Professor Marisa S. Otegui



b	Marisa S. Otegui Assistant Professor of Botany Ph.D. University of La Plata , 1996 Office: 224 Birge Hall Phone: 608- 265-5703 Email: otegui@wisc.edu Cell biology; intracellular membrane traffic; cytokinesis; endosperm development Lab Homepage

blank	I am interested in structural and functional aspects of multivesicular bodies (MVBs) in plant cells. MVBs are endosomes that consist of a limiting membrane and internal vesicles. The internal vesicles arise from invaginations of the limiting membrane and carry membrane proteins targeted for degradation in the lysosome/vacuole. MVBs play a crucial role in both the endocytic and the secretory pathways of all eukaryotic cells, sorting proteins for degradation or recycling, downregulating receptors, and mediating the transport of proteins to the vacuole/lysosome. Thus, MVB functions are tightly related to cell signaling, differentiation, and transport of vacuolar cargoes. The endosomal invagination process is unique because, unlike most vesiculation processes characterized to date, the vesiculating membrane buds away from the cytoplasm. This invagination process requires the concentration of membrane proteins into specific membrane domains and the initiation of budding into the MVB lumen. In yeast and mammalian cells, ubiquitination of receptors is now well established as a signal for sorting into internal vesicles of MVBs. In addition, multiprotein complexes called e ndosomal s orting c omplexes r equired for t ransport I, II, and III (ESCRT-I, -II, -III) are also required for protein sorting into MVB internal vesicles. In plants, MVBs have been adapted to serve both the same functions as in yeast and mammalian cells as well as unique plant functions. In particular, plant MVBs appear to play a central role in the transport and processing of vacuolar storage proteins and recycling of membrane removed from the cell plate during cytokinesis. We are addressing questions such as: How many classes of endosomal compartments exist in plants? How do MVBs arise? What steps in proteolytic protein processing take place in MVBs? What are their functions in different cell types? What is the molecular machinery responsible for MVB sorting and invagination? Are receptors targeted to MVB ubiquitinated? Research areas: 1- Identification of different types of MVBs and their cargoes in plant cells and the analysis of their specific function during cytokinesis. 2- Analysis of the specific roles of MVBs in storage protein transport in seeds. 3- Analysis of the molecular basis of MVB functions in plants and identification of ortologues of ESCRT subunits in Arabidopsis thaliana. Approach We use a variety of multidisciplinary techniques, including cryofixation/freeze substitution, electron and confocal microscopy, dual-axis electron tomography, immunolabeling, subcellular fractionation, RT-PCR, and expression of GFP fusion proteins and heterologous proteins in Arabidopsis thaliana . Recent Publications Martínez, D.E., Costa, M.L., Otegui, M.S., Guiamet, J.J. 2008. Senescence-associated vacuoles of tobacco leaves are involved in the degradation of chloroplast proteins. The Plant Journal (in press). Otegui, M.S., Spitzer, C. 2008. Plant endosomal functions. Traffic (in press). Tian, Q., Olsen, L., Sun, B., Lid, S.E., Brown, R., Lemmon, B.E., Fosnes, K., Gruis, F., Opsahl-Sorteberg, H.-G., Otegui, M.S. and Olsen, O.-A. 2007. Subcellular localization and functional domain studies of DEFECTIVE KERNEL 1 in maize and Arabidopsis thaliana suggest a model for aleurone cell fate specification involving CRINKLY 4 and SUPERNUMERARY ALEURONE LAYER 1. The Plant Cell 19: 3127-3145. Holding, D.R., Otegui, M.S., Li, B., Meeley, R., Dam, T., Hunter, B.G., Jung, R., Larkins, B.A. 2007. The maize Floury1 gene encodes a novel ER protein involved in zein protein-body formation. The Plant Cell 19: 2569-2582. Kobayashi, K, Otegui, M.S., Krishnakumar, S., Mindrinos, M., Zambryski, P. 2007. INCREASED SIZE EXCLUSION LIMIT 2 encodes a DEVH box RNA helicase that regulates plasmodesmata during Arabidopsis embryogenesis. The Plant Cell 19: 1885-1897. Haas, T.J., Sliwinski, M.K., Martínez, D.E., Preuss, M., Ebine, K., Ueda, T., Nielsen, E., Odorizzi, G., and Otegui, M.S. 2007. The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LIP5. The Plant Cell 19:1295-312. Otegui, M.S., Austin, J.R. II. 2007. Visualization of membrane-cytoskeletal interactions during plant cytokinesis. Methods in Cell Biology 79:221-40. Otegui, M.S. 2007. Endosperm cell walls: formation, composition, and functions. In: Olsen, O.A. Endosperm: development and molecular biology. Springer-Verlag, Heidelberg, Germany. Otegui, M.S., Herder, R., Schulze, J., Jung, R., Staehelin, L.A. 2006. A population of prevacuolar compartments acts as proteolytic processing stations for seed storage proteins in Arabidopsis thaliana. The Plant Cell 18: 2567-2581. Chaiwongsar, S., Otegui, M.S., Jester, P.J., Monson, S.S., Krysan, P.J. 2006. The protein kinase genes MAP3Ke1 and MAP3Ke2 are required for pollen viability in Arabidopsis thaliana. The Plant Journal 48:193-205. Petruccelli, S., Otegui, M.S., Lareu, F., Trandinhthanhlien, O., Fitchette, A.-C., Circosta, A., Rumbo, M., Bardor, M., Carcamo, R., Gomord, V., Beachy, R. 2006. A KDEL tagged monoclonal antibody is efficiently retained in the reticulum endoplasmic in leaves but is both partially secreted and sorted to protein storage vacuoles in seeds. Plant Biotechnology Journal 4: 511-527. Preuss, M.L., Schmitz, A.J., Thole, J.M. Bonner, H.K.S., Otegui, M.S., Nielsen, E. 2006. A role for the RabA4b effector protein, PI-4Kbeta1, in polarized expansion of root hair cells in Arabidopsis. Journal Cell Biology 172:991-8. Otegui, M.S., Verbrugghe, K.J., Skop A.R. 2005. Midbodies and phragmoplasts: analogous structures involved in cytokinesis. Trends in Cell Biology 15: 404-13 Otegui, M.S., Noh, Y.S., Martinez, D., Vila Petroff, M., Staehelin, L.A., Amasino, R.M., Guiamet, J.J. 2005. Senescence-associated vacuoles with intense proteolytic activity develop in leaves of Arabidopsis and soybean. Plant J. 41: .831-844. Otegui, M.S., Capp, R., Staehelin, L.A. 2002. Developing seeds of Arabidopsis thaliana store different minerals in two types of vacuoles and in the ER. The Plant Cell 14: 1311-1327. Otegui, M.S., Mastronarde, D., Kang, B-H., Bednarek, S.Y., Staehelin, L.A. 2001. Three-dimensional analysis of syncytial type cell plates during endosperm cellularization visualized by high resolution electron microscopy. The Plant Cell 13: 2033-2051. Structural Plant Biology \| Botany Department © 2000 University of Wisconsin Department of Botany Last updated: October 27, 2004

I am interested in structural and functional aspects of multivesicular bodies (MVBs) in plant cells. MVBs are endosomes that consist of a limiting membrane and internal vesicles. The internal vesicles arise from invaginations of the limiting membrane and carry membrane proteins targeted for degradation in the lysosome/vacuole. MVBs play a crucial role in both the endocytic and the secretory pathways of all eukaryotic cells, sorting proteins for degradation or recycling, downregulating receptors, and mediating the transport of proteins to the vacuole/lysosome. Thus, MVB functions are tightly related to cell signaling, differentiation, and transport of vacuolar cargoes.

The endosomal invagination process is unique because, unlike most vesiculation processes characterized to date, the vesiculating membrane buds away from the cytoplasm. This invagination process requires the concentration of membrane proteins into specific membrane domains and the initiation of budding into the MVB lumen. In yeast and mammalian cells, ubiquitination of receptors is now well established as a signal for sorting into internal vesicles of MVBs. In addition, multiprotein complexes called e ndosomal s orting c omplexes r equired for t ransport I, II, and III (ESCRT-I, -II, -III) are also required for protein sorting into MVB internal vesicles.

In plants, MVBs have been adapted to serve both the same functions as in yeast and mammalian cells as well as unique plant functions. In particular, plant MVBs appear to play a central role in the transport and processing of vacuolar storage proteins and recycling of membrane removed from the cell plate during cytokinesis. We are addressing questions such as: How many classes of endosomal compartments exist in plants? How do MVBs arise? What steps in proteolytic protein processing take place in MVBs? What are their functions in different cell types? What is the molecular machinery responsible for MVB sorting and invagination? Are receptors targeted to MVB ubiquitinated?

Research areas:

1- Identification of different types of MVBs and their cargoes in plant cells and the analysis of their specific function during cytokinesis.

2- Analysis of the specific roles of MVBs in storage protein transport in seeds.

3- Analysis of the molecular basis of MVB functions in plants and identification of ortologues of ESCRT subunits in Arabidopsis thaliana.

Approach

We use a variety of multidisciplinary techniques, including cryofixation/freeze substitution, electron and confocal microscopy, dual-axis electron tomography, immunolabeling, subcellular fractionation, RT-PCR, and expression of GFP fusion proteins and heterologous proteins in Arabidopsis thaliana .

Recent Publications

Martínez, D.E., Costa, M.L., Otegui, M.S., Guiamet, J.J. 2008. Senescence-associated vacuoles of tobacco leaves are involved in the degradation of chloroplast proteins. The Plant Journal (in press).

Otegui, M.S., Spitzer, C. 2008. Plant endosomal functions. Traffic (in press).

Tian, Q., Olsen, L., Sun, B., Lid, S.E., Brown, R., Lemmon, B.E., Fosnes, K., Gruis, F., Opsahl-Sorteberg, H.-G., Otegui, M.S. and Olsen, O.-A. 2007. Subcellular localization and functional domain studies of DEFECTIVE KERNEL 1 in maize and Arabidopsis thaliana suggest a model for aleurone cell fate specification involving CRINKLY 4 and SUPERNUMERARY ALEURONE LAYER 1. The Plant Cell 19: 3127-3145.

Holding, D.R., Otegui, M.S., Li, B., Meeley, R., Dam, T., Hunter, B.G., Jung, R., Larkins, B.A. 2007. The maize Floury1 gene encodes a novel ER protein involved in zein protein-body formation. The Plant Cell 19: 2569-2582.

Kobayashi, K, Otegui, M.S., Krishnakumar, S., Mindrinos, M., Zambryski, P. 2007. INCREASED SIZE EXCLUSION LIMIT 2 encodes a DEVH box RNA helicase that regulates plasmodesmata during Arabidopsis embryogenesis. The Plant Cell 19: 1885-1897.

Haas, T.J., Sliwinski, M.K., Martínez, D.E., Preuss, M., Ebine, K., Ueda, T., Nielsen, E., Odorizzi, G., and Otegui, M.S. 2007. The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LIP5. The Plant Cell 19:1295-312.

Otegui, M.S., Austin, J.R. II. 2007. Visualization of membrane-cytoskeletal interactions during plant cytokinesis. Methods in Cell Biology 79:221-40.

Otegui, M.S. 2007. Endosperm cell walls: formation, composition, and functions. In: Olsen, O.A. Endosperm: development and molecular biology. Springer-Verlag, Heidelberg, Germany.

Otegui, M.S., Herder, R., Schulze, J., Jung, R., Staehelin, L.A. 2006. A population of prevacuolar compartments acts as proteolytic processing stations for seed storage proteins in Arabidopsis thaliana. The Plant Cell 18: 2567-2581.

Chaiwongsar, S., Otegui, M.S., Jester, P.J., Monson, S.S., Krysan, P.J. 2006. The protein kinase genes MAP3Ke1 and MAP3Ke2 are required for pollen viability in Arabidopsis thaliana. The Plant Journal 48:193-205.

Petruccelli, S., Otegui, M.S., Lareu, F., Trandinhthanhlien, O., Fitchette, A.-C., Circosta, A., Rumbo, M., Bardor, M., Carcamo, R., Gomord, V., Beachy, R. 2006. A KDEL tagged monoclonal antibody is efficiently retained in the reticulum endoplasmic in leaves but is both partially secreted and sorted to protein storage vacuoles in seeds. Plant Biotechnology Journal 4: 511-527.

Preuss, M.L., Schmitz, A.J., Thole, J.M. Bonner, H.K.S., Otegui, M.S., Nielsen, E. 2006. A role for the RabA4b effector protein, PI-4Kbeta1, in polarized expansion of root hair cells in Arabidopsis. Journal Cell Biology 172:991-8.

Otegui, M.S., Verbrugghe, K.J., Skop A.R. 2005. Midbodies and phragmoplasts: analogous structures involved in cytokinesis. Trends in Cell Biology 15: 404-13

Otegui, M.S., Noh, Y.S., Martinez, D., Vila Petroff, M., Staehelin, L.A., Amasino, R.M., Guiamet, J.J. 2005. Senescence-associated vacuoles with intense proteolytic activity develop in leaves of Arabidopsis and soybean. Plant J. 41: .831-844.

Otegui, M.S., Capp, R., Staehelin, L.A. 2002. Developing seeds of Arabidopsis thaliana store different minerals in two types of vacuoles and in the ER. The Plant Cell 14: 1311-1327.

Otegui, M.S., Mastronarde, D., Kang, B-H., Bednarek, S.Y., Staehelin, L.A. 2001. Three-dimensional analysis of syncytial type cell plates during endosperm cellularization visualized by high resolution electron microscopy. The Plant Cell 13: 2033-2051.