Characterization of the interaction between diferric transferrin and transferrin receptor 2 by functional assays and atomic force microscopy.

Transferrin receptor 2 (TfR2), a homologue of the classical transferrin receptor 1 (TfR1), is found in two isoforms, alpha and beta. Like TfR1, TfR2alpha is a type II membrane protein, but the beta form lacks transmembrane portions and therefore is likely to be an intracellular protein. To investigate the functional properties of TfR2alpha, we expressed the protein with FLAG tagging in transferrin-receptor-deficient Chinese hamster ovary cells. The association constant for the binding of diferric transferrin (Tf) to TfR2alpha is 5.6x10(6) M(-)(1), which is about 50 times lower than that for the binding of Tf to TfR1, with correspondingly reduced rates of iron uptake. Evidence for Tf internalization and recycling via TfR2alpha without degradation, as in the TfR1 pathway, was also found. The interaction of TfR2alpha with Tf was further investigated using atomic force microscopy, a powerful tool used for investigating the interaction between a ligand and its receptor at the single-molecule level on the living cell surface. Dynamic force microscopy reveals a difference in the interactions of Tf with TfR2alpha and TfR1, with Tf-TfR1 unbinding characterized by two energy barriers, while only one is present for Tf-TfR2. We speculate that this difference may reflect Tf binding to TfR2alpha by a single lobe, whereas two lobes of Tf participate in binding to TfR1. The difference in the binding properties of Tf to TfR1 and TfR2alpha may help account for the different physiological roles of the two receptors.

Single molecular dynamic interactions between glycophorin A and lectin as probed by atomic force microscopy.

Glycophorin A (GpA) is one of the most abundant transmembrane proteins in human erythrocytes and its interaction with lectins has been studied as model systems for erythrocyte related biological processes. We performed a force measurement study using the force mode of atomic force microscopy (AFM) to investigate the single molecular level biophysical mechanisms involved in GpA-lectin interactions. GpA was mounted on a mica surface or natively presented on the erythrocyte membrane and probed with an AFM tip coated with the monomeric but multivalent Psathyrella velutina lectin (PVL) through covalent crosslinkers. A dynamic force spectroscopy study revealed similar interaction properties in both cases, with the unbinding force centering around 60 pN with a weak loading rate dependence. Hence we identified the presence of one energy barrier in the unbinding process. Force profile analysis showed that more than 70% of GpAs are free of cytoskeletal associations in agreement with previous reports.

Elastic membrane heterogeneity of living cells revealed by stiff nanoscale membrane domains.

Many approaches have been developed to characterize the heterogeneity of membranes in living cells. In this study, the elastic properties of specific membrane domains in living cells are characterized by atomic force microscopy. Our data reveal the existence of heterogeneous nanometric scale domains with specific biophysical properties. We focused on glycosylphosphatidylinositol (GPI)-anchored proteins, which play an important role in membrane trafficking and cell signaling under both physiological and pathological conditions and which are known to partition preferentially into cholesterol-rich microdomains. We demonstrate that these GPI-anchored proteins reside within domains that are stiffer than the surrounding membrane. In contrast, membrane domains containing the transferrin receptor, which does not associate with cholesterol-rich regions, manifest no such feature. The heightened stiffness of GPI domains is consistent with existing data relating to the specific condensation of lipids and the slow diffusion rates of lipids and proteins therein. Our quantitative data may forge the way to unveiling the links that exist between membrane stiffness, molecular diffusion, and signaling activation.

Receptor trafficking and AFM.

Adaptation of a cell behavior to the environment is possible due to the biochemical and physical information that is transmitted through molecular receptor present at the cell surface. Regulation of receptor distribution and trafficking is thus a key feature to allow cells to properly respond to extracellular signals. Many of the molecular mechanisms that support receptor trafficking occurs at a submicrometric scale and are highly dynamic. Because of its exceptional resolution and its piconewton sensitivity, atomic force microscope (AFM) is a powerful tool to study the trafficking of individual receptors in living cells under near-physiological conditions. In this review, we first describe the general principles of the AFM that allow the detection of single ligand-receptor interaction. We then turn to early studies that demonstrated the ability of AFM to detect individual receptors and map their distribution on the surface of living cell. Finally, we discuss how AFM in combination with optical imaging tools allow the simultaneous investigation of cellular biophysical properties and receptor-trafficking dynamics at the nanometer scale.

Exploring transferrin-receptor interactions at the single-molecule level.

Interaction between the iron transporter protein transferrin (Tf) and its receptor at the cell surface is fundamental for most living organisms. Tf receptor (TfR) binds iron-loaded Tf (holo-Tf) and transports it to endosomes, where acidic pH favors iron release. Iron-free Tf (apo-Tf) is then brought back to the cell surface and dissociates from TfR. Here we investigated the Tf-TfR interaction at the single-molecule level under different conditions encountered during the Tf cycle. An atomic force microscope tip functionalized with holo-Tf or apo-Tf was used to probe TfR. We tested both purified TfR anchored to a mica substrate and in situ TfR at the surface of living cells. Dynamic force measurements showed similar results for TfR on mica or at the cell surface but revealed striking differences between holo-Tf-TfR and apo-Tf-TfR interactions. First, the forces necessary to unbind holo-Tf and TfR are always stronger compared to the apo-Tf-TfR interaction. Second, dissociation of holo-Tf-TfR complex involves overcoming two energy barriers, whereas the apo-Tf-TfR unbinding pathway comprises only one energy barrier. These results agree with a model that proposes differences in the contact points between holo-Tf-TfR and apo-Tf-TfR interactions.

Elastic properties of the cell surface and trafficking of single AMPA receptors in living hippocampal neurons.

Although various approaches are routinely used to study receptor trafficking, a technology that allows for visualizing trafficking of single receptors at the surface of living cells remains lacking. Here we used atomic force microscope to simultaneously probe the topography of living cells, record the elastic properties of their surface, and examine the distribution of transfected alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA)-type glutamate receptors (AMPAR). On nonstimulated neurons, AMPARs were located in stiff nanodomains with high elasticity modulus relative to the remaining cell surface. Receptor stimulation with N-methyl-D-aspartate (NMDA) provoked a permanent disappearance of these stiff nanodomains followed by a decrease (53%) of the number of surface AMPARs. Blocking electrical activity before NMDA stimulation recruited the same number of AMPARs for internalization, preceded by the loss of the stiff nanodomains. However, in that case, the stiff nanodomains were recovered and AMPARs were reinserted into the membrane shortly after. Our results show that modulation of receptor distribution is accompanied by changes in the local elastic properties of cell membrane. We postulate, therefore, that the mechanical environment of a receptor might be critical to determine its specific distribution behavior in response to different stimuli.

Phosphorylation of glutamate receptor interacting protein 1 regulates surface expression of glutamate receptors.

The number of synaptic alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors (AMPARs) controls the strength of excitatory transmission. AMPARs cycle between internal endosomal compartments and the plasma membrane. Interactions between the AMPAR subunit GluR2, glutamate receptor interacting protein 1 (GRIP1), and the endosomal protein NEEP21 are essential for correct GluR2 recycling. Here we show that an about 85-kDa protein kinase phosphorylates GRIP1 on serine 917. This kinase is present in NEEP21 immunocomplexes and is activated in okadaic acid-treated neurons. Pulldown assays and atomic force microscopy indicate that phosphorylated GRIP shows reduced binding to NEEP21. AMPA or N-methyl-D-aspartate stimulation of hippocampal neurons induces delayed phosphorylation of the same serine 917. A wild type carboxy-terminal GRIP1 fragment expressed in hippocampal neurons interferes with GluR2 surface expression. On the contrary, a S917D mutant fragment does not interfere with GluR2 surface expression. Likewise, coexpression of GluR2 together with full-length wild type GRIP1 enhances GluR2 surface expression in fibroblasts, whereas full-length GRIP1-S917D had no effect. This indicates that this serine residue is implicated in AMPAR cycling. Our results identify an important regulatory mechanism in the trafficking of AMPAR subunits between internal compartments and the plasma membrane.

Interactions between NEEP21, GRIP1 and GluR2 regulate sorting and recycling of the glutamate receptor subunit GluR2.

Trafficking of AMPA-type glutamate receptors (AMPAR) between endosomes and the postsynaptic plasma membrane of neurons plays a central role in the control of synaptic strength associated with learning and memory. The molecular mechanisms of its regulation remain poorly understood, however. Here we show by biochemical and atomic force microscopy analyses that NEEP21, a neuronal endosomal protein necessary for receptor recycling including AMPAR, is associated with the scaffolding protein GRIP1 and the AMPAR subunit GluR2. Moreover, the interaction between NEEP21 and GRIP1 is regulated by neuronal activity. Expression of a NEEP21 fragment containing the GRIP1-binding site decreases surface GluR2 levels and delays recycling of internalized GluR2, which accumulates in early endosomes and lysosomes. Infusion of this fragment into pyramidal neurons of hippocampal slices induces inward rectification of AMPAR-mediated synaptic responses, suggesting decreased GluR2 expression at synapses. These results indicate that NEEP21-GRIP1 binding is crucial for GluR2-AMPAR sorting through endosomes and their recruitment to the plasma membrane, providing a first molecular mechanism to differentially regulate AMPAR subunit cycling in internal compartments.

Gradient of rigidity in the lamellipodia of migrating cells revealed by atomic force microscopy.

Changes in mechanical properties of the cytoplasm have been implicated in cell motility, but there is little information about these properties in specific regions of the cell at specific stages of the cell migration process. Fish epidermal keratocytes with their stable shape and steady motion represent an ideal system to elucidate temporal and spatial dynamics of the mechanical state of the cytoplasm. As the shape of the cell does not change during motion and actin network in the lamellipodia is nearly stationary with respect to the substrate, the spatial changes in the direction from the front to the rear of the cell reflect temporal changes in the actin network after its assembly at the leading edge. We have utilized atomic force microscopy to determine the rigidity of fish keratocyte lamellipodia as a function of time/distance from the leading edge. Although vertical thickness remained nearly constant throughout the lamellipodia, the rigidity exhibited a gradual but significant decrease from the front to the rear of the lamellipodia. The rigidity profile resembled closely the actin density profile, suggesting that the dynamics of rigidity are due to actin depolymerization. The decrease of rigidity may play a role in facilitating the contraction of the actin-myosin network at the lamellipodium/cell body transition zone.