I work as part of the Mike Williamson group (Department of Molecular Biology and Biotechnology) and Kathryn Ayscough lab (Department of Biomedical Science) at the University of Sheffield. I have a BSc in Biochemistry that was obtained in the Molecular Biology and Biotechnology Department of the University of Sheffield. During my third undergraduate year I completed a python-based project. This experience was what encouraged me to apply for a PhD project that included a heavy computational element.
This project shall primarily focus on Las17; the yeast homologue of the human protein WASP. The WASP family play an important role during Clathrin Mediated Endocytosis (CME). Invagination of the membrane during CME relies on actin polymerisation. Las17 can activate the Arp2/3 complex, which can in turn nucleate actin and drive this polymerisation. However, Arp2/3 can only nucleate new actin filaments from the side of pre-existing filaments (termed “mother filaments”). Yeast lacks an extensive cortical actin meshwork and so the process by which these mother filaments arrive to the site of endocytosis (the “endocytic patch”) is not well understood. More recent studies by the Ayscough group have found that the central, proline-rich region of Las17 may nucleate actin. Furthermore, Las17 is recruited to the patch 20 seconds before Arp2/3 and its arrival ‘commits’ the patch to completing endocytosis. This has led to the hypothesis that Las17 not only activates Arp2/3 directly, but also indirectly through nucleating the initial filaments from which Arp2/3 branches.
The central proline-rich region of Las17 contains two arginine pairs and 8 polyproline tracts, whereby a track is defined as a sequence containing five or more consecutive proline residues. These arginines and tracks are now thought to play a role in actin-binding. It is not surprising that many SH3 domains can also bind these tracks considering the important role that proline plays in defining their core binding sites. However, what is interesting is that many of these SH3-containing proteins are recruited to the patch shortly after Las17s arrival. One such protein, Sla1, has been shown to competitively outcompete actin from the two arginine pairs. The composition of this cloud changes throughout endocytic progression as more proteins are recruited to the patch.
A model for Las17 regulation
This information has helped further develop a model for Las17 catalysed, actin nucleation. In this model, Las17 associates with Sla1 in the cytosol to form a complex capable of stably inhibiting actin nucleation (termed the SLAC complex by the Di Pietro group). The Sla1-Las17 interaction may be disrupted upon recruitment to the endocytic patch leaving the polyproline region free. Actin and SH3 domains compete to bind the polyproline tracks. The SH3 domain-contains proteins can be more simplistically thought of as a ‘cloud’ of SH3s. As endocytosis progresses, the composition of the ‘SH3 cloud’ and thus the ‘clouds’ efficacy for inhibition changes. The model suggests that actin may successfully outcompete the SH3 domains at some point during the 20 second interval between Las17 and Arp2/3 recruitment. This system, defined by many competing binding events, may function to control and restrict the activity of Las17 nucleation to prevent unwanted polymerisation in the cell.
The aim of my project is to simulate competition between Las17, actin and SH3 domains to investigate the model outlined above. Agent based modelling retains the positions and each protein throughout the simulation time, allowing spatiotemporal effects to be simulated. Such a method may prove useful considering the complexity of the system and the occurrence of both tandem SH3-domains (e.g. the three SH3 domains of Sla1) and tandem binding motifs (Las17 polyproline tracks). The program will use numerous experimentally characterised binding events to predict their combined effect in the system. Microscale thermophoresis will be one such method used to characterise SH3-Las17 and actin-Las17 binding. This method is capable of measuring the binding affinity of interactions. Expanding our knowledge both of CME-linked nucleators and the regulatory mechanisms that restrict their activity may help further develop our understanding of the endocytic process.