Production, characterisation and use of fluorescent markers in the study of plant nuclear envelope dynamics

The nuclear envelope (NE) is one of the least studied membranes in plant cells. Genes encoding NE protein homologues are absent from currently available sequenced plant genomes. To produce a specific marker for the plant NE and in view of previous positive results with heterologously expressed proteins in plant cells, mammalian NE proteins were considered in order to find a marker for the plant NE. Green fluorescent protein (GFP) was chosen to label the protein of interest as it provides a non-invasive method of monitoring protein location and movement in vivo. The lamin B receptor (LBR) is the most extensively studied of the mammalian inner NE (INE) proteins. It is an INE protein that binds lam in B, chromatin and chromatin-associated proteins. A LBR-GFPs construct was produced and placed in a plant expression vector. Transient expression of the LBR-GFPs protein in tobacco leaf epidermal cells showed labelling of the NE, with minimal labelling of the endoplasmic reticulum (ER). The construct was used to produce stably transformed tobacco plants and tobacco BY -2 cells. NE labelling was observed in the majority of tissues stably expressing the protein, with NE location confirmed by electron microscopy. In BY -2 cells the construct showed NE location during interphase, with co-localisation with an ER marker, sporamin signal peptide YFP-HDEL (sp Y~P:"HDEL) during mitosis. Similar labelling of the ER with NE proteins is seen in mammalian cells during division. This, in combination with the targeting and retention of LBR-GFPs at the plant NE suggests a conservation of mechanisms 'for INE targeting and retention in plant and animal NEs. Preliminary evaluation of the nature of LBR retention at the NE was conducted using a set of LBR-GFPs mutants. Labelling of the NE was perturbed with some of the mutants, indicating that similar domains contribute to LBR retention in plant and ,animal cells.

Grantor dc:publisher

Oxford Brookes University

Year dc:date

2004

Author dc:creator

• Irons, Sarah L.

Contributors dc:contributor

• Evans, David
• Brandizzi, Fede

Statement dc:rights

• All rights reserved

Language dc:language

en

DOI dc:identifier

https://doi.org/10.24384/k2hz-z080

OAI identifier oai:identifier

tle:a90da521-0136-466e-a37c-7fe6128e1cc2:d6bd9758-527a-46cd-bfe2-c433766e8fca:1