Deciphering the Composition of the Plant Nuclear Pore Complex

The Nuclear Pore Complex (NPC) is an essential conduit for the nucleo-cytoplasmic transport of proteins and RNA and is comprised of 30 nucleoporin proteins repeated in 8-fold symmetry around a central channel [1]. In the Parry lab we have isolated a both single and higher order Arabidopsis nucleoporin (nup) mutants that display a range of growth defects including early flowering, increased disease susceptibility and altered responses to hormones and abiotic stresses [2, 3]. Currently we know nothing about how removal of specific NUPs changes the composition of the entire NPC.

The primary aim of the project is to use protein biochemistry and microscopy to assess how the NPC is composed in these mutant plants. This will inform us about the interactions that occur within the plant NPC and identify relationships that might be exploited in future research. In addition we will develop tagged-NUPs and NUP-specific antibodies to importantly identify novel plant specific components of the NPC.

The secondary aim of the project is to use developing molecular resources (such as the RevGenUK TILLING service) to investigate the composition of the NPC in other commercially important plant species. The Parry lab (and others) have a growing understanding of the NUPs that are important for Arabidopsis and Medicago development. We aim to transfer this knowledge of how NUPs influence growth phenotypes into other plant species including other Brassicas and the grasses. Due to its crucial role in maintaining cell homeostasis, genetic manipulation of the NPC will offer future opportunities to alter plant growth for the common good. This research will underpin that future work by increasing the knowledge of the NPC whilst providing the successful student a range of highly relevant cell-biology skills.


Training:
The successful student will learn a wide range of techniques in molecular and cell biology. Maintaining knowledge of these skills in the next generation of researchers critically underpins the bioinformatics revolution occurring in the Biological sciences. As part of the project, the student will travel to Durham University to take advantage of their expertise in cell imaging.
- Plant Growth Techniques: Student will gain expertise growing Arabidopsis, Brassica, Legumes and Grasses performing a range of appropriate bioassays.
- Molecular Biology Techniques: gene cloning, nucleic acid extraction, protein expression, development of specific antibodies against plant nucleoporins and immunoprecipation of nucleoporin sub-complexes.
- Con-focal Microscopy: Immunolocalisation of NPC components in a range of plant tissues and plant cell culture. Analysis of the interaction of NPC components both with each other and with other members of the nuclear membrane proteome using FRAP and related techniques. This will be undertaken in collaboration with the Liverpool Cell Imagine Centre.
- Electron Microscopy: A key component of this project involves use of specific antibodies and transgenic lines to precisely localize NUPs in a variety of mutant backgrounds by TEM. Dr Goldberg is a world leader in the imaging of the nuclear membrane and therefore sample preparation, trouble shooting and imaging will take place in the Durham Bio-Imaging Centre.