A lattice light-sheet microscopy-informed mathematical model of macropinocytosis

Session

Microscopy to Modelling

Authors

Judith Lutton (5), Helena Coker (3), Peggy Paschke (1), Jason King (4), Till Bretschneider (5), Robert Kay (2)

Affiliations

1. Beatson Institute for Cancer Research
2. MRC Laboratory of Molecular Biology
3. University of Oxford
4. University of Sheffield
5. University of Warwick

Keywords

Macropinocytosis, Mathematical modelling, Light-sheet microscopy, Cell biology

Abstract text

Macropinocytosis is a process by which cells can take in the surrounding medium by forming concave structures, known as cups, on the cell surface membrane and sealing off the cups to form vesicles inside the cell. This process provides a mechanism for antigen uptake in immune cells and feeding in cancer cells¹. Despite its medical importance, there are many unanswered questions on the mechanisms underlying macropinocytosis, including how cups form and close. We address these points by analysing microscopy movies of this process in Dictyostelium cells. The result of this analysis suggests a mechanism for cup formation and closure, which we test using a mathematical model informed by the microscopy results.

 

Dictyostelium cells expressing fluorescent reporters for actin and its regulators were recorded undergoing macropinocytosis on a lattice light-sheet microscope. Utilising lattice light-sheet microscopy enabled us to capture cells at high 3D spatial ( 0.104 µm in-plane pixel size, 0.162 µm plane separation) and temporal (1-3 seconds per frame) resolutions. A custom-built image analysis pipeline, including steps for whole-cell segmentation² and cell surface analysis was used to identify and analyse PIP3-rich domains on the surface, which are known to be present in the membrane inside the cups³. This pipeline enabled us to plot fluorescence distributions within the cup, providing insight into the localisation of the reporters during macropinocytosis. Additionally, the cup geometry was measured over time to characterise the physical changes in the cup, from formation to closure.

 

The key results from these experiments were (a) increased presence of actin polymerisation agents at the lip of the cup, (b) persistently elevated levels of actin present along the length the cup, and (c) when a PIP3 domain stops expanding, the cup lengthens and the lip comes together to close the cup. In addition, two distinct mechanisms of closure were observed to occur at similar rates: lip closure, where actin polymerisation at the lip of the cup drives the membrane together; and base closure, where a vesicle forms below the lip without the direct action of actin polymerisation.

 

These results allowed us to construct a mathematical model of the cell membrane, based on a model previously developed to investigate bleb formation⁴. The model uses a 2D contour to represent the cell membrane, subject to forces resulting from the Helfrich model for membrane bending. Macropinocytosis is modelled by applying an actin polymerisation force driving the membrane outwards at the edge of a simulated PIP3 domain. The PIP3 domain is modelled as an area on the surface that expands from a point until stopping with a maximum area. Domain area is calculated by assuming rotational symmetry about the centre of the domain. From this simplified formulation, the model replicate lip closures, and produces the conditions that allow base closure to occur. Additionally, our model predicts that changes in membrane tension could be a mechanism for determining if and how a cup can close.

References

1. Bloomfield, G., and Kay, R.R. (2016). Uses and abuses of macropinocytosis. J Cell Sci 129, 2697-2705. 10.1242/jcs.176149. 

2. Lutton, E.J., Collier, S., and Bretschneider, T. (2021). A Curvature-Enhanced Random Walker Segmentation Method for Detailed Capture of 3D Cell Surface Membranes. Ieee T Med Imaging 40, 514-526. 10.1109/Tmi.2020.3031029. 

3. Araki, N., Johnson, M.T., and Swanson, J.A. (1996). A role for phosphoinositide 3- kinase in the completion of macropinocytosis and phagocytosis by macrophages. J Cell Biol 135, 1249-1260.

4. Collier, S., Paschke, P., Kay, R.R., and Bretschneider, T. (2017). Image based modeling of bleb site selection. Scientific reports 7, 6692. 10.1038/s41598-017- 06875-9.