How the ESCRT-III-like protein Vipp1 assembles polymeric super-structures to mitigate membrane stress

In all living systems, membranes are used to separate the inside of the cell from the outside environment. Membranes are also used to shape cells internally so that different areas can form specialist compartments with distinct roles. In cells, membranes are dynamic requiring continual remodelling for many processes including cell division for growth or membrane trafficking for the movement of cargo. In order to remodel the membrane, cells have evolved specialist protein families to undertake this physical work.

One of the most important membrane remodelling families are ESCRT-III proteins. They are universal in eukaryotes (cells like our own). ESCRT-III proteins are so ancient that they have ancestors in some archaea from which eukaryotes later evolved. Recently, in an exciting discovery, we showed that ESCRT-III proteins also exist in bacteria (PspA) and in cyanobacteria (Vipp1). This is important as it showed that an ESCRT-III-like protein was present in the last universal common ancestor of all cells (LUCA) and that all evolutionary domains including bacteria, archaea and eukaryotes have depended on ESCRT-III-like proteins to shape membrane since the earliest attempts at life.

ESCRT-III-like proteins undertake many essential functions. In humans, they are essential for the final separation of dividing cells and membrane repair. They are also implicated in many diseases including viral invasion, bacterial infection, cancer and neurodegeneration such as dementia and Huntington's disease. Due to its role in membrane protection, PspA is a driver of anti-microbial resistance (AMR) and bacterial pathogenesis.

In this proposal we study Vipp1, which is found in all cyanobacteria, algae and plants. We know that Vipp1 is important as gene knockout is usually lethal. This is due to abnormal formation of the thylakoid membranes where photosynthesis is undertaken. What we still do not know is what Vipp1 does in the cell and what its membrane remodelling duties are. Currently, we think that Vipp1 proteins assemble together to build superstructures that include rings, helical filaments and flat scaffolds that somehow shape and support membrane possibly in regions of high stress where the integrity of the membrane is physically or chemically threatened. The overall goal of this proposal is to understand the mechanism for how Vipp1 builds these superstructures and uses them to do mechanical work on the membrane. Vipp1 also represents a tractable system which can show us the universal mechanistic principles underlying how PspA and more complicated ESCRT-III systems work and cause disease. Finally, Vipp1 modification in engineered cyanobacteria facilitates high yields of fatty acids for both nutritional and anti-inflammatory use. In future biotechnological application, similar Vipp1 modification may facilitate the production of other useful molecules such as biofuels in cyanobacteria.

Aims:

1) to understand how Vipp1 builds and switches between different superstructures so as to shape, stabilise and repair membrane. Specifically, a powerful form of electron microscopy will allow us to visualize the precise position of the Vipp1 atoms within helical filaments so we can learn about their 3D structure and chemistry. 2D planar filament architecture when attached to membrane will be deduced at lower resolution. Understanding how Vipp1 builds different structural forms lies at the heart of its membrane remodelling capabilities.

2) to explore how Vipp1 superstructures have the ability to sculpt membrane in a simplified 'in vitro' environment. By mixing Vipp1 with both membrane and Vipp1 binding proteins (VBPs), we aim to reconstitute any membrane cutting, joining or stabilising events that may represent what Vipp1 does in the cell.

3) to find other proteins in the cell that attach to Vipp1 and changes how it functions. Such VBPs may shift the way Vipp1 builds or disassembles superstructures and how it remodels membrane.

Membrane remodelling and repair are essential for all cells. Systems that undertake these functions include Vipp1/IM30 in photosynthetic plastids, PspA in bacteria, and ESCRT-III in eukaryotes. In an exciting discovery, we recently showed that Vipp1, PspA and ESCRT-III form an ancient membrane remodelling superfamily with broad function, from membrane repair in bacteria, cell division in archaea to endosome biogenesis in humans. Understanding how these proteins work has broad implication for cyanobacteria/plant biology and biotechnology, and human pathologies such as viral infection, antimicrobial resistance (AMR), and neurodegeneration.

Here we aim to understand the mechanism that underlies Vipp1 membrane remodelling in cyanobacteria. We aim to provide a molecular context for how Vipp1 stabilises membranes, mitigates membrane stress and promotes thylakoid biogenesis. ESCRT-III systems are complex with multiple components often forming composite filaments. Vipp1 represents the exciting potential of a readily tractable model by which to dissect the conserved and underlying mechanistic principles for how ESCRT-III-like proteins sculpt and exert force on the membrane.

We have three aims that utilise electron and light microscopy, biophysics and biochemistry:

1) to understand how Vipp1 builds polymeric superstructures such as helical and planar filaments, and how Vipp1 morphs between them. The comparison of these superstructures will give insight into how ESCRT-III-like proteins flex, bind and deform membrane.

2) to describe the dynamics of Vipp1-mediated membrane remodelling. A simplified cell-like system will be reconstituted in vitro with Vipp1 mixed with Vipp1 binding proteins and membrane, and the effects quantitatively measured.

3) to investigate how Vipp1 is regulated by finding novel VBPs in Nostoc punctiforme. We aim to test the hypothesis that Vipp1 may form composite filaments and co-complex with NTPases to modulate assembly dynamics.