SPATIAL AND TEMPORAL ASPECTS OF SIGNALLING

TERM PAPER (BTY-302)

Submitted By : Rajeev Kumar Roll No. : A-14 Reg. No. : 10812767 Section : K7803

Submitted To : Mr. Prashant Singh

ACKNOWLEDGEMENT
I am very thankful to my teacher Mr Prashant Singh Kumar who thought me worthy of handling this project and helped me in lot in term paper on Spatial and temporal aspects of signalling. Without his able guidance this work would have been impossible. Rajeev k7803 A14

Table of contents:
1) Introduction 2) Spatial and temporal control in intracellular signaling
3)

Spatial and temporal signaling regulation

4) Techniques for measuring spatial and temporal aspects of signaling 5) Spatial and temporal aspects of cellular calcium signaling 6) Spatial and temporal expression patterns of two type I receptors for bone morphogenetic proteins 7) Coupled Spatial and Non-Spatial Simulations of ErbB1 Signaling Pathways
8)

Spatial and temporal aspects of the chromosome and DNA replication

9) Conclusion 10) References

Introduction
As new techniques are developed to measure intracellular messengers it becomes increasingly apparent that there is a remarkable spatial and temporal organization of cell signalling. Cells possess a small discrete hormone-sensitive pool of inositol lipid. In some cells such as Xenopus oocytes and Limulus photoreceptors this phosphoinositide signalling system is highly concentrated in one region of the cell, so establishing localized calcium gradients. Another example is the hydrolysis of inositol lipids in eggs at the point of sperm entry resulting in a localized increase in Ins(1,4,5)P3 and calcium which spreads like a wave throughout the egg. In hamster eggs this burst of calcium at fertilization recurs at 1-3 min intervals for over 100 min, a

particularly dramatic example of spontaneous activity. Spontaneous oscillations in intracellular calcium exist in many different cell types and are often induced by agonists that hydrolyse inositol lipids. We have made a distinction between oscillations that are approximately sinusoidal and occur at a higher frequency where free calcium is probably continuously involved in the oscillatory cycle and those where calcium falls to resting levels for many seconds between transients. In the former case, the oscillations are thought to be induced through a cytoplasmic oscillator based on the phenomenon of calcium-induced calcium release. Such oscillations can be induced in Xenopus oocytes after injection with Ins(1,4,5)P3. A receptor-controlled oscillator based on the periodic formation of Ins(1,4,5)P3 is probably responsible for the generation of the widely spaced calcium transients. The function of such calcium oscillations is currently unknown. They may be a reflection of the feedback interactions that operate to control intracellular calcium. Another possibility emerged from observations that in some cells the frequency of calcium oscillations varied with agonist concentration, suggesting that cells might employ these oscillations as a way of encoding information. One advantage of using such a frequency-dependent mechanism may lie in an increase in fidelity, especially at low agonist concentrations. Whatever these functions might be, it is clear that uncovering the mechanisms responsible for such oscillatory activity will greatly enhance our understanding of the relation between the phosphoinositides and calcium signalling.

Spatial and Temporal Control in intracellular signaling

Cells integrate many inputs through complex networks of interacting signaling pathways. Systems approaches as well as computer-aided reductionist approaches attempt to "untangle the wires" and gain an intimate understanding of cells. But "understanding" any system is just the way that the human mind gains the ability to predict behavior. Computer simulations are an alternative way to achieve this goalquite possibly the only way for complex systems. We have new tools to probe large sets of unknown interactions, and we have amassed enough detailed information to quantitatively describe many functional modules. Cell physiology has passed the threshold: the time to begin modeling is now. As cell biology entered the 21st century, a convergence of factors promised to revolutionize the way state-of-the-art research is done. On the one hand, there has been an explosion of new data: some from "-omic" technologies and other

high-throughput methods and some from new imaging and in vivo technologies that can reveal molecular interactions in a real and quantitative way . One common theme is the fact that to properly leverage these data, we need computers to process them and to help us use them for generating and testing hypotheses. Traditional bioinformatics is being transformed and complemented by the expanding systems biology and the emergent computational cell biology. However, the scope of intracellular signaling networks is increasingly difficult to delineate, given the multiple molecular interactions present inside the cell. Thus it is not clear where to draw the boundary between signaling networks on the one hand and either metabolic networks or generegulatory networks on the other. We do not attempt to make such clear distinctions here, and we will mainly use the more global view of modeling and simulating intracellular molecular networks. We conclude that the field of cell biology is ripe for widespread use of computational approaches and that the best avenue for progress is to combine top-down, systemslevel modeling with bottom-up, detailed quantitative modeling.

Regulation in temporal and spatial signaling :

Spatially and temporally evolving collective dynamics act critically to coordinate multicellular development. In general, periodic phenomena are prevalent in transcriptional regulation - for example, in circadian rhythms , Msn transcription factor regulation in yeast and the pulsatile response of NF-B and p53 in tissue culture cells following stimulation. Oscillations seem to be a universal mode of regulation for morphogenetic cell movements and gene transcription that requires fine spatial and temporal coordination. Calcium waves are observed during convergent extension in Xenopus and are believed to coordinate cell movement . In the case of somitogenesis, where segmentation is periodic, Notch and Wnt signaling is coupled to periodic expression of the Notch components themselves. It is expected that the functions of molecular networks will become apparent only when put into the context of such multicellular organization in time and space. Biologically relevant readouts with a temporal and spatial resolution are thus

the final layer needed to connect high-throughput genomics data obtained at the molecular and cellular level to higher organizational and functional levels. A classic experimental paradigm in developmental biology begins with a mutant phenotype and then asks which aspects of development are altered. The goal is to relate structure to function, first at the molecular, then the cellular, and finally the whole-organism level. The current richness of information for a few model organisms is testimony to the success of this approach. With the explosion of genome sequences, it is becoming realistic to rapidly map out relations between genotype and molecular level phenotype using large-scale assays at the level of transcription and translation. Efforts to complement such bottom-up approaches by high-throughput screens based on observational phenotypes at the cellular level have recently been reported in yeast, nematode, and cells in tissue culture.

Techniques for measuring spatial and temporal aspects of signaling :

Fluorescence microscopy of cells loaded with fluorescent, Ca(2+)-sensitive dyes is used for measurement of spatial and temporal aspects of Ca(2+) signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca(2+)reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca(2+)-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging

chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca(2+) response as % change in fluorescence is obtained.

Spatial and temporal aspects of cellular calcium signaling :

Cytosolic Ca2+ signals are often organized in complex temporal and spatial patterns, even under conditions of sustained stimulation. In this review we discuss the mechanisms and physiological significance of this behavior in nonexcitable cells, in which the primary mechanism of Ca2+ mobilization is through (1,4,5)IP3-dependent Ca2+ release from intracellular stores. Oscillations
of cytosolic free Ca2+ are a common form of temporal organization; in the spatial domain, these oscillations may take the form of waves that propagate throughout the cell or they

may be restricted to specific subcellular regions. These patterns of Ca2+ signaling result from the limited range of cytoplasmic Ca2+ diffusion and the feedback regulation of the pathways responsible for Ca2+ mobilization. In addition, the spatial organization of changes appears to depend on the strategic distribution of Ca2+ stores within the cell. One type of Ca2+ oscillation is baseline spiking, in which discrete Ca2+spikes occur with a frequency, but not amplitude, that is determined by agonist dose. Most current evidence favors a model in which
baseline Ca2+ spiking results from the complex interplay between Ca2+ and (1,4,5)IP3 in

regulating the gating of (1,4,5)IP3-sensitive intracellular Ca2+ channels. Sinusoidal

Ca2+oscillations represent a mechanistically distinct type of temporal organization, in which

agonist dose regulates the amplitude but has no effect on oscillation frequency. Sinusoidal
Ca2+ oscillations can be explained by a negative feedback effect of protein kinase C on the

generation of (1,4,5)IP3 at the level of phospholipase C or its activating G-protein. The physiological significance of [Ca2+]i oscillations and waves is becoming more established with the observation of this behavior in intact tissues and by the recognition of Ca2+-dependent

processes that are adapted to respond to frequency-modulated oscillator y Ca2+signals. In

some cells, these C2+i signals are targeted to control processes in limited cytoplasmic

domains, and in other systems Ca2+ waves can be propagated through gap junctions to coordinate the function of multicellular systems.

Spatial and temporal expression patterns of two type I receptors for bone morphogenetic proteins :
Bone morphogenetic proteins (BMPs) are multifunctional proteins structurally related to transforming growth factor-beta (TGF beta) and activin that can induce cartilage and bone growth in vivo. Members of the TGF beta superfamily exert their biological effects via heteromeric serine/threonine kinase complexes of type I and type II receptors. We previously obtained six different type I receptors, termed activin receptor-like kinase-1 (ALK-1) to -6. ALK5 is a TGF beta type I receptor, ALK-2 and ALK-4 are activin type I receptors, and ALK-3 and ALK-6 are type I receptors for osteogenic protein-1 (OP-1)/bone morphogenetic protein-7 (BMP7) and BMP-4. Here we report the complementary DNA cloning of the mouse homolog of ALK3, which is highly conserved between mouse and man. ALK-3 messenger RNA (mRNA) is ubiquitously expressed in various adult mouse tissues, whereas ALK-6 mRNA is only found in brain and lung. The distribution of ALK-3 and ALK- 6 mRNA in the postimplantation mouse embryo [6.5-15.5 days postcoitum (pc)] was studied by in situ hybridization. ALK-3 was nearly ubiquitously expressed throughout these stages of development, but was notably absent in the liver. In contrast, ALK-6 showed a more restricted expression pattern. ALK-6 mRNA was absent in early postimplantation embryos, was detected first in 9.5 days pc embryos, and persisted until 15.5 days pc. In midgestation embryos, ALK-6 transcripts were detected in mesenchymal precartilage condensations, premuscle masses, blood vessels, central nervous system, parts of the developing ear and eye, and epithelium. The expression in sites of developing cartilage and bone supports the idea that ALK-3 and -6 are receptors for BMPs in vivo. In addition, the expression of these genes in many soft tissues suggests broader functions for BMPs in embryogenesis.

Coupled Spatial and Non-Spatial Simulations of ErbB1 Signaling Pathways : The ErbB family of receptors activates intracellular signaling pathways that control cellular proliferation, growth, differentiation and apoptosis. Given these central roles, it is not surprising that overexpression of the ErbB receptors is often associated with carcinogenesis. Therefore, extensive laboratory studies have been devoted to understanding the signaling events associated with ErbB activatioSystems biology has contributed significantly to our current understanding of ErbB signaling networks. However, although computational models have grown in complexity over the years, little work has been done to consider the spatialtemporal dynamics of receptor interactions and to evaluate how spatial organization of membrane receptors influences signaling transduction. Herein, we explore the impact of spatial organization of the epidermal growth factor receptor (ErbB1/EGFR) on the initiation of downstream signaling. We describe the development of an algorithm that couples a spatial stochastic model of membrane receptors with a nonspatial stochastic model of the reactions and interactions in the cytosol. This novel algorithm provides a computationally efficient method to evaluate the effects of spatial heterogeneity on the coupling of receptors to cytosolic signaling partnersMathematical models of signal transduction rarely consider the contributions of spatial organization due to high computational costs. A hybrid stochastic approach simplifies analyses of the spatio-temporal aspects of cell signaling and, as an example, demonstrates that receptor clustering contributes significantly to the efficiency of signal propagation from ligand-engaged growth
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factor receptorsTop of Form

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Bottom of FormThe ErbB family of receptors activates intracellular signaling pathways that control cellular proliferation, growth, differentiation and

apoptosis. Given these central roles, it is not surprising that overexpression of the ErbB receptors is often associated with carcinogenesis. Therefore, extensive laboratory studies have been devoted to understanding the signaling events associated with ErbB activation. Systems biology has contributed significantly to our current understanding of ErbB signaling networks. However, although computational models have grown in complexity over the years, little work has been done to consider the spatial-temporal dynamics of receptor interactions and to evaluate how spatial organization of membrane receptors influences signaling transduction. The ErbB family of receptors, under normal physiological conditions, regulate key cellular processes such as growth, proliferation and differentiation. Overexpression of these receptors deregulates normal cellular function and is a contributing factor to tumorigenesis. There are four members of the ErbB family (ErbB1, ErbB2, ErbB3 and ErbB4) and each family member has its own unique ligand specificity, kinase activity and spatial organization on the membrane. In our current study, we have focused solely on the epidermal growth factor receptor (typically abbreviated ErbB1 or EGFR) and the ErbB1 activation of ERK, which is a mitogen activated protein kinase. Ligand binding to ErbB1 stabilizes a conformation of the extracellular domain that allows receptor dimerization. Dimerized receptors are active tyrosine kinases, capable of transautophosphorylation. Phosphorylation of receptor cytoplasmic tails results in recruitment of SH2-containing adaptor and signaling proteins, such as Grb2, Sos, and Shc, that form a signaling scaffold to activate ERK. Advanced microscopy techniques have demonstrated that membrane properties, such as transient confinement zones and corrals, may restrict and govern the spatialtemporal dynamics of lipids and membrane protein. The challenge is to develop computational approaches that can account for membrane spatial heterogeneity and evaluate the impact on signal propagation. Spatial modeling has been implemented in many scientific disciplines, including physics, material sciences, chemistry, engineering and biological systems. Spatial MC platforms are particularly powerful numerical simulation tools for studying the dynamics of membrane components.

Spatial and temporal aspects of the chromosome and DNA replication:

Cells invest significant resources in the faithful replication and partitioning of chromosomes. Chromosome duplication involves assembly of a large complex of proteins at an origin (at 0 on the B. subtilis circular chromosome) and processive replication in both directions away from the origin. Our work studying replication in vivo indicated that during replication, the DNA moves to the DNA replication machinery (the replisome), gets duplicated, and then moves away. This is in contrast to models in which the replication machinery moves along the DNA. To gain insight into the spatial and temporal organization of the replication cycle, we visualized replication origins and the replication machinery (replisomes) inside live cells. Our analysis indicates that the location of the origin at the time of replication initiation establishes the position of the replisome. Also, it appears that sister replication forks are not intimately associated with each other throughout the replication cycle. This work indicates that there as yet uncharacterized factors involved in chromosome positioning and orientation.

Conclusion
Proper understanding of the mechanisms of disease as well as effective design and creation of new therapies for disease and aging is hampered by the complexity of cellular interactions. We made a point at the beginning that the essence of understanding is just the ability to predict a systems behavior. Whether this is achieved by finding some (more-or-less simple) sets of rules or laws that govern the system (e.g., "mechanisms") or by simulating a whole network on a computer is ultimately an academic question. Given the complex weave of intracellular interactions governed by many nonlinear and often stochastic relationships, the computer-based approach appears to be the only practical solution.

References:
Csete ME and Doyle JC. Reverse engineering of biological complexity

Fell DA. Understanding the Control of Metabolism. London Endy D and Brent R. Modelling cellular behavior Csete ME and Doyle JC. Reverse engineering of biological complexity