The cytoskeleton, composed of actin filaments, intermediate filaments, and microtubules, is a highly dynamic supramolecular network actively involved in many essential biological mechanisms such as cellular structure, transport, movements, differentiation, and signaling. As a first step to characterize the biophysical changes associated with cytoskeleton functions, we have developed finite elements models of the organization of the cell that has allowed us to interpret atomic force microscopy (AFM) data at a higher resolution than that in previous work. Thus, by assuming that living cells behave mechanically as multilayered structures, we have been able to identify superficial and deep effects that could be related to actin and microtubule disassembly, respectively. In Cos-7 cells, actin destabilization with Cytochalasin D induced a decrease of the visco-elasticity close to the membrane surface, while destabilizing microtubules with Nocodazole produced a stiffness decrease only in deeper parts of the cell. In both cases, these effects were reversible. Cell softening was measurable with AFM at concentrations of the destabilizing agents that did not induce detectable effects on the cytoskeleton network when viewing the cells with fluorescent confocal microscopy. All experimental results could be simulated by our models. This technology opens the door to the study of the biophysical properties of signaling domains extending from the cell surface to deeper parts of the cell.

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.

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.

Measuring the biophysical properties of macromolecular complexes at work is a major challenge of modern biology. The protein complex composed of vesicle-associated membrane protein 2, synaptosomal-associated protein of 25 kDa, and syntaxin 1 [soluble N-ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) complex] is essential for docking and fusion of neurotransmitter-filled synaptic vesicles with the presynaptic membrane. To better understand the fusion mechanisms, we reconstituted the synaptic SNARE complex in the imaging chamber of an atomic force microscope and measured the interaction forces between its components. Each protein was tested against the two others, taken either individually or as binary complexes. This approach allowed us to determine specific interaction forces and dissociation kinetics of the SNAREs and led us to propose a sequence of interactions. A theoretical model based on our measurements suggests that a minimum of four complexes is probably necessary for fusion to occur. We also showed that the regulatory protein neuronal Sec1 injected into the atomic force microscope chamber prevented the complex formation. Finally, we measured the effect of tetanus toxin protease on the SNARE complex and its activity by on-line registration during tetanus toxin injection. These experiments provide a basis for the functional study of protein microdomains and also suggest opportunities for sensitive screening of drugs that can modulate protein-protein interactions. E xocytosis is the process in which an intracellular membranebound vesicle fuses with the plasma membrane, leading to the release of the vesicle content into the extracellular space. One of the most extensively studied forms of exocytosis is the chemical synaptic transmission used by nervous system cells to communicate. A better understanding of this mechanism is a key to understanding synaptic transmission and synaptic plasticity, learning, and memory.The current hypothesis suggests soluble N-ethyl-maleimidesensitive factor attachment protein receptor (SNARE) proteins are mediators of membrane fusion (1-4). In this model, vesicleassociated membrane protein 2 (VAMP 2) is located on the vesicle membrane whereas syntaxin 1 and synaptosomalassociated protein of 25 kDa (SNAP-25) reside in the target membrane (Fig. 1A). Before the fusion process, these proteins assemble into a highly thermostable ternary complex (5-7), which consists of a coiled-coil bundle of four ␣-helices (one from syntaxin 1, one from VAMP 2, and two from SNAP-25) bound in a parallel alignment (8-10). Whether the SNARE complex triggers fusion directly or indirectly is still controversial (11). The ''zipper'' model proposes that the complex zips from the membrane distal amino termini to the proximal carboxyl termini, thus bringing the vesicle and the membrane in a close configuration that might induce fusion (4, 10). The sequence of steps leading to fusion is still not clear. The SNARE complex could exist in a partially zipped conformation (12) and complete zip...