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Research



Introduction

The plant endoplasmic recticulum (ER) is a dynamic organelle responsible for not only producing secretory proteins and lipids, but regulating cellular calcium. It is connected to many other organelles (mitochondria, chloroplasts, cytoskelton and plasma membrane to name a few). It is a vital organelle, but there are large gaps in our knowledge about it. Questions that are yet to be answered include:

How are the dynamics and the structure of the ER regulated?

How does ER form affect function?

These questions I hope to start answering during the course of my PhD project. I will be using Arabidopsis thaliana seeds to investigate the above questions.

Why seeds? 70% of human protein consumption comes from seeds, either directly from the seeds we eat (such as rice and wheat), or indirectly from eating the animals that we feed with grains. This means that seeds are a highly important source of nutrients in out diet.The protein inside seed is produced by the ER, making it a vital organelle.

In plant cells the ER has a very distict morphology due to the vacuole pushing the network to the top of the cell. This means we can study the shape of the ER with comparative ease. The two main network structures are sheets and tubules. It has been proposed that these two structures have different functional roles.

Two protein familes, reticulon and atlastin, are known to influence the ER morphology. By finding proteins that interact with the seed specific isoforms of reticulon (RTNL B13) and atlastin (RHD3 RL2) we will gain a greater understanding of how they are regulated, and consequently how the ER shape is regulated. This will also gives us an insight into how the form of the ER affects the function. This information could then be used to create a more effective ER, increasing production of proteins inside seeds.

Aims


1. Finding proteins interacting with the seed specific reticulon and atlastin through co-immunoprecipitation

2. Confirming these interactions with FRET-FLIM analysis

3. Observing ER morphology change in mutants and knockouts of these protein interactors

4. Analysing ER morphology quantitatively through confocal microscopy



Proteins of Interest

Reticulons - Small, trans-membrane proteins with 4 proposed transmembrane domains forming a 'W' shape. These transmembrane domains are longer than normal membrane proteins, and it has been suggested that they insert diagonally into the membrane, causing the lipid bilayer to bend into tubules. There is also a putative ampiphatic helix, which may assist in the bending of the ER membrane.

RTN13 Colour

Atlastins - Large, GTPases that fuse ER membranes together by forming homodimers. They do this via a GTP hydrolysis cycle, which causes conformational changes in the protein which brings membranes together and fuses them. This fusion brings tubules together into three way junctions, forming the connected ER network.

RHD3 Colour