Direct in vivo monitoring of sarcoplasmic reticulum Ca2+ and cytosolic cAMP dynamics in mouse skeletal muscle

Rudolf, Rüdiger; Magalhães, Paulo; Pozzan, Tullio

doi:10.1083/jcb.200601160

Cited by 118 publications

(152 citation statements)

References 39 publications

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“…To determine the absolute values of [Ca 2+ ] within the TG we used the in vitro calibration procedures described previously for other organelles (27). Given that luminal pH affects the calibration procedure, the TG luminal pH was also determined in intact cells using a null point method (SI Text S2).…”

Section: Pozzan@unipditmentioning

confidence: 99%

Unique characteristics of Ca ²⁺ homeostasis of the trans -Golgi compartment

Lissandron

Podini

Pizzo

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Taking advantage of a fluorescent Ca 2+ indicator selectively targeted to the trans -Golgi lumen, we here demonstrate that its Ca 2+ homeostatic mechanisms are distinct from those of the other Golgi subcompartments: ( i ) Ca 2+ uptake depends exclusively on the activity of the secretory pathway Ca 2+ ATPase1 (SPCA1), whereas the sarco-endoplasmic reticulum Ca 2+ ATPase (SERCA) is excluded; ( ii ) IP 3 generated by receptor stimulation causes Ca 2+ uptake rather than release; ( iii ) Ca 2+ release can be triggered by activation of ryanodine receptors in cells endowed with robust expression of the latter channels (e.g., in neonatal cardiac myocyte). Finally, we show that, knocking down the SPCA1, and thus altering the trans -Golgi Ca 2+ content, specific functions associated with this subcompartment, such as sorting of proteins to the plasma membrane through the secretory pathway, and the structure of the entire Golgi apparatus are dramatically altered.

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Section: Pozzan@unipditmentioning

confidence: 99%

Unique characteristics of Ca ²⁺ homeostasis of the trans -Golgi compartment

Lissandron

Podini

Pizzo

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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121

131

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“…Regarding the data obtained at 22 [19] to near 1 mM [20], with most of the obtained figures around 400-800 M [21][22][23][24][25][26][27]. On the other hand, NMR measurements [21], skeletal muscle cells [25,27] and rabbit heart cells [28] The new probe has also a very important technical and experimental advantage over the previous aequorin probes. All the [Ca 2+ ] ER measurements carried out before with these probes were performed in cells previously depleted of Ca 2+ , because the high Ca 2+ content of the ER precluded reconstitution with the coelenterazine cofactor.…”

Section: Discussionmentioning

confidence: 90%

Ca2+ homeostasis in the endoplasmic reticulum measured with a new low-Ca2+-affinity targeted aequorin

et al. 2013

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a b s t r a c tWe use here a new very low-Ca 2+ -affinity targeted aequorin to measure the [Ca 2+ ] between 0.1 and 3 mM, and was only partially affected by mitochondrial membrane depolarization. Instead, it was significantly reduced by loading cells with chelators, and the fast chelator BAPTA was much more effective than the slow chelator EGTA. This suggests that local [Ca 2+ ] microdomains connecting the store operated Ca 2+ channels with the ER Ca 2+ pumps may be important during refilling.

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“…Intraperitonial injection of Rompun (Bayer) and Zoletil 100 (Laboratoires Virbac) was used for anesthesia. Transfection was as described (41,46). For Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Emission signals of CFP and YFP were simultaneously recorded. The ratio between the fluorescence intensities emitted from the two chromophores can be conveniently used to dynamically monitor the level of FRET and thus [cAMP] (36,41). In control muscles, the NMJ localization was easily determined by the accumulated RIα-EPAC fluorescence signals.…”

Section: Turnover Of Achrs Is Dependent On Nerve Input and An Intactmentioning

confidence: 99%

Myosin Va cooperates with PKA RIα to mediate maintenance of the endplate in vivo

Röder¹,

Choi²,

Reischl

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Myosin V motor proteins facilitate recycling of synaptic receptors, including AMPA and acetylcholine receptors, in central and peripheral synapses, respectively. To shed light on the regulation of receptor recycling, we employed in vivo imaging of mouse neuromuscular synapses. We found that myosin Va cooperates with PKA on the postsynapse to maintain size and integrity of the synapse; this cooperation also regulated the lifetime of acetylcholine receptors. Myosin Va and PKA colocalized in subsynaptic enrichments. These accumulations were crucial for synaptic integrity and proper cAMP signaling, and were dependent on AKAP function, myosin Va, and an intact actin cytoskeleton. The neuropeptide and cAMP agonist, calcitonin-gene related peptide, rescued fragmentation of synapses upon denervation. We hypothesize that neuronal ligands trigger local activation of PKA, which in turn controls synaptic integrity and turnover of receptors. To this end, myosin Va mediates correct positioning of PKA in a postsynaptic microdomain, presumably by tethering PKA to the actin cytoskeleton.cAMP | muscle | neuromuscular junction | microdomain | synaptic plasticity R ecently, myosin V motor proteins were found to be crucial for the plasticity of central and peripheral synapses by facilitating, respectively, the recycling of AMPA (1-3) and acetylcholine receptors (AChRs) (4). The neuromuscular junction (NMJ) is the synapse between motor neurons and skeletal muscle fibers. Even though it is formed during ontogenesis and then maintained over the years (5), half-lives (t 1/2 ) of a few days are typical for its protein constituents, such as the AChR (6). The high enrichment of AChRs in the postsynapse is regulated by accurate targeting and turnover of this receptor. Vesicular transport is employed for this purpose, using different routes of exocytosis, endocytosis, and recycling (7-9). We identified myosin Va as the first motor protein involved in AChR trafficking and have found it to facilitate its recycling (4). Down-regulation of myosin Va led to a reduced t 1/2 of AChRs (4) similar to pathological conditions, such as denervation (6, 10-13) or dystrophy (14). A shortened t 1/2 of AChRs accompanies morphological changes of the NMJ: while NMJs in mouse muscles are normally elliptic and pretzel-shaped (15), they elongate and fragment under pathological conditions (16,17).Previous studies have revealed a major role of cAMP signaling in controlling the t 1/2 of AChRs (12, 18). cAMP is formed upon activation of G protein-coupled receptors and mediates its effects mainly through protein kinase A (PKA). Notably, stimulation of cAMP synthesis rescued AChR lifetime in denervated (12,19) and dystrophic muscles (14). Several groups have described antagonistic functions of protein kinase C and PKA on AChR persistence and synaptic strength (18,20,21): although activation of protein kinase C destabilizes AChRs, PKA activity protects this receptor from such destabilization (18). In this context, the neuropeptide and cAMP agonist, α-calcitonin gene-rel...

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Direct in vivo monitoring of sarcoplasmic reticulum Ca2+ and cytosolic cAMP dynamics in mouse skeletal muscle

Cited by 118 publications

References 39 publications

Unique characteristics of Ca ²⁺ homeostasis of the trans -Golgi compartment

Unique characteristics of Ca ²⁺ homeostasis of the trans -Golgi compartment

Ca2+ homeostasis in the endoplasmic reticulum measured with a new low-Ca2+-affinity targeted aequorin

Myosin Va cooperates with PKA RIα to mediate maintenance of the endplate in vivo

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Direct in vivo monitoring of sarcoplasmic reticulum Ca2+ and cytosolic cAMP dynamics in mouse skeletal muscle

Cited by 118 publications

References 39 publications

Unique characteristics of Ca 2+ homeostasis of the trans -Golgi compartment

Unique characteristics of Ca 2+ homeostasis of the trans -Golgi compartment

Ca2+ homeostasis in the endoplasmic reticulum measured with a new low-Ca2+-affinity targeted aequorin

Myosin Va cooperates with PKA RIα to mediate maintenance of the endplate in vivo

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Unique characteristics of Ca ²⁺ homeostasis of the trans -Golgi compartment

Unique characteristics of Ca ²⁺ homeostasis of the trans -Golgi compartment