Transcript Calcium Imaging and Caged compounds

Calcium Imaging and Caged
Compounds
Roger Thompson
BSC 5936
Spring 2005
Introduction
• Calcium cannot be visualized directly
• Specific molecules are used that have optical
properties which change upon interacting with
calcium
• Calcium concentrations can change in milliseconds
• Calcium acts as a universal 2nd messenger
• Calcium regulates cellular processes such as
muscle contraction, fertilization, cell division,
blood clotting, and synaptic transmission
Fluorescence
• Requires molecules called fluorophores or
fluorescent dyes
• Properties include: absorbance, lifetime, intensity,
& spectra
• Is the result of a three stage process
• 3 stages are:
– Excitation
– Excited-state lifetime
– Fluorescence emission
Instrumentation
• Detection system
–
–
–
–
Fluorophore
Wavelength filters
Detector
Excitation source
• Types of instruments
– Spectrofluorometer
– Fluorescence Microscope
– Flow cytometer
Chemical Fluorescent Indicators
Selection Criteria
• Calcium Concentration range
– Near dissociation constant (Kd) (best)
– Detectable at 0.1Kd to 10Kd
• Delivery method
• Measurement
– Quantitative or qualitative
– Ion concentration
– Instruments
– Sources of noise
• Indicator’s light intensity
• Other physiological parameters
– Simultaneous patch-clamp
Ultraviolet Wavelength
Excitation Fluorescent Indicators
• Fura-2, Indo-1 and derivatives
• Quin-2 and derivatives
• Intermediate Ca2+-binding affinity
– Fura-4F, Fura-5F & Fura-6F; plus
– Benzothiaza-1 & 2
• Low-affinity indicators
– Fura-FF, BTC, Mag-Fura-2, Mag-Fura-5, and
Mag-Indo-1
Visible Wavelength Excitation
Fluorescent Indicators
• Fluo-3, Rhod-2 and derivatives
• Low-affinity indicators
– Fluo-5N, Rhod-5N, X-Rhod-5N and derivatives
• Calcium Green, Calcium Orange, Calcium
Crimson
• Oregon Green 488 BAPTA indicators
• Fura Red indicator
• Calcein
Ratiometric vs. NonRatiometric
• Ratiometric
– Includes indo-1 and fura-2
– Allows for correction of differences of path length and
accessible volume in three dimensional specimens
– Indicator has 2 excitable wavelengths
• Indo-1 at 405- and 485 nm
• Nonratiometric
– Includes fluo-3, rhod-2 and the calcium green class
Bioluminescent Calcium
Indicators
Bioluminescence
•
•
•
•
Light produced by biological organisms
Low intensity
Assays are sensitive and free of background
One example is Aequorin
–
–
–
–
Photoprotein isolated from luminescent jellyfish
Is not exported or secreted; not compartmentalized
Usually microinjected into cells
Has 3 Ca2+ binding sites
2+
Ca -binding
Photoproteins
• Visible bioluminescence by an
intramolecular reaction in the presence of
calcium
• Simple instrument requirements
• Not affected by photobleaching
• Ex: Obelin and Aequorin
• Problems
– Loading, detection and calibration
Green Fluorescent Protein-based
• Photosensitive proteins synthesized by the
jellyfish Aequorea victoria
• Can be cloned and fused with DNA
• Provides marker for gene expression and
protein localization in living organisms
Dye-loading Procedures
• Ester Loading
– Derivatized with an
AM (acetoxymethyl)
ester
– Passively diffuses
through plasma
membrane
– Subject to
compartmentalization
or incomplete
hydrolysis
• Microinjection
– Intermittent injection
of indicator dissolved
in cytosolic-like
solution via glass
micropipette
– Invasive technique
• Diffusion from PatchClamp pipettes
– Passive microinjection;
i.e. perforated patch
technique using
substance such as
Nystatin
• Diffusion through Gap
Junctions
– Retrograde perfusion
w/ a low Ca2+ solution
containing collagenase
and proteases followed
by mechanical
dissociation of cells
• ATP-induced
permeabilization
– Extracellular ATP
induces cation flux and
increases permeability
of the plasma
membrane via ATP4receptor
• Hyposmotic Shock
Treatment
– Several washes in Ca2+
free solution, followed
by hyposmotic solution
containing indicator
– Can kill cells
• Gravity Loading
– Ca2+ free solution wash
– Centrifugation
– Incubation with
indicator
– Centrifugation
• Scrape Loading
– Cultured cells are
scraped from dish
while in buffer
containing indicator
– Scraping rips holes in
membrane
• Lipotransfer Delivery
Method
– Uses membrane
permeant cationic
liposomes containing
indicator/dye
• Fused Cell Hybrids
– Photoproteins are
loaded by fusing cells
and human erythrocyte
“ghosts” contain the
photoprotein in a
medium containing
Sendai virus
Potential Problems of Ca2+
Indicators and their Solutions
• Intracellular buffering
– Indicator can alter
[Ca2+]i when loaded in
high concentration
• Cytotoxicity
– Can damage some
types of cells
– Can affect redox
metabolism or cell
proliferation
• Autofluorescence
– Collagen fibers and
calcifications can give
off autofluorescence
– Pyridine nucleotides
may also. These
include NADH, NADP,
FAD, and FMN
• Bleaching
– Too much illumination
or photodamage
– Can be diminished by
adding O2 or an
antioxidant
• Compartmentalization
– Indicator becomes
trapped within some
intracellular organelles
and is not
homogeneous
throughout the cell
• Binding to Other Ions
& Proteins
– Indicators may bind to
intracellular proteins
and alter their
fluorescent properties
including changes in
spectrum, kinetics, or
Kd
• Dye Leakage
– Indicators may leak
from the cytosol into
the extracellular
medium
– Is regulated by anion
transporter system
– Can be inhibited by
probenecid,
sulfinpyrazone or low
temperature
Techniques for measuring
2+
Ca
Optical Techniques
• Multiparameter digitized video microscopy
• Confocal laser scanning microscopy
– Scans specimen collecting emitted fluorescence thru a pinhole
• Two-photon excitation laser scanning microscopy
•
– One fluorophore is excited by two individual photons simultaneously
Pulsed-laser imaging for rapid Ca2+ gradients
– Allows submicron spatial resolution and msec temporal resolution
• Time-resolved fluorescence lifetime imaging
microscopy (FLIM)
– (  ) time in excited state to ground state
• Photomultiplier tube
• Flow cytometry
– Flowing stream suspension
Non-Optical Techniques
• Electrophysiology
– Currents generated by Ca2+-dependent ion channels
• Ca2+-selective electrodes
– Ion-complexing ligand in a liquid lipophilic membrane
• Vibrating Ca2+-selective probe
– Ion-selective; non-invasive; located w/in microns of the
cell surface; measures ion flux
Caged Compounds
Characteristics
• Photosensitive chelators of ions or
substances such as Ca2+, H+, ATP, or IP3
• Exist as an inactive form due to
combination with radicals such as the
nitrophenyl group
• “chemically caged”
• Activated by exposure to UV illumination
• Release of Ca2+ in sec or msec
Ratiometric intracellular calcium
imaging in the isolated beating
rat heart using indo-1
fluorescence
Eerbeek etal., 2004
J. Appl. Physiol. 97:2042-2050.
Purpose
• Describes an optical ratio imaging setup and
an analysis method for the beat-to-beat
Cai2+ videofluorescence images of an indo-1
loaded, isolated Tyrode-perfused beating rat
heart.
• Possible to register different temporal Cai2+
transients together with left ventricle
pressure changes.
Why?
• Because abnormalities in intracellular
calcium (Cai2+) handling has been
implicated as the underlying mechanism in
a large number of pathologies in the heart;
i.e., contraction, electrophysiological
properties, mitochondrial function, heart
failure, and cardiac hypertrophy.
How?
• Used indo-1. Requires a single-excitation
wavelength of light and two emission wavelengths
to be recorded
• Visualization of Cai2+ changes in time and space
• A multiviewer (filters/dichroic mirrors) split
emission light into two wavelengths; projected the
two mages on a CCD chip of a CCD camera
Fig. 1. A: schematic diagram of the dualwavelength videofluorometric
measurement system. The 365-nm
excitation light, which is provided by a
100-W Mercury (Hg) arc lamp, is selected
by a 365-nm filter (UG-1 filter), DCLP
390 is a dichroic mirror in the beam
splitter, which is translucent for
wavelengths higher than 385 nm. B: the
dichroic mirrors DCLP 455 are
translucent for wavelengths higher than
455 nm. Both light paths use filters
405_+ 10nm and 485 _+ 20 nm,
respectively. Both images are projected
simultaneously on the cathode (I) of a
second-generation image intensifier tube
by a macro lens charge-coupled device
(CCD) of a cooled video camera. BP,
band-pass filter; LVP, left ventricular
pressure.
Fig. 2. A: a hear image is shown on which
the 5 X 5 matrix pattern (spatial resolution
1.8 mm) is superimposed to illustrate how
the spatial dependency of the cytosolic
calcium (Cai2+) was analyzed. The whole
matrix, including the uppermost left part, is
covering the left ventricle of the heart.
Symbols are the same as Fig. 6 (symbols
are associated with that heart position).
RP, reference point for the image
processing. B: the three traces show the
indo-1 ratio signals of the 3 selected areas
during a whole heart cycle before filtering.
Fig. 3. NADH autofluorscence (NADH /
uranyl) measured at 450 nm, during the
first 100 ms of the heart cycle, before and
after a mimicked loading procedure.
Fig 4. Two images are
shown at 485 nm. A:
NADH image (487nm).
B: the same heart after
loading with indo-1 at the
same wavelength.
Fig. 5. Image at both
wavelengths (405 and 485
nm) and the ratio image
after a small lesion
(pinching the tissue) on the
epicardium of an indo-1loaded heart.
Fig. 6. Time course of the indo-1 ratio () and the left
ventricular pressure (0) during a whole heart cycle of 200 ms
(pacing frequency 300/min). The indo-1 ratio is corrected for
the autofluorescence.
Fig. 7. Time course of the indo-1 ratio of
the 3 squares o,•, and  (positions
shown in Fig. 2A) produced with a 5 X 5
matrix (spatial resolution 1.8 mm) during
a heart cycle of 200 ms (pacing
frequency 300/min). A and B with
stimulation of the right atrium, and C and
D, with direct stimulation of the left
ventricle, show the time course of the
indo-1 ratio. B and D after addition of
2,3-butanedione monoxine (BDM) (=
diacetyl monoxine DAM). Inset in A and
C shows the schematic heart with the
position of the 3 squares in the matrix
and in C also the position of the
stimulation electrode (stim).
Fig. 8. A and B: Time course of the indo-1 ratio at
different locations in 2 different hearts (positions
shown in each matrix) produced with a 15 X 15
matrix (spatial resolution 0.6 mm) during the first
100 ms of the heart cycle (pacing frequency
300/min). In the horizontal direction a delay in
calcium transients is present from left to right.
Fig. 9. A: steps taken from the acquired image from the
CCD to a detailed analysis of calcium transients in a small
portion of the left ventricle. The splitting of the CCD output
into 2 images, representing the 405- and 485-nm
wavelengths, is performed by using the 2 reference points
(see MATERIALS AND METHODS). The contrast and brightness
of the ratio image (image at 405 nm divided by the image at
485 nm) is optimized to show more clearly the region of
interest. It is clear that the ratio method is effective in
canceling out inhomogeneities in the fluorescence at the
same wavelengths. The area of interest is 4.2 by 4.2 mm and
was analyzed by dividing it in 7 parts in either the horizontal
or vertical direction. B: results of the analysis in both the
horizontal (vertical binned) and vertical (horizontal binned)
direction from 1 heart. In the horizontal direction, a delay in
calcium transients is present from the left (1) to the right (7).
This delay is absent in the vertical direction.
Results
• Cai2+ transients show that Cai2+ activation
propagates horizontally from the left to right
during sinus rhythm or from the stimulus
site during direct left ventricle stimulation.
• The indo-1 ratiometric video technique
allows the imaging of ratio changes of Cai2+
with a high temporal (1 ms) and spatial
(0.6mm) resolution in the beating heart
Inhibition of inositol 1,4.5trisphosphate-induced Ca2+
release by cAMP-dependent
protein kinase in a living cell
Tertyshnikova & Fein, 1998
PNAS 95:1613-1617
Hypothesis
• Is the principal mechanism of cAMPdependent inhibition of Ca2+ mobilization
by inhibition of IP3-induced Ca2+ release or
by stimulation of Ca2+ removal from the
cytoplasm?
2+
Ca
& cAMP
• Ubiquitous intracellular 2nd messengers
• Believed to modulate each other
• cAMP can potentiate or inhibit agonistinduced Ca2+ elevation depending on cell
type
How tested?
• Used caged IP3, caged Ca2+, and caged
cAMP to rapidly elevate their [C]i
• Carbacyclin = elevates cAMP (via G proteindependent activation of adenylyl cyclase) = inhibits IP3
• KT5720 and IP20 used as inhibitors of
carbacyclin
• SNP (sodium nitoprusside) = inhibits cGMP
s
Results
• Elevation of cAMP inhibits IP3-induced
Ca2+ release in an intact cell but does not
affect the removal of Ca2+ from the
cytoplasm.
• Inhibition is mediated by cAMP Protein
Kinase.