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Membrane shaping, cytoskeletal and nuclear envelope (NE) proteins in NE dynamics during cell division: a study that will develop and use correlative super resolution light and electron microscopy (sCLEM)


About This PhD Project

Project Description

Scientific aims: the nuclear envelope (NE) is a central cellular hub with roles in ageing, cancer migration and degenerative diseases. NE functions in transcription/translation control and cell/nuclear organisation are established as the NE reassembles after cell division. How this complex structure is put together determines how it functions. Therefore understanding the assembly process is essential for understanding its functions and the role of the assembly process and individual components in disease. However, little is known about NE assembly mechanisms. This will be a systematic study testing roles of individual proteins in specific assembly steps, using a combination of the latest microscopies.

Technical aims: this is a unique opportunity to learn, develop and combine cutting-edge advanced light and electron microscopy (EM) techniques, in conjunction with CRISPR-Cas9 gene editing methods.

Project plan: The project centres on mechanisms of NE reassembly after mitosis and is based on our proteomics study that identified candidate proteins involved in the targeting, transport and binding of membranes to the chromosomes, their fusion with each other, and membrane remodelling over the chromosome surface. Cells will be genetically edited to express candidate proteins that are tagged with fluorescent proteins for super resolution light microscopy/live imaging (3D SIM and PALM), or with tags for inducible knock-down. We will study the effects of manipulating candidate proteins as cells progress through NE reassembly in telophase. Cells will be imaged with super resolution light microscopy to determine precise locations of candidate proteins and how they associate with each other. The same cell will then be instantly frozen by high pressure freezing (HPF) and processed for thin section EM, automated 3D EM, or high resolution scanning EM. By correlating fluorescence signals with structural context from the EM we can determine exactly where candidate proteins are located and how this changes during NE assembly. By combining localisation, interaction and perturbation studies with high resolution and 3D structural analysis, we can test a holistic model for the assembly and establishment of the NE.

Technical training and development: Exciting developments in light and electron microscopy have lead to a need to bridge the information gap between the two types of imaging. This can be realized by super resolution correlative light/electron microscopy (sCLEM). This project will be an opportunity to advance this field. The student will receive training in both essential skills (such as molecular biology, cell culture) and more specialised, but sort after, skills, such as advanced microscopy, including scanning and transmission electron microscopy, immunogold-labelling, super resolution light microscopy and sCLEM.

Through recent major grants, the department has new state-of-the-art equipment for correlative cryo preparation techniques for electron microscopy, a high resolution SEM, several super resolution light microscopy techniques and suitable computer facilities and software for image processing and analysis.

Candidates should have a demonstrable passion for cell biology and a desire to be involved in development of cutting-edge imaging techniques.

References

LaJoie D, Ullman KS. 2017 Coordinated events of nuclear assembly. Curr Opin Cell Biol. 46:39-45.
Radulovic M, Stenmark H. 2018 ESCRTs in membrane sealing. Biochem Soc Trans. 46:773-778.
Fiserova J, Spink M, Richards SA, Saunter C, Goldberg MW. 2014 Entry into the nuclear pore complex is controlled by a cytoplasmic exclusion zone containing dynamic GLFG-repeat nucleoporin domains. J Cell Sci. 127:124-36.
Palmer SE, Smaczynska-de Rooij II, Marklew CJ, Allwood EG, Mishra R, Johnson S, Goldberg MW, Ayscough KR. 2015 A dynamin-actin interaction is required for vesicle scission during endocytosis in yeast. Curr Biol. 25:868-78.
Fišerová J, Richardson C, Goldberg MW. 2016 Immunoelectron Microscopy of Cryofixed Freeze-Substituted Yeast Saccharomyces cerevisiae. Methods Mol Biol.;1474:243-58.
de Castro IJ, Budzak J, Di Giacinto ML, Ligammari L, Gokhan E, Spanos C, Moralli D, Richardson C, de Las Heras JI, Salatino S, Schirmer EC, Ullman KS, Bickmore WA, Green C, Rappsilber J, Lamble S, Goldberg MW, Vinciotti V, Vagnarelli P. 2017 Repo-Man/PP1 regulates heterochromatin formation in interphase. Nat Commun. 8:14048.

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