Tubulins are conserved cytoskeletal proteins with essential roles in intracellular transport, cell division, cell migration and neuronal synapse connectivity.Humans have multiple tubulin genes, and different isotypes. Microtubules are assembled from dimers of α- and β-tubulin. Although proteins within the tubulin superfamily show a high degree of similarity, the phenotypic differences associated with variants in the various tubulin isotypes suggests that each tubulin has a distinctive function. Mutations in human tubuins have been found in patients with a range of brain malformations and other deficits, and are collectively known as tubulinopathies. De novo mutaions in TubulinA3D were found in patients with the degenerative eye disease, Keratoconus.How TubA3D functions in the eye is not understood.In this project, the student will generate corneal cell culture, organoid and animal models of TubA3D mutations and study progression of the disease through high resolution live imaging. The findings from this project have the potential to lead to early diagnostic tests which can aid the development of new therapeutic interventions for the disease.

• Genome editing in zebrafish and cell cultur
• Analysis of zebrafish mutant embryos and cultured cells/organoid
• High resolution in vivo time lapse imagin
• Computational analysis of imaging data

Understanding cell biology means exploring subcellular organization in 3D and locating important proteins at high resolution. Correlative light-electron microscopy (CLEM) is a powerful technique to do this, since we can combine the specificity and dynamics of fluorescence light microscopy (LM) with the high resolution and cellular context of electron microscopy (EM). We are developing genetically encoded tools that allow us to track intracellular events using CLEM. This project will apply our existing tags to new cell biological questions and develop the next generation of CLEM tools. Our lab is interested in several cell biological processes and the probes are useful for all of them. These processes include: cell division, membrane trafficking and cell migration and invasion in cancer.

During your PhD you would:

1.Apply FerriTagging technology to track cellular events by live-cell fluorescence microscopy and visualisation by electron microscopy.

2.Make cell biological discoveries. Our primary interests are in microtubule-associated proteins during mitosis and proteins involved in membrane trafficking.

3.Develop new genetically encoded probes for CLEM that are fluorescent and electron-dense.

In this project you will primarily use multi-modal imaging (correlating light microscopy and electron microscopy images) and cell culture. The development of the probes will require molecular biology and cell manipulation methods. All projects in the lab involve quantitative unbiased analysis using automated computational methods.

Mechanical (physical) forces play a critical role in many physiological processes during development and adult life (e.g. wound healing), and in various diseases (e.g. cancer). Forces can regulate changes in cell size, shape, movement and proliferation and are therefore essential drivers for organ/tissue morphogenesis and patterning during embryonic development. Cell fate decisions in the embryo are typically regulated by morphogens and transcription factors causing modification of genes characterizing distinct cell identities. Cell proliferation is usually tightly controlled through the cell division cycle and transcription factors which can respond to diverse signals such as DNA damage, growth factors and cell density. However, recent findings in cultured stem cells and in vivo suggest that mechanical signals have profound effects on cell fate and cell division decisions. Notably, it was shown that extrinsic forces can be transduced from the membrane to the nucleus thereby modulating processes such as chromatin remodelling and gene expression. However, the precise mechanisms of how forces impact on cell fate specification and cell division regulation remain unclear. Zebrafish embryonic stem (ES) cells have proven to be an excellent model system to study cell mechanosensing and response due to the sensitivity to mechanical stimuli and the versatility of genetic lineage markers available. This project aims to investigate the molecular and cellular mechanisms controlling cell fate and division decisions in pluripotent ES cells under the influence of extrinsic forces. The project will lead to a better understanding of how physical cues can regulate cell fate and division decisions during embryonic development and will enhance our knowledge of what strategies cells use to drive developmental programs under physiological and pathological conditions.

To address these questions, you will use interdisciplinary methodologies from molecular, cell and developmental biology, live imaging, biophysics and computational tools to observe and quantify processes in vivo and ex vivo.

Following methodologies and skills could be acquired in this project: 1) Training to work with a model organism (zebrafish); 2) molecular biology: working with DNA and mRNA; 3) microinjection of 1-cell stage zebrafish embryos; 4) live imaging of biological processes in vivo on different scales using advanced microscopy, such as spinning disc, confocal and multiphoton microscopy; 5) isolation of ES cells from the embryo for ex vivo culturing; 6) biophysical cell manipulations using a dynamic cell confiner and cell stretcher; 7) transcriptomics, RNAseq; 8) computational image processing and analysis using appropriate software such as Fiji and Matlab; 9) Quantification of results using statistics software (R, Prism or similar).

The vertebrate skeletal muscle forms in distinct segments during development. Each segment consists of striated and aligned muscle fibres. The muscle fibres themselves are differentiated into fast and slow muscle populations. Defects in formation of the skeletal muscle results in a plethora of human diseases. Yet, our understanding of how muscle forms into a very robust structure remains poorly understood.

In this project, we will take advantage of the imaging capabilities of zebrafish to generate live movies of skeletal muscle formation. We will focus on two important questions: (1) what role does actin play in formation of skeletal muscle; and (2) how do the different cell populations interact to ensure robust spatial organisation of the tissue?

For the first part, we have exciting provisional data on how actin dynamics evolve during the process of skeletal muscle formation. There appear to be distinct modes of actin action, that combined ensure precise tissue formation. The student will build on these results in a range of suitable mutant backgrounds. In the second part, we have recently published single cell tracks of how the cell fibres move during skeletal muscle formation (Mendieta-Serrano et al. Dev Cell 2022). However, how the different fibre types interact mechanically remains poorly understood. We will dissect the 3D cell shape changes during the muscle fibre rearrangements to build up a more thorough understanding of how the cellular mechanical

environment adapts during muscle formation. Relatedly, we will quantify changes in nucleus shape and correlate this with cell behaviours.

This PhD project offers an exciting opportunity to leverage recent advances in live imaging and genetics to gain unprecedented insights into how a critical organ forms during development, with potential impact on human development.

This project is a truly interdisciplinary project, ideally suited for either (a) a trained biologist interested in developing their expertise in quantitative approaches or (b) a physicist or engineer (preferably with some optics experience) interested in applying the latest microscopy approaches to important biological systems.

Methodologies required as part of the project:

1)Zebrafish genetics. This will include learning to perform crossing and injections.

2)Live imaging. We will utilise spinning disc, multiphoton microscopy and lattice light-sheet microscopy to gain a subcellular view of how the muscle initially forms

3)Image analysis. Taking data from our microscopy and generating quantitative data is essential. In particular, we want to understand the specific developmental time when morphological changes occur

4)Mechanobiology approaches to understanding cell function and morphology

The Saunders lab is a highly interdisciplinary environment, with biologists and physicists working together to tackle major questions concerning how organs form. This project offers a motivated student the opportunity to learn a breadth of techniques in quantitative biology that have broad applicability.

Microtubules are one of the key components of the cellular skeleton, providing both structural integrity and serving as the tracks for intracellular long-distance transport. Microtubules also form the mitotic spindle and their dynamic assembly and disassembly drives chromosome segregation. Thus, microtubule are dynamically rearranging polymers that form different arrays to fulfil vitally important tasks in the cell. My lab studies how microtubule arrays self-organise with the help of motorised and passive crosslinking proteins in the context of motile cells and differentiating skeletal muscle cells. We use a combination of quantitative live cell imaging approaches and biochemical experiments to directly test mechanistic hypotheses.

Your PhD project could address one of the following questions:

(1)How is the number and position of microtubule minus ends controlled? This is an open question in skeletal muscle cells, which do no longer have a centrosome and nucleation of new microtubules plays an important role in rearranging microtubule arrays.

(2)How are actin and microtubule arrays integrated? In skeletal muscle cells both cytoskeletal filament systems form paraxial arrays, how they influence each other is largely unknown. Testable hypotheses are that microtubule assembly could follow actin bundles or that actin could be transported and aligned along microtubules.

(3)How is microtubule dynamics regulated at the cell cortex? Selective stabilisation of microtubule ends at certain regions of the cell cortex – such as at the protruding edge in migrating cells or the tips in skeletal muscle cells – is thought to contribute to the formation of polarised microtubule arrays. Careful quantitative observation and identification of the cellular machinery controlling this process is required to understand both microtubule array organisation itself and its interdependence with cell shape.

Any of the three projects will enable the better understanding of how microtubule arrays are formed and maintained in healthy cells and allow us to identify what goes wrong in patients with muscular dystrophies that show abnormal microtubule density and organisation.

You will culture human cells and generate genome-edited cell lines either to tag endogenous proteins with fluorescent markers or to manipulate protein expression (i.e. generate knockouts or make mutations). You will use a variety of live cell imaging techniques from widefield fluorescence to lattice light sheet microscopy to record multi-colour timelapse images and use image analysis tools to segment, track and quantify imaging data. Depending on the project, you might also do biochemical and biophysical assays using purified proteins.

For students with a background in mathematics or physical sciences there are many opportunities to apply their quantitative skills to these research projects and develop mathematical models and/or new analysis tools.

Macrophages are highly migratory immune cells capable of engulfing and removing dead cells, debris and pathogens. Macrophages, also known as hemocytes constitute 95% of the immune cell population in Drosophila Melanogaster, are highly migratory and bear striking similarities to their mammalian counterparts in migratory behaviour, function and ontogeny¹ making it an excellent model system to understand embryonic macrophage migration. Sterile injuries in Drosophila embryos result in hemocyte recruitment to wounds in a process which is highly similar to vertebrate inflammatory response and are involved in phagocytosis and removal of apoptotic cells. Hemocytes also secrete a large repertoire of extracellular matrix proteins such as Collagen and Laminin which are essential during embryogenesis. Thus hemocytes are integral during embryogenesis and essential during adult life similar to vertebrate macrophages. Our lab is currently interested in understanding the contribution of hemocytes to organogenesis as well as repair and regeneration using interdisciplinary approaches integrating cell biology, genetics, biophysics and cutting-edge microscopy. Vertebrate macrophages are known to be highly effective at performing different and sometimes even opposing functions depending on the context; an example is the pro and anti-tumor functions exhibited by macrophages. We believe that understanding how Drosophila hemocytes respond to different physiological and pathological conditions would enable us to better understand and devise better intervention strategies to counter or enhance vertebrate macrophage functions during disease.

This project will study the mechanistic details of how macrophages are recruited to specific sites and work to understand the hierarchy and kinetics of macrophage functions at these sites. We will aim to answer these overarching aims through these specific objectives:

1.Understand the biophysical and biochemical cues that drive macrophage recruitment to developing organs and sites of repair. 2. Decipher the functional differences exhibited by hemocytes in diverse environments such as developing kidneys and sites of wounding and repair.

Drosophila genetics, optogenetics, live imaging of embryos using a confocal and two photon microscope, FACS sorting, ex vivo migration assays, biophysical manipulations, quantitative image analysis and mathematical modelling.