Reconstructing cell surface dynamics from lightsheet microscopy data

Recent advances in light microscopy have made it possible to acquire fast high-quality 3D scans of single cells. The new techniques are so gentle that live cells can be followed over long periods of time without disrupting their normal behavior. The possibility to accurately reconstruct the outer cell envelope in 3D opens a new window on two distinct biological processes we are investigating, where the cell envelope bulges either outwards or inwards, in a very distinct manner.

During blebbing, the first of these two processes, the cell envelope rapidly bulges outwards in form of a blister. It is an important mechanism found in cell locomotion, where blebs are directed to the cell front, for example in immune cells chasing pathogens, spreading tumour cells, and during development of organisms.
Macropinocytosis, the second mechanism we are investigating, is the technical term for cell drinking. The outer cell envelope bulges inwards to form a cup like structure. In a process that is not yet understood, the lip of this cup closes, and fluid that previously surrounded the cell is being internalized. Macropinocytosis plays important roles in taking up nutrients for feeding, supporting for example the high demand of fast growing tumour cells. It also is relevant for sampling antigens by immune cells, and can be exploited by pathogens to invade cells. Contrary, it can be exploited as a route of entry for drugs into cells.

Blebbing and macropinocytosis share commonality in that they both involve the shaping of the cell envelope under physical forces that are generated by molecules interacting with the envelope. These molecules belong to what is called the cellular cytoskeleton, a structure that is not static but needs to be constantly remodeled. Using a similar approach to tackle both problems, we will acquire detailed 3D scans of the cell envelope and image components of the cytoskeleton and its regulators that have previously been shown to play a role in blebbing and macropinocytosis.

Advanced image analysis will then be used to generate spatial maps of the molecular events and deformations of the cell envelope, and how they change over time. These maps will be used to construct mathematical and computer models that help us to infer how molecular events relate to forces acting on the cell envelope. Previous models developed by us successfully predicted where on the cell envelope blebs are going to form, but lack of high quality 3D image data prevented us to enquire further how blebs grow and stop for example.

We will take advantage of the new, fast 3D microscopy to investigate the mechanisms that lead to the bulging out or of the bulging in, of the cell envelope in Dictyostelium discoideum. Dictyostelium discoideum is an amoeboid type of non-animal cell in which blebbing and macropinocytosis have been well studied. Asking whether one of our previous discoveries, namely that bleb site localisation in Dictyostelium largely depends on cell geometry, also applies to animal cells, we will conduct a small study in blebbing cells during early development of zebrafish, where blebbing is known to play an important role.

Long term goal of the project is to contribute to a better understanding of how the dynamic cytoskeleton can be programmed to fulfill so many different functions, ranging from cell locomotion and cell eating, to the immune response. And, how we could exploit this, for example to prevent entry of pathogens into cells or enhance the uptake of drugs.

Cell membrane deformations underpin diverse cellular functions such as bleb driven amoeboid cell locomotion, or the take-up of fluid through macropinosomes. Light-sheet microscopy opens a new window on these large-scale and relatively fast-moving cellular structures, which previously were difficult to follow over time by confocal or spinning disc microscopy because of speed, resolution or photo-toxicity limitations. Here, we will combine dual-view inverted selective plane illumination microscopy (diSPIM), advanced image analysis and statistical modelling, to determine the signaling, cytoskeletal, geometric and physical mechanisms that shape blebs and macropinocytic cups. Dictyostelium discoideum is an excellent model to study the latter two.

We previously identified a new mechanism where blebs are induced in regions of negative curvature. Image based modelling successfully predicted where blebs form, but lack of high-quality 3D image data denied answers to important questions like, what stops bleb expansion, or what determines the exact timing of blebs? diSPIM provides 3D cell scans in unprecedented quality to answer these questions, and develop advanced 3D biophysical models to infer the forces acting on the membrane. Asking whether curvature dependent blebbing applies to animal cells, too, we will complement the work with a study of blebbing cells during zebrafish gastrulation.

We will map reporter dynamics and membrane evolution during macropinocytosis, and analyze the dependency of key regulatory events using selected signalling and cytoskeletal mutants, and inhibitors. We will distinguish between mechanisms for cup closure by analysing myosin-II recruitment to the cup lip and effects of myosin-II knockouts, and measuring the actin distribution in cup walls. Using the same modelling approach as will be developed for blebbing, we will attempt to explain the physical forces acting on the cell membrane during different stages of cup formation and closure

We are fortunate to have access to one of currently only three of the first generation of commercially available Dual Inverted Selective Plane Illumination Microscopes (diSPIM) in the UK. diSPIM makes it possible to image live cells in 3D over long periods of time, in unprecedented quality, and is a major step change when compared to current laser scanning or spinning disk confocal microscopy. Thus our project will draw attention from other institutions and facilities who follow latest trends in imaging technologies, and potential users.

Main stakeholders to benefit from our research are:


Microscopy companies
3i - Intelligent Imaging Solutions producer of the first commercially available diSPIM microscopes will as part of our project develop new technology to enhance the current platform. Thus our project will directly contribute to development of new microscopy products for the market, potentially resulting in the generation of intellectual property, new business and new jobs.

Employers in IT, pharma and academia
We will train postdoctoral researchers in a cross-disciplinary environment which will acquire expert knowledge of biology and a diverse set of skills in computational image analysis, 3D visualization and modelling, as well as in transferable skills. These skills are highly sought after in a number of employment sectors, such as IT, pharma and academia.

Biomedical researchers and medical doctors
Macropinocytosis, one of the processes we are studying under the current grant, has huge implications for drug uptake and cancer cell biology. The Kay laboratory is already actively collaborating on macropinocytosis with scientists in the pharma sector. We expect that our work will be influential in setting research objectives and strategies in this area.

Generation and exploitation of intellectual property
Through development of novel software for 3D cell imaging we will generate intellectual property that will be freely available to academic users. We will employ a dual license scheme with the potential to generate revenue from commercial users.

Computer Science in Warwick and beyond
The project will be a great test bed for working with large volumes of new 3D image data in a production environment. We will actively seek new collaborations with computer science experts in data storage, image compression, and high performance computing to tackle the challenges involved in handling of this data. The project will be very attractive for Computer Science 3rd year and MSc project students to work on selected problems in a real world application.

Communications / Outreach
Results of our research will be disseminated through journal publication, presentations at conferences, and press releases. We will engage with the public through Warwick's very successful Christmas lectures and Warwick 'Computer Science Ambassadors', which will present our work in schools to attract the next generation of researchers. Images and analysis from this project will make ideal material, illustrating how biological science and computer analysis have converged.