extracellular matrix (ECM), of plant cells is composed of a complex arrangement of carbohydrates and protein that is intimately involved in developmental processes and interactions with changing environments. Despite detailed descriptions of the cell wall macromolecules and their importance, the method by which these structures are attached to the cell plasma membrane and how signals are transmitted between these compartments has not until recently been discerned, even though there has been direct visualization of such connections.
Cell walls have essential functions in development as they help to either define or modulate the cell's shape and size. The interaction between cells and the influence of invading organisms and wounding must also necessarily involve cell wall molecules as these are the first region of exposure. The primary cell wall of angiosperms is laid down through the ordered secretion of cellulose fibers by plasma membrane associated cellulose synthase. Xyloglucans, and often similar complex carbohydrates are thought to be layered onto the cellulose network. In some way, the pectic rhamno- and polygalacturonic acids are added to form a seemingly organized structure that is termed the cell wall. The pectins are thought to form a jelly-like matrix through which the more rigid cellulose fibers run. A number of abundant proteins such as AGPs, GRPs and HGRPs are associated with the carbohydrate complexes, but their role remains controversial. The expansion of the newly synthesized wall appears to be dependent upon a series of carbohydrate specific glyconases, and a family of tissue specific expansins. The esterification of pectins and the role of calcium have also been suggested to play a role in controlling the expansion, movement or remodeling of the cell wall. Despite these and other detailed descriptions of the cell wall macromolecules, the method by which these structures are attached to the cell plasma membrane has not been discerned.

WAKs

We have discovered a family of 5 Arabidopsis thaliana protein kinases, designated WAKs for Wall Associated Kinases, that likely provide a physical and a signaling continuum between the cell wall and the cytoplasm. All five WAKs contain a cytoplasmic protein kinase domain and span the plasma membrane to extend into the cell wall a domain that contains cysteine rich repeats.

Electron Micrograph with gold labeled WAK on cell surface and cell wall

WAK Function: Turgor Control

Angiosperm cell walls or ECM are composed of a complex of cellulose, hemicellulose, and a more flexible pectin that forms a scaffold that includes proteins. As the turgor of a plant cell increases, the membrane pushes against the ECM. Regulated cell wall loosening allows the creation of increased cell size, and the formation of cell shapes that form the plan of the entire plant. As a cell enlarges, there needs to be a mechanism for the cell to increase the turgor within to maintain pressure upon the cell wall. The cell Wall Associated Kinases (WAKs) of Arabidopsis are linked to pectin with an amino terminal domain that contains epidermal growth factor repeats. The five WAKs span the plasma membrane of most cells and also have a serine threonine kinase domain within the cytoplasm. Plants with a 50% reduction in WAK protein have reduced amounts of cell elongation implicating these receptors in sensing of ECM activity. A null mutation in one of the five WAKs leads to arrest of Arabidopsis seedling growth in the absence of exogenously supplied sugar and appropriate salts, indicating a need for sufficient osomotic conditions. The wak mutation causes a reduction in levels vacuolar invertase activity which is thought to control the turgor of a cell, and hence regulate cell expansion. The WAK receptor is responsible for the activation of the invertase gene. Thus WAKs may sense cell wall expansion by their attachment to pectin, and provide a mechanism to maintain turgor as the cell enlarges.

WAK Biogenesis; Assembly within the cell The Arabidopsis thaliana Wall Associated Kinases (WAKs) bind to pectin with an extracellular domain and also contain a cytoplasmic protein kinase domain. WAKs are required for cell elongation, and modulate sugar metabolism. This work shows that in leaf protoplasts a WAK1-GFP fusion accumulates in a cytoplasmic compartment that contains pectin. The WAK compartment contains markers for the Golgi, the site of pectin synthesis. The migration of WAK1-GFP to the cell surface is far slower than that of a cell surface receptor not associated with the cell wall, is influenced by the presence of fucose side chains on one or more unidentified molecules that might include pectin, and is dependant upon cellulose synthesis on the plasma membrane. WAK is cross linked into a detergent insoluble complex within the cytoplasmic compartment before it appears on the cell surface, and this is independent of fucose modification or cellulose synthesis. Thus the assembly and cross linking of WAKs may begin at an early stage within a cytoplasmic compartment rather than in the cell wall itself, and is coordinated with surface cellulose synthesis.

Selected publications:

Kohorn, B.D. 1993. Identification of cDNAs Encoding Proteases of Known Specificity Using a Cleavable Gal4 Protein. Methods. 5: 156-160
Smith, T.A., B.D. Kohorn. 1994. Mutations in a signal sequence for the thylakoid membrane identify multiple protein transport pathways and nuclear suppressors. J. Cell Biol. 126:365-374.
He, Z.H., M. Fujiki, and B.D. Kohorn. 1996 A cell wall associated recpetor-like kinase. J. Biol. Chem. 271: 19789-19793 (PDF reprint)
Baillet, B. and B.D. Kohorn. 1996. Hydrophobic core but not amino terminal charged residues are required for translocation of an integral thylakoid membrane protein in vivo. J. Biol. Chem. 271:18371-18374 (PDF reprint)
Bernd, K., Perret, M. and B.D. Kohorn. 1997. Protein translocation in chloroplasts; contributions from Chlamydomonas. In the Molecular Biology of Chlamydomonas. Eds. Rochaix, Goldschmidt-Clermont and Merchant. in press
Bernd, K. and B.D. Kohorn. 1998. Tips; six chlamydomonas nuclear suppressors that permit the translocation of proteins with mutant thylakoid signal sequences . Genetics 149:1293-1301
He, Z.H, He, D., and B.D. Kohorn. 1998. Requirement for the induction of a cell wall associated receptor kinase for survival during the pathogen response. The Plant J. 14: 55-63
Snyders, S and B.D. Kohorn. 1999. TAKs, Thylakoid Membrane Protein Kinases Associated with Energy Transduction J. Biol. Chem. 1999 274: 9137-9140
He, Z.H., Cheeman, I., He, D. and B.D. Kohorn. 1999. A cluster of cell wall assocaited kinase genes are expressed in specific tissues of Arabidopsis. Plant Mol Biol 39: 1189-1196
Kohorn, B.D. 1999. Shuffling the deck; plant signaling plays a club. Trends in Cell Biol. 9: 381-383.
Kohorn, B.D. 2000. Plasma membrane-cell wall contacts. Plant Physiology 124:21-38
Wagner, TA, and Kohorn, B.D. 2001. Wall associated kinases, WAKs, are expressed throughout development and are required for cell expansion. Plant Cell 13:303-18
Anderson, C.M., Wagner, T.A., He, Z.H., He, D., and Kohorn, B.D. 2001. WAKs : Cell wall associated kinases linking the cytoplasm to the extracellular matrix. Plant Mol. Biol47:197-206.
Anderson, K.A. and Kohorn, B.D. 2001. Inactivation of Arabidopsis SIP1 leads to reduced levels of sugars and drought tolerance. J. Plant Physio 158. 1215-12
Kohorn B.D. 2001. Cell Wall Associated Kinases. Curr. Opin. Cell Biol. 13:529-533
Snyders, S. and Kohorn, B.D. 2001.Disruption of thylakoid kinase activity TAK1 leads to alteration of light energy transduction. J. Biol. Chem. 276:32169-32176
Kohorn, B.D., Kobayashi, M, Johansen, S., Riese, J., Huang, L-F., Koch K., Fu, S., Dotson, A., and Byers, N, (2006). An Arabidopsis Cell Wall Associated Kinase Required for Invertase Activity and Cell Growth. The Plant Journal 46:307-316.
Kohorn, B.D., Kobayashi, M, Johansen, S., Fischer, A, Byers, N,. (2006) Wall Associated Kinase 1 is Crosslinked in Endomembranes and Transport To The Cell Surface Requires Correct Cell Wall Synthesis. Journal Cell Science 119: 2282-2290.