Post-tetanic increase in the fast-releasing synaptic vesicle pool at the expense of the slowly releasing pool.

Post-tetanic potentiation (PTP) at the calyx of Held synapse is caused by increases not only in release probability (P(r)) but also in the readily releasable pool size estimated from a cumulative plot of excitatory post-synaptic current amplitudes (RRP(cum)), which contribute to the augmentation phase and the late phase of PTP, respectively. The vesicle pool dynamics underlying the latter has not been investigated, because PTP is abolished by presynaptic whole-cell patch clamp. We found that supplement of recombinant calmodulin to the presynaptic pipette solution rescued the increase in the RRP(cum) after high-frequency stimulation (100 Hz for 4-s duration, HFS), but not the increase in P(r). Release-competent synaptic vesicles (SVs) are heterogeneous in their releasing kinetics. To investigate post-tetanic changes of fast and slowly releasing SV pool (FRP and SRP) sizes, we estimated quantal release rates before and 40 s after HFS using the deconvolution method. After HFS, the FRP size increased by 19.1% and the SRP size decreased by 25.4%, whereas the sum of FRP and SRP sizes did not increase. Similar changes in the RRP were induced by a single long depolarizing pulse (100 ms). The post-tetanic complementary changes of FRP and SRP sizes were abolished by inhibitors of myosin II or myosin light chain kinase. The post-tetanic increase in the FRP size coupled to a decrease in the SRP size provides the first line of evidence for the idea that a slowly releasing SV can be converted to a fast releasing one.

In vivo assessment of artery smooth muscle [Ca2+]i and MLCK activation in FRET-based biosensor mice.

The cellular mechanisms that control arterial diameter in vivo, particularly in hypertension, are uncertain. Here, we report a method that permits arterial intracellular Ca(2+) concentration ([Ca(2+)](i)), myosin light-chain kinase (MLCK) activation, and artery external diameter to be recorded simultaneously with arterial blood pressure (BP) in living mice under 1.5% isofluorane anesthesia. The method also enables an assessment of local receptor activity on [Ca(2+)](i), MLCK activity, and diameter in arteries, uncomplicated by systemic effects. Transgenic mice that express, in smooth muscle, a Ca(2+)/calmodulin-activated, Förster resonance energy transfer (FRET)-based "ratiometric", exogenous MLCK biosensor were used. Vasoactive substances were administered either intravenously or locally to segments of exposed femoral or cremaster arteries. In the basal state, mean BP was approximately 90 mmHg, femoral arteries were constricted to 65% of their passive diameter, MLCK fractional activation was 0.14, and [Ca(2+)](i) was 131 nM. Phenylephrine (300 ng/g wt iv) elevated mean BP transiently to approximately 110 mmHg, decreased heart rate, increased femoral artery [Ca(2+)](i) to 244 nM and fractional MLCK activation to 0.24, and decreased artery diameter by 23%. In comparison, local application of 1.0 muM phenylephrine raised [Ca(2+)](i) to 279 nM and fractional MLCK activation to 0.26, and reduced diameter by 25%, but did not affect BP or heart rate. Intravital FRET imaging of exogenous MLCK biosensor mice permits quantification of changes in [Ca(2+)](i) and MLCK activation that accompany small changes in BP. Based on the observed variance of the FRET data, this method should enable the detection of a difference in basal [Ca(2+)](i) of 29 nM between two groups of 12 mice with a significance of P < 0.05.

The myosin C-loop is an allosteric actin contact sensor in actomyosin.

Actin and myosin form the molecular motor in muscle. Myosin is the enzyme performing ATP hydrolysis under the allosteric control of actin such that actin binding initiates product release and force generation in the myosin power stroke. Non-equilibrium Monte Carlo molecular dynamics simulation of the power stroke suggested that a structured surface loop on myosin, the C-loop, is the actin contact sensor initiating actin activation of the myosin ATPase. Previous experimental work demonstrated C-loop binds actin and established the forward and reverse allosteric link between the C-loop and the myosin active site. Here, smooth muscle heavy meromyosin C-loop chimeras were constructed with skeletal (sCl) and cardiac (cCl) myosin C-loops substituted for the native sequence. In both cases, actin-activated ATPase inhibition is indicated mainly by the lower V(max). In vitro motility was also inhibited in the chimeras. Motility data were collected as a function of myosin surface density, with unregulated actin, and with skeletal and cardiac isoforms of tropomyosin-bound actin for the wild type, cCl, and sCl. Slow and fast subpopulations of myosin velocities in the wild-type species were discovered and represent geometrically unfavorable and favorable actomyosin interactions, respectively. Unfavorable interactions are detected at all surface densities tested. Favorable interactions are more probable at higher myosin surface densities. Cardiac tropomyosin-bound actin promotes the favorable actomyosin interactions by lowering the inhibiting geometrical constraint barriers with a structural effect on actin. Neither higher surface density nor cardiac tropomyosin-bound actin can accelerate motility velocity in cCl or sCl, suggesting the element initiating maximal myosin activation by actin resides in the C-loop.

Ligand-dependent equilibrium fluctuations of single calmodulin molecules.

Single-molecule force spectroscopy allows superb mechanical control of protein conformation. We used a custom-built low-drift atomic force microscope to observe mechanically induced conformational equilibrium fluctuations of single molecules of the eukaryotic calcium-dependent signal transducer calmodulin (CaM). From this data, the ligand dependence of the full energy landscape can be reconstructed. We find that calcium ions affect the folding kinetics of the individual CaM domains, whereas target peptides stabilize the already folded structure. Single-molecule data of full length CaM reveal that a wasp venom peptide binds noncooperatively to CaM with 2:1 stoichiometry, whereas a target enzyme peptide binds cooperatively with 1:1 stoichiometry. If mechanical load is applied directly to the target peptide, real-time binding/unbinding transitions can be observed.

Visualization of synaptic vesicle movement in intact synaptic boutons using fluorescence fluctuation spectroscopy.

Not much is known about the mobility of synaptic vesicles inside small synapses of the central nervous system, reflecting a lack of methods for visualizing these dynamics. We adapted confocal spot detection with fluctuation analysis to monitor the mobility of fluorescently labeled synaptic vesicles inside individual boutons of cultured hippocampal neurons. Using Monte Carlo simulations we were able to propose a simple quantitative model that can describe vesicle mobility in small hippocampal boutons under resting conditions and different pharmacological treatments. We find that vesicle mobility in a time window of 20 s can be well described by caged diffusion (D approximately 5 x 10(-5) microm(2)/s, cage sizes of approximately 50 nm). Mobility can be upregulated by phosphatase blockage and increased further by actin disruption in a dose-dependent manner. Inhibition of the myosin light chain kinase slows down vesicle mobility 10-fold, whereas other kinases like protein kinase C (PKC), A (PKA), and calmodulin kinase II (caMKII) do not affect mobility in unstimulated boutons.

Rapid actin transport during cell protrusion.

Transformed rat fibroblasts expressing two variants of green fluorescent protein, each fused to beta-actin, were used to study actin dynamics during cell protrusion. The recently developed FLAP (fluorescence localization after photobleaching) method permits the tracking of one fluorophore after localized photobleaching by using the other as a colocalized reference. Here, by visualizing the ratio of bleached to total molecules, we found that actin was delivered to protruding zones of the leading edge of the cell at speeds that exceeded 5 micrometers per second. Monte Carlo modeling confirmed that this flow cannot be explained by diffusion and may involve active transport.

Energetic cost of activation processes during contraction of swine arterial smooth muscle.

1. The objective of this study was to partition the increase in ATP consumption during contraction of swine carotid arterial smooth muscle estimated from suprabasal oxygen consumption (suprabasal JO2) and lactate release (Jlactate) into a component associated with cross-bridge cycling (JX) and one reflecting activation (JA). 2. Two experimental approaches-varying length under constant activation, and varying activation at a long length (1.8 times the optimal length for force development (Lo)) where force generation is minimal-revealed a linear dependence of JO2 and activation energy (JA) on cross-bridge phosphorylation. Protocols inducing a large increase in myosin regulatory light chain (MRLC) phosphorylation at 1.8 Lo resulted in significant elevations of JO2 and marked reductions in the economy of force maintenance. Our evidence suggests that this is primarily due to the increased cost of cross-bridge phosphorylation. 3. The extrapolated estimate of JA during maximal K(+)-induced depolarization made by varying length was 16%, while at 1.8 Lo it was 33% of the suprabasal JO2 at Lo. Calculated activation energies ranged from 17 to 45% of the suprabasal JO2 at Lo and from 72 to 87% of the suprabasal JO2 at 1.8 Lo under stimulation conditions that varied steady-state MRLC phosphorylation from 15 to 50%. 4. The results suggest that the kinetics of cross-bridge phosphorylation-dephosphorylation can rival those of cross-bridge cycling during isometric contractions in swine arterial smooth muscle.