LifeScienceConnect - Home_Index

Colin Hopkins’ Research Group

Futter, C. Collinson, L M. Backer, J and Hopkins, C.R. The regulation of inward vesiculation by PI3-kinase in Multivesicular endosomes. J Cell Biology 155, 1251 - 1263. 2001

Hume A. N, Collinson, L.M. Rapak, A. Gomes, A. O. Hopkins C.R. and Seabra, M. Rab27a regulates the peripheral distribution of melanosomes in melanocytes.
J Cell Biol.152:795-808.2001

Marks B, Stowell MH, Vallis Y, Mills IG, Gibson A, Hopkins CR, McMahon HT. GTPase activity of dynamin and resulting conformation change are essential for endocytosis.Nature;410:231-5. 2001

Ford M.J., Pearse B. M. F., Higgins M. K., Vallis Y., Owen D. J.  Gibson  A., Hopkins C. R. Evans P. R. and McMahon H. T.Simultaneous binding of PtdIns(4,5)P2 and clathrin byAP180 in the nucleation of clathrin lattices on membranes" Science 291, 1051 - 1053, 2001

A Gibson, CE Futter, S Maxwell, EH Allchin, M Shipman, J-P Kraehenbuhl, D. Domingo, G Odorizzi, IS Trowbridge and CR Hopkins. Sorting mechanisms regulating membrane protein traffic in the apical transcytotic pathway of polarized MDCK cells. J. Cell Biol. 143: 81-94. 1998

My group is interested in the molecular mechanisms of membrane boundaries.

Our current projects are concerned with:

(1) Mechanisms on the cell surface which collect trafficking proteins such as receptors for growth factors and G protein-linked receptors into specialised domains which then pinch off and carry them into the cell.

(2) Sorting mechanisms on intracellular membranes that route internalized proteins to sites of signal transduction and/or degradation.

One of the most significant recent insights into the mechanisms of signal transduction is that lipids of cellular membranes are organized into discrete domains and that the movements of proteins within, between and below these domains are key events in the transfer of regulatory signals into cells (see paper by K. Simons in Nature Reviews in Molecular Cell Biology, October, 2000).

Our current electron microscope studies on the cell surface are mapping the distribution of individual receptors for growth factor receptors (e.g. EGFR) and G-protein linked receptors (e.g. beta-adrenergic receptors). Using adhaerent cells treated with detergent at low temperature we have now shown that these proteins occupy separate domains which correlate with sphingolipid-rich rafts, caveolae and regions occupied by clathrin lattices. In a parallel project we are analysing the composition of these detergent resistant domains using 2D gels and mass spectrometry.

A related project is correlating fluorescence and electron microscope studies to follow integral membrane proteins in lipid raft domains on moving lymphocytes.

Using transmission EM of surface replicas with video microscopy of contrast-enhanced images of living cells we have developed methods which employ 5-10nm dia gold particles (carrying ligand or specific antibody) to localise cell surface receptors. Most of these receptors have a Stoke's radius in the size range of these particles so that individual receptors and their spatial re-arrangements (such as dimerization on ligand binding) can be mapped (Hopkins, (Cell 1985), de Brabander et al (Cytoskel and Cell Motil.1998),). More recently methods have been developed in which proteins (e.g. clathrin) can be localised on the cytoplasmic surface of the plasma membrane (in living cells with green fluorescent protein (GFP) tags and with gold conjugates by EM, (Miller et al 1991). We have also developed EM methods (Stinchcombe et al, J. Cell Biol., 1996 (using the ability of horseradish peroxidase to cross-link the content of internalised vesicles) which allow the formation of free coated vesicles (which takes about 1 minute to complete) to be followed at intervals of 5-10 seconds

By combining these techniques the sequences of the molecular re-arrangements which drive endocytosis are being analysed as follows:

The processes of dimerization and incorporation into clathrin lattices which occur when epidermal growth factor receptors (EGFR) bind their cognate ligand are being mapped and the role of the accessory proteins (EPS 15 and the synaptojanin) known to be involved in these processes are being analysed by microinjecting competitive peptides and antibodies.

In collaboration with Dr H. McMahon (MRC Laboratory of Molecular Cell Biology, Cambridge) the role of dynamin in the formation of clathrin coated vesicles and caveolae during the internalisation of transferrin receptors (TfR), EGFR and GPCRs is being studied (see Science 291, 1051 - 1053, 2001)

As part of the studies on the recycling of membrane in migrating fibroblasts described below projects dissecting the role of the actin cytoskeleton in the internalization of trafficking receptors behind the leading edge of moving cells are also being undertaken.

The insertion of recycling membrane in migrating cells

Using migrating chick fibroblasts we have shown that internalised transferrin receptors are routed through the peri-centriolar endosome and returned to the cell surface at the edge of the leading lamellaepodium (Hopkins et al J. Cell Biology, 1994). This routing is believed to identify the path followed by bulk membrane flow currently thought to be an integral part of all forms of directed cell migration. In present studies we are developing methods for loading the peri-centriolar compartment of the endosome with fluorescent probes so that we can follow the vesicular transport process that delivers recycling membrane to the leading edges of migrating cells. In collaboration with Dr I.S. Trowbridge of the Salk Institute, San Diego we have developed inducible expression systems which selectively inhibit the activity of the small GTPases known to regulate trafficking through the peri-centriolar compartment. In collaboration with Dr Jim Goldenring we are focussing on the distribution of Rabs 11a and 11b and dissecting their role in packaging and directing recycling vesicles to the cell surface.

In the longer term we hope to extend these studies on migrating fibroblasts to leucocytes and cultured hippocampal neurones.

In a series of previously published studies (Hopkins et al J. Cell Biology, 1990, Felder et al, Cell 1990, Futter et al, J. Cell Biology, 1996, Futter et al. J.Cell Biology, in Press) we have shown that EGF receptors bind their cognate ligand and internalise to multivesicular bodies (MVB-endosomes). Within these endosomes a process of inward vesiculation removes the EGF/EGFR complexes from the perimeter membrane. It is important to note that all previously studied sorting mechanisms which employ membrane vesiculation invaginate towards rather than away from the cytoplasm those operating in the MVB are, therefore, fundamentally distinctive. This inward vesiculation step is, nevertheless, probably the major cellular mechanism for removing integral membrane proteins for degradation via the lysosome pathway.

Using high resolution video-microscopy we have shown that the MVB in which this inward vesiculation takes place arise from a tubular reticulum (Hopkins et al, Nature 1990) and by devising a method which blocked the fusion of the MVB with lysosomes (Futter et al JCB1996) we have now been able to show that the inward invagination process employs a specific phosphatidylinositol kinase (PI(3)P kinase) and that its purpose is to remove the signalling receptor complex from its substrates in the cytoplasm rather than delivery to the lysosome (Futter et.al. J.Cell Biol., In the Press).

Our current studies of the remodelling that gives rise to mature MVB and leads to their fusion with lysosomes indicate that HRS-2 (a protein with a FYVE domain which binds to PI(3)P) is also involved with this specialised invagination process and we are currently developing probes which will allow us to investigate the other candidate proteins being (identified by yeast studies, Stenmark and Aasland 1999) operating at this stage of the endocytic pathway of mammalian cells. However, we have recent evidence that there are proteins located on the MVB perimeter membrane (such as AP3) which would be expected to be involved in the formation of cytoplasmic vesicles (i.e requiring membrane domains to pinch off into the cytoplasm).

Most recently there are reports that clathrin (but not AP1) is also located on the MVB perimeter membrane and it appears, therefore, that the MVB endosome vacuole occupies a key position in the regulation of membrane protein trafficking. Located at a cross-roads between endocytic and exocytic pathways it controls the surface expression of the many proteins that continuously recycle across the cell surface and for growth factor receptors like those for EGF it also determines functional longevity.

High resolution microscopical techniques are the methods of choice for studying MVB function because of their pleiomorphic form and their plasticity in response to changes in traffic flow. In our future studies we will use these methods to follow the differential processing of receptors like EGFR, and pIgR in cells in which the selected vesiculation steps are blocked. To avoid grossly distorting the endosome system experience suggests that microinjection of competing peptides in conjunction with confocal microscopy (and correlative EM) is the best, currently available, approach.

Mouse models of the Hermansky-Pudlak Syndrome (HPS) are very useful for analysing the molecular mechanisms underlying packaging, movement and exocytosis of specialised, lysosome-like vacuoles such as the melanin granules of melanocytes or platelets and dense granules. We are studying melanosome biogenesis and cellular distribution in cultured cells derived from these model systems. In this work we are expressing proteins tagged with fluorescent labels and/or with peroxidase so that correlative light and electron microscope studies can be made. In a recent collaborative study with Miguel Seabra's group (published in the Journal of Cell Biology February 2001) we observed that a Ras-like GTPase, Rab27a decorates melanosomes and co-localises with melanosome resident proteins and myosinV. When dominant-interfering Rab27a mutants were expressed in pigmented cells, we observed a redistribution of pigment granules with perinuclear clustering. these studies suggest a model whereby Rab27a co-operates with myosinVa in the determination of the peripheral distribution of melanosomes within melanocytes.

Movement of transcytotic vesicles in polarised epithelial cells

In a recent studies (Gibson et al, JCB, 1998, Futter et al JCB 1998) we showed that polymeric IgA receptors being transcytosed across a polarised monolayer of MDCK cells become concentrated in endosome vacuoles and that, in the presence of the receptor occupied with ligand, these vacuoles produce apically-directed tubules which carry the receptor towards the apical surface of the polarised monolayer. Confirming studies published by many other groups we also showed that this apical transcytosis was microtubule dependent and currently we are studying the interaction between pIgR-loaded endosomes and microtubules in a cell free system. This system is using video-microscopy to measure the movement of fluorescently labelled endosomes carrying wild type and mutated pIgR. It should allow the molecular interactions involved in endosome movement to be dissected in molecular detail. Based on these cell-free studies we then plan to study the apical delivery of transcytotic vesicles in intact cells using two photon confocal microscopy and internal reflection microscopy.

In the longer term we also plan to use this approach to block selectively the participation of microtubule-associated proteins (eg the dynactin complex) and the membrane-related proteins of the actin cytoskeleton (N-WASP and Arp2/3 complex proteins) thought to be the most likely components involved in the movement of endosome vesicles (Qualmann et al JCB 2000).