Investigating the Role of Novel Nuclear Envelope-Associated Plant Proteins in Nuclear Dynamics

Ohio State University Research Foundation -Do Not Use, Columbus OH

Investigators

Abstract

ABSTRACT A striking feature of the nucleus of multicellular eukaryotes is its ability to completely disassemble and re-assemble during the course of cell division. This process may allow higher organisms to have a structurally highly complex nuclear interior during interphase, but not have such structures interfere with the condensation and partitioning of chromosomes during cell division. Such nuclear dynamics require the regulated reversibility of interactions between chromatin and proteins of the nuclear envelope and probably involve dynamic changes of nucleoskeletal structures. Since plants and animals are most likely derived from different unicellular ancestors that underwent intranuclear mitosis, one expects that nuclear assembly and disassembly have evolved twice. Presently, their molecular mechanisms are poorly understood in any system. The long-term goal of this research is to understand the mechanism, the function in interphase, and the dynamics during cell cycle of chromatin-nuclear envelope interactions in plants. Two novel, interacting plant proteins (MFP 1 and MAF 1) have been identified that are associated both with a filamentous nuclear framework called the nuclear matrix and with the nuclear envelope. MFP 1 is a plant-specific, filament-like protein, which binds specifically to matrix attachment regions (MARs), specific DNA segments which have been proposed to be involved in chromatin organization. In vitro, its MAR-binding activity is regulated by phosphorylation. The hypothesis is that MFP1 is involved in attaching chromatin to the plant interphase nuclear envelope and that MAF 1 is involved in this process through interaction with MFP 1. The specific questions asked in this project are: 1) How does the localization of MFP 1 and MAF 1 change during cell cycle? The strategy employs dual-color imaging of co-expressed MFP 1 and MAF 1 fusion proteins with GFP spectral variants in synchronized cell cultures. The results will show whether the localization of the two proteins functionally correlates with nuclear envelope dynamics. 2) How does the depletion of MFP 1 and MAF 1 alter nuclear, cell, and plant phenotypes? To this end, insertional knock-out mutations of MFP 1 and MAF 1 will be identified in Arabidopsis. They are expected to cause informative cellular and possibly developmental phenotypes, which will allow conclusions about the in vivo function of the proteins. 3) Is phosphorylation of MFP1 involved in reversible regulation of DNA-binding during cell cycle? The pattern of phosphorylated MFP1 isoforms and their MAR-binding activity will be compared at different cell-cycle stages. The effect of overexpressing a constitutively DNA-binding MFP 1 isoform on cellular and plant phenotypes will be investigated. These data will indicate whether reversible MFP1 phosphorylation is a means to regulate the interaction of chromatin with the nuclear envelope. Together, the answers to these questions will allow the evaluation of the role of MFP 1 and MAF 1 in chromatin-nuclear envelope interactions in plants. Because of the possibility that nuclear dissociation and re-association have evolved twice in plants and animals, any molecular information about the plant nucleus is likely to add novel concepts to our understanding of a process which is currently enigmatic in any organism. In addition, the discovery of plant-specific aspects of cellular organization could lead to the development of new classes of broad-spectrum herbicides.