Cooperative and Stochastic Calcium Releases from Multiple Calcium Puff Sites Generate Calcium Microdomains in Intact HeLa Cells

Nakamura, Hideki; Bannai, Hiroko; Inoue, Takafumi; Michikawa, Takayuki; Sano, Masaki; Mikoshiba, Katsuhiko

doi:10.1074/jbc.m111.311399

Cited by 6 publications

(9 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, and consistent with our analyses of HEK and HeLa cells ( Keebler and Taylor, 2017 ; Thillaiappan et al, 2017 ), the sites at which Ca 2+ puffs occurred in the 40 s after photo-release of iIP 3 (33.8±2.7 sites, n =15 cells; Fig. 2 I) were about ten-times more abundant than reported in studies using different cell types ( Nakamura et al, 2012 ; Smith and Parker, 2009 ; Thomas et al, 2000 ). Our detection of more Ca 2+ release sites is not due to mis-identification of Ca 2+ puffs but probably arises from improved detection through automated analysis and longer effective recordings ( Keebler and Taylor, 2017 ).…”

Section: Resultssupporting

confidence: 90%

All three IP3 receptor subtypes generate Ca2+ puffs, the universal building blocks of IP3-evoked Ca2+ signals

Mataragka

Taylor

2018

Journal of Cell Science

View full text Add to dashboard Cite

All three subtypes of inositol 1,4,5-trisphosphate receptor (IP3R) are intracellular Ca2+ channels that are co-regulated by IP3 and Ca2+. This allows IP3Rs to evoke regenerative Ca2+ signals, the smallest of which are Ca2+ puffs that reflect the coordinated opening of a few clustered IP3Rs. We use total internal reflection microscopy (TIRF) microscopy to record Ca2+ signals in HEK cells expressing all three IP3R subtypes or a single native subtype. Ca2+ puffs are less frequent in cells expressing one IP3R subtype, commensurate with them expressing fewer IP3Rs than wild-type cells. However, all three IP3R subtypes generate broadly similar Ca2+ puffs with similar numbers of IP3Rs contributing to each. This suggests that IP3R clusters may be assembled by conserved mechanisms that generate similarly sized clusters across different IP3R expression levels. The Ca2+ puffs evoked by IP3R2 had slower kinetics and more prolonged durations, which may be due to IP3 binding with greater affinity to IP3R2. We conclude that Ca2+ puffs are the building blocks for the Ca2+ signals evoked by all IP3Rs.

show abstract

Section: Resultssupporting

confidence: 90%

All three IP3 receptor subtypes generate Ca2+ puffs, the universal building blocks of IP3-evoked Ca2+ signals

Mataragka

Taylor

2018

Journal of Cell Science

View full text Add to dashboard Cite

show abstract

“…The densities of Ca 2+ initiation sites (number/µm 2 ) detected during a 10-s period of stimulation are similar in HEK293 and HeLa cells ( Fig. 2 C), but the sites were at least 10-fold more abundant than observed in other studies ( Table 1 ), although none of these studies examined effects of extracellular stimuli on HEK293 cells ( Nakamura et al, 2012 ; Smith and Parker, 2009 ; Thomas et al, 2000 ). The unexpectedly large number of Ca 2+ puffs is unlikely to be due to the automated analysis mistakenly identifying puffs because the temporal and spatial profiles of the Ca 2+ puffs in HeLa and HEK293 cells are similar to those reported previously ( Fig.…”

Section: Resultssupporting

confidence: 53%

“…S1B " type="url"/> ). However, the sites at which these Ca 2+ puffs occurred were about 10-fold more abundant than reported previously for other cells ( Nakamura et al, 2012 ; Smith and Parker, 2009 ; Thomas et al, 2000 ). In HEK293 cells, for example, we estimate the number of sites to be 65±7 sites/cell ( Fig.…”

Section: Discussioncontrasting

confidence: 53%

“…Ca 2+ puffs evoked by either caged IP 3 (ci-IP 3 ) or by IP 3 produced after activation of endogenous signalling pathways have been observed in numerous cell types. A common theme is the suggestion that Ca 2+ puffs recur at the same small number of subcellular locations and that these sites often initiate global cytosolic Ca 2+ signals ( Bootman et al, 1997a ; Kasai et al, 1993 ; Marchant and Parker, 2001 ; Nakamura et al, 2012 ; Olson et al, 2012 ; Rooney et al, 1990 ; Simpson et al, 1997 ). However, most quantitative analyses of Ca 2+ puffs that have used total internal reflection fluorescence microscopy (TIRFM) have also been restricted to a single cell type (SH-SY5Y neuroblastoma cells) responding to photolysis of ci-IP 3 (e.g.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Endogenous signalling pathways and caged IP3 evoke Ca2+ puffs at the same abundant immobile intracellular sites

Keebler

Taylor

2017

Journal of Cell Science

View full text Add to dashboard Cite

The building blocks of intracellular Ca2+ signals evoked by inositol 1,4,5-trisphosphate receptors (IP3Rs) are Ca2+ puffs, transient focal increases in Ca2+ concentration that reflect the opening of small clusters of IP3Rs. We use total internal reflection fluorescence microscopy and automated analyses to detect Ca2+ puffs evoked by photolysis of caged IP3 or activation of endogenous muscarinic receptors with carbachol in human embryonic kidney 293 cells. Ca2+ puffs evoked by carbachol initiated at an estimated 65±7 sites/cell, and the sites remained immobile for many minutes. Photolysis of caged IP3 evoked Ca2+ puffs at a similar number of sites (100±35). Increasing the carbachol concentration increased the frequency of Ca2+ puffs without unmasking additional Ca2+ release sites. By measuring responses to sequential stimulation with carbachol or photolysed caged IP3, we established that the two stimuli evoked Ca2+ puffs at the same sites. We conclude that IP3-evoked Ca2+ puffs initiate at numerous immobile sites and the sites become more likely to fire as the IP3 concentration increases; there is no evidence that endogenous signalling pathways selectively deliver IP3 to specific sites.

show abstract

“…96,97 The ER lumen is one site of Ca 2þ storage, in addition to extracellular space. The plumes (microdomains 105,106 ) of Ca 2þ ions entering specific regions of the cytosol are transient under normal conditions, owing to the activity of transmembrane pumps/exchangers that return Ca 2þ to the extracellular space and the ER. Despite the transiency of Ca 2þ microdomains and despite the buffering of Ca 2þ by Ca 2þ -binding proteins such as calmodulin, 107 increased concentrations of Ca 2þ inside microdomains suffice for a local activation of calpains.…”

Section: The Fragment Generation Hypothesismentioning

confidence: 99%

Augmented generation of protein fragments during wakefulness as the molecular cause of sleep: a hypothesis

Varshavsky

2012

Protein Science

View full text Add to dashboard Cite

Despite extensive understanding of sleep regulation, the molecular-level cause and function of sleep are unknown. I suggest that they originate in individual neurons and stem from increased production of protein fragments during wakefulness. These fragments are transient parts of protein complexes in which the fragments were generated. Neuronal Ca 21 fluxes are higher during wakefulness than during sleep. Subunits of transmembrane channels and other proteins are cleaved by Ca 21 -activated calpains and by other nonprocessive proteases, including caspases and secretases. In the proposed concept, termed the fragment generation (FG) hypothesis, sleep is a state during which the production of fragments is decreased (owing to lower Ca 21 transients) while fragment-destroying pathways are upregulated. These changes facilitate the elimination of fragments and the remodeling of protein complexes in which the fragments resided. The FG hypothesis posits that a proteolytic cleavage, which produces two fragments, can have both deleterious effects and fitness-increasing functions. This (previously not considered) dichotomy can explain both the conservation of cleavage sites in proteins and the evolutionary persistence of sleep, because sleep would counteract deleterious aspects of protein fragments. The FG hypothesis leads to new explanations of sleep phenomena, including a longer sleep after sleep deprivation. Studies in the 1970s showed that ethanol-induced sleep in mice can be strikingly prolonged by intracerebroventricular injections of either Ca 21 alone or Ca 21 and its ionophore (Erickson et al., Science 1978;199:1219-1221 Harris, Pharmacol Biochem Behav 1979;10:527-534; Erickson et al., Pharmacol Biochem Behav 1980;12:651-656). These results, which were never interpreted in connection to protein fragments or the function of sleep, may be accounted for by the FG hypothesis about molecular causation of sleep.

show abstract

Cooperative and Stochastic Calcium Releases from Multiple Calcium Puff Sites Generate Calcium Microdomains in Intact HeLa Cells

Cited by 6 publications

References 37 publications

All three IP3 receptor subtypes generate Ca2+ puffs, the universal building blocks of IP3-evoked Ca2+ signals

All three IP3 receptor subtypes generate Ca2+ puffs, the universal building blocks of IP3-evoked Ca2+ signals

Endogenous signalling pathways and caged IP3 evoke Ca2+ puffs at the same abundant immobile intracellular sites

Augmented generation of protein fragments during wakefulness as the molecular cause of sleep: a hypothesis

Contact Info

Product

Resources

About