Mechanisms mediating intracellular sorting of Roundabout

Lead Research Organisation: King's College London
Department Name: MRC Ctr for Developmental Neurobiology

Abstract

We are dependent on our nervous system functioning correctly for us to move, think, learn, speak and control our bodies. To do this all our nerve cells must connect up in the brain and to the parts of the body they control. Most of this 'wiring together' happens during the growth and development of the embryo in pregnancy. To do this each nerve cell must extend a long process, called an axon, over large distances and through complex environments to find and connect to its appropriate partners. Each axon is guided when to turn and which way to grow to reach its partner by sensing specific chemicals or molecular 'cues' in different parts of the body. These signals are detected by 'receptor' proteins at the tip of the growing axon. The activity of these receptors change as the axons grow into different regions, We want to find the molecules that work to control the activity of these receptors as they guide axons along their pathways. We know already that many of the same molecules and receptors in mammals are also present in smaller animals like the fruitfly Drosophila where they do the same job but on a simpler scale. We are using Drosophila to characterise how one of these receptors called Roundabout is regulated in a subset of neurons. We know that Roundabout must be precisely regulated for axons to grow correctly. Although we have identied one key regulator of the Roundabout protein, a protein called Commissureless we do not know precisely how this molecule acts to regulate Roundabout. We plan to use Drosophila as a model system where we can watch exactly how the Roundabout and Commissureless molecules behave in the axons as they grow. This will give us information on the dynamics of the process. We also use Drosophila as a model system to rapidly identify and test the role of further molecules that may act in this process, e.g. those that bind to Commissureless. By using Drosophila we can reduce the need to sacrifice large numbers of mice in research. Once we have found out how these molecules work in Drosophila we will inform other researchers so that the molecules can be tested in other model systems. We need this information both to learn how the nervous system is made and to find out what molecules might be useful in helping us to repair neural injuries or diseases that lead to paralysis or neural degeneration. Unfortunately mammals cannot repair nerve damage that occurs in the brain, our hope is that by identifying the molecules that were originally used to drive and direct nerve cell growth in the embryo we can re-supply these molecules to help nerve cell regeneration in people.

Technical Summary

Cells must accurately regulate their ability to send and receive signals during normal homeostasis and development. Regulation of receptor protein trafficking is an important mechanism, whereby proteins are localised to different compartments or regions in the cell, to modulate their activity. It has been identified in both mice and Drosophila that the regulated spatial distribution of Robo proteinsin commissural axons is crucial for their ability to navigate the midline of the central nervous system (CNS). In Drosophila, Commissureless acts on the Roundabout receptor to regulate its cellular distribution. As neurons approach the midline Commissureless sorts Roundabout to an intracellular location whereas after the axons cross the midline Commissureless activity is reduced and Roundabout reaches the cell surface. Once Roundabout is at the cell surface it acts to receive the midline derived repellent signal Slit. Little is known about how this mechanism is regulated and we propose to study how Roundabout is distributed in the growing axons during the development of the CNS. We propose to examine in more detail the localisation of the proteins in neurons as they approach and cross the midline using time-lapse studies. This will allow us to identify the nature of the intracellular compartment occupied by Commissureless as neurons extend along their pathway and the precise dynamics of Commissureless and Roundabout trafficking. We also propose to identify additional components of this mechanism using an affinity chromatography approach to identify molecules that bind a region of Comm essential for its function. Although it was previously suggested that the ubiquitin ligase Nedd4 may be a key mediator of Comm activity in commissural neurons this has been challenged and we have confirmed that Nedd4 may not be essential for this process. We therefore seek to identify additional proteins that function with Comm to regulate Robo protein distribution.

Publications

10 25 50
 
Description We have discovered that specific molecules are trafficked to and from the surface of nerve cells as they extend towards their targets during the wiring of the nervous system. This allows neurons to make decisions as they grow in order that they connect correctly to from neural circuits or networks. The funding allows us to develop tools and imaging method to visualize the movement and spatial organization of specific molecules in time. This allowed us to investigate candidate molecules that mediate this trafficking. This work was published in two publications.
Exploitation Route Others can use our findings to begin to elucidate the molecular mechanisms that traffic neuronal proteins and how they influence neuronal outgrowth and guidance.
Sectors Pharmaceuticals and Medical Biotechnology

 
Description BBSRC Project Grant (Mechanisms mediating axon outgrowth in the Drosophila CNS)
Amount £419,505 (GBP)
Funding ID BB/K002031/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2013 
End 12/2015
 
Title Drosophila research tools and reagents 
Description Transgenic lines and mutants made available to community 
Type Of Material Biological samples 
Year Produced 2007 
Provided To Others? Yes  
Impact Use in publications 
 
Description Study of axon guidance mechanisms 
Organisation Trinity College Dublin
Country Ireland 
Sector Academic/University 
PI Contribution Ability to study function of novel genes implicated in axon guidance in the Drosophila model system.
Collaborator Contribution Provision of reagents
Impact The extracellular leucine-rich repeat superfamily; a comparative survey and analysis of evolutionary relationships and expression patterns. PMID: 17868438
 
Title Comm as method to modulate neuronal function 
Description The amount of active Robo expressed on a cell is modified by modulating the effective amount of a Comm polypeptide in contact with the cell, whereby the amount of expressed active Robo is modulated inversely with the modulation of the effective amount of the Comm polypeptide in contact with the cell. In a particular embodiment, the Comm polypeptide is provided to the cell by exogenously in a pharmaceutically acceptable composition. In another aspect, the invention provides methods of screening for agents which modulate Robo-Comm interactions. These methods generally involve forming a mixture of a Robo-expressing cell, a Comm polypeptide and a candidate agent, and determining the effect of the agent on the amount of Robo expressed by the cell 
IP Reference WO1999025833 
Protection Patent granted
Year Protection Granted 2006
Licensed Commercial In Confidence
Impact None thusfar
 
Description School Visit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach Local
Primary Audience Schools
Results and Impact Presentation to Year 10, 11, 12 students on scientific research and career opoortunities in science.

Increase in knowledge of what a career in science involves for a group of school children.
Year(s) Of Engagement Activity 2006,2007,2008,2009
 
Description Science Week Presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Approximately 120 students, parents and members of public attend a science week presentation at a sixth form school. The presentation was followed by a lively question and answer discussion at which school pupils and members of the public participated. The teachers present reported a great level of interest from the pupils, while I was personally thanked by several memners of the public on an interesting and engaging presentation.
Year(s) Of Engagement Activity 2017
 
Description University Open Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Open day discussing research opportunities to undergraduate students
Year(s) Of Engagement Activity 2013,2014,2015