HARVEY T McMAHON
Date of birth: 31/8/65
Marital status: Married+2 boys
Work address: Medical Research Council,
Laboratory of Molecular Biology,
Hills Road, Cambridge CB2 2QH, England
Tel. No.: +44 1223 402311
Telefax No.: +44 1223 402310
Present Position: Tenured Group Leader
Web pages: http://www.endocytosis.org
1983-1987 Trinity College, Dublin B.A.Mod in Biochemistry, First Class Honours
1987-1990 Dundee University, Scotland. Ph.D in Neurochemistry
HONOURS and AWARDS:
Associate of the London College of Music- A.L.C.M. piano
EMBO member 2005
1990-1991 Postdoctoral researcher with Prof. David Nicholls, Dundee University, Dundee, Scotland.
Worked on the pharmacology and mechanics of glutamate transmitter release. During my PhD with Prof Nicholls I demonstrated biphasic kinetics of glutamate exocytosis with sustained depolarisation and proposed that the two phases were due to docked and reserve vesicle pools. I also observed a more efficient release of transmitter with calcium channel opening than with non-specific calcium entry and thus provided evidence for a direct coupling mechanism between calcium entry and vesicle release. Both of these observations have been reinforced in many subsequent papers in the literature. I introduced the use of (inward rectifying) potassium channel blockers as a means to mimic action potentials and achieve transmitter release in isolated nerve terminals. This is now also a standard protocol for this preparation. I showed the mechanism of action of alpha-latrotoxin in inducing glutamate release, the different inhibitory effects of tetanus and botulinium toxins on glutamate exocytosis, and the effects of anoxia and ischaemia on reversal of glutamate transporters. All of this work culminated in the writing of a review for BBA in 1991 on The Bioenergetics of Transmitter Release. This review covered many of the bioenergetic considerations that were a theme throughout the work of my PhD. I was awarded a medal for this work and gave an ESN honorary lecture in Dublin 1992. I also demonstrated the techniques developed during these years at a number of international neuroscience workshops.
1991-1995 Howard Hughes Research Fellow with Prof. Thomas S?dhof, UTSW, Dallas
Here I worked on the idea that synaptic mechanisms are not unique but simply specialisations of more ubiquitous processes. In line with this I cloned Cellubrevin (a homologue of synaptobrevin) and showed its involvement in vesicle trafficking outside the synapse. I went on to work on the SDS resistance of the SNARE complex and the differential sensitivities of SNARE proteins to tetanus and botulinum toxins when present in complexes (in collaboration with Heiner Niemann), and thus explained the distinct in vivo effects of the toxins. The SDS resistance of the SNARE complex is now used as a standard protocol for those working on synaptic vesicle fusion mechanisms. I then followed up the SDS resistance of the SNARE complex by noting an additional protein doublet that co-migrated with synaptobrevin on SDS-PAGE and was thus missed as part of the SNARE complex. I sequenced and cloned the proteins and called them complexin A and B. I went on to work on the function of complexins in the regulation of SNARE assembly and vesicle release. Finally I started to make a number of knock-out mouse models and completed one for synaptophysin.
1May1995- Staff scientist and group leader at the Medical Research Council Laboratory of Molecular Biology, Cambridge, England
Here I changed from exocytosis to start working on molecular mechanisms of endocytosis.
Tenured in 2000.
Overriding interests after tenure: Cell membranes are in a constant state of flux. The plasma membrane needs to accommodate major changes in cell shape during motility and during differentiation and division. In addition, the plasma membrane has to accommodate more localised and dynamic changes during invagination and evagination of local areas of membrane. In the cell, membrane bound compartments are constantly merging with each other and budding off to produce distinct microenvironments. My laboratory is interested in many aspects of this choreography of membrane dynamics. We try to cover all aspects of function from physiology to biochemistry, right down to aspects of protein biophysics and structure.
In 1996 we started our studies on synaptic vesicle retrieval making the first observations on the calcium dependence of clathrin-mediated retrieval and the involvement of a calcium-dependent phosphatase (Marks and McMahon, Current Biology, 1998). To understand how process this might work we started a dissection of this pathway and over the past few years we have been able to discover the roles, and reconstitute the activities, of a number of proteins involved in clathrin-coated vesicle retrieval. We have shown how adaptors target accessory proteins to sites of endocytosis (Owen et al papers, Wigge et al papers). This involved producing the first structures of appendage domains found on many coat proteins. We have also solved the first structure of an ANTH domain and shown its function in membrane binding. We showed that the ANTH containing protein, AP180, recruits the clathrin-coat to sites of endocytosis in the synapse (Ford et al, Science, 2001). Previously it was believed that AP2 adaptors were the principal clathrin recruitment proteins. This work also introduced a lipid monolayer assay. Following on from ANTH domains we solved the structure of an ENTH domain bound to phosphatidyl-inositol (4,5) bisphosphate headgroup. We have shown that epsin ENTH domain is actively involved in shaping the membrane (Ford et al, Nature, 2002; Mills et al, JCB, 2003). Ligand binding to the ENTH domain induces the folding of an amphipathic helix that inserts into the membrane. Membrane insertion and resulting membrane bending occurs in concert with clathrin polymerisation which stabilises the invagination. This domain is also present in other proteins and we have thereby defined families of proteins capable of sculpting membranes. This work was followed by the structural and functional characterisation of the BAR domain of amphiphysin which can sense membrane curvature (Peter et al Science, 2004, see below). We have also worked on the molecular mechanism of dynamin and proposed that it functions like a 'loaded spring' in vesicle scission (Stowell et al, Nature Cell Biol, 1999; Marks et al, Nature, 2001). Our basic observation here was a change in the dynamin helix pitch on GTP hydrolysis. We have reconstituted various stages of endocytosis and have used the information gained from crystallography and biophysical studies to test the hypothesised mechanisms of synaptic vesicle retrieval in neurons (Jockusch et al, Neuron, 2005).
Our current 2 major topic of research are as follows:
As we began to understand how clathrin organises and stabilises membrane invaginations and cargo recruitment, we recognised the need for more active membrane bending to create high curvature vesicles. Thus we went in search of proteins that could orchestrate membrane budding, fission and fusion. This led us to the study of amphipathic helix insertion into membranes. We first noted that amphipathic helices of ENTH domains only fold on binding to membranes and that this promoted membrane curvature. We now find a similar folding of amphipathic helices in response to membrane binding for a wide range of proteins that effect membrane curvature and show that this is a general principle used in sculpting membranes. We have used a range of biophysical parameters to study this process, combined with crystallographic and microscopic observations and reconstitutions.
We also solved the crystal structure of the first BAR domain. This module is found in a wide variety of membrane trafficking proteins and dimerises on membrane binding. The membrane binding face is concave and thus prefers membranes that make maximum use of the protein?s intrinsic curvature. We refer to this domain as a curvature-stabilising domain and we are aware of a number of other BAR-like domains that likely perform similar functions. We are currently using electron spin resonance, calorimetry and different monolayer and bilayer techniques to gain more insight into the workings of these domains. Given the central role that membrane curvature regulation must play in making vesicles, we are testing whether we can define new endocytic and trafficking pathways by the study of membrane curvature sculpting proteins. In a study underway at the moment we show that the BAR domain protein, endophilin, is a molecular component of the elusive ?fast-pathway? of synaptic vesicle retrieval. We are also nearing the completion of a study on alternative endocytic pathways in fibroblasts.
Another major current topic in the lab is on the functional organisation of protein networks. Given our early observations on the structure and interactions of adaptor appendage domains with short peptide motifs, we set out to understand how these might function in a cellular context. An initial paper on this subject has been published (Praefcke et al, EMBO, 2004) and others are on the way. These studies have changed the way in which we view the coordination and control of these processes. Thus we have gone back to understanding how exocytosis may proceed in synapses, and we are also applying the same rationale to cell motility, dendritic spine morphogenesis and neural plasticity.
This work has benefited enormously from close and very productive collaborations, most particularly with Phil Evans at the MRC and Ralf Langen at USC.
For details of current research interests see : http://www.endocytosis.org/
Lectures given within the last 8 months:
Gordon Research Conference ?Proteins?: June2005
Gordon Research Conference ?Molecular Membrane Biology? Membrane Bending/Fission: 10-15July2005
Royal Netherlands Academy of Arts and Science, 2005 Academy colloquium: Lipids moving centre stage: 7Oct2005
FEBS advanced course: Harden Conference, Ambleside: Coincidence Detection 15Aug2005
EMBL course Heidelberg: 26Aug2005
Caltech Los Angeles, Biology Division: 9Dec2005
American Society for Cell Biology, San Francisco: chair of session and lecturer 10-14Dec2005
Children's Medical Research Institute, Sydney: 1Feb2006
Australian Neuroscience Society annual meeting symposium lecture: 2Feb2006
Institute for Molecular Bioscience, Brisbane: 7Feb2006
Max Planck Institute for Biochemistry Munich graduate lecture programme: 21Feb2006
Cologne Spring Meeting: 1-3March2006
McMahon, H.T. and Gallop J.L. (2005) Membrane curvature and mechanisms of dynamic cell remodelling. Nature 438, 590-596. (invited review)
Exploratory review on the role of proteins in dynamically changing membrane conformation and the downstream effects of these changes. We address different mechanisms of curvature generation and how these interact to orchestrate membrane trafficking.
Higgins, M and McMahon, H.T. (2005) In vitro reconstitution of discrete stages of dynamin-dependent endocytosis. Methods Enzymology 404, 597-611.
Gallop, J.L., Butler, P.J.G. and McMahon H.T. (2005) Endophilin and CtBP/BARS are not acyl transferases in endocytosis or Golgi fission. Nature 438, 675-678.
Both endophilins and CtBP/BARS have been proposed to have lysophosphatidic acid acyl transferase (LPAAT) activities that can make phosphatidic acid in membranes. For endophilins this activity is thought to change the bilayer asymmetry in such a way that negative membrane curvature at the neck of a budding vesicle will be stabilised. Here we show that the LPAAT activities are contaminants associating with these proteins during purification. This now prompts a re-evaluation of the functions of these proteins.
Jochusch, W.J., Praefcke, G.J.K., (McMahon, H.T. and Lagnado, L.) (2005) Clathrin-dependent and clathrin-independent retrieval of synaptic vesicles in retinal bipolar cells. Neuron 46, 869-878.
Endocytosis in central neurons can frequently be separated into two kinetic components. Here we show that the fast component in retinal bipolar cells (time constant of 1s) is clathrin-independent and dynamin dependent. The slow component (time constant of 10s) is AP2 adaptor, clathrin and dynamin-dependent and thus is the canonical clathrin-mediated endocytic pathway.
Carlton, J.G., Bujny, M.V., Peter, B.J., Oorschot, V.M.J., Rutherford, A., Arkell, R.S., Klumperman, J., McMahon, H.T. and Cullen, P.J. (2005) Sorting nexin-2 is associated with tubular elements of the early endosome, but is not essential for retromermediated endosome-to-TGN transport. J. Cell Sci. 118, 4527-4539.
A continuation of the collaboration with Pete Cullen, Bristol, on the role of sorting-nexins in membrane trafficking. The BAR domain of sorting-nexin2 does not tubulate membranes but is sensitive to curvature and in vivo sorting-nexin2 localises to tubular elements of endosomes.
Gallop, J.L. and McMahon H.T. (2005) BAR domains and membrane curvature: bringing your curves to the BAR. Biochem. Soc. Symp. 72, 223-231.
Do BAR domains create membrane subdomains and how can BAR domains influence membrane trafficking? This review covers the significance of, and ways in which, BAR domains function in various contexts.
Saint-Pol, A., Yelamos, B., Amessou, M., Mills, I.G., Dugast, M., Tenza, D., Schu, P., Antony, C., McMahon, H.T., Lamaze, C. and Johannes, L. (2004) Clathrin adaptor epsinR is required for retrograde sorting on early endosomal membranes. Dev. Cell 6, 525-538.
This paper builds on our 2003 epsinR paper by Ian Mills et al, where we found that epsinR overexpression had a trafficking defect in the uptake of TGN46 antibodies. We show that epsinR is a component of a vesicle budding step in retrograde transport from endosomes to the TGN. This pathway is used for retrieval of cargo (including TGN38/46 and mannose-6-phosphate receptors) from endosomes and Shiga toxin can also traffic to the TGN via this pathway.
Praefcke, G.J.K., Ford, M.G.J., Schmid, E.M., Olesen, L.E., Gallop, J.L., Peak-Chew, S-Y., Vallis, Y., Madan Babu, M., Mills, I.G. and McMahon, H.T. (2004) Evolving nature of the AP2 a-appendage hub during clathrin-coated vesicle endocytosis. EMBO J. 23, 4371-4383. (see also cover of same issue, and Highlight in Science 306, p1103)
This is the first of a series of network papers on trying to understand the making of a clathrin-coated vesicle as a ?series of many parallel processes?. In protein interaction networks, hubs are proteins that have disproportionately large numbers of interaction partners. For these hubs to function in a biological process it would seem that there must be sequential or spatial ordering of the interactions. In this paper, where we treat clathrin-mediated endocytosis as a module of a network, we uncover how the AP2 alpha-appendage works as an interaction hub after being concentrated at sites of endocytosis. These concentrated appendages provide a multivalent binding platform (hub) for interaction partners. Thus the partners are represented according to their relative affinities and concentrations. In this paper we also discuss the biological basis for the temporal nature of this hub and for the directionality imposed by the system. This has implications for all biological networks where similar principles are likely to underlie their functions.
Carlton, J., Bujny, M., Peter, B.J., Oorschot, V.M., Rutherford, A., Mellor, H., Klumperman, J., McMahon, H.T. and Cullen, P.J. (2004) Sorting nexin-1 mediates tubular endosome-to-TGN transport through coincidence sensing of high- curvature membranes and 3-phosphoinositides. Current Biol. Oct 26;14(20):1791-800. (see also Research Roundup ?Snx1 hugs the curves? in JCB 2004 167, p581)
Arising from our observations that many sorting-nexin proteins have BAR domains (see Peter et al 2004 below) we tested sorting nexin1 for membrane binding and curvature sensing and made appropriate mutations. We then collaborated with Peter Cullen (Bristol University) to test the in vivo function of sorting-nexin1 in membrane trafficking. This paper reinforces our previous observation of the necessity for a lipid selective domain and a curvature sensing domain to work in tandem to localize proteins to high curvature on membranes (in this case endosomes) in the cell. This paper also demonstrates that overexpression of this sorting-nexin leads to extreme tubulation of endosomes which disrupts all trafficking through this compartments, but an RNAi experiment shows that knock-down of the protein specifically disrupts the trafficking of mannose-6-phosphate receptors to the TGN and thus we propose that this receptor goes though via a tubule transported which is formed/stabilized by sorting-nexin 1.
Tsuboi, T., McMahon, H.T. and Rutter, G.A. (2004) Mechanisms of dense core vesicle recapture following "kiss and run" ("cavicapture") exocytosis in insulin-secreting cells. J. Biol. Chem. 279 47115-47124.
This paper is the result of a collaboration with Guy Rutter in Bristol University showing the dynamin-dependence of large-dense cored vesicle endocytosis. This shows that the not only clathrin-mediated retrieval of vesicles but also the ?kiss and run? form of endocytosis is dynamin dependent. The stabilization and closure of the fusion pore, through which transmitter is release, required the GTPase activity of dynamin.
McMahon, H.T. and Mills, I.G. (2004) COP and clathrin-coated vesicle budding: different pathways, common approaches. Current Opinions in Cell Biol. 16, 379-391.
The remarkable similarities of appendage domains of COP and clathrin adaptors led us to look closely at the overall evolutionary relationships between COP and clathrin-coated vesicles. This review covers cargo recruitment, membrane curvature mechanisms and coat polymerization showing that common paradigms underpin these diverse pathways.
Praefcke, G.J.K. and McMahon, H.T. (2004) The Dynamin Superfamily: Universal membrane tubulation and fission molecules? Nature Reviews in Molecular Cell Biology 5, 133-147.
Here for the first time we define and classify the dynamin ?Superfamily? of proteins. All members have GTPase, oligomerisation and membrane targeting domains. The superfamily is subdivided into classical dynamins, dynamin-like proteins, Optic atrophy proteins (OPA1), Mx proteins and GBPs/atlastins. In this review we propose a common mechanism leading to membrane tubulation and/or fission that encompasses all superfamily members.
Peter, B.J., Kent, H.M., Mills, I.G., Vallis, Y., Butler, P.J.G., Evans, P.R. and McMahon, H.T. (2004) BAR Domains as Sensors of Membrane Curvature: the Amphiphysin BAR Structure. Science 303, 495-499. published online in Science Express Nov 26, 2003. (see cover and perspective in same issue and reviews in Current Biology and EMBO Reports)
In this paper we present the crystal structure of the BAR (Bin/Amphihysin/Rvs) domain from Drosophila amphiphysin. The protein is a crescent-shaped dimer which binds to highly curved negatively charged membranes. Unexpectedly, we found structural similarity with arfaptin2, a G-protein effector which also binds and curves membranes. This similarity allowed us to search the sequence database where we found BAR domains in many proteins, including nadrins, sorting-nexins, oligophrenins, centaurins, ICA69, and PICK1. In some of these proteins (N-BARs) the BAR is adjacent to an N-terminal amphipathic helix, and these N-BAR proteins can drive membrane curvature in vitro and in vivo. In other proteins the BAR is adjacent to a PH, PX, or other lipid binding domain, and in this context the BAR can sense membrane curvature, showing a strong preference for highly curved membranes (diameter <80nm). This curvature sensing effect is also seen with BAR domains alone, and is likely due to the curved shape of the broad membrane binding face. We propose that BAR domains act together with lipid binding domains to function as coincidence detectors for the correct degree of curvature and the correct lipid composition, and this will precisely localise proteins and any associated enzymatic activities to membrane sub-domains. The universal and minimal BAR domain is a dimerisation, membrane binding, and curvature sensing module found in many protein families.
Stahelin, R.V., Long, F., Peter, B.J., Murray, D. DeCamilli, P., McMahon, H.T. and Cho, W. (2003) Contrasting membrane interaction mechanisms of AP180 ANTH and epsin ENTH domains. J. Biol. Chem. 278, 28993-28999.
This is a biophysical paper including measurements of the interactions of ANTH and ENTH domains with membranes, providing an explanation for the different properties of these domains.
Mills, I.G., Praefcke, G.J.K., Vallis, Y., Peter, B.J., Olesen, L.E., Gallop, J.L., Butler, P.J.G., Evans, P.R. and McMahon, H.T. (2003) EpsinR: an AP1/clathrin interacting protein involved in vesicle trafficking. J. Cell Biol. 160, 213-222.
If epsin is important for membrane bending under the clathrin lattice then there must be an epsin homologue for AP1/clathrin budding events. Here we describe a ubiquitous epsin1 homologue, which we call epsinR (for epsin-related protein). Endogenous epsinR in COS cells shows a TGN localisation and also puncta in the cytoplasm. Overexpression of lipid binding mutants cause disruption of the TGN to lysosome trafficking pathway (cathepsinD now goes to the cell surface) and also disruption of plasma membrane to TGN trafficking.
In addition to the cell biology in this paper we characterised epsinR binding to gamma-adaptin in the AP1 complex and to clathrin. We were the first to identify and characterise a gamma-adaptin binding sequence present in epsinR, gamma-synergin and Eps15. We identify the strong consensus sequence as being DFxDF where, in many cases, only one of the D residues is present.
Ford, M.G.J., Mills, I.G., Peter, B.J., Vallis, Y., Praefcke, G.J.K., Evans, P.R. and McMahon, H.T. (2002) Curvature of clathrin-coated pits driven by epsin. Nature article 419, 361-366. (see also News and Views and highlights in Nature Cell Biology, Nature reviews in molecular cell biology, and a mini review in Cell)
This paper shows that epsin is able to deform membranes by binding to PtdIns(4,5)P2 and inserting an amphipathic helix into the membrane. In conjunction with clathrin polymerisation we propose that this drives the curvature of the nascent coated pit. We present the crystal structure of epsin ENTH domain bound to PtdIns(4,5)P2 with mutagenesis of the PtdIns(4,5)P2 binding site and the hydrophobic surface of helix '0'. PtdIns(4,5)P2 binding mutants are not recruited to the plasma membrane and inhibit AP2 adaptor localisation at the plasma membrane. This results in the inhibition of clathrin-mediated endocytosis. We also present the effect of epsin in tubulating liposomes and of epsin + clathrin in forming clathrin-coated pits on lipid monolayers.
Kent, H.M., McMahon, H.T., Evans, P.R., Benmerah, A. and Owen, D.J. (2002) g-adaptin appendage domain: Structure and binding site for Eps15 and g-synergin. Structure 10, 1139-1148.
In this paper we present the crystal structure of the gamma-adaptin appendage domain which is part of the AP1 complex. The appendage is very similar to both the alpha and beta appendages of the AP2 complex that we previously published except for the absence of a platform domain where the DxF motifs bind. We further identify an interaction site on the gamma appendage for gamma ligands by point mutagenesis.
Graham, M.E., O?Callaghan, D.W., McMahon, H.T. and Burgoyne, R.D. (2002) Dynamin-dependent and dynamin-independent processes contribute to the regulation of single vesicle release kinetics and quantal size. PNAS 99, 7124-7129.
Higgins, M.K. and McMahon, H.T. (2002) Snap shots of clathrin-mediated endocytosis. TIBS 27, 257-263. A review of electron microscopy techniques as applied to endocytosis.
Ford, M.G.J., Pearse, B., Higgins, M., Vallis, Y., Owen, D., Gibson, A., Hopkins, C.R., Evans, P.R. and McMahon, H.T. (2001) Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 causes nucleation of clathrin lattices on membranes. Science 291, 1051-1055. (see cover and highlight in same issue)
This paper presents the crystal structure of CALM ANTH domain bound to PtdIns(4,5)P2 and demonstrates the critical role of AP180/CALM in clathrin recruitment to lipid membranes. A new assay has been developed whereby clathrin polymerisation by AP180 on lipid monolayers can be visualised by electron microscopy. This paper highlights the role of PtdIns(4,5)P2 in the membrane in the initial stages of clathrin assembly/vesicle formation.
Marks, B., Stowell, M., Vallis, Y., Mills, I., Gibson, A., Hopkins, C.R. and McMahon, H.T. (2001) GTPase activity of dynamin and resulting conformation change are essential for endocytosis. Nature 410, 231-235. (see picture story in Nature Structural Biology, April issue 8, 301)
This paper shows that dynamin is a mechanochemical molecule and not a regulator of endocytosis (as had been previously proposed). We have made the first GTPase 'dead' mutants of dynamin and show that these do not accelerate endocytosis as predicted by models of classical GTPases, but instead vesicle scission is arrested and the vesicle neck is extended over time, eventually filling the cells with tubulated/invaginated plasma membrane. The effects of GTPase mutants of dynamin are monitored by electron microscopy and demonstrate that the change in pitch of the dynamin helix on GTP hydrolysis is prevented by the GTPase dead mutation. We also identified another important mutation in this paper that prevents the conformational change in dynamin while maintaining a wild type GTP hydrolysis rate. As predicted from a mechanochemical model for dynamin's action, this mutant gives a severe endocytic block.
Razzaq, A., Robinson, I. M., McMahon, H. T., Skepper, J. N., Su, Y., Zelhof, A. C., Jackson, A. P., Gay, N. J., O'Kane, C. J. (2001) Amphiphysin is necessary for organization of the excitation-contraction coupling machinery of muscles, but not for synaptic vesicle endocytosis in Drosophila. Genes and Development. 15, 2967-79.
Deletion of the amphiphysin gene in Drosophila led to a defect in excitation-contraction coupling in muscle. This was analysed and we found that the muscle T-tubule system is not correctly developed. We show that this form of amphiphysin can tubulate lipids and suggest that this function is essential in the formation of the tubule network. This paper also highlights that amphiphysin function is not restricted to the synapse and also that it is not restricted to its interaction with dynamin. Thus amphiphysin has a different role in muscle than in the synapse. In the fly there does not appear to be a neuronal form of amphiphysin.
Reim, K., Mansour, M., Varoqueaux, F., McMahon, H.T., S?dhof, T., Brose, N., Rosenmund, C. (2001) Complexins regulate a late step in Ca-dependent neurotransmitter release. Cell. 104, 71-81.
I cloned the complexins in 1995 and showed how they bound to the core SNARE complex (see paper below). I followed this up by starting to make mouse knock-outs of these proteins to determine their role in exocytosis. I handed over this project to Nils Brose to generate the mice and analyse the phenotypes while I moved into the endocytosis field. This paper is the culmination of the mice work and confirms our previous conclusion that complexins are indeed involved in a late stage of exocytosis. However the surprising result is that the mice have an altered calcium sensitivity of exocytosis implying a further role for complexins here.
Wigge, P. and McMahon, H.T. (2000) Synaptic vesicle recycling: multiple pathways and functions in the nervous system (p204-214) in Endocytosis issue (ed Mark Marsh) of Frontiers in Molecular Biology series published by Oxford University Press (eds B.D.Hames and D.M.Glover).
A book chapter on the possible implication of endocytosis in synaptic function and synaptic plasticity.
Owen, D.J., Vallis, Y., Pearse, B.M.F., McMahon, H.T. and Evans, P.R (joint corresponding author) (2000) The structure and function of the B2-adaptin appendage domain. EMBO J. 19, 4216-4227.
Following on from our structure determination of the alpha-adaptin appendage domain we here present the crystal structure of the beta2-adaptin appendage domain. We demonstrate that despite a similar fold the ligand binding site has a different ligand specificity. The major binding partner for beta2-adaptin is clathrin and this paper shows that beta2-adaptin has at least 2 binding sites (one on the hinge and one on the appendage). This combination leads to very efficient polymerisation of clathrin into cages in vitro and we propose that this is part of the role of the beta2-appendage domain.
Marsh, M. and McMahon, H.T. (1999) The Structural Era of Endocytosis. Science, 285, 215-220.
This was the first of a large number of reviews that cover the implication of structural analysis of endocytic proteins for our understanding of the biology.
Owen, D.J., Vallis, Y., Noble, M.E.M., Hunter, J.B., Dafforn, T.R., Evans, P.R., and McMahon, H.T. (1999). A structural explanation for the binding of multiple ligands by the alpha-adaptin appendage domain. Cell 97, 805-815.
Here we presented the first crystal structure of the binding domain for accessory proteins on adaptors. We identified the binding site and a short peptide motif that binds to this site. This allowed us to identify new ligands for this domain simply by database trawling. We proposed that the appendage domain of adaptors is the linch-pin for clathrin-mediated endocytosis, recruiting most of the accessory proteins via the same binding site. We do not know how this process is temporally regulated but the strength of interactions of the many ligands varies dramatically.
Stowell, M.H.B., Marks, B., Wigge, P., and McMahon, H.T. (1999) Nucleotide-dependent conformational changes in dynamin: Evidence for a mechanochemical molecular spring. Nature Cell Biol. 1, 27-32.
This was the first demonstration of dynamin's GTPase activity being stimulated under physiological conditions. We found that lipid nanotubules containing PtdIns(4,5)P2 could stimulate the GTPase activity by over 1000-fold. By EM we were able to observe a 2-fold increase in the pitch of the dynamin helix on GTP hydrolysis. This led us to propose a model by which this extension of the helix would force a nascent vesicle away from the parent membrane. This is a cooperative process and the energy from GTP hydrolysis of dynamin molecules in the helix is more than sufficient to break the membrane. Our subsequent Nature paper (2001) tested this hypothesis with several key mutants. The model stands the test of time but we would still like a direct measurement of the force produced by GTP hydrolysis.
Vallis, Y., Wigge, P., Marks, B., Evans, P.R., and McMahon, H.T. (1999) Importance of the pleckstrin homology domain of dynamin in clathrin-mediated endocytosis. Current Biol. 9, 257-260. (see also review in next issue)
In this paper we modelled the head group of PtdIns(4,5)P2 into the PH domain of dynamin and made some mutations to prevent binding. The key mutant K535A in the context of full-length dynamin was then shown to not support endocytosis of transferrin and EGF into cells. The in vivo recruitment of dynamin by amphiphysin was also tested in this paper where we show that cells expressing dynamin K535A could be partially rescued by a further mutation of the amphiphysin binding site of dynamin.
McMahon, H.T. (1999). Endocytosis: An assembly protein for clathrin cages. Current Biol. 9, R332-335.
The first and only review to date on the protein AP180.
Owen, D.J., Wigge, P. Vallis, Y., Moore, J.D.A., Evans, P.R. and McMahon H.T. (1998) Crystal structure of the amphiphysin-2 SH3 domain and its role in the prevention of dynamin ring formation. EMBO J. 17, 5273-5285.
In this paper we presented the crystal structure of the major dynamin-binding domain of amphiphysin-2. This study allowed us to make specific mutations in dynamin that tested the role of amphiphysin in the recruitment of dynamin to sites of endocytosis (see Current Biology paper 1999). We also found that when dynamin was bound to amphiphysin it was held in a non-polymerised state and thus could be recruited at high concentrations to the neck of a forming vesicle awaiting the signal for release and helix formation and thus activation of its GTPase activity.
Marks, B. and McMahon, H.T. (1998) Calcium triggers calcineurin-dependent synaptic vesicle recycling in mammalian nerve terminals. Current Biol. 8, 740-749.
This was our initial entry into endocytosis with a study to find out if endocytosis is differently regulated to exocytosis. We found that calcium is the trigger for endocytosis (just as had been previously known for exocytosis). We showed that a target for the calcium was the calcium-dependent phosphatase, calcineurin. This phosphatase has a lower affinity for calcium than the exocytic trigger and so will sense ?residual calcium? after exocytosis (calcium that diffuses away from the local calcium increases at sites of exocytosis) and therefore fits with the fact that endocytosis can occur in response to exocytosis in the living synapse. We found that by manipulation we could trigger endocytosis without exocytosis by using a low calcium stimulus, and by using an inhibitor of the calcineurin (cyclosporin) we could trigger exocytosis without endocytosis. We furthermore confirmed that the major target of calcineurin was the protein apparatus of clathrin-mediated endocytosis. This was the study that started us out on a path of wanting to understand the molecular machinery of clathrin-mediated endocytosis so that we could determine its regulation and specificity.
Wigge, P. and McMahon, H.T. (1998) The Amphiphysin Family and their role in Endocytosis at the Synapse. TINS 21, 339-344.
Wigge, P. and McMahon, H.T. (1998) Amphiphysins, in Guidebook to the Cytoskeletal and Motor Proteins published by Oxford University Press (eds Thomas E. Kreis and Ron Vale).
Wigge, P., Vallis, Y. and McMahon, H.T. (1997) Inhibition of receptor-mediated endocytosis by the amphiphysin SH3 domain. Current Biol. 7, 554-560.
In this paper we demonstrated the use of the dynamin binding partner amphiphysin as a dominant-negative for clathrin-mediated endocytosis. We also showed that we could rescue the phenotype by overexpression of dynamin. We still receive many more requests for this reagent than the more specific reagents that we have subsequently developed.
McMahon, H.T., Wigge, P. and Smith, C. (1997) Clathrin interacts specifically with amphiphysin and is displaced by dynamin. FEBS Lett. 413, 319-322.
This was the first demonstration of clathrin binding to amphiphysin.
Wigge, P., K?hler, K., Vallis, Y., Doyle, C.A., Owen, D., Hunt, S.P. and McMahon, H.T. (1997) Amphiphysin heterodimers: Potential role in clathrin-mediated endocytosis. Mol. Biol. Cell 8, 2003-2015.
Here we cloned a second form of amphiphysin (plus splice-variants) which is found enriched in the brain along with amphiphysin I (as a heterodimer) and in muscle (as a homodimer). This paper characterises the brain distribution, synaptic localisation and dimerisation characteristics.
McMahon, H.T., Bolshakov, V.Y., Janz, R., Hammer, R.E., Siegelbaum, S. and S?dhof, T.C. (1996) Synaptophysin, a major synaptic vesicle protein, is not essential for transmitter release. PNAS 93, 4760-4764.
Mouse knock-out of the prominent synaptic vesicle protein, synaptophysin. Surprisingly the mice were still viable.
McMahon, H.T., Missler, M., Li,.C. and S?dhof, T.C. (1995) Complexins: Cytosolic proteins that regulate SNAP receptor function. Cell 83, 111-119.
McMahon, H.T. and S?dhof, T.C. (1995) Synaptic core complex of synaptobrevin, syntaxin and SNAP25 forms a high affinity aSNAP binding site. J. Biol. Chem. 270, 2213-2217.
Hayashi, T., McMahon, H.T., Yamasake, S., Binz, T., Hata, Y., S?dhof, T. C. and Niemann, H. (1994) Synaptic vesicle membrane fusion complex: Action of Clostridial neurotoxins on assembly. EMBO J. 13, 5051-5061.
Ullrich, B., Li, C., Zhang, J.Z., McMahon, H.T., Anderson, R.G.W., Geppert, M. and S?dhof, T.C. (1994) Functional properties of multiple synaptotagmins in brain. Neuron 13, 1020.
McMahon, H.T., Ushkaryov, Y.A., Edelmann, L., Link, E., Binz, T., Niemann, H., Jahn, J. and S?dhof, T.C. (1993) Cellubrevin is a ubiquitous tetanus-toxin substrate homologous to a putative synaptic vesicle fusion protein. Nature 364, 346-349. (see also News and Views)
Link, E., McMahon, H., Fischer von Mollard, G., Yamasaki, S., Niemann, H., S?dhof, T.C. and Jahn, R. (1993) Cleavage of cellubrevin by tetanus toxin does not affect fusion of early endosomes. J.Biol.Chem. 268, 18423-18426.
Robinson, P.J., Sontag, J-M., Liu, J-P., Fyske, E.M., Slaughter, C., McMahon, H.T. and S?dhof, T.C. (1993) Dynamin GTPase regulated by protein kinase C phosphorylation in nerve terminals. Nature 365, 163-166. (see also News and Views)
McMahon, H.T. and Nicholls, D.G. (1993) Barium-evoked glutamate release from guinea-pig cerebrocorical synaptosomes. J.Neurochem. 61, 110-115.
McMahon, H.T., Foran, P., Dolly, J.O., Verhage, M., Wiegant, V.M. and Nicholls, D.G. (1992) Tetanus and Botulinum toxins type A and B inhibit glutamate, GABA, aspartate and met-enkephalin release from synaptosomes: Clues to the locus of action. J.Biol.Chem. 267, 21338-21343.
McMahon, H.T. and Nicholls, D.G. (1991) The Bioenergetics of Neurotransmitter Release (invited review). BBA 1059, 243-264.
McMahon, H.T. and Nicholls, D.G. (1991) Transmitter glutamate release from isolated nerve terminals: evidence for biphasic release and triggering by localized Ca2+. J. Neurochem. 56, 86-94.
Verhage, M., McMahon, H.T. Boomsna, Wiegant and Nicholls, D.G. (1991) Release of amino acids, cholecystokinin and noradrenaline from isolated hippocampal terminals show differing preferences for localised versus delocalised Ca2+ entry. Neuron 6, 517-524.
McMahon, H.T. and Nicholls, D.G. (1990) The relationship between cytoplasmic free Ca2+ and the release of glutamate from synaptosomes. Biochem. Soc. Trans. 18, 375-377.
McMahon, H.T., Rosenthal, L., Meldolesi, J and Nicholls, D.G. (1990) a-Latrotoxin releases both vesicular and cytoplasmic glutamate from isolated nerve terminals. J. Neurochem. 55, 2039-2047.
McMahon,H.T. and Nicholls, D.G. (1990) Glutamine and aspartate loading of guinea pig cerebral cortical synaptosomes: a re-evaluation of effects on Ca2+-dependent excitatory amino acid release. J. Neurochem. 54, 373-380.
Kauppinen, R.A., McMahon, H.T., and Nicholls, D.G. (1990) The effects of experimental anoxia on glycolysis, energy status, cytosolic free calcium, and glutamate release from isolated nerve terminals, in Cerebral Ischaemia and Calcium (Hartmann A. and Kuschinsky W. eds.) Springer, Berlin.
McMahon, H.T., Barrie, A.P., Lowe, M. and Nicholls, D.G. (1989) Glutamate release from guinea pig synaptosomes: Extra-cellular glutamate may exert a positive feedback by reuptake-induced depolarisation rather than receptor activation. J. Neurochem. 53, 71-79.
Tibbs, G., Barrie, A.P., Van Mieghem F., McMahon, H.T. and Nicholls, D.G. (1989) Repetitive action potentials in isolated nerve terminals in the presence of 4-aminopyridine: effects on cytosolic free Ca2+ and glutamate release. J. Neurochem. 53, 1693-1699.
Kauppinen, R.A., McMahon H.T., and Nicholls, D.G. (1988) Calcium-dependent and calcium-independent glutamate release, energy status and cytosolic free calcium concentration in isolated nerve terminals following in vitro hypoglycaemia and anoxia. Neuroscience, 27, 175-182.
McMahon, H.T., Kauppinen, R. and Nicholls, D.G. (1988) The energetics of Ca-dependent release from synaptosomes and the effect of experimental anoxia, hypoglycaemia and ischaemia. Biochem. Soc. Trans. 16, 880-881.
Nicholls, D.G., Barrie, A.P., McMahon, H.T., Tibbs, G. and Wilkinson, R. (1988) Mechanisms of Glutamate Exocytosis from Isolated Nerve Terminals. In, Nato ASI Series H: Cell Biology: "Receptors, Membrane Transport and Signal Transduction" 29, 147-161.