Cells employ a variety of linear motors, such as myosin, kinesin and RNA polymerase, which move along and exert force on a filamentous structure. But only one rotary motor has been investigated in detail, the bacterial flagellum (a complex of about 100 protein molecules). We now show that a single molecule of F1-ATPase acts as a rotary motor, the smallest known, by direct observation of its motion. A central rotor of radius approximately 1 nm, formed by its gamma-subunit, turns in a stator barrel of radius approximately 5nm formed by three alpha- and three beta-subunits. F1-ATPase, together with the membrane-embedded proton-conducting unit F0, forms the H+-ATP synthase that reversibly couples transmembrane proton flow to ATP synthesis/hydrolysis in respiring and photosynthetic cells. It has been suggested that the gamma-subunit of F1-ATPase rotates within the alphabeta-hexamer, a conjecture supported by structural, biochemical and spectroscopic studies. We attached a fluorescent actin filament to the gamma-subunit as a marker, which enabled us to observe this motion directly. In the presence of ATP, the filament rotated for more than 100 revolutions in an anticlockwise direction when viewed from the 'membrane' side. The rotary torque produced reached more than 40 pN nm(-1) under high load.
The enzyme F1-ATPase has been shown to be a rotary motor in which the central gamma-subunit rotates inside the cylinder made of alpha3beta3 subunits. At low ATP concentrations, the motor rotates in discrete 120 degrees steps, consistent with sequential ATP hydrolysis on the three beta-subunits. The mechanism of stepping is unknown. Here we show by high-speed imaging that the 120 degrees step consists of roughly 90 degrees and 30 degrees substeps, each taking only a fraction of a millisecond. ATP binding drives the 90 degrees substep, and the 30 degrees substep is probably driven by release of a hydrolysis product. The two substeps are separated by two reactions of about 1 ms, which together occupy most of the ATP hydrolysis cycle. This scheme probably applies to rotation at full speed ( approximately 130 revolutions per second at saturating ATP) down to occasional stepping at nanomolar ATP concentrations, and supports the binding-change model for ATP synthesis by reverse rotation of F1-ATPase.
Long-term potentiation (LTP) of synaptic strength occurs during learning and can last for long periods, making it a probable mechanism for memory storage. LTP induction results in calcium entry, which activates calcium–calmodulin-dependent protein kinase II (CaMKII). CaMKII subsequently translocates to the synapse, where it binds to the NMDA-type glutamate receptors and produces potentiation by phosphorylating principal and auxiliary subunits of AMPA-type glutamate receptors. These processes all are localized to stimulated spines and account for the synapse specificity of LTP. In the later stages of LTP, CaMKII has a structural role in enlarging and strengthening the synapse.
Ca2+ / Calmodulin-dependent kinase II (CaMKII) plays a central role in long-term potentiation (LTP), which underlies some forms of learning and memory. Here we monitored the spatiotemporal dynamics of CaMKII activation in individual dendritic spines during LTP using 2-photon fluorescence lifetime imaging microscopy in combination with 2-photon glutamate uncaging. Induction of LTP and associated spine enlargement in single spines triggered transient (∼ 1 min) CaMKII activation restricted to the stimulated spines. CaMKII in spines was specifically activated by NMDA receptors and L-type voltage sensitive calcium channels, presumably via nanodomain Ca2+ near the channels, in response to glutamate uncaging and depolarization, respectively. The high degree of compartmentalization and channel specificity of CaMKII signalling allow stimuli-specific spatiotemporal patterns of CaMKII signalling and may be important for synapse-specificity of synaptic plasticity.
A single molecule of F1-ATPase, a portion of ATP synthase, is by itself a rotary motor in which a central gamma subunit rotates against a surrounding cylinder made of alpha3beta3 subunits. Driven by three catalytic betas, each fueled with ATP, gamma makes discrete 120 degree steps, occasionally stepping backward. The work done in each step is constant over a broad range of imposed load and is close to the free energy of hydrolysis of one ATP molecule.
The brain is complex and dynamic. The spatial scales of interest to the neurobiologist range from individual synapses (approximately 1 microm) to neural circuits (centimeters); the timescales range from the flickering of channels (less than a millisecond) to long-term memory (years). Remarkably, fluorescence microscopy has the potential to revolutionize research on all of these spatial and temporal scales. Two-photon excitation (2PE) laser scanning microscopy allows high-resolution and high-sensitivity fluorescence microscopy in intact neural tissue, which is hostile to traditional forms of microscopy. Over the last 10 years, applications of 2PE, including microscopy and photostimulation, have contributed to our understanding of a broad array of neurobiological phenomena, including the dynamics of single channels in individual synapses and the functional organization of cortical maps. Here we review the principles of 2PE microscopy, highlight recent applications, discuss its limitations, and point to areas for future research and development.
The Rho family of GTPases play important roles in morphogenesis of dendritic spines1–3 and synaptic plasticity4–9 by modulating the organization of the actin cytoskeleton10. Here, we monitored the activity of Rho GTPases, RhoA and Cdc42, in single dendritic spines undergoing structural plasticity associated with long-term potentiation (LTP) using 2-photon fluorescence lifetime imaging microscopy (2pFLIM)11–13. When long-term volume increase was induced in a single spine using 2-photon glutamate uncaging14,15, RhoA and Cdc42 were rapidly activated in the stimulated spine. These activities decayed over ~5 min, and were then followed by a phase of persistent activation lasting more than 30 min. Although active RhoA and Cdc42 were similarly mobile, their activity patterns were different. RhoA activation diffused out of the stimulated spine and spread over ~5 μm along the dendrite. In contrast, Cdc42 activation was restricted to the stimulated spine, and exhibited a steep gradient at the spine necks. Inhibition of the Rho-Rock pathway preferentially inhibited the initial spine growth, whereas the inhibition of the Cdc42-Pak pathway blocked the maintenance of sustained structural plasticity. RhoA and Cdc42 activation depended on Ca2+/calmodulin-dependent kinase (CaMKII). Thus, RhoA and Cdc42 relay transient CaMKII activation13 to synapse-specific, long-term signalling required for spine structural plasticity.
In neurons, individual dendritic spines isolate N-methyl-d-aspartate (NMDA) receptor-mediated calcium ion (Ca2+) accumulations from the dendrite and other spines. However, the extent to which spines compartmentalize signaling events downstream of Ca2+ influx is not known. We combined two-photon fluorescence lifetime imaging with two-photon glutamate uncaging to image the activity of the small guanosine triphosphatase Ras after NMDA receptor activation at individual spines. Induction of long-term potentiation (LTP) triggered robust Ca2+-dependent Ras activation in single spines that decayed in approximately 5 minutes. Ras activity spread over approximately 10 micrometers of dendrite and invaded neighboring spines by diffusion. The spread of Ras-dependent signaling was necessary for the local regulation of the threshold for LTP induction. Thus, Ca2+-dependent synaptic signals can spread to couple multiple synapses on short stretches of dendrite.
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