Detail of Fig7_180702_SypHy_20Hz200APs



Project
Title
Live imaging of hippocampal neurons expressing synaptophysin as control
Description
Live imaging of hippocampal neurons expressing synaptophysin as control
Release, Updated
2022-03-31
License
CC BY
Kind
Image data
File Formats
uncompressed TIFF
Data size
72.7 MB

Organism
Mus musculus ( NCBI:txid10090 )
Strain(s)
-
Cell Line
-

Datatype
-
Molecular Function (MF)
-
Biological Process (BP)
synaptic vesicle recycling ( GO:0036465 )
Cellular Component (CC)
plasma membrane ( GO:0005886 )
Biological Imaging Method
time lapse microscopy ( Fbbi:00000249 )
X scale
0.108 micrometer/pixel
Y scale
0.108 micrometer/pixel
Z scale
-
T scale
1 second for each time interval

Image Acquisition
Experiment type
-
Microscope type
-
Acquisition mode
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Contrast method
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Microscope model
-
Detector model
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Objective model
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Filter set
-

Summary of Methods
See details in Ono Y, et. al. (2019) Sci Rep., 9(1):s41598-019-40557-y.
Related paper(s)

Yoshiyasu Ono, Yasunori Mori, Yoshihiro Egashira, Kenta Sumiyama, Shigeo Takamori (2019) Expression of plasma membrane calcium ATPases confers Ca(2+)/H(+) exchange in rodent synaptic vesicles., Scientific reports, Volume 9, Number 1, pp. 4289

Published in 2019 Mar 12 (Electronic publication in March 12, 2019, midnight )

(Abstract) Ca(2+) transport into synaptic vesicles (SVs) at the presynaptic terminals has been proposed to be an important process for regulating presynaptic [Ca(2+)] during stimulation as well as at rest. However, the molecular identity of the transport system remains elusive. Previous studies have demonstrated that isolated SVs exhibit two distinct Ca(2+) transport systems depending on extra-vesicular (cytosolic) pH; one is mediated by a high affinity Ca(2+) transporter which is active at neutral pH and the other is mediated by a low affinity Ca(2+)/H(+) antiporter which is maximally active at alkaline pH of 8.5. In addition, synaptic vesicle glycoprotein 2 s (SV2s), a major SV component, have been proposed to contribute to Ca(2+) clearance from the presynaptic cytoplasm. Here, we show that at physiological pH, the plasma membrane Ca(2+) ATPases (PMCAs) are responsible for both the Ca(2+)/H(+) exchange activity and Ca(2+) uptake into SVs. The Ca(2+)/H(+) exchange activity monitored by acidification assay exhibited high affinity for Ca(2+) (Km ~ 400 nM) and characteristic divalent cation selectivity for the PMCAs. Both activities were remarkably reduced by PMCA blockers, but not by a blocker of the ATPase that transfers Ca(2+) from the cytosol to the lumen of sarcoplasmic endoplasmic reticulum (SERCA) at physiological pH. Furthermore, we rule out the contribution of SV2s, putative Ca(2+) transporters on SVs, since both Ca(2+)/H(+) exchange activity and Ca(2+) transport were unaffected in isolated vesicles derived from SV2-deficient brains. Finally, using a PMCA1-pHluorin construct that enabled us to monitor cellular distribution and recycling properties in living neurons, we demonstrated that PMCA1-pHluorin localized to intracellular acidic compartments and recycled at presynaptic terminals in an activity-dependent manner. Collectively, our results imply that vesicular PMCAs may play pivotal roles in both presynaptic Ca(2+) homeostasis and the modulation of H(+) gradient in SVs.
(MeSH Terms)

Contact
Shigeo Takamori , Doshisha University , Graduate School of Brain Science , Laboratory of Neural Membrane Biology
Contributors

OMERO Dataset
OMERO Project
Source