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Mechanisms of protein nanoscale clustering
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ScienceDirect Mechanisms of protein nanoscale clustering Jesse Goyette1,2 and Katharina Gaus1,2 Due to recent technical developments in microscopy, huge advances have been made in our understanding of the architecture of the cell membrane. It is now well appreciated that nanoscale clustering is a common feature of membrane proteins. Many of these clusters have been implicated in signal initiation and integration platforms. However, the mechanisms that mediate the dynamic nanoscale arrangement of membrane proteins are not fully understood and could involve lipid domains, electrostatic interactions between proteins and lipid, protein scaffolding as well as purely mechanical processes. In this review we summarise these mechanisms giving rise to dynamic nanoscale protein reorganisation in the plasma membrane with reference to recent examples of immune receptor clustering to illustrate general principles. Addresses 1 EMBL Australia Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney 2052, Australia 2 ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, Sydney 2052, Australia Corresponding author: Gaus, Katharina ([email protected])
Current Opinion in Cell Biology 2016, 44:86–92 This review comes from a themed issue on Cell Architecture Edited by Terry Lechler and Rafael Carazo Salas For a complete overview see the Issue and the Editorial Available online 22nd September 2016 http://dx.doi.org/10.1016/j.ceb.2016.09.004 0955-0674/# 2016 Elsevier Ltd. All rights reserved.
Protein clustering in the picket fence era With the advent of super-resolution microscopy, new insights into the organisation of membrane proteins have [5_TD$IF]emerged with clusters ranging from nanoscale, transient interactions to micrometre scale, stable and immobile structures. For example in immune receptor signalling, a large and diverse group of protein have been reported to reside in clusters[6_TD$IF], including[7_TD$IF]: Ras proteins [1], syntaxin-1a [2], IL2Ra, KIR2DS1, KIR2DL1 [3[1_TD$IF]], T cell receptor [4], B Cell Receptor [5], LAT [6,7], FceRI [8], multiple glycosylphosphatidylinositol anchored proteins (GPIAPs), LFA-1 [9] and DC-SIGN [10,11]. Indeed, as more evidence accumulates examples of proteins that are not described as clustered become scarce. This has led some authors to propose that clustering may be a common feature of membrane receptors [12], but is it a universal feature? Current Opinion in Cell Biology 2017, 44:86–92
Electron microscopy studies were among the first to report non-random groupings of proteins on the scale of nanometres, tens of nanometres and on a much larger scale of protein islands interspersed by protein poor regions [13–15]. More recently, super-resolution microscopy has provided supporting evidence for the protein island architecture [16] where most cell surface proteins were organised into clusters a few hundred nanometres in size. In this study by Saka et al., some proteins had a preference for the centre of assemblies, others for the edge, and some showed no preferential localisation, suggesting that for some proteins some kind of specificity of localisation remained [16]. These ‘protein islands’ were bordered by cortical actin cytoskeleton and were disrupted by cholesterol depletion. This study is one of the clearest demonstrations of the architecture predicted by the ‘picket fence’ model of membrane biology (Figure 1a), as outlined elegantly by Kusumi et al. [17]. Briefly, the picket fence model states that the cortical actin cytoskeleton (fences), and transmembrane proteins anchored to it (pickets), separate the membrane into segments. Within these segments proteins diffuse freely but are sterically restricted from crossing between segments by the membrane cytoskeleton, which acts as a diffusion barrier. The picket fence model, and recent studies supporting it, provides an explanation for the predominance of protein clustering — a transmembrane protein with a random distribution in the cell membrane is always sterically excluded from regions occupied by the actin cytoskeleton (Figure 1b). Thus the protein is localised within corrals, which are on the scale of the diffraction limit (200 nm). When imaged with super-resolution microscopy the heterogeneity in localisation will be auto-correlated on the scale of these corrals [16]. If this autocorrelation is described as ‘clustering’ or ‘nanoclustering’ then it seems likely to be a universal feature of surface membrane proteins.
Mechanisms of protein clustering Mechanisms that lead to clustering of some but not all membrane proteins can be broadly grouped into mechanisms driven by lipid-phase separation, phospholipid charge interactions, polyvalent cross-linking and mechanical mechanisms. There are other mechanisms such as membrane curvature, which are not included here but reviewed elsewhere [18–21]. Lipid domains
Protein organisations under cholesterol depletion conditions are often interpreted in the context of the lipid raft www.sciencedirect.com
Mechanisms of protein nanoscale clustering Goyette and Gaus 87
Figure 1
(a)
liquid order-like domains form only after immobilisation of long acyl chain lipids in one leaflet [25]. In support of this, phase separation was observed in supported lipid bilayers when either lipid order or lipid disorder favouring lipids were pinned to actin filaments [32].
(b)
‘Picket fence’ architecture
Localisation of protein of interest Current Opinion in Cell Biology
Non-random distribution of proteins caused by underlying membrane architecture. (a) Picket fence architecture with cortical actin cytoskeleton (black lines), actin-binding transmembrane proteins (red dots) and randomly distributed membrane proteins (yellow and blue dots). (b) Because of steric exclusion from areas occupied by the cytoskeleton, when a randomly distributed protein of interest (blue dots) is imaged with super-resolution microscopy or electron microscopy the distribution appears non-random.
It should be pointed out that immobilisation of GPI-APs by antibodies micro-patterned on cover glass was not sufficient to recruit different species of GPI-AP [33]. Further, Raghupathy et al.’s molecular dynamics simulations revealed local GPI-AP protein density to be a critical parameter for the formation of a liquid order domain [25[8_TD$IF]]. Thus it is likely that the density of GPI-AP clustering induced by surface-immobilised antibody (10–45 nm nearest neighbour distance) was insufficient to induce large-scale lipid phase separation. Thus there may be an inherent difference of ligand-induced or antibody-induced receptor clustering and coalescence of proteins through weak ectodomain interactions that may synergise with weak lipid interactions [31]. Electrostatic interactions between proteins and lipids
model, which states that nanoscale (10–200 nm) lipidmediated phase separation serves to compartmentalise proteins in the plasma membrane, although these treatments also affect other aspects of membrane biology, including the cytoskeleton [22]. The lipid raft model continues to be highly controversial [23], with many authors adopting the view that the influence of membrane proteins on lipid distribution is more important that the converse [21,24]. Recent results present an intriguing aspect of lipid domains, namely the transbilayer coupling of clustered GPI-APs on the outer leaflet with points of cortical actin cytoskeleton interaction on the inner leaflet [25]. Previous results using fluorescence resonance energy transfer [26], single-particle tracking, near-field scanning microscopy [27] and photoactivation localisation microscopy [28,29] established that GPI-APs form transient nanoclusters that are in equilibrium with non-clustered monomers. These clusters are dependent on cholesterol and the actin cytoskeleton [28,30,31]. Cross-linking of GPI-APs results in larger clusters that co-localise with membrane-associated actin [28]. As GPI-APs localise exclusively on the outer leaflet of the plasma membrane the question arose how GPI-APs clusters are coupled to actin on the inner leaflet. Using a range of GPI analogues, Raghupathy et al. propose that nanoclustering is dependent on long acyl chains in GPI anchors that can interdigitate with long acyl chains of phosphatidylserine in the inner leaflet creating liquid order-like domains [25]. Thus immobilisation and clustering of GPI-APs leads to clustering of phosphatidylserine (PS), which in turn forms anchor sites for the cytoskeleton (Figure 2a). Molecular dynamics simulations support a model in which cholesterol-dependent www.sciencedirect.com
Activating immune receptors pair with membrane-bound signalling adaptors through compatible charge residues in their transmembrane domains [34]. In contrast, inhibitory immune receptors have endogenous signalling motifs, lack transmembrane charge residues and do not associate with membrane adaptor molecules [35]. Recent evidence suggests that an inhibitory and activatory natural killer immune receptor pair display different nanoscale organisation, with the activatory receptor KIR2DS1 displaying denser nanoclusters than the inhibitory receptor KIR2DL1 [3]. These receptors share significant homology in their ectodomains and differ mainly in their transmembrane and cytoplasmic regions, suggesting that these regions are important for differences in clustering. Indeed, the transmembrane lysine of KIR2DS1 pairs with the two transmembrane aspartic acid residues in the dimeric adaptor DAP12 and mutation of this lysine residue to alanine led to a decrease in cluster density and size. Conversely mutation of isoleucine to lysine at a similar position in the transmembrane domain of KIR2DL1 led to increased cluster size and density [3]. These results suggest that charge interaction between receptors can fine-tune the properties of clusters, in particular packing density within clusters. The charged head groups of phospholipids also play an important role in membrane protein organisation. In the plasma membrane negatively charged PS and the lower abundance phosphatidylinositol-4,5-bisphosphate (PIP2) localise preferentially to the inner leaflet and are important for the localisation of proteins with domains containing stretches of polybasic residues [36–38]. These electrostatic lipid–protein interactions can drive the assembly of domains that separate from neutral lipids in artificial liposomes and mediate syntaxin-1A and PIP2 Current Opinion in Cell Biology 2017, 44:86–92
88 Cell architecture
Figure 2
(a)
GPI-AP cluster at actin pinning point
Actin pinning
Liquid order-like domain formation
GPI-AP clustering
(b)
Depolarisation-induced PS clustering
K-Ras clustering and signalling
Current Opinion in Cell Biology
Phospholipid-dependent protein clustering mechanisms. (a) Transbilayer coupling between GPI-AP protein clustering and actin cytoskeleton pinning points. Clustering of GPI-APs (blue ovals) in the outer leaflet of the membrane forms local liquid order domains with long acyl chain phospholipids in the outer leaflet (coloured black) that interdigitate with long acyl-chained PS molecules on the inner leaflet (coloured red), forming binding sites for actin-cytoskeleton pinning proteins (depicted as green pinning proteins and purple actin filament). Conversely actin-pinning proteins on the inner leaflet cause clustering on GPI-APs on the outer leaflet. (b) Clustering of K-Ras. Depolarisation induces clustering of PS (coloured red) on the inner leaflet of the plasma membrane through an as yet undetermined mechanism. Charge interactions between PS and the poly-basic region of K-Ras (blue region on green sphere) lead to co-clustering and signalling (indicated by red arrows).
co-clustering in neurons [2]. These interactions can show surprising specificity, as exemplified by the Ras protein family in which different members form transient nanoclusters with different species of anionic lipids [1,39].
of the PS-binding protein K-Ras from the plasma membrane to endomembranes [38]. Ca2+ can also directly interact with negatively charged lipids, which can cause dissociation of the PS-binding basic rich regions in the cytoplasmic tails of the T cell receptor [41].
Cation concentrations can have a dramatic effect on anionic lipid organisation and protein–lipid interactions, which can have consequences for protein clustering and localisation. Raising intracellular Ca2+ levels causes externalisation of PS to the outer leaflet and hydrolysis of PIP2 by phospholipase C [40] and the associated decrease in inner leaflet charge causes a redistribution
Interestingly recent results show that plasma membrane depolarisation leads to the rapid clustering of PS and a slower clustering of PIP2, but does not affect other negatively charged phospholipids such as phosphatidic acid or phosphatidylinositol-3,4,5-triphosphate [1]. K-Ras also clustered in response to membrane depolarisation, which resulted in downstream ERK signalling [1]. The
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Mechanisms of protein nanoscale clustering Goyette and Gaus 89
related protein H-Ras, which does not bind PS, did not cluster and depleting PS eliminated depolarisation-induced K-Ras clustering, suggesting that the PS clustering causes K-Ras clustering rather than the converse (Figure 2b). The mechanism behind clustering of PS in response to depolarisation is unclear but could have important consequences for other PS-binding proteins. Polyvalent cross-linking by ligands and adaptors
Cross-linking of cell surface receptors by polyvalent ligands is perhaps the most obvious method of inducing membrane protein clustering. Similarly, cross-linking of raft-favouring lipids with proteins can lead to long-range lipid ordering and phase separation [42]. Recently, it was shown that adaptor molecules binding to the cytoplasmic tails of membrane proteins could also induce large-scale domain formation. Su et al. [43] reconstituted the membrane-anchored adaptor LAT into lipid bilayers that formed large clusters when the soluble cytoplasmic signalling adaptors Grb2 and Sos1 were added. Cluster formation required phosphorylation of multiple tyrosines on LAT, which are binding sites for the SH2 domain of Grb2, and required both SH3 domains of Grb2, which interact with multiple proline-rich regions on Sos1. Formation of such multi-protein complex assemblies around multivalent adaptor molecules is a common feature in membrane receptor signal transduction pathways, and may serve to sterically restrict access of inhibitory proteins as well as amplify signalling [43,44,45]. Mechanical mechanisms
An interesting and underappreciated area of membrane protein biology is the effect of mechanical forces, membrane fluctuation and steric effects on membrane protein organisation (Figure 3). Interactions occurring at cell–cell interfaces experience forces that do not occur for protein– protein interactions in solution and these forces have consequences for the clustering of surface receptors. Over two decades ago it was noted that the dimensions of important immune receptors such as T cell receptor/ peptide MHC and CD28/CD80 were much smaller than other highly abundant T cell surface molecules such as CD43 and CD45 [46]. The bending stiffness of the plasma membrane and the compressional resistance of abundant large surface molecules resist the close apposition of cell membranes required for interaction of these receptor–ligand pairs. The resulting energy penalty of bending the membrane and/or large surface molecules to accommodate the transmembrane binding pairs has three theoretical consequences: firstly, it places force on the receptor–ligand bond, increasing the dissociation rate exponentially with applied force for typical slip bonds [47] or, for the less common catch-bond, decreasing the dissociation rate with moderate force through an allosteric mechanism [48]. In the case of integrins, which display catch-bond behaviour, this can enhance interactions [49]. www.sciencedirect.com
Secondly, the energy penalty of closely apposed membranes causes the segregation of large surface molecules from small receptor-ligand interactions [45,50–52,53] (Figure 3a). This effect was clearly demonstrated in a reductionist system utilising giant unilamellar vesicles coated in binding and non-binding fluorescent proteins of different dimensions [53]. In the absence of a cellular machinery, segregation of non-binding proteins from vesicle interfaces was driven by the energy penalty of membrane bending and occurred for proteins as little as 5 nm larger than the interacting pair. That this mechanism is biologically relevant is clear from the many studies reporting segregation of large surface phosphatases from immune receptor–ligand engagement sites [35,51]. One example is the efficient phagocytosis of IgG opsonised targets by macrophages, which involves engagement of Fcg receptors and exclusion of the large phosphatase CD45 from the contact interface. Recent evidence suggests that integrins and the actin cytoskeleton may help support the process of CD45 exclusion as integrins and the actin cytoskeleton are recruited around engaged and clustered Fcg receptors, contributing to the diffusion barrier [44]. It is likely this mechanism helps to enlarge the region of tight apposition and thus allows the phagocytic cup to grow on poorly opsonised phagocytic targets. The third consequence of the coupling of two fluctuating membranes through receptor–ligand pairs is the forces that promote clustering of compatible receptor–ligand bonds [50,54,55] (Figure 3a). This property may be important for signal integration, where colocalisation and coclustering of different receptor species is important [35]. Ko¨hler et al. demonstrated that matching ectodomain sizes is required for optimal colocalisation and signal integration between activatory and inhibitory receptors [56], which is consistent with the idea that passive reorganisation leads to colocalisation of compatible ectodomain sizes. In addition to the mechanical mechanisms described thus far, the stiffness of the glycocalyx has intriguingly emerged as an organiser of receptor clustering. In a series of elegant experiments, Paszek et al. demonstrated that a thick, stiff glycocalyx impeded overall binding of integrins but enhances the degree of integrin clustering [49]. This occurred because a thick glycocalyx holds integrinbinding sites too distant for integrins to efficiently reach ligands in the extracellular matrix, except in those regions that already contain at least one integrin interaction, which locally holds the membrane close enough for further interactions to occur nearby (Figure 3b). This results in funnelling of new integrin bonds to sites of existing bonds, resulting in clustered integrin bonds, focal adhesion assembly and enhanced growth and survival of cells [49]. Although this ‘kinetic trapping’ mechanism was described for integrins, the principle is likely to apply to interactions between any cell surface receptor that interacts with a surface immobilised ligand. Current Opinion in Cell Biology 2017, 44:86–92
90 Cell architecture
Figure 3
(a)
Segregation and clustering to minimise membrane bending
(b)
‘Kinetic trapping’ around initial bond
Current Opinion in Cell Biology
Mechanical mechanisms of membrane receptor clustering. (a) Due to repulsion by the glycocalyx (depicted as large glycosylated transmembrane proteins) the plasma membrane must bend to accommodate interactions between small receptor–ligand pairs at cell–cell interfaces (depicted as blue receptors and green ligands). The bending rigidity of the membrane introduces an energetic penalty (depicted as red regions in the membrane), which induces clustering of ligand–receptor pairs and exclusion of large proteins to reduce the overall membrane deformation. (b) A thick glycocalyx resists close apposition of the cell membrane with the extracellular matrix (pink strands). Initial interactions of integrin (blue molecules) with the extracellular matrix stabilise close contact points making further bonds more likely to occur nearby within the same close contact point. This leads to clustered bond formation and focal adhesion assembly.
Conclusion
References and recommended reading
The mechanisms of membrane protein clustering outlined in this review are by no means exhaustive and are meant only to illustrate interesting recent examples of our state of understanding. The picture emerging from these recent advances is one of dynamic membrane protein distribution arising from the interplay between the cytoskeleton, lipid phases, electrostatic interactions and mechanical forces. Future work building on these principles with more nuanced investigation of how temporal dynamics, density and coclustering of proteins links to functional consequences will greatly enhance our understanding of membrane biology.
Papers of particular interest, published within the period of review,
Acknowledgements KG acknowledges funding from the ARC Centre of Excellence in Advanced Molecular Imaging (CE140100011) and National Health and Medical Research Council of Australia (1059278, 1037320). Current Opinion in Cell Biology 2017, 44:86–92
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ScienceDirect Mechanisms of protein nanoscale clustering Jesse Goyette1,2 and Katharina Gaus1,2 Due to recent technical developments in microscopy, huge advances have been made in our understanding of the architecture of the cell membrane. It is now well appreciated that nanoscale clustering is a common feature of membrane proteins. Many of these clusters have been implicated in signal initiation and integration platforms. However, the mechanisms that mediate the dynamic nanoscale arrangement of membrane proteins are not fully understood and could involve lipid domains, electrostatic interactions between proteins and lipid, protein scaffolding as well as purely mechanical processes. In this review we summarise these mechanisms giving rise to dynamic nanoscale protein reorganisation in the plasma membrane with reference to recent examples of immune receptor clustering to illustrate general principles. Addresses 1 EMBL Australia Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney 2052, Australia 2 ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, Sydney 2052, Australia Corresponding author: Gaus, Katharina ([email protected])
Current Opinion in Cell Biology 2016, 44:86–92 This review comes from a themed issue on Cell Architecture Edited by Terry Lechler and Rafael Carazo Salas For a complete overview see the Issue and the Editorial Available online 22nd September 2016 http://dx.doi.org/10.1016/j.ceb.2016.09.004 0955-0674/# 2016 Elsevier Ltd. All rights reserved.
Protein clustering in the picket fence era With the advent of super-resolution microscopy, new insights into the organisation of membrane proteins have [5_TD$IF]emerged with clusters ranging from nanoscale, transient interactions to micrometre scale, stable and immobile structures. For example in immune receptor signalling, a large and diverse group of protein have been reported to reside in clusters[6_TD$IF], including[7_TD$IF]: Ras proteins [1], syntaxin-1a [2], IL2Ra, KIR2DS1, KIR2DL1 [3[1_TD$IF]], T cell receptor [4], B Cell Receptor [5], LAT [6,7], FceRI [8], multiple glycosylphosphatidylinositol anchored proteins (GPIAPs), LFA-1 [9] and DC-SIGN [10,11]. Indeed, as more evidence accumulates examples of proteins that are not described as clustered become scarce. This has led some authors to propose that clustering may be a common feature of membrane receptors [12], but is it a universal feature? Current Opinion in Cell Biology 2017, 44:86–92
Electron microscopy studies were among the first to report non-random groupings of proteins on the scale of nanometres, tens of nanometres and on a much larger scale of protein islands interspersed by protein poor regions [13–15]. More recently, super-resolution microscopy has provided supporting evidence for the protein island architecture [16] where most cell surface proteins were organised into clusters a few hundred nanometres in size. In this study by Saka et al., some proteins had a preference for the centre of assemblies, others for the edge, and some showed no preferential localisation, suggesting that for some proteins some kind of specificity of localisation remained [16]. These ‘protein islands’ were bordered by cortical actin cytoskeleton and were disrupted by cholesterol depletion. This study is one of the clearest demonstrations of the architecture predicted by the ‘picket fence’ model of membrane biology (Figure 1a), as outlined elegantly by Kusumi et al. [17]. Briefly, the picket fence model states that the cortical actin cytoskeleton (fences), and transmembrane proteins anchored to it (pickets), separate the membrane into segments. Within these segments proteins diffuse freely but are sterically restricted from crossing between segments by the membrane cytoskeleton, which acts as a diffusion barrier. The picket fence model, and recent studies supporting it, provides an explanation for the predominance of protein clustering — a transmembrane protein with a random distribution in the cell membrane is always sterically excluded from regions occupied by the actin cytoskeleton (Figure 1b). Thus the protein is localised within corrals, which are on the scale of the diffraction limit (200 nm). When imaged with super-resolution microscopy the heterogeneity in localisation will be auto-correlated on the scale of these corrals [16]. If this autocorrelation is described as ‘clustering’ or ‘nanoclustering’ then it seems likely to be a universal feature of surface membrane proteins.
Mechanisms of protein clustering Mechanisms that lead to clustering of some but not all membrane proteins can be broadly grouped into mechanisms driven by lipid-phase separation, phospholipid charge interactions, polyvalent cross-linking and mechanical mechanisms. There are other mechanisms such as membrane curvature, which are not included here but reviewed elsewhere [18–21]. Lipid domains
Protein organisations under cholesterol depletion conditions are often interpreted in the context of the lipid raft www.sciencedirect.com
Mechanisms of protein nanoscale clustering Goyette and Gaus 87
Figure 1
(a)
liquid order-like domains form only after immobilisation of long acyl chain lipids in one leaflet [25]. In support of this, phase separation was observed in supported lipid bilayers when either lipid order or lipid disorder favouring lipids were pinned to actin filaments [32].
(b)
‘Picket fence’ architecture
Localisation of protein of interest Current Opinion in Cell Biology
Non-random distribution of proteins caused by underlying membrane architecture. (a) Picket fence architecture with cortical actin cytoskeleton (black lines), actin-binding transmembrane proteins (red dots) and randomly distributed membrane proteins (yellow and blue dots). (b) Because of steric exclusion from areas occupied by the cytoskeleton, when a randomly distributed protein of interest (blue dots) is imaged with super-resolution microscopy or electron microscopy the distribution appears non-random.
It should be pointed out that immobilisation of GPI-APs by antibodies micro-patterned on cover glass was not sufficient to recruit different species of GPI-AP [33]. Further, Raghupathy et al.’s molecular dynamics simulations revealed local GPI-AP protein density to be a critical parameter for the formation of a liquid order domain [25[8_TD$IF]]. Thus it is likely that the density of GPI-AP clustering induced by surface-immobilised antibody (10–45 nm nearest neighbour distance) was insufficient to induce large-scale lipid phase separation. Thus there may be an inherent difference of ligand-induced or antibody-induced receptor clustering and coalescence of proteins through weak ectodomain interactions that may synergise with weak lipid interactions [31]. Electrostatic interactions between proteins and lipids
model, which states that nanoscale (10–200 nm) lipidmediated phase separation serves to compartmentalise proteins in the plasma membrane, although these treatments also affect other aspects of membrane biology, including the cytoskeleton [22]. The lipid raft model continues to be highly controversial [23], with many authors adopting the view that the influence of membrane proteins on lipid distribution is more important that the converse [21,24]. Recent results present an intriguing aspect of lipid domains, namely the transbilayer coupling of clustered GPI-APs on the outer leaflet with points of cortical actin cytoskeleton interaction on the inner leaflet [25]. Previous results using fluorescence resonance energy transfer [26], single-particle tracking, near-field scanning microscopy [27] and photoactivation localisation microscopy [28,29] established that GPI-APs form transient nanoclusters that are in equilibrium with non-clustered monomers. These clusters are dependent on cholesterol and the actin cytoskeleton [28,30,31]. Cross-linking of GPI-APs results in larger clusters that co-localise with membrane-associated actin [28]. As GPI-APs localise exclusively on the outer leaflet of the plasma membrane the question arose how GPI-APs clusters are coupled to actin on the inner leaflet. Using a range of GPI analogues, Raghupathy et al. propose that nanoclustering is dependent on long acyl chains in GPI anchors that can interdigitate with long acyl chains of phosphatidylserine in the inner leaflet creating liquid order-like domains [25]. Thus immobilisation and clustering of GPI-APs leads to clustering of phosphatidylserine (PS), which in turn forms anchor sites for the cytoskeleton (Figure 2a). Molecular dynamics simulations support a model in which cholesterol-dependent www.sciencedirect.com
Activating immune receptors pair with membrane-bound signalling adaptors through compatible charge residues in their transmembrane domains [34]. In contrast, inhibitory immune receptors have endogenous signalling motifs, lack transmembrane charge residues and do not associate with membrane adaptor molecules [35]. Recent evidence suggests that an inhibitory and activatory natural killer immune receptor pair display different nanoscale organisation, with the activatory receptor KIR2DS1 displaying denser nanoclusters than the inhibitory receptor KIR2DL1 [3]. These receptors share significant homology in their ectodomains and differ mainly in their transmembrane and cytoplasmic regions, suggesting that these regions are important for differences in clustering. Indeed, the transmembrane lysine of KIR2DS1 pairs with the two transmembrane aspartic acid residues in the dimeric adaptor DAP12 and mutation of this lysine residue to alanine led to a decrease in cluster density and size. Conversely mutation of isoleucine to lysine at a similar position in the transmembrane domain of KIR2DL1 led to increased cluster size and density [3]. These results suggest that charge interaction between receptors can fine-tune the properties of clusters, in particular packing density within clusters. The charged head groups of phospholipids also play an important role in membrane protein organisation. In the plasma membrane negatively charged PS and the lower abundance phosphatidylinositol-4,5-bisphosphate (PIP2) localise preferentially to the inner leaflet and are important for the localisation of proteins with domains containing stretches of polybasic residues [36–38]. These electrostatic lipid–protein interactions can drive the assembly of domains that separate from neutral lipids in artificial liposomes and mediate syntaxin-1A and PIP2 Current Opinion in Cell Biology 2017, 44:86–92
88 Cell architecture
Figure 2
(a)
GPI-AP cluster at actin pinning point
Actin pinning
Liquid order-like domain formation
GPI-AP clustering
(b)
Depolarisation-induced PS clustering
K-Ras clustering and signalling
Current Opinion in Cell Biology
Phospholipid-dependent protein clustering mechanisms. (a) Transbilayer coupling between GPI-AP protein clustering and actin cytoskeleton pinning points. Clustering of GPI-APs (blue ovals) in the outer leaflet of the membrane forms local liquid order domains with long acyl chain phospholipids in the outer leaflet (coloured black) that interdigitate with long acyl-chained PS molecules on the inner leaflet (coloured red), forming binding sites for actin-cytoskeleton pinning proteins (depicted as green pinning proteins and purple actin filament). Conversely actin-pinning proteins on the inner leaflet cause clustering on GPI-APs on the outer leaflet. (b) Clustering of K-Ras. Depolarisation induces clustering of PS (coloured red) on the inner leaflet of the plasma membrane through an as yet undetermined mechanism. Charge interactions between PS and the poly-basic region of K-Ras (blue region on green sphere) lead to co-clustering and signalling (indicated by red arrows).
co-clustering in neurons [2]. These interactions can show surprising specificity, as exemplified by the Ras protein family in which different members form transient nanoclusters with different species of anionic lipids [1,39].
of the PS-binding protein K-Ras from the plasma membrane to endomembranes [38]. Ca2+ can also directly interact with negatively charged lipids, which can cause dissociation of the PS-binding basic rich regions in the cytoplasmic tails of the T cell receptor [41].
Cation concentrations can have a dramatic effect on anionic lipid organisation and protein–lipid interactions, which can have consequences for protein clustering and localisation. Raising intracellular Ca2+ levels causes externalisation of PS to the outer leaflet and hydrolysis of PIP2 by phospholipase C [40] and the associated decrease in inner leaflet charge causes a redistribution
Interestingly recent results show that plasma membrane depolarisation leads to the rapid clustering of PS and a slower clustering of PIP2, but does not affect other negatively charged phospholipids such as phosphatidic acid or phosphatidylinositol-3,4,5-triphosphate [1]. K-Ras also clustered in response to membrane depolarisation, which resulted in downstream ERK signalling [1]. The
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Mechanisms of protein nanoscale clustering Goyette and Gaus 89
related protein H-Ras, which does not bind PS, did not cluster and depleting PS eliminated depolarisation-induced K-Ras clustering, suggesting that the PS clustering causes K-Ras clustering rather than the converse (Figure 2b). The mechanism behind clustering of PS in response to depolarisation is unclear but could have important consequences for other PS-binding proteins. Polyvalent cross-linking by ligands and adaptors
Cross-linking of cell surface receptors by polyvalent ligands is perhaps the most obvious method of inducing membrane protein clustering. Similarly, cross-linking of raft-favouring lipids with proteins can lead to long-range lipid ordering and phase separation [42]. Recently, it was shown that adaptor molecules binding to the cytoplasmic tails of membrane proteins could also induce large-scale domain formation. Su et al. [43] reconstituted the membrane-anchored adaptor LAT into lipid bilayers that formed large clusters when the soluble cytoplasmic signalling adaptors Grb2 and Sos1 were added. Cluster formation required phosphorylation of multiple tyrosines on LAT, which are binding sites for the SH2 domain of Grb2, and required both SH3 domains of Grb2, which interact with multiple proline-rich regions on Sos1. Formation of such multi-protein complex assemblies around multivalent adaptor molecules is a common feature in membrane receptor signal transduction pathways, and may serve to sterically restrict access of inhibitory proteins as well as amplify signalling [43,44,45]. Mechanical mechanisms
An interesting and underappreciated area of membrane protein biology is the effect of mechanical forces, membrane fluctuation and steric effects on membrane protein organisation (Figure 3). Interactions occurring at cell–cell interfaces experience forces that do not occur for protein– protein interactions in solution and these forces have consequences for the clustering of surface receptors. Over two decades ago it was noted that the dimensions of important immune receptors such as T cell receptor/ peptide MHC and CD28/CD80 were much smaller than other highly abundant T cell surface molecules such as CD43 and CD45 [46]. The bending stiffness of the plasma membrane and the compressional resistance of abundant large surface molecules resist the close apposition of cell membranes required for interaction of these receptor–ligand pairs. The resulting energy penalty of bending the membrane and/or large surface molecules to accommodate the transmembrane binding pairs has three theoretical consequences: firstly, it places force on the receptor–ligand bond, increasing the dissociation rate exponentially with applied force for typical slip bonds [47] or, for the less common catch-bond, decreasing the dissociation rate with moderate force through an allosteric mechanism [48]. In the case of integrins, which display catch-bond behaviour, this can enhance interactions [49]. www.sciencedirect.com
Secondly, the energy penalty of closely apposed membranes causes the segregation of large surface molecules from small receptor-ligand interactions [45,50–52,53] (Figure 3a). This effect was clearly demonstrated in a reductionist system utilising giant unilamellar vesicles coated in binding and non-binding fluorescent proteins of different dimensions [53]. In the absence of a cellular machinery, segregation of non-binding proteins from vesicle interfaces was driven by the energy penalty of membrane bending and occurred for proteins as little as 5 nm larger than the interacting pair. That this mechanism is biologically relevant is clear from the many studies reporting segregation of large surface phosphatases from immune receptor–ligand engagement sites [35,51]. One example is the efficient phagocytosis of IgG opsonised targets by macrophages, which involves engagement of Fcg receptors and exclusion of the large phosphatase CD45 from the contact interface. Recent evidence suggests that integrins and the actin cytoskeleton may help support the process of CD45 exclusion as integrins and the actin cytoskeleton are recruited around engaged and clustered Fcg receptors, contributing to the diffusion barrier [44]. It is likely this mechanism helps to enlarge the region of tight apposition and thus allows the phagocytic cup to grow on poorly opsonised phagocytic targets. The third consequence of the coupling of two fluctuating membranes through receptor–ligand pairs is the forces that promote clustering of compatible receptor–ligand bonds [50,54,55] (Figure 3a). This property may be important for signal integration, where colocalisation and coclustering of different receptor species is important [35]. Ko¨hler et al. demonstrated that matching ectodomain sizes is required for optimal colocalisation and signal integration between activatory and inhibitory receptors [56], which is consistent with the idea that passive reorganisation leads to colocalisation of compatible ectodomain sizes. In addition to the mechanical mechanisms described thus far, the stiffness of the glycocalyx has intriguingly emerged as an organiser of receptor clustering. In a series of elegant experiments, Paszek et al. demonstrated that a thick, stiff glycocalyx impeded overall binding of integrins but enhances the degree of integrin clustering [49]. This occurred because a thick glycocalyx holds integrinbinding sites too distant for integrins to efficiently reach ligands in the extracellular matrix, except in those regions that already contain at least one integrin interaction, which locally holds the membrane close enough for further interactions to occur nearby (Figure 3b). This results in funnelling of new integrin bonds to sites of existing bonds, resulting in clustered integrin bonds, focal adhesion assembly and enhanced growth and survival of cells [49]. Although this ‘kinetic trapping’ mechanism was described for integrins, the principle is likely to apply to interactions between any cell surface receptor that interacts with a surface immobilised ligand. Current Opinion in Cell Biology 2017, 44:86–92
90 Cell architecture
Figure 3
(a)
Segregation and clustering to minimise membrane bending
(b)
‘Kinetic trapping’ around initial bond
Current Opinion in Cell Biology
Mechanical mechanisms of membrane receptor clustering. (a) Due to repulsion by the glycocalyx (depicted as large glycosylated transmembrane proteins) the plasma membrane must bend to accommodate interactions between small receptor–ligand pairs at cell–cell interfaces (depicted as blue receptors and green ligands). The bending rigidity of the membrane introduces an energetic penalty (depicted as red regions in the membrane), which induces clustering of ligand–receptor pairs and exclusion of large proteins to reduce the overall membrane deformation. (b) A thick glycocalyx resists close apposition of the cell membrane with the extracellular matrix (pink strands). Initial interactions of integrin (blue molecules) with the extracellular matrix stabilise close contact points making further bonds more likely to occur nearby within the same close contact point. This leads to clustered bond formation and focal adhesion assembly.
Conclusion
References and recommended reading
The mechanisms of membrane protein clustering outlined in this review are by no means exhaustive and are meant only to illustrate interesting recent examples of our state of understanding. The picture emerging from these recent advances is one of dynamic membrane protein distribution arising from the interplay between the cytoskeleton, lipid phases, electrostatic interactions and mechanical forces. Future work building on these principles with more nuanced investigation of how temporal dynamics, density and coclustering of proteins links to functional consequences will greatly enhance our understanding of membrane biology.
Papers of particular interest, published within the period of review,
Acknowledgements KG acknowledges funding from the ARC Centre of Excellence in Advanced Molecular Imaging (CE140100011) and National Health and Medical Research Council of Australia (1059278, 1037320). Current Opinion in Cell Biology 2017, 44:86–92
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