Organisation of actin waves and cups by differential GTPase activity

The cells that make up all organisms are highly dynamic, and often must change shape to perform their function. This is achieved through their cytoskeleton, where individual molecules of a protein called actin can be assembled (polymerised) into filaments to generate force and push the cell surface outwards. We are interested in the mechanisms that regulate where and when actin is polymerised to produce protrusions with specific functions.

In particular, we are interested in a process known as macropinocytosis, where cells use actin to extend cup-shaped structures that can capture and internalise large volumes of fluid from their environment. Cells need to do this for many reasons. The most fundamental is to feed, capturing nutrients to sustain growth, which is used by both simple organisms such as amoebae, as well as cancer cells. In higher animals, macropinocytosis has also been adapted by immune cells to sample their environment and detect foreign bodies and both viruses and bacteria can also exploit macropinocytosis as a way into host cells.

Remarkably, the cup-shaped protrusions required for macropinocytosis occur spontaneously on the cell surface and self-assemble without any external spatial signals or template. How this is achieved is a major unanswered question and the main focus of this proposal.

Similar structures can also form on the bottom surface of the cell. However, because they are physically restricted by the surface below, they propagate as flat waves until they eventually collide with the cell periphery. There, they generate a protrusion that drive cell migration. Both macropinocytic cups and these basal waves have comparable structure and dynamics, so we propose they form by the same mechanisms. By studying both structures in parallel, we will identify the general principles that dictate how actin is coerced to polymerise in specific locations to make specific structures.

Previous work has suggested that cups form by generating a patch of a specific phospholipid (PIP3) on the cell surface that is able to direct actin polymerisation to its periphery. This generates a ring of protrusion, providing a mechanism to extrude a cup shape. An identical organisation in 2D is observed in basal waves. Restriction of actin to the edge of the PIP3 domain is fundamental to form both structures, but how this is achieved is completely unknown. Identifying this mechanism is our main objective.

Our previous work led us to propose a new model to generate a ring of actin polymerisation, based upon the relative activities of two different regulatory proteins: Rac, which is an activator of actin polymerisation, and Ras which we propose leads to inhibition. We observed that whilst both Ras and Rac activities coincide with the PIP3 domains observed in cells, Rac alone extends slightly further. Our main hypothesis is that this peripheral ring where only Rac is active defines where actin polymerises and generates the cup shape and basal waves. We will test this model and determine the mechanisms by which Ras and/or PIP3 inhibit actin polymerisation in the centre of the cup.

To achieve this, we need to take advantage of the latest advances in microscopy. This will allow us to study these rapidly moving and highly dynamic structures in 3D for the first time. This new technology also requires new analytical methods, so an important part of this project is to develop novel computational tools that will be of general use to the scientific community as well as providing important new insights into how cups form.

Combined, this work will provide new general insights into how protrusions are organised with specific relevance to macropinocytosis and cell migration.

Our main aim is to understand how actin-driven protrusions are coerced into specific shapes. Specifically, we focus on the formation of macropinocytic cups that enable cells to engulf extracellular fluid. This serves diverse roles in both normal physiology and disease; it allows immune cells to survey their environment for antigens but also provides nutrients to cancer cells and allows pathogens and prions to spread.

The dynamics of macropinocytic cups appears identical to the travelling actin waves that spontaneously form on the cell base and have been proposed to drive migration. This indicates a universal mechanism, but how they self-organise without any spatial cues is unknown. By studying cups and basal waves in parallel we will uncover the underlying general principles.

The common key feature is the formation of a ring of actin encircling a central membrane domain of active Ras and phosphatidylinositol(3,4,5) trisphosphate (PIP3). How this is achieved is completely unknown and is our main question. Our previous work demonstrated that Rac, an upstream regulator of actin polymerisation, overlays the Ras/PIP3 domain but extends 1-2um further. This has led to our primary hypothesis that the protrusive ring is defined by the annulus where only Rac is active.

We will test this model and identify the molecular mechanisms that prevent actin polymerisation in the core Ras/PIP3 domain. This will be achieved by both cutting-edge 3D imaging of cellular models and in vitro reconstitution allowing us to uncover how signals coordinate in intact cells, then biochemically test the mechanism and identify new components.

Our focus is actin organisation, but both Ras and Rac have important additional roles and are implicated in several diseases. How these different small GTPase families functionally interact is poorly understood. We therefore hope to provide an important advance in GTPase crosstalk, in addition to the fundamental mechanisms that shape protrusions.