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Dissecting the role of SPIN90 in cellular morphogenesis

Reference	BB/V007483/1
Principal Investigator / Supervisor	Dr Guillaume Charras
Co-Investigators / Co-Supervisors
Institution	University College London
Department	London Centre for Nanotechnology
Funding type	Research
Value (£)	473,701
Status	Current
Type	Research Grant
Start date	01/04/2021
End date	30/09/2024
Duration	42 months

Abstract

Cell shape changes are governed by mechanical changes in the cortex, a submembranous network of actomyosin. Actin filaments within the cortex are produced by two nucleators generating distinct network organisations: the Arp2/3 complex makes dendritic networks of filaments while mDia1 generates linear arrays. One potential mechanism to control cortex mechanics involves regulation of architecture through changes in actin nucleator activity. One class of proteins called Nucleation Promoting Factors (NPFs) is involved in regulating nucleators alongside RhoGTPases. We identified a cortical NPF, SPIN90, that can coordinate the activity of Arp2/3 and mDia1. SPIN90 appears essential for division, profoundly alters the organisation of cortical networks, controls the mechanics of cells and is ubiquitously expressed. In aim 1, I will use proximity labelling to identify interactors of SPIN90. For this, we will fuse SPIN90 to a biotin ligase, TurboID. This enzyme generates a cloud of activated biotin that reacts with proteins in close proximity to SPIN90 (<10nm). After cell lysis, biotinylated proteins are identified by mass spectrometry. SPIN90 depletion leads to cell death and multinucleation but its precise spatiotemporal localization is unclear. Aim 2 will examine localization of SPIN90 in processes such as mitosis and migration using microscopy. Importantly, we will identify when and where SPIN90 interacts with Arp2/3 and mDia1 using bimolecular fluorescence complementation. Our published data demonstrates that depleting SPIN90 leads to an increase in cortical stiffness in metaphase cells that is not due to a change in actin density or phospho-myosin. This suggests that stiffening results from changes in cortex organisation. Aim 3 will use atomic force microscopy and optogenetics to establish how SPIN90 activation induces changes in cortex architecture at the molecular scale. We will use numerical simulation to correlate cortex architecture and mechanics.

Summary

One of the most striking properties of living cells is their ability to change shape to fulfil their function, such as when they divide, migrate, and differentiate. Cell shape changes are governed by mechanical changes that occur in a thin layer of biopolymer situated below the membrane, known as the cortex. Polymers within the cortex are produced by specialized proteins, known as nucleators. Two of these are present in the cortex forming distinct network organisations: the Arp2/3 complex makes arborescent networks while mDia1 generates linear arrays of filaments. Changes in cortical mechanics can originate from changes in motor protein activity or cortex architecture, which arise from changes in polymer length or network organization. While we know a lot about how myosin activity alters cortex mechanics, we know much less about how changes in architecture do. One potential mechanism to control cortex architecture involves regulation of nucleators. Yet, little is known about the molecular mechanisms of coordination between nucleators. One class of proteins called Nucleation Promoting Factors (NPFs) is involved in regulating nucleators. We identified several cortical NPFs that can interact with multiple nucleators, making them prime candidates to mediate crosstalk. One of these, SPIN90, appears essential in division and development, profoundly alters the organisation of F-actin networks, controls the mechanics of cells and its mRNA is ubiquitously expressed. Thus, it may play a general role as an effector of cell shape change. However, we currently know very little about where and when SPIN90 acts in cell shape change, its interactors, and how it induces cortical reorganization. Indeed, to date, only ~30 papers have examined its function. I propose to investigate SPIN90 function during cell shape change focusing on the following aims: 1) Determine the interactors of SPIN90 2) Investigate the spatiotemporal role of SPIN90 in processes involving cell shape change 3) Examine how SPIN90 controls cortical network architecture and cell mechanics My lab is uniquely placed to investigate these questions because of our expertise on the genesis of the cell cortex, cell mechanics, and cytoskeletal organisation. Objective 1 will identify interactors of SPIN90. For this, we will fuse SPIN90 to a biotin ligase, TurboID. This enzyme generates a cloud of activated biotin that reacts with proteins in close proximity to SPIN90. Biotinylated proteins can be isolated and identified by mass spectrometry. Although our previous work showed that removal of SPIN90 led to cell death, where and when it acts during cell shape changes remains unclear. Aim 2 will examine a role for SPIN90 in processes of cell shape change using fluorescence imaging. Importantly, we will identify when and where SPIN90 interacts with each nucleator using approaches in which fluorescence only occurs upon interaction between SPIN90 and one of its interactors. Our published data demonstrates that depleting SPIN90 leads to a significant increase in cortical stiffness in dividing cells. This stiffening may result from changes in cortex organisation. Aim 3 will establish how SPIN90 activation induces changes in cortex architecture at the molecular scale. For that, it will use atomic force microscopy, a technique which enables imaging of the cortex with nm-resolution and characterization of cell mechanics. We will use numerical simulations to determine how changes in cortex architecture correlate with changes in mechanics. In summary, this project will determine how SPIN90 coordinates nucleator activity to control cell mechanics for cell shape change. In addition, we will identify how SPIN90 integrates with other cytoskeleton remodelling pathways during cell division and migration.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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