Proj Report Fin1final

1. Introduction: Cells display an enormous variety of oscillatory phenomena based on fluctuation in intracellular calcium concentration. Such oscillations fall into two main categories depending on whether the oscillations originate from calcium influx (membrane oscillator) or from internal stores (cytosolic oscillator).The main focus of the current study is the cytosolic calcium oscillators. Such cytosolic calcium oscillators are frequently associated with stimuli that act through phosphoinositide pathway and are divided into 4 main categories based on their kinetics and the maximum amplitude: namely blip, puff, global saltatoric wave and global sustained wave. The global calcium waves are very rich signals in that they possess enormous variation in frequency and amplitude but elementary calcium signals have no less functional significance in spite of their similar frequency and amplitudes. The elementary calcium waves can raise the local calcium ion concentration to very high levels which is the basis of their action in most cell types. Some examples of processes that are directly controlled by elementary Ca2+ signals include the release of synaptic and secretory vesicles, the activation of ion channels, mitochondrial energy metabolism and the generation of nuclear-specific Ca2+ signals. There are several advantages to using elementary Ca2+ signals, rather than global increases in Ca2+, to control such processes. For example, as the elementary Ca2+ signals have only a limited spatial range, and the Ca2+ concentration declines sharply with distance from the site of origin, regulation of cellular activities relies on close localization of the Ca2+ channels and their targets. This allows Ca2+ to have a highly specific effect. In addition, elementary Ca2+ signals can have a rapid effect at relatively low energy cost to the cells, in contrast to global Ca2+ changes. The rapidity of signaling through elementary events is evident, for example, in synaptic transmission where voltage-operated channels located next to synaptic vesicles trigger exocytosis by providing high-intensity pulses of Ca2+. In addition to controlling the local functions of cells, elementary Ca2+ signals are responsible for the generation of global Ca2+ signals such as waves and oscillations. Essentially, global Ca2+ signals arise via the coordinated recruitment of many elementary

1

Ca2+ release and entry channels. The mechanisms by which this is achieved, and the balance between Ca2+ influx and release, are cell-type specific. For example, in many types of non-excitable cell, Ca2+ release provides the main component of the signal, whereas excitable cells often rely on both Ca2+ entry and release to varying degrees. In non-excitable cells, the co-ordination of Ca2+ release is usually achieved through the autocatalytic process of Ca2+-induced Ca2+ release. The usual sequence of events involves an elementary Ca2+ signal in one region of the cell providing a triggering pulse of Ca2+ that diffuses outwards to recruit neighboring channels. This sets up a Ca2+ wave that spreads in a regenerative manner throughout the cell. Such waves travel relatively slowly (about 10–100 mm/sec), meaning that the synchronization of Ca2+ release necessary to create a global signal takes about 1 second in cell types using this mechanism. In those cases where cells are connected by gap junctions, Ca2+ waves can also spread from one cell to the next, thus coordinating the activity of groups of cells within a tissue. Here, we have studied the different types of calcium waves focusing on the global variants by applying histamine as an agonist for activating the phosphoinositide pathway to generate the calcium waves. Different calcium signals have different physiological implications. Interconvertibility of calcium signals can lead to altered physiological functions. In the current work physiological conditions were varied to alter the nature of the calcium waves. This with further studies can give us a very useful tool of tuning the intracellular calcium waves with possibilities of changing the physiological function of the calcium signals as well. 2. A Brief Literature Review: The multiple roles of calcium, which controls birth, life and death, were identified in the late 19 century, when Sydney Ringer discovered that Ca2+ ions control heart contractions, regulate fertilization and development of tadpole and determine survival of fishes (Ringer 1883a, b, 1886, 1890; Ringer and Sainsbury 1894). In late 1960-es/ early 1970-es it has been recognized that cell death is associated with an increase in cell calcium content

2

(Farber 1981) , and in 1974 the key role of excessive plasmalemmal Ca2+ entry and subsequent Ca2+ overload in triggering ischemic death of

cardiomyocytes was

hypothesized (Fleckenstein et al., 1974). Several years later massive translocation of Ca2+ from the extra cellular space into neural cells was identified in ischemic cerebellum (Nicholson et al., 1977). In the following 30 years the role of Ca 2+ overload and cell Ca2+ dyshomeostasis in initiation and regulation of various death pathways was firmly established (Nicotera et al., 1992; Orrenius and Nicotera, 1994; Choi, 1995; Kristian and Siesjo, 1998; Ermak and Davies 2002; Eisner et al., 2006). 2.1. Generation of Calcium Ion Flux within the Cell: 2.1.1. Calcium Ion Homeostasis within CellFundamentally, calcium homeostasis is a result of coordinated transportation of Ca2+ ions through several sets of membranes, which delineate distinct cellular compartments; these compartments maintain very different Ca2+ concentrations, and have a specific role in both physiological and pathological Ca2+ signaling (Petersen et al., 1994; Berridge et al., 2003; Carafoli 2004; Verkhratsky 2005). These compartments are represented by the cytosol, endoplasmic reticulum which is often connected with nuclear envelope and complex Golgi, mitochondria and the nucleus (Figure 1). Although each of these compartments has its own Ca2+ homeostatic pattern, they all rely on Ca2+ movements across relevant membranes, which are governed by relatively restricted number of Ca 2+ channels and Ca2+ transporters. Calcium-binding proteins, whose Ca2+ affinities vary, between 30-100 nM and .5-1.2 mM represent second important element, which controls Ca2+ traffic within the said compartments. The Ca2+ binding proteins also act as Ca2+ sensors, which control cellular biochemistry and execute cellular reactions. Calcium fluxes between different cellular compartments occur either by diffusion down the concentration gradient, or by active energy dependent transport against the latter.

3

Figure 1. Compartmentalization of cellular Ca2+ homeostasis (Berridge et al., 2000).

The Ca2+ concentration gradients, build across different membranes are, as a rule, quite steep, which ensures the high signal to noise of Ca2+ signaling system; simultaneously these steep gradients present a danger of rapid Ca2+ overload upon disruptions of Ca2+ homeostasis. The extra cellular Ca2+ concentration ([Ca2+]o) varies within the range of 1.2–1.5 mM. The cytosolic Ca2+ concentration (or [Ca2+ ]i) is 4 orders in magnitude lower than extracellular concentration being set around 30–100 nM. Free Ca2+ concentration in the lumen of endoplasmic reticulum ([Ca2+]L) can reach 0.5–1.0 mM, being thus comparable with [Ca2+]o. Mitochondria have another degree of complexity, added by an electrical gradient between cytosol and mitochondrial matrix, which can reach up to 200 mV, and thus favors Ca2+ influx down the electrogenic gradient.

Figure 2. Molecular cascades responsible for Ca2+ homeostasis and Ca2+ signaling (Berridge et al., 2000).

4

2.1.2. Sources of Ca2+ Ca2+ signals generally result from the opening of Ca2+ channels or the activity of Ca2+ transporters. These are located either on the plasma membrane, or inside the cell on the endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR). The plasma membrane Ca2+ channels can be divided into different types, according to their activation mechanism: 1. Voltage-operated channels, 2. Receptor-operated channels, 3. Mechanically-activated channels and 4. The so-called ‘store-operated channels’, which are opened following the depletion of internal Ca2+ stores. Ca2+ release from the ER and SR occurs via three types of channel. Of these, inositol 1, 4, 5-trisphosphate (IP3) receptors (IP3Rs) and ryanodine receptors (RyRs) are the best characterized. A third type of channel, known as SCaMPER (sphingolipid Ca 2+-releasemediating protein of endoplasmic reticulum) seems to release Ca2+ in response to an increase in intracellular sphingolipid concentrations. The differential expression of these Ca2+ entry or release channels allows cells to respond to a diverse range of stimuli and produce Ca2+ signals that are tissue specific. When activated, both Ca2+ entry and Ca2+ release channels introduce Ca2+ into the cytoplasm. As these channels are only open for a short time, however, they only introduce brief pulses of Ca2+ that form a small plume around the mouth of the channel before diffusing into the cytoplasm (Figure 3). For some of the Ca2+ entry and release channels, these localized plumes of Ca2+ have been visualized using confocal microscopy of living cells. Such Ca2+ increases have been recorded in vastly different cell types, prompting the realization that these so-called ‘elementary’ Ca2+ signals represent the basic building blocks of Ca2+ signaling.

5

Figure3. A typical elementary event resulting from the localized release of Ca2+ (red) from channels (ryanodine or IP3 receptors; colored blue) located in the endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR). (a) Quiescent state. (b) A group of channels open to form a spark or puff. (c) The channels shut and Ca2+ diffuses into the cytoplasm.(Bootman and Lipp,2001).

2.1.3. Elementary Ca2+ signalsHeLa cells displayed multiple levels of Ca2+ signaling in response to histamine stimulation, one of the ways to activate the IP3Rs and the phosphoinositide pathway leading to release of calcium from intracellular stores: fundamental “Ca 2+ blips” arising from the gating of single InsP3R, intermediate Ca2+ puffs reflecting the coordinated recruitment of a cluster of InsP3Rs, and propagating Ca2+ waves. Ca2+ blips were distinguished from Ca2+ puffs by their kinetics and their amplitude. Ca2+ release events were referred to as blips if they reached maximal amplitude within 130 ms and when they had amplitudes of, ≤40 nM. Ca2+ release events were regarded as Ca2+ puffs when they reached their maximal amplitudes within 360 ms and when their amplitude was ≤150 nM. Similar criteria were used to identify elementary events in Xenopus oocytes (Parker and Yao, 1996). 2.1.4. Generation of Global Ca2+ wavesThe calcium signals that are familiar to most physiologists are the global calcium signals arising from the synchronized release of calcium from a large proportion of the intracellular channels. Because of the potent negative feedback effect of calcium, each channel is open for a brief period, which means that global responses depend upon synchronization of the elementary events described earlier. Cells appear to have evolved 6

two mechanisms to co-ordinate these elementary events (Bootman & Berridge, 1995). Firstly, channel opening can be evoked nearly simultaneously (i.e. within milliseconds) by being tightly coupled to an action potential in the plasma membrane as occurs in skeletal, cardiac, and some smooth muscle cells. Secondly, the channels co-ordinate their own activity through the regenerative process of CICR (Calcium Induced Calcium Release). This process, which is present in nonexcitable cells (e.g. HeLa cell), is much slower because the synchronization signal is calcium itself diffusing from one channel to the next, usually in the form of a calcium wave taking several seconds to traverse a typical cell. 2.1.5. Intracellular calcium ion buffering in mitochondriaCells maintain a calcium ion homeostasis by a coordinated action of intracellular calcium stores which are mainly ER and mitochondria. The calcium ions released from ER during the propagation of a wave are eventually buffered by mitochondria, back into ER, and in equilibration with the extracellular medium. The uptake of calcium ions by mitochondria is a function of Δψm (mitochondrial membrane potential). The uptake pathway is an electrophoretic mechanism (Ca2+ uniporter) driven by the electrochemical potential gradient across the inner mitochondrial membrane obeys the Nernst’s equation. Calcium ion uptake by the mitochondria decreases the membrane potential (from -200 mV-0 mV) and can cause complete depolarization at higher concentrations. The mitochondria in turn, by virtue of buffering the calcium ions may modulate the spatio-temporal properties of regenerative calcium waves. Mitochondria have been implicated in the determination of wave characteristics in cultured oligodendrocytes and astrocytes (Simpson and Russell, 1996; Simpson et al., 1998), and the velocity of cytosolic Ca2+ wave propagation is increased after up-regulation of mitochondrial function in Xenopus oocytes (Jouaville et al., 1995). The impact of mitochondrial calcium uptake on the spatio-temporal characteristics of intracellular Ca2+ signals has been controversial (Gunter, 1994). The high capacity, low affinity ( Kd for Ca2+ = 5–10 mM) mitochondrial Ca2+ uptake pathway has often been considered relevant in the regulation of cytosolic Ca2+ only in response to pathological Ca2+ elevations (Carafoli, 1987).

7

3. Objectives of the Current Study: •

Generation of regenerative calcium waves by chemical induction with histamine.

•

Mode of propagation of Calcium transients and its effect on the nature of the signal.

•

Alteration of the mode of propagation of calcium waves to alter the nature of the wave.

•

Linking different physiological phenomenon with the spatio-temporal pattern of calcium waves (saltatoric and continuous).

4. Materials and Methods: 4.1. Preparation of PDMS microchannels: Microfluidic devices offer significant benefits to chemical and biological analysis, including portability for point-of-care testing, disposability and minimal reagent consumption, analysis time and low cost of manufacture. Recent moves towards the use of soft materials, such as poly(dimethylsiloxane) (PDMS) offer additional advantages of chemical resistance, biocompatibility, ease of fabrication and flexibility in application. In terms of PDMS microfluidic device fabrication two conditions dominate: (a) The fabrication of the microchannels and (b) The sealing of the microchannels to the substrate. 4.1.1. Fabrication of microchannels: The microfluidic cell culture device was fabricated by conventional rapid prototyping methods. Photomasks were designed using a computer aided design (CAD) program (AutoCAD, Autodesk) and were printed on an acetate sheet with a commercial 1200 dpi printer (LaserJet 8000 N, Hewlett–Packard) by employing a regular transparency film. The master was fabricated by contact photolithography using SU8–2075 resist (Microchem Corp, Newton, MA, USA). The photoresist was spin coated on a piranha solution (H2SO4 : H2O2 = 1 : 1 by volume)-cleaned medical grade glass slide at 500 rpm 8

for 10 s, followed by a speed of 3000 rpm for 20 s. The coated photoresist was exposed to ultra-violet (UV) light through the photomask for 30 s. Subsequently, it was developed using SU8 developer (Microchem Corp, Newton, MA, USA) and was cleaned with isopropyl alcohol. A mixture of PDMS base to cross-linker (Sylgard 184, Dow Corning) in ratio 10: 1 w/w was poured onto the SU-8 mold, degassed, heated at 90ºC for 20 min and was subsequently peeled off the mold. Inlet and outlet ports were punched by a blunt end 18-gauge needle. 4.1.2. Bonding of microchannels to glass substrates: The PDMS microchannels were oxidized with Piranha solution (H2SO4 : H2O2 = 1 : 1 by volume). 40 mm glass coverslips were also oxidized in the same procedure. The oxidation procedure resulted in the removal of methyl groups and freed the SiO- groups for bonding. In the acidic condition the SiO- groups acquire a proton and two SiOH groups bond with each other with the release of one molecule of water. The oxidized glass and PDMS surfaces were then pressed upon each other and kept in hot air oven at 65ºC for 20 minutes for the bonding to take place. The edges of the microchannel were then hand painted with mixture of PDMS with cross-linker to add rigidity to the system. Two reservoirs were made by cutting the base of 200μL tips. The PDMS mixture was applied on the edges of the reservoirs taking care not to put PDMS mixture inside them. Then the reservoirs were carefully placed on the microchannel centering the inlet and outlet ports. It was then kept at 65ºC for 30 minutes fro the bonding to take place. 4.2. HeLa Cell culture: Human cervical carcinoma cell line HeLa was maintained in Minimum Essential Medium (MEM, HyClone, India) supplemented with 10% heat inactivated fetal calf serum under similar conditions. This was incubated in a CO2 incubator (Heraeus, Germany) at 37 ◦C and 5% CO2. For the experiment confluent HeLa cells were trypsinized for 5 minutes at 37ºC. The trypsin was neutralized by addition of complete media. The cells were then precipitated by centrifuging at 2000rpm for 10 minutes. The media was carefully poured off. Resuspension of the cells were done by adding 5 ml of complete media. 20 μL of cell

9

suspension along with 80μL were added to each of the reservoir of the microchannels. The microchannels were then kept in a CO2 incubator (Heraeus, Germany) at 37 ◦C and 5% CO2. After 8 hours the media was replaced with fresh media (MEM, complete) in each of the microchannel. 4.3. Generation of calcium signal and its visualization: 4.3.1. Fluo-3/AM loading bufferCells were loaded with Fluo-3/AM (Sigma) prior to agonist stimulation. Fluo-3 binds specifically with calcium ions and fluoresces at emission maxima of 530 nm. Composition of loading buffer145mM NaCl, 5mM KCl, 1 mM MgCl2, 1.2 mM CaCl2, 10 mM HEPES, 10 mM Glucose, pH 7.4 at 37ºC (Merritt et al.,1990). The buffer was prepared weighing each component very carefully. The pH was checked and adjusted by adding 1 M NaOH. After preparation the buffer was equilibrated at 37ºC. 4.3.2. Loading of Fluo-3/AM in HeLa cells1mM stock solutions of Fluo-3/AM were prepared in DMSO. 2 μM solutions of Fluo3/AM were prepared from stock by diluting in the Fluo-3 loading buffer. HeLa cells were incubated with 2μM Fluo-3 solution for 30 minutes at 37ºC. After that cells were washed with PBS and allowed an additional de-esterification time of 30 minutes. After that cells were taken for the calcium imaging. 4.4. Confocal Imaging of calcium transients: All the imaging experiments performed at 20-22ºC with acquisition rate of 5-6 Hz in the Olympus Fluoview FV1000 Confocal Laser Scanning Microscope. Images of HeLa cells were taken with 40X objective.

10

4.4.1. Measurement of fluorescence4.4.1a. Preparation of Histamine solutionA 10 mM histamine (Sigma) stock solution was prepared in the calcium buffer. Aliquots of 0.2-1 μM were made and used for induction of calcium signals in the HeLa cells through the phosphoinositide pathway which gave the [F] 4.4.1b. Measurement of [F]minThe basal level of fluorescence that the cell gives under the excitation wavelength of 505 nm and emission wavelength of 530 nm is taken as [F]min. 4.4.1c. Measurement of [F]maxA saponin (Sigma) solution of 0.1 mg/ml prepared in the calcium buffer was added along with 2 μM Fluo-3 solution. After an incubation period of 30 minutes the fluorescence was measured to get [F]max. 4.5. Measurement of mitochondrial membrane potential: Cells were loaded with a cationic carbocyanine dye JC-1 (5, 5’, 6, 6’-tetrachloro- 1, 1, 3, 3’-tetraethylbenzimidazolylcarbocyanine

iodide)

(Sigma)

prior

to

membrane

depolarization. In monomer condition JC-1 has excitation maxima of 488 nm and emission maxima of 515 nm while in normal cells, JC-1 enters the mitochondria and forms multimeric J aggregates which have an excitation maxima of 545 nm and emission maxima of 580 nm. The plasma membrane potential was disrupted with a high K + containing buffer prior to loading of the dye so that its effect is nullified. 4.5.1. High K+ buffer composition137 mM KCl, 3.6 mM NaCl, 0.5 mM MgCl2, 1.8 mM CaCl2, 4 mM Hepes, 1 mg/ml sucrose at pH 7.0 (Reers et al,1991). 4.5.2. Loading of JC-1 in HeLa cells1 mM stock solution of JC-1 was prepared in DMSO. 2μM solutions of JC-1 were prepared by diluting in 1 ml complete MEM media. HeLa cells were incubated with the high K+ buffer for 30 minutes at 37ºC (Reers et al, 1991) to disrupt the plasma membrane potential. Cells were subsequently washed with PBS and incubated with the JC-1 solution for 10 minutes at 37 ºC. The cells were immediately taken for imaging by confocal microscope.

11

4.5.3. Measurement of Fluorescence4.5.3. a. Measurement of [F]max The J aggregate fluorescence observed in normal cells stained with JC-1 with their plasma membrane depolarized by high K+ buffer was considered as the [F]max. 4.5.3.b. Measurement of [F]minCells were incubated with CCCP (Carbonyl cyanide 3-chlorophenylhydrazone) for 30 minutes at 37ºC to depolarize the mitochondrial membrane after being treated with the high K+ buffer. 10 mM CCCP solution was prepared in DMSO from which a 10 μM solution was made by dilution in incomplete MEM media (Reers et al, 1991). 4.5.3.c. Measurement of [F]HeLa cells loaded with JC-1 were stimulated with 1μM histamine solution to release Ca2+ from ER and subsequently deplete the mitochondrial membrane potential. Resulting fluorescence intensity of J aggregates was taken as [F]. Normalized fluorescence intensity of JC-1 = {[F]-[F]min}/ {[F]max-[F]min}. 4.6. Buffering of intracellular calcium ionThe increased level of calcium ions within the cell after administration of sucrose was brought down to normal basal level by buffering with a. 0.5 M NaCl solution with incubation time of 30 minutes at 37ºC. b. 0.1 μg/ml of saponin solution with incubation time of 10 minutes followed by incubation with 0.01mM EGTA at 37ºC. 4.7. Analysis with Matlab: All analysis was done with Matlab software version 6.5 (Mathworks Inc.). 5. Results: 5.1. Generation of Elementary and Global Calcium Signal through Histamine stimulation: Histamine concentration ranging from 0.1-1 μM was used to study the generation of calcium waves (Bootman, 1997).A particular region of interest was selected and the average intensity in that region was measured using the series analysis with the Olympus Fluoview Software. The images were then analyzed with Matlab v6.5. Figure 3 shows a

12

global calcium wave generated by 0.5 μM of histamine concentration, whereas figure 4 shows a train of global waves generated by 0.2 μM histamine concentration.

Figure 4. Representative plot of Global calcium waves induced by histamine (0.5 μM).

Figure 5. Representative plot of Global and elementary calcium waves generated with stimulation by histamine (0.2μM).

13

5.2.1. Alteration of nature of Calcium Waves under altered physiological conditionsDifferent concentrations of sucrose (500mM-1M) in PBS were applied to alter the intracellular diffusion coefficient. After incubation for 30 minutes at 37ºC with the sucrose solutions the HeLa cells were subjected to stimulation by histamine. Instead of saltatoric increase in calcium concentration in the cytoplasm we obtained sustained increase in the intracellular calcium concentration. Another important observation was that the basal level of intracellular calcium concentration was higher than normal at 240280 nM.

Figure 6a.

Representative plot of Application of histamine (0.2 μM) under increased sucrose

concentration (1 M) leading to altered nature of the calcium wave (frames 1-2000).

14

Figure 6b. Representative plot of Decrease in the low amplitude global wave followed by a further increase which died down subsequently (frames 2100-4000).

Figure 6c. Representative plot of application of histamine (0.2 μM) under increased sucrose concentration (500 mM) leading to altered nature of the calcium wave (frames 1-2000).

15

Figure 6d. Representative plot of Application of histamine (0.2 μM) under increased sucrose concentration (500 mM) leading to altered nature of the calcium wave (frames 2100-4000).

5.2.2. Alteration of Nature of Calcium Waves under altered chemical conditionsHeLa cells were incubated with Cytochalasin D (Sigma) at 37ºC to alter the equilibrium condition of actin polymerization and shift it towards the dissociation. Cytochalasin D has been reported to cause alteration in various motile activities of eukaryotic cells. It was observed that the effect of cytochalasin D on the propagation and the nature of calcium waves were similar to that of applying sucrose to cells. Sustained increase in levels of intracellular calcium was obtained instead of the saltatoric behavior and the basal level here was also high (higher than sucrose at 400nM).

16

Figure 7a. Representative plot of Calcium waves induced by histamine (0.5 μM) under the application of Cytochalasin D (frames 1-1500).

Figure 7b. Representative plot of Sustained increase in the intracellular calcium concentration by histamine (0.5 µM) under the application of Cytochalasin D (frames 2500-4000).

17

5.3. Contour plot analysis of intracellular Calcium waves: Increase in the intracellular concentration of calcium induced by histamine under normal and different physiological conditions, were mapped by analyzing the Confocal microscopy images in Matlab v6.5.

Figure 8a. Representative plot of spatial shift of the local calcium concentration increases (blips) during the propagation of a regenerative global calcium wave (time lapse of frames- 0.188 sec) on application of histamine (0.5μM).

18

Figure 8b. Representative contour plot of cells showing diffusion of calcium ion in subsequent frames on stimulation by histamine (0.5μM).

5.4.1. Alteration of MMP (mitochondrial membrane potential) by calcium waves: HeLa cells were incubated with calcium loading buffer ([Ca2+] =1.2mM) to fully equilibrate and standardize the extracellular condition. After 10 minutes of loading JC-1 the cells were taken for imaging by confocal microscope (roundtrip scan was employed).

19

The histamine (0.5 µM) was applied to the cells just before imaging was started.

Fig- 9a

Fig-9b

Fig 9. Representative plot of JC-1 fluorescence intensity (F) with time, fig 9a (frames 1-2250) and fig 9b (frames 2251-4500) with a gap of 1 min for instrumental adjustment

5.4.2. Alteration of MMP with modified calcium waves: 0.5 mM sucrose solution was used to convert the saltatoric waves to continuous type waves as described earlier. 2 μM solution of JC-1 was added to the cells prior to stimulation with histamine and imaging stages.

20

Fig-10a.

Fig-10b.

Fig-10c. Fig 10. Representative plot of JC-1 fluorescence intensity (F) with time after treatment with 0.5μM sucrose for 30 min. Fig10a (frames1-1200), 10b (frames1201-2400), 10c (frames2401-2900) with 1 min gap for instrumental adjustment.

For normal cell with histamine treatment, red fluorescence of JC-1 decreases but green remains constant or decreases. Conversely, for sucrose treated cells J aggregate intensity of JC-1 decreases with simultaneous ‘increase’ in green fluorescence. 5.4.3. Variation of MMP under normal and altered calcium wave: 21

The variation of mitochondrial membrane potential was plotted with the normalized fluorescence intensity mentioned by the formula in methods section. [F]max was found to be 200, [F]min was observed to be 15. HeLa cells were incubated with 0.5 mM sucrose solution to alter the nature of the calcium waves as described earlier. Under the altered physiological situation the change in Δψ was monitored

Fig-11a

Fig-11b

Fig-11. Relationship of JC-1 J aggregate fluorescence intensity with corresponding mitochondrial membrane potential. Fig-11a. MMP under normal physiological condition, Fig-11b.MMP after incubation with 0.5 mM sucrose.

.

6. Discussions:

22

Multiple levels of calcium signaling were observed in HeLa cells in response to histamine stimulation. Elementary Ca2+ blips arising from the gating of single InsP3 Rs (Figure 3), as well as intermediate calcium puffs could be observed (Figure 5). Calcium blips were distinguished from puffs distinctly by their kinetics and amplitude. Since blips and puffs were sometimes observed during the stimulation of the same cell and under the same histamine concentration these (Bootman etal., 1997) activities are not necessarily linked to actual IP3 concentrations. However, although different elementary Ca2+ signals could be evoked by a fixed histamine concentration (Figure 4, 5), increasing the agonist concentration enhanced the probability of a transition from non-regenerative to regenerative global Ca2+ waves. Regenerative responses (Figure 4, 5) take place when cellular calcium concentrations reach a threshold level. The frequency and amplitude of the elementary events increase in the pacemaker phase to reach the threshold level in cells where a global wave could be observed (Figure 5), (Bootman et al., 1997).But to understand the different release phenomena under different concentrations of agonist, we have to look to the structure of ligand-gated Inositol 3 Phosphate Receptor which is mainly implicated in the calcium release phenomena in non-excitable cells (e.g. HeLa). It contains 3 different domains: a. IP3 binding domain: IP3 binds to this domain and renders it excitable by local calcium concentrations. b. CICR domain: Calcium Induced Calcium Release or binding domain, it is principally responsible for release of calcium from ER into cytoplasm. c. Calcium Inhibitory Domain: This domain gets activated under high local calcium concentrations and leads to inhibition of calcium release providing a feedback mechanism to maintain the intracellular calcium level within a specific maximum concentration. When higher histamine concentration was applied (Figure 4), it led to the generation of saturating level of IP3 inside the cell. This in turn activated the available InsP3 Rs which resulted in release of all the intracellular calcium reserve at once. The result was a high amplitude saltatoric global calcium wave (Figure 4).

23

In the case of lower agonist concentrations the generation of IP3 was not at the saturating level, therefore all the cellular calcium reserve were not emptied at once as is evident from the maximum amplitude of the first peak in Figure 4 which is much lower than Figure 5. Though different cells can have differing levels of calcium inhibition concentration, saltatoric global calcium waves were observed with decreasing amplitude reinforcing the hypothesis that cellular reserve of IP3 is limited. Intracellular propagation of calcium waves is primarily a function of diffusion. Incubation of cells with sucrose leads to the increase in anomalous sub-diffusion(α) which leads to increase in effective diffusion coefficient of cellular fluid (Deff) (Guigas et al. 2007). Increased diffusion ensures that local concentration of calcium does not quickly (within 5-10 sec) reach to an inhibitory concentration so that the calcium induced inhibition receptors are not activated before the calcium is diffused out to neighboring InsP3Rs and activate them. This also results in less binding time of calcium ions with the CICRs increasing the time needed to saturate the receptors. Thereby increasing the tmax of the calcium wave (time needed to reach the maximum amplitude) to almost 300 seconds. This results in the sustained release of calcium from ER thus producing another kind of global calcium wave (Figure 6) with the increase in calcium concentration reaching the maximum amplitude in time more than thrice in magnitude of the saltatoric wave. The saltatoric and sustained global waves have different physiological significance (Berridge et al.2000,Dupont et al,2007) with saltatoric waves generally seen in normal cells and sustained waves during fertilization. So, this can be a significant step towards altering the function of calcium signals through physiological tuning. The global calcium waves are composed of elementary release events which are highly coordinated in time, space and frequency domains. The global wave is generated when the elementary events behave in a cooperative manner and leads to a very high increase in overall intracellular calcium concentration. For the cooperative nature of the fundamental release events to take place the frequency or the amplitude of the events should increase above a threshold level. The propagation of calcium ions to neighboring regions of the release site within a required time is thus necessary for the wave to get the global nature. The propagation of local increase in calcium as seen from (Figure 8) seems

24

to be taking place without diffusion in some cases, because there is no definite trace of diffusion as is evident from the Figure 8. Our proposition is that the propagation is a function of both diffusion and convection. To validate the hypothesis HeLa cells were treated with Cytochalasin D which blocks the intracellular convection by shifting the equilibrium towards actin de-polymerization. The result were same to that of increasing the diffusion inside the cell (Figure 7a).The basal level of calcium was higher indicating to the activation of p53 pathway by Cytochalasin D (Guigas et al.2007).The cellular calcium signal propagation can thus be described as convection-diffusion process. To describe a convection-diffusion process we take the dimension less number: Peclet Number (Pe) = UR/D, where U = Mean convective velocity of fluid. R = Radius of a tube assuming the wave propagates through a micro or nano circular channel. D = Diffusion coefficient. For a constant Peclet number an increase in the diffusion coefficient or a decrease in convection will generate the same nature of calcium wave propagation. We can see from Figure 6c and 7b that the net effects of increasing the diffusion coefficient or decreasing the convective velocity were similar. To study the effect of alteration of calcium waves on their physiological functions, mitochondrial membrane potential of the cells was probed. Mitochondrial membrane potential (around -200mV) is a function of Ca2+ ion concentration as it depletes with increase in intracellular calcium level as mitochondria takes up the positively charged Ca2+ ions to buffer it out of the cytosol. Mitochondrial membrane potential is measured using the slow redistributive cationic dye JC-1. Partitioning of JC-1 in cell and cellular organelles strongly depends upon ionic strength and membrane potential. If basal Ca2+ (intracellular) is maintained, JC-1 preferentially distributes within mitochondria and if it exceeds its critical J-aggregate forming concentration J-aggregates (emission maxima 580 nm) are formed, as suggested by the high red fluorescence at time t = 0 sec in Fig-9. However, in elevated basal levels of Ca 2+, which is taken up by mitochondria, JC-1 J- aggregates de-polymerize and redistribute

25

itself in cytosol as observed at time t >0 sec in Fig-9 when stimulation with histamine induced a rise in intracellular and intra-mitochondrial calcium level. The rate limiting step for concentration dependent monomer <–> H aggregate <–> J aggregate formation is the diffusion across the membrane (Reers et al, 1991). In normal condition the Ca2+ wave generated by histamine is of saltatoric type, characterized by a sharp increase and subsequent restoration of Ca2+ level (Fig-4). In this framework, mitochondria existing only in the vicinity of wave are majorly affected and depolarized yielding de-agglomeration of JC-1 J aggregates to JC-1 monomers and the J aggregate intensity drops due to loss of membrane potential and steep increase in ionic strength. However, as the total reaction time of the calcium release and subsequent uptake event is too short (of the order of 10-30 sec as in Fig-4) to allow breaking and partitioning of JC-1 monomers into cytosol through the membrane by means of diffusion, the elevated level of intra-mitochondrial JC-1 monomers form kinetically favored Haggregates with disallowed transition to ground state and no fluorescence (Smiley et al, 1991) which are subsequently quenched; instead of staying in JC-1 monomer state. Cells thus display almost constant level of green monomer intensity (Fig-9). For sucrose treated cells, beyond histamine excitation intracellular Ca2+ level sustains at an elevated level for a prolonged time course, a phenomenon intrinsic to global continuous waves. In this case, JC-1 monomers can be released into the cytosol preventing excessive accumulation of JC-1 monomers within mitochondria. Thus, formation of H-aggregates is prevented. Subsequently, the resultant effect is experimentally perceived with concurrent decrease of red fluorescence and most noticeably, increase of green fluorescence intensity (Fig-10). It is interesting to observe that with time green fluorescence intensity overwhelms the red fluorescence, an event that is never observed in normal (sucrose untreated) histamine treated cells. Mitochondria thus exert a negative feedback on the spatio-temporal patterning of the Ca2+ waves by buffering it out of cytosol. Ca2+ ions in turn activate various proteins that are implicated in regulating multiple signaling pathways. So, the nature of the wave and its localization in effect determines major cellular physiological events.

26

7. Future works: •

Establishing the relationship between different Ca2+ waves and expression of proapoptotic factors.

•

Determination of diffusion coefficient of HeLa cell and the amount of contribution of convection and diffusion in intracellular calcium signal propagation.

•

Developing a convection-diffusion-reaction model for calcium ion generation and propagation.

•

Study of calcium wave generated through mechanical stimulation by shear stress.

•

Developing a 2 cell-Y channel system as a tool for studying gap junction based intercellular communication and related cellular phenomena.

•

Probing alteration of membrane asymmetry and its relation with Ca2+ waves.

8. References: Berridge, M. J., Galione, A., 1988. Cytosolic calcium oscillators. FASEB J.2, 3074-3082. Berridge, M. J., Lipp, P., Bootman, M. D., Calcium signalling.2001. Current Biology.9, 157-159. Bootman, M. D., Berridge, M. J., 1995. The Elemental Principles of Calcium Signaling. Cell.83, 675-678. Bootman, M. D., Berridge, M. J., Lipp, P., 1997.Cooking with Calcium: The Recipes for Composing Global Signals from Elementary Events.Cell.91, 367–373.

27

Bootman, M. D., et al., 2001. Calcium signalling- an overview. Seminars in Cell and Developmental Biology.12, 3-10. Burlando, B., Bonomo, M., Fabbri, E., Dondero, F., Viarengo, A., 2003. Hg 2+ signaling in trout hepatoma (RTH-149) cells: involvement of Ca 2+ -induced Ca 2+ release. Cell Calcium.34, 285-293. Carafoli, E., 2004. Calcium-mediated cellular signals: a story of failures. TRENDS in Biochemical Sciences.29, 371-379. Dupont, G., Combettes, L., Leybaert, L., 2007.Calcium Dynamics: Spatio‐Temporal Organization from the Subcellular to the Organ Level. International Review of Cytology.261, 193-245. Ermak, G., Davies, K.J.A.,2001. Calcium and oxidative stress: from cell signaling to cell death. Molecular Immunology.38, 713-721. Guigas, G., Kalla, C., Weiss, M., 2007. Probing the Nanoscale Viscoelasticity of Intracellular Fluids in Living Cells. Biophysical Journal.93,316-323. Hajno´czky, G., P.Thomas, A.P., 1997. Minimal requirements for calcium oscillations driven by the IP3 receptor. The EMBO Journal.16, 3533-3543. Hanson, C.J., Bootman, M.D., Roderick, H.L., 2004. Cell Signalling: IP3 Receptors Channel Calcium into Cell Death. Current Biology.14,933-935. Majeed, M., Krause, K., Clark, R.A., Kihlström, E., Stendahl, O., 1999.Journal of Cell Science.112, 35-44.

28

Merritt, J.E., McCarthy, S.A., Davies, M. P.A., Moores, K.E., 1990. Use of fluo-3 to measure cytosolic Ca2+ in platelets and neutrophils. Biochem. J.269, 513-519. Montell, C., 2005. The Latest Waves in Calcium Signaling. Cell.122,157-163. Uhlen, P., 2004. Spectral analysis of calcium oscillations. Science's STKE.pl15, 1-12. Verkhratsky, A., 2007. CALCIUM AND CELL DEATH. In: Carafoli, E., Brini, M., (eds.), Calcium signaling and disease. Springer, UK, 465-480.

29