Role%20of%20Elementary%20Calcium%20Puffs%20in%20Generating%20Cellular%20Calcium%20Oscillations

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Role of Elementary Calcium Puffs in Generating
Cellular Calcium Oscillations

• Ian Parker
• Dept. Neurobiology Behavior
• UC Irvine

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Ca2 signaling
Resting Ca 2 in cytosol maintained at 50 nM
by actions of pumps exchangers. So, only a
little Ca entering the cytosol will give big (a
few mM) increase in concentration. Cytosolic Ca
can Activate membrane ion channels Regulate
enzyme activity Modulate protein
function Regulate gene expression Kill cells!
(necrotic apoptotic death) It has functions in
virtually all cells of the body. Christened a
life and death messenger
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?
out
plasma membrane
in
Ca2 sensitive processes

Ca2
IP3

IP3 receptor
?
endoplasmic reticulum
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Xenopus laevis and her oocyte
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In the presence of IP3, positive and negative
feedback of Ca2 on the IP3R generates
repetitive, regenerative waves
Ca2 waves in a whole oocyte
Ca2
Ca2
e.r.
time
time
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Experimental methods

• Fast (ms) , high-resolution (ca. 250 nm) imaging
  of intracellular calcium signals using
  fluorescent indicator dyes and confocal
  microscopy
• Precise control of intracellular IP3 by
  photorelease from caged IP3.

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Local Ca2 puffs in the Xenopus oocyte
z
x
y
x
20 mm
20 mm
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Elementary calcium release eventsHighly
localized, stochastic calcium signals
representing Ca2 liberation through.Single
IP3R channels (blips)Coordinated opening of
several (tens?) of IP3R grouped tightly within
clusters (Puffs)
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distance
Linescan imaging of puffs
3.5
0
F/F0
10mm
time
UV flash
250ms
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Model of local global IP3 - evoked Ca2 signals
local Ca2 signal
global Ca2 signal
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The spread of Ca2 during elementary events is
consistent with passive diffusion from a point
(lt 250 nm) source.
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Calcium signals show a continuum of sizes,
probably resulting from stochastic variation in
both number and duration of channel openings
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Variability in puff durations
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Puffs show rectangular kinetics, with durations
appreciable longer than mean IP3R channel open
lifetime
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Hypothetical patterns of IP3R channel activity
that may underlie puffs
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Puff occurrence is stochastic
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Coupling between puff sites to generate calcium
wavesfire-diffuse-fire model
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Saltatory propagation of calcium waves between
puff sites
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Coupling between puff sites to generate waves is
strongly affected by calcium buffers
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Triggering of calcium waves by puffsone puff
is not enough.
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Method for rapidly changing IP3 and Ca2 in
the intact cell
laser scan line
Globally elevate IP3
Locally elevate Ca2
widefield photorelease of IP3
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IP3
Ca2
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The amount of calcium in a single puff is too
small to have a significant probability of
triggering a wave
Calcium responses evoked by laser zaps
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Role of puffs in setting frequency of repetitive
calcium waves
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Puffs occur between, and trigger, repetitive
calcium waves
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Despite stochastic occurrence of puffs, waves
show regular periodicity
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Failure of puffs to trigger wave is not because
cytosol is inexcitable
zap
zap
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Triggering of wave depends on accumulation of
basal Ca from several puffs
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Waves are initiated preferentially at focal
puff sites
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Focal wave initiation sites show higher
sensitivity to IP3
Focal site
Non-focal site
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Conclusions
IP3-dependent Ca2 signals are ordered
hierarchically
Ca2
IP3
local
cellular
molecular
stochastic
periodic
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Why are Ca2 release channels organized in
clusters?

• Autonomous functioning of separate clusters
  allows graded cellular response despite inherent
  regenerative nature of Ca2 -induced Ca2 release
• Local (puff) and global (wave) signals can
  convey different signals even in the same cell.
  In smooth muscle local signals cause relaxation,
  global waves cause contraction.
• Channel clustering may optimize calcium signaling
  i.e. give large Ca2 response to weak stimulus.
• Different cells may tailor cluster-cluster
  interactions (by varying spacing and/or Ca2
  buffering) to suit their particular functions
  e.g. bias toward local or global signals.

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Credits
Jonathan Marchant Nick Callamaras