Rab proteins, microtubule motors and the organisation of the endocytic pathway

Lead Research Organisation: University of Manchester
Department Name: Life Sciences

Abstract

The behaviour of cells within a tissue is controlled by their environment. Amongst the most important signals that cells receive are in the form of circulating small proteins called growth factors. These bind to specific receptors that are found on the surface of cells. Binding of growth factors causes the receptors to alter their pattern of interactions with many molecules inside the cell that control cell growth. In this way growth factor receptors act as essential bridges between the cell exterior and interior to stimulate proliferative, or mitogenic responses. In order to prevent such mitogenic responses continuing endlessly, which would lead to uncontrolled cell division, the growth factor receptor must be inactivated shortly after the growth factor binds. This is achieved by removing the activated receptor from the cell surface and sending it to a specialised compartment within the cell, where it can be degraded. This compartment is called the lysosome. Movement of the receptor from the cell surface to the lysosome involves the receptor being sequestered into regions of the cell surface membrane that invaginate and pinch off to form spherical packages, or vesicles, within the cell interior. These vesicles move in a directed fashion to the lysosome, via a number of intermediate compartments. The situation becomes more complicated when it is realised that the route that growth factor receptors take to the lysosome is also followed part of the way by other types of receptor, which are engaged in taking up nutrients. These receptors are returned to the cell surface from the intermediate compartments so that they can participate in further rounds of nutrient uptake. Hence, at a critical point along the pathway towards the lysosome, these receptors are selected away. The aim of this project is to understand how this diversion takes place.

Technical Summary

Cell surface receptors that have been internalised are sorted within the early endosome, either entering a recycling pathway back to the cell surface or being transported to the lysosome in order to be degraded. It is essential that this separation occur efficiently, to minimise the loss of recycling receptors to the degradative pathway. Transport to the lysosome is characterised by the maturation of the vacuolar regions of early endosomes into late endosomes, which are able to fuse directly with the lysosome. This project will explore the possibility that a sensing mechanism helps delay this maturation until all recycling receptors have been delivered from the vacuolar endosome into associated recycling tubules, thus ensuring that none of these receptors enter the lysosome. We will focus on two key aspects of this potential regulatory step.
Firstly, since Rab GTPases are fundamental to organising functional domains within the endocytic pathway, we will examine whether there is co-ordination between Rab proteins participating in lysosomal transport and those involved in recycling. Specifically, we will examine whether vacuolar endosomes lose Rab5 and acquire Rab7, an event linked to their maturation to late endosomes, only after Rab4 and Rab11, which specialise in recycling, have been released into neighbouring membranes. Assuming this is the case, we will test whether Rab4 and Rab11 either directly or indirectly control the activation and membrane recruitment of Rab7.
Secondly, we will investigate the ability of microtubule motors to maintain close spatial links between the recycling and degradative pathways and thereby maximise the efficiency of cargo sorting. To this end, we will investigate which combination of plus and minus end-directed microtubule motors are responsible for the movement and localisation of each of the endosomal Rab domains. Based on our preliminary findings that cytoplasmic dynein moves Rab5-positive endosomal vacuoles to the perinuclear region, we will investigate whether dynein is also responsible for localising Rab4 and Rab11-positive membranes to this region. We will then examine which plus end-directed microtubule motor is responsible for moving Rab4 and Rab11-positive membranes to the periphery, concentrating in the first place on the activities of kinesin 1 and kinesin 2.
These collaborative studies, based on preliminary investigations and our extensive experience of endosomal trafficking and microtubule motors, will rely heavily on our proven ability to image endocytic trafficking in live cultured cells at very high temporal resolution in multiple fluorescence channels.

Publications

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Allan VJ (2011) Cytoplasmic dynein. in Biochemical Society transactions

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Brownhill K (2009) Molecular motors and the Golgi complex: staying put and moving through. in Seminars in cell & developmental biology

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Granger E (2014) The role of the cytoskeleton and molecular motors in endosomal dynamics. in Seminars in cell & developmental biology

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Kenwright DA (2012) First-passage-probability analysis of active transport in live cells. in Physical review. E, Statistical, nonlinear, and soft matter physics

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Rogers SS (2010) The first passage probability of intracellular particle trafficking. in Physical chemistry chemical physics : PCCP

 
Description BBSRC project grant; dynein mechanochemistry
Amount £600,000 (GBP)
Funding ID BB/H017828/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2010 
End 10/2013
 
Description MRC Research Grant (Dynamics and Function of Early Endosomes)
Amount £822,000 (GBP)
Funding ID G0900930 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 11/2009 
End 04/2014
 
Title Colocalisation software 
Description This is a new method to analyse fluorescence images for object-based colocalisation of structures. The analysis uses PolyParticleTracker to identify all structures in each fluorescent channel with an accuracy of approximately 20 nm. The colocalisation software then identifies the proportion of each particle within a channel that colocalises with particles in other fluorescent channels. The analysis allows the user to set parameters such as pixel distance thresholds between particles. The analysis allows the user to perform colocalisation analysis on several thousand particles. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact We have used the tool to analyse the distriction and colocalisation of subpopulations of early endosomes. 
 
Description Physics collaboration 
Organisation University of Manchester
Department School of Physics and Astronomy Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution We have imaged GFP-Rab5 endosomes under various experimental conditions in order to generate large datasets for Dr Waigh and his colleagues to analyse.
Collaborator Contribution Dr Waigh and his colleagues have provided software for tracking particle motion and have generated novels ways of analysing particle motion in cells.
Impact 1 research paper
Start Year 2009
 
Description Tom Waigh 
Organisation University of Manchester
Department School of Physics and Astronomy Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution Using GFP-Rab5 to label early endosomes, we have used our MRC-funded microscopy equipment to image endosomes at very high temporal resolution in living cells. This has provided a dataset from which Dr Waigh has been able to optimise his tracking and analysis software. This has allowed us to establish imaging and tracking systems that allow us to follow virtually every single moving early endosome in live cells. We have also developed algorithms that allow us to describe, in global terms, how the population of endosomes behave.
Collaborator Contribution Our work describes the movement of endosomes and the importance of this movement for endosomal trafficking and signalling. Dr Waigh has designed novel algorithms that has allowed us to track accurately and analyse the movement of tens of thousands of endosomes. Our combined research will be an important step forward for the field.
Impact Publications: 20358070 Rogers Salman S (Apr, 2010) The first passage probability of intracellular particle trafficking., Physical chemistry chemical physics : PCCP 12, 15, 3753-61. We have extended this collaboration to successfully apply to the MRC for further funding (G0900930) and also have a project grant from the BBSRC.
Start Year 2007
 
Description School Visit Manchester 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact Conducting mock UCAS interviews

Prepared students for University applications
Year(s) Of Engagement Activity 2008,2009