Biomolecular imaging using atomic force microscopy (AFM)
We study the structure and function of proteins at the single-molecule level using atomic force microscopy (AFM).
AFM imaging. The sample is mounted on mica, and the tip is scanned across it in a raster pattern. A laser beam is bounced off the back of the cantilever and is reflected by a mirror onto a split photodiode. When the tip encounters an object on the surface of the mica, it becomes deflected, causing the position of the laser beam on the photodiode to change. The scanner moves in the Z-dimension to restore the beam to its original position. The XYZ movements of the scanner are analysed by a computer in order to generate the image. Imaging can be carried out either in air or under fluid.
Activation-induced structural changes in ionotropic receptors
We are using used AFM imaging to visualize activation-induced structural changes in ionotropic receptors reconstituted into a lipid bilayer. At the moment, we are focusing on ionotropic glutamate receptors, which include NMDA, kainate and AMPA receptors. We have shown that in the absence of agonist, the extracellular domain of the GluN1/GluN2A NMDA receptor protrudes from the bilayer surface by 8.6 nm. In the presence of the co-agonists glycine and glutamate, the height of the extracellular region falls to 7.3 nm. Fast-scan AFM imaging, combined with UV photolysis of caged glutamate, permits the detection of a rapid reduction in the height of individual NMDA receptors. The reduction in height does not occur in the absence of glycine or in the presence of the NMDA receptor antagonist D-AP5, indicating that the observed structural change is caused by receptor activation. These results represent the first demonstration of an activation-induced effect on the structure of the NMDA receptor at the single-molecule level. A change in receptor size following activation could have important functional implications, in particular by affecting interactions between the NMDA receptor and its extracellular synaptic partners. We are now studying the behaviour of the kainate receptor, another member of the ionotropic glutamate receptor super-family.
Fast-scan imaging of NMDA receptor height shifts. Frames were captured at 1 Hz. A single receptor was monitored in buffer containing 100 μM glycine and 100 μM caged-glutamate. UV irradiation occurred at 107 s. A fitted Gaussian curve is overlaid on each histogram.
Cross-talk between membrane receptors is a mechanism used widely by many cell types to modulate responses to a complex environment. Although of major importance, the underlying molecular mechanisms are still poorly understood. Our objective is to investigate the changes in configuration that occur when two ionotropic receptors physically interact. In a collaborative project with Dr. Nelson Barrera (Pontificia Universidad Católica de Chile, Santiago, Chile), we are using fast-scan AFM to study the interaction between P2X and 5-HT3 receptors in an attempt to unravel the molecular basis of their cross-inhibition.
The interaction of the sigma-1 receptor with ionotropic receptors and ion channels
The sigma-1 receptor is widely expressed in the central nervous system, where it has a neuroprotective role in ischaemia and stroke, and an involvement in schizophrenia. The sigma-1 receptor interacts functionally with a variety of ion channels, including the NMDA receptor. We showed that the sigma-1 receptor binds directly and specifically to GluN1 subunits within GluN1/GluN2A NMDA receptor heterotetramers. This interaction likely accounts for at least some of the observed modulatory effects of sigma-1 receptor ligands on the NMDA receptor.
In collaboration with Dr. Olivier Soriani (University of Nice), we investigated the interaction between the sigma-1 receptor and the Nav1.5 voltage-gated Na+ channel, which has been implicated in promoting the invasiveness of cancer cells. AFM imaging demonstrated that the sigma-1 receptor binds to Nav1.5 with four-fold symmetry. Hence, each set of six transmembrane regions in Nav1.5 likely constitutes a sigma-1 receptor binding site. Interestingly, two known sigma-1 receptor ligands, haloperidol and (+)-pentazocine disrupted the sigma-1 receptor/Nav1.5 interaction both in vitro and in living cells. At the moment, we are looking at the interaction between the sigma-1 receptor and the Orai1 channel, that is responsible for store-operated Ca2+ entry, and trying to elucidate the mechanisms by which drugs perturb the interactions between the sigma-1 receptor and its ion channel targets.
The sigma-1 receptor decorates Nav1.5 with four-fold symmetry. Images of isolated Nav1.5 particles decorated by one, two, three or four Sig1R particles. Images are 75 nm on each side; height range, 2 nm.
The mechanism underlying the interaction of urinary exosomes with the primary cilium
Autosomal recessive polycystic kidney disease (ARPKD), the most common cause of hereditary childhood PKD, is caused by mutations in the gene PKHD1, which encodes the protein fibrocystin/polyductin (FCP). PC1, PC2 and FCP are believed to be present on the primary cilium. Here, the PC1/PC2 complex senses tubular fluid flow. The function of FCP is less clear, but it too complexes with PC2. PC1, PC2 and FCP are also expressed in exosomes, small vesicles present in urine. Exosomes are derived from intraluminal vesicles of multi-vesicular bodies (MVBs), and are released into the renal tubule when the MVBs fuse with the apical membrane of the tubular epithelial cells. They can mediate transfer of protein expression between cells, and they are known to play a crucial role in left-right axis determination in the embryonic node. Exosomes interact specifically with primary cilia. Significantly, exosomes from ARPKD mice decorate cilia much more extensively than exosomes from WT mice, suggesting that the binding is normally followed by fusion or endocytosis, a step that might be inhibited when FCP is mutated, thus accounting for the increased decoration. In a project funded by Kidney Research UK, and in collaboration with Prof. Fiona Karet (University of Cambridge), we are currently studying the mechanisms underlying the interaction of urinary exosomes with the primary cilium.
AFM imaging of the primary cilium. Images of isolated primary cilia that have flattened out on the mica substrate. Scale bar, 500 nm; height scale, 20 nm.
The structure and behaviour of synaptotagmin
Synaptotagmin 1 is the major Ca2+ sensor for membrane fusion during neurotransmitter release. The cytoplasmic domain of synaptotagmin consists of two C2 domains, C2A and C2B. On binding Ca2+, the tips of the two C2 domains rapidly and synchronously penetrate lipid bilayers. In collaboration with Dr. Ed Chapman (University of Wisconsin-Madison), we have used AFM to show that C2A and C2B physically interact with one another, and that this interaction is critical to the ability of synaptotagmin to drive neurotransmitter release. For example, the substitution of the flexible linker between the two C2 domains by a rigid 18-proline rod visibly separates the two C2 domains and at the same time almost abolishes the activity of the protein in neurons.
Structure of synaptotagmin. Images of wildtype C2AB and a mutant in which the flexible linker between the two C2 domains has been replaced by a rigid 18-proline rod. Scale bar, 20 nm.
We are currently imaging various other mutants that affect the behaviour of synaptotagmin, to shed light on the relationship between structure and function.
Interactions of proteins with lipid bilayers
We are interested in how proteins interact with, and remodel, lipid bilayers. As an example, we have recently been studying the Sar1 GTPase, in collaboration with Dr. Anjon Audhya (University of Wisconsin-Madison). Sar1 plays a critical role in membrane bending, recruitment of coat components, and nascent vesicle formation at the endoplasmic reticulum. How these events are appropriately coordinated remains poorly understood. Using fast-scan AFM imaging, we showed that in response to addition of GTP, Sar1 was able to rapidly remodel a lipid bilayer, resulting in its systematic removal from the mica substrate. Moreover, membrane removal occurred from the highly curved edges of the bilayer, indicating that Sar1 can sense membrane curvature.
Sar1-mediated remodelling of a lipid bilayer. Sequential images are shown, after addition of GTP at time zero. Scale bar, 250 nm.
Protection of teeth against dental caries
Dental caries is a major public health issue worldwide, with approximately 35% of the world’s population currently suffering from dental caries of their permanent teeth. Water fluoridation has been shown to be an effective mechanism for combating caries, and artificial water fluoridation is implemented in many countries, reaching 5.7% of the world’s population. Fluoride is also added to toothpastes, with the result that populations are exposed to fluoride both topically and systemically. Although the relative benefits of fluoride have been thoroughly demonstrated, water fluoridation has remained a controversial issue since its introduction in the 1940s, largely as a result of uncertainty about potential toxic effects. In collaboration with Prof. Marilia Buzalaf (University of São Paulo, Brazil), we are investigating the effects of fluoride on the structure, function and proteome of epithelia. In particular, AFM is being used to assess the effects of fluoride on the mechanical properties of the epithelial cells.
In a related project, we are testing the ability of candidate proteins to protect enamel against acid-induced damage. AFM is being used to screen the surface roughness of enamel samples after various treatments. We are also using AFM force spectroscopy to measure the forces of interaction of single protein molecules with enamel.
Current lab members
|Fahim Kadir||Pia Jeggle||Flavia Amadeu de Oliveira|