Photolysis Of Caged Calcium Research Articles

Activation of skeletal muscle is controlled by a dual-filament mechano-sensing mechanism

Contraction of skeletal muscle is triggered by a transient rise in intracellular calcium concentration leading to a structural change in the actin-containing thin filaments that allows binding of myosin motors from the thick filaments. Most myosin motors are unavailable for actin binding in resting muscle because they are folded back against the thick filament backbone. Release of the folded motors is triggered by thick filament stress, implying a positive feedback loop in the thick filaments. However, it was unclear how thin and thick filament activation mechanisms are coordinated, partly because most previous studies of the thin filament regulation were conducted at low temperatures where the thick filament mechanisms are inhibited. Here, we use probes on both troponin in the thin filaments and myosin in the thick filaments to monitor the activation states of both filaments in near-physiological conditions. We characterize those activation states both in the steady state, using conventional titrations with calcium buffers, and during activation on the physiological timescale, using calcium jumps produced by photolysis of caged calcium. The results reveal three activation states of the thin filament in the intact filament lattice of a muscle cell that are analogous to those proposed previously from studies on isolated proteins. We characterize the rates of the transitions between these states in relation to thick filament mechano-sensing and show how thin- and thick-filament-based mechanisms are coupled by two positive feedback loops that switch on both filaments to achieve rapid cooperative activation of skeletal muscle.

Structural dynamics of myosin motors and troponin in demembranated cardiac trabeculae activated by photolysis of caged calcium

The contractility of cardiac muscle is controlled by a dual-filament mechanism in which calcium-dependent structural changes in the thin filament gate the access of the myosin motors to the actin-binding sites, whereas regulatory structural changes in the thick filament modulate the number of motors available for the actomyosin interaction. Here we investigated the role of those structural changes in determining the kinetics of force generation during maximal activation by photolysis of caged calcium (NP-EGTA) in demembranated rat cardiac trabeculae at 27°C with lattice compression by Dextran, conditions in which the native OFF state of the thick filament is preserved in the absence of calcium.

Structural Kinetics of the RLC Domain of Myosin during Activation of Skeletal Muscle Fibers by Photolysis of Caged-Calcium

Recent fluorescence polarization studies in skeletal muscle fibers using bifunctional rhodamine (BSR) probes on the C-lobe of the regulatory light chain (RLC) of myosin showed that the steady-state calcium-sensitivity and co-operativity of the orientation change of the RLC C-lobe are higher than those of force (Fusi et al., Nat Commun 2016,doi:10.1038/ncomms13281). We have now investigated the in situ kinetics of the conformational change of the RLC during isometric contraction of a muscle fiber triggered by a rapid (>104 s−1) increase in [Ca2+] following UV-photolysis of caged-calcium (NP-EGTA). We monitored by polarized fluorescence changes in the orientation of the E-helix of a BSR-labeled RLC exchanged into demembranated fibers from rabbit psoas muscle during activation by photolysis of NP-EGTA, under conditions which preserve the physiological resting structure of the thick filament (T=25°C, 5% Dextran, 2.45 µm sarcomere length). After photolysis force increased with a sigmoidal time course similar to that of an isometric tetanus in an intact muscle fiber. The change in the order parameter P2 of the RLC probe, associated with it becoming more perpendicular to the fiber axis, also had a sigmoidal time course. The fractional change in P2 was faster than that of force. The P2 change was 25% complete 20ms after photolysis when activation of the thin filament is already maximal (T=12°C, Fusi et al., PNAS 2014, 111:4626-31) but force had developed to only 10% of its plateau value (T0). The P2 change was essentially complete by ca 70ms after photolysis when force was only 60% T0. These results show that the change in RLC orientation associated with the activation of the myosin motors is slower than thin-filament activation but faster than force generation. Supported by MRC, UK.

Calcium-Dependent Synaptic Vesicle Trafficking Underlies Indefatigable Release at the Hair Cell Afferent Fiber Synapse

Sensory hair cell ribbon synapses respond to gradedstimulation in a linear, indefatigable manner, requiring that vesicle trafficking to synapses be rapid and nonrate-limiting. Real-time monitoring of vesicle fusion identified two release components. The first was saturable with both release rate and magnitude varying linearly with Ca(2+), however the magnitude was too small to account for sustained afferent firing rates. A second superlinear release component required recruitment, in a Ca(2+)-dependent manner, of vesicles not in the immediate vicinity of the synapse. The superlinear component had a constant rate with its onset varying with Ca(2+) load. High-speed Ca(2+) imaging revealed a nonlinear increase in internal Ca(2+) correlating with the superlinear capacitance change, implicating release of stored Ca(2+) in driving vesicle recruitment. These data, supported by a mass action model, suggest sustained release at hair cell afferent fiber synapse is dictated by Ca(2+)-dependent vesicle recruitment from a reserve pool.

A Fresh View on Astrocyte Regulation of Blood Flow

Neural activity leads to a localized increase in blood flow to accommodate the increased metabolic demand. Although astrocytes (whose endfeet appose cerebral blood vessels) have long been suspected of playing a leading role in this functional hyperemia, exactly what they do and how they do it has been controversial (see Rossi). Takano et al . used calcium imaging and photolysis of caged calcium to investigate the role of astrocytes in regulating local microcirculation in the somatosensory cortex of anesthetized adult mice. After confirming that the calcium indicator Rhod-2 was preferentially taken up by astrocytes, the authors loaded Rhod-2 and caged calcium into astrocytes and visualized the vasculature with fluorescein-5-isothiocyanate-conjugated dextran (FITC-dextran). Localized photolysis of caged calcium in the endfeet of astrocytes in contact with arteries led to an increase in endfoot calcium concentration (which sometimes propagated back to the astrocyte body) associated with rapid vasodilatation (measured as an increase in arterial cross-sectional area). Pharmacological analysis indicated that this vasodilatation primarily involved phospholipase A 2 (PLA 2 )-dependent generation of arachidonic acid and its subsequent metabolism by cyclooxygenase 1 (COX1). Electrical stimulation (to elicit local increases in neural activity) also stimulated an increase in astrocyte calcium concentration and vasodilatation, as well as increasing blood flow. Pharmacological analysis indicated that this activity-mediated hyperemia depended in part on the activation of mGluR5 metabotropic glutamate receptors (which are found on astrocytes) and the PLA 2 -COX1 pathway. Thus, the authors conclude that astrocytes contribute to local vasodilatation in response to neural activity through a pathway involving the calcium- and PLA 2 -dependent release of COX1 metabolites. T. Takano, G.-F. Tian, W. Peng, N. Lou, W. Libionka, X. Han, M. Nedergaard, Astrocyte-mediated control of cerebral blood flow. Nat. Neurosci. 9 , 260-267 (2006). [PubMed] D. J. Rossi, Another BOLD role for astrocytes: Coupling blood flow to neural activity. Nat. Neurosci. 9 , 159-161 (2006). [PubMed]

Astrocytes Regulate Cerebral Blood Flow

Mulligan and MacVicar used two-photon laser scanning microscopy to selectively increase intracellular calcium concentration ([Ca 2+ ] i ) in astrocytes surrounding the arterioles in brain slices. Localized flash photolysis of caged calcium was used to increase [Ca 2+ ] i in astrocytes identified by expression of green fluorescent protein (GFP) from an astrocyte-specific promoter. When a large amplitude increase in [Ca 2+ ] i occurred in the astrocyte endfeet (the part that surrounds the blood vessel), arteriole constriction was observed. The extent of arteriole constriction correlated with the number of endfeet of the astrocyte into which the increase in [Ca 2+ ] i propagated and vasoconstriction occurred after the increase in astrocyte [Ca 2+ ] i . Noradrenergic neurons form synapses on the astrocyte endfeet, and norepinephrine (NE) causes vasoconstriction and reduced cerebral blood flow. Bath application or local application of NE produced large increases in [Ca 2+ ] i within astrocyte endfeet and caused vasoconstriction. Vasoconstriction stimulated by astrocytic calcium photolysis was blocked by pharmacologic inhibition of astrocytic phospholipase A 2 (PLA 2 ) or pharmacologic inhibition of smooth muscle production of the eicosanoid 20-hydroxy eicosatetraenoic acid (20-HETE). 20-HETE blocks calcium-activated K + channels and leads to membrane depolarization and smooth muscle calcium influx and contraction. The authors propose that increased astrocytic [Ca 2+ ] i activates PLA 2 and produces arachidonic acid, which diffuses into the vasculature smooth muscle and is converted into the vasoconstrictive 20-HETE. Peppiatt and Attwell discuss this new model in the context of previously proposed mechanisms for regulation of cerebral blood flow. S. J. Mulligan, B. A. MacVicar, Calcium transients in astrocyte endfeet cause cerebrovascular constrictions. Nature 431, 95-99 (2004). [Online Journal] C. Peppiatt, D. Attwell, Feeding the brain. Nature 431, 137-138 (2004). [Online Journal]

Examining synaptotagmin 1 function in dense core vesicle exocytosis under direct control of Ca2+.

We tested the long-standing hypothesis that synaptotagmin 1 is the Ca2+ sensor for fast neurosecretion by analyzing the intracellular Ca2+ dependence of large dense-core vesicle exocytosis in a mouse strain carrying a mutated synaptotagmin C2A domain. The mutation (R233Q) causes a twofold increase in the KD of Ca2+-dependent phospholipid binding to the double C2A-C2B domain of synaptotagmin. Using photolysis of caged calcium and capacitance measurements we found that secretion from mutant cells had lower secretory rates, longer secretory delays, and a higher intracellular Ca2+-threshold for secretion due to a twofold increase in the apparent KD of the Ca2+ sensor for fast exocytosis. Single amperometric fusion events were unchanged. We conclude that Ca2+-dependent phospholipid binding to synaptotagmin 1 mirrors the intracellular Ca2+ dependence of exocytosis.

Mechanisms governing dendritic gamma -aminobutyric acid (GABA) release in the rat olfactory bulb

In the olfactory bulb, synaptic transmission between dendrites plays an important role in the processing of olfactory information. Glutamate released from the dendrites of principal mitral cells excites the dendritic spines of granule cells, which in turn release gamma-aminobutyric acid (GABA) back onto mitral cell dendrites. Slow N-methyl-d-aspartate (NMDA) receptors on granule dendrites are particularly effective in driving this reciprocal dendrodendritic inhibition (DDI), raising the possibility that calcium influx through NMDA receptors may trigger GABA exocytosis directly. In this study, I show that NMDA receptor activation is not an absolute requirement and that DDI can be evoked solely by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors when granule cell excitability is increased or under conditions that slow AMPA receptor kinetics. In physiological extracellular Mg(2+), DDI elicited by photolysis of caged calcium in mitral dendrites is blocked by cadmium and toxins to N- and P/Q-type voltage-gated calcium channels. DDI is largely unaffected after granule dendrites have been loaded with the slow calcium chelator EGTA, suggesting a tight coupling between the site of calcium influx and the release machinery governing GABA exocytosis. These results indicate that voltage-gated calcium channels play an essential role in dendritic GABA release during reciprocal feedback inhibition in the olfactory bulb.

Mechanisms governing dendritic gamma-aminobutyric acid (GABA) release in the rat olfactory bulb.

In the olfactory bulb, synaptic transmission between dendrites plays an important role in the processing of olfactory information. Glutamate released from the dendrites of principal mitral cells excites the dendritic spines of granule cells, which in turn release gamma-aminobutyric acid (GABA) back onto mitral cell dendrites. Slow N-methyl-d-aspartate (NMDA) receptors on granule dendrites are particularly effective in driving this reciprocal dendrodendritic inhibition (DDI), raising the possibility that calcium influx through NMDA receptors may trigger GABA exocytosis directly. In this study, I show that NMDA receptor activation is not an absolute requirement and that DDI can be evoked solely by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors when granule cell excitability is increased or under conditions that slow AMPA receptor kinetics. In physiological extracellular Mg(2+), DDI elicited by photolysis of caged calcium in mitral dendrites is blocked by cadmium and toxins to N- and P/Q-type voltage-gated calcium channels. DDI is largely unaffected after granule dendrites have been loaded with the slow calcium chelator EGTA, suggesting a tight coupling between the site of calcium influx and the release machinery governing GABA exocytosis. These results indicate that voltage-gated calcium channels play an essential role in dendritic GABA release during reciprocal feedback inhibition in the olfactory bulb.

Rapid Coupling of Calcium Release to Depolarization inLimulus polyphemusVentral Photoreceptors as Revealed by Microphotolysis and Confocal Microscopy

Microphotolysis and confocal microscopy were used to investigate the timing of calcium release and of the electrical response in Limulus polyphemus ventral photoreceptors. The fluorescent dyes Fluo-3 and Calcium Green-5N were used to monitor local Ca2+ elevations. Photolysis of caged inositol trisphosphate (InsP3) close to the plasma membrane of the light-sensitive rhabdomeral (R-) lobe resulted in Ca2+ elevation within 10-20 msec, 20-45 msec before the physiological response to light normally would be detected. Inward ionic current flow and depolarization followed InsP3-induced calcium release within 2.5 +/- 3.3 msec. Voltage-clamping the cells and removal of extracellular Ca2+ did not affect the timing of the Ca2+ elevation that followed the photolysis of caged InsP3 or its relationship to the electrical response. In contrast to the physiological response to light, which only released calcium within the R-lobe, photolysis of InsP3 elevated Cai in both lobes, although with much greater effect in the R-lobe, as compared with the bulk of the A-lobe, suggesting the presence of InsP3-sensitive calcium stores in both lobes. Photolysis of caged calcium [o-nitrophenyl EGTA (NPE)] at the edge of the R-lobe activated an inward ionic current within 1.8 +/- 0.7 msec. This NPE-induced current reversed at a membrane potential of 10 +/- 6 mV in the range typical of that of the light-activated current under physiological conditions. Calcium release, therefore, activates an inward current rapidly enough to contribute to the electrical response to light.

Photolysis of caged calcium using a low-cost flash unit: efficacy analysis with a calcium selective electrode

Photolysis of caged calcium (Nitr5 ®, Calbiochem) can be used to study calcium dependent processes such as excitation-contraction coupling and muscular mechanics. Expensive high energy light sources are routinely used for UV light exposure, but this study describes an alternative low cost xenon flash unit constructed in our laboratory. A 300 J short arc xenon flash lamp (Heimann) was mounted in an elliptical reflector and driven by a modified Metz 60 CT 4 photoflash unit up to 240 J input energy and 4 ms flash duration. A 20 μl cuvette containing a test solution was placed in a complementary elliptical reflector. An ion selective calcium electrode was used to measure the free calcium concentration [Ca 2+] before and after flash in test solutions containing 1.00 mM Nitr5 in combination with different added [Ca 2+]s. Using this technique we estimated that 1 flash on 1.00 mM Nitr5 increased the free [Ca 2+] from 10 −7 to 1.1 × 10 −5 M. When the added [Ca 2+] was less than 2.3 × 10 −4 M, the used Nitr5 behaved as a strong calcium chelator because 23% of it was unloaded with calcium. It is concluded that a physiologically relevant change in free [Ca 2+] can be evoked by photolysis of Nitr5 using a low cost (approximately $1500) xenon flash unit, and that ion selective Ca electrodes can be adequately used to monitor the resulting changes in [Ca 2+].

Thin filament activation by photolysis of caged-calcium in skinned muscle fibres

Thin filament activation by photolysis of caged-calcium in skinned muscle fibres