Cellular Biophysics SS Prof. Manfred Radmacher

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1 SS Manfred Radmacher Ch. 12 Systems Biology Let's recall chemotaxis in Dictiostelium synthesis of camp excretion of camp external camp gradient detection cell polarity cell migration 2

2 Single cells are interacting with the other cells synthesis of camp excretion of camp external camp gradient detection cell polarity cell 1 via diffusion cell migration We have a complex network of interaction at different levels other cells 3 Networks tend to be complex: camp processing in Dictyostelium Peter N. Devreotes et al. Annu. Rev. Cell Dev. Biol :22 4

3 Life's complexity pyramid genome proteome metabolome Large-scale organization cells Functional modules interacting cells ATP ADP ATP ADP ATP ADP organisms Regulatory motifs Metabolic pathways eco-systems mrna ATP Genes Proteins Metabolites Processing Execution Life s Complexity Pyramid Zoltán N. Oltvai and Albert-László Barabási SCIENCE VOL OCTOBER Genome regulation is also part of these metabolic networks Figure Outline of the mechanism of the circadian clock in Drosophila cells. The central feature of the clock is the periodic accumulation and decay of two gene regulatory proteins, Tim (short for timeless, based on the phenotype of a gene mutation) and Per (short for period). These proteins are translated in the cytosol, and, when they have accumulated to critical levels, they form a heterodimer. This heterodimer is transported into the nucleus where it regulates a number of genes in concert with the clock. The Tim-Per heterodimer also represses the tim and per genes, creating a feedback system that causes the levels of Tim and Per to rise and fall periodically. In addition to this transcriptional feedback, the clock depends on the phosphorylation and subsequent degradation of the Per protein, which occurs in both the nucleus and the cytoplasm and is regulated by an additional clock protein, Dbt (short for double-time). This degradation imposes delays in the periodic accumulation of Tim and Per, which are crucial to the functioning of the clock. For example, the accumulation of Per in the cytoplasm is delayed by the phosphorylation and degradation of free Per monomers. Steps at which specific delays are imposed are shown in red. Entrainment (or resetting) of the clock occurs in response to new light-dark cycles. Although most Drosophila cells do not have true photoreceptors, light is sensed by intracellular flavoproteins, and it rapidly causes the destruction of the Tim protein, thus resetting the clock. From: Alberts et al "Molecular Biology of the Cell" Ch. 09 Pattern Formation - Spatial & Temporal Oscillations 6

4 Complete signaling pathway diagram of Dictiostelium 7 The complete metabolome of a cell is breath taking 8

5 Luckily man has developed tools to cope with complex networks 9 Signaling Pathways are basically systems for signal-processing Input Output 10

6 Modules are known from electronics 11 From an information processing point of view the hardware used is often irrelevant From molecular to modular cell biology Leland H. Hartwell, John J. Hopfield, Stanislas Leibler and Andrew W. Murray NATURE VOL 402 SUPP 2 DECEMBER

7 What counts is the processing of information 13 Networks and graphs are very general concepts Network Motifs: Simple Building Blocks of Complex Networks R. Milo,1 S. Shen-Orr,1 S. Itzkovitz,1 N. Kashtan,1 D. Chklovskii,2 U. Alon1* 25 OCTOBER 2002 VOL 298 SCIENCE 14

8 Networks and graphs are very general concepts Network Motifs: Simple Building Blocks of Complex Networks R. Milo,1 S. Shen-Orr,1 S. Itzkovitz,1 N. Kashtan,1 D. Chklovskii,2 U. Alon1* 25 OCTOBER 2002 VOL 298 SCIENCE 15 Some motifs can be found in very different networks Network Motifs: Simple Building Blocks of Complex Networks R. Milo,1 S. Shen-Orr,1 S. Itzkovitz,1 N. Kashtan,1 D. Chklovskii,2 U. Alon1* 25 OCTOBER 2002 VOL 298 SCIENCE 16

9 Classification of Networks: directed & undirected n. NETWORK BIOLOGY: UNDERSTANDING THE CELL S FUNCTIONAL ORGANIZATION Albert-László Barabási* & Zoltán N. Oltvai NATURE REVIEWS GENETICS VOLUME 5 FEBRUARY Classification of Networks: directed & undirected n. Parameters to describe nodes: Parameters to describe links: k:!! kin:!! kout:! number of links number of incoming links number of outgoing links lab:!! distance between node A and B NETWORK BIOLOGY: UNDERSTANDING THE CELL S FUNCTIONAL ORGANIZATION Albert-László Barabási* & Zoltán N. Oltvai NATURE REVIEWS GENETICS VOLUME 5 FEBRUARY

10 Classification of Networks: directed & undirected n. Parameters to describe networks: averages!! <k>, <k in>, <k out>, <l> distributions:! P (k) etc. scale-free networks:! P(k) k -" NETWORK BIOLOGY: UNDERSTANDING THE CELL S FUNCTIONAL ORGANIZATION Albert-László Barabási* & Zoltán N. Oltvai NATURE REVIEWS GENETICS VOLUME 5 FEBRUARY Different classes of networks Clustering coefficient C C = 2 ni / k (k-1) where n I is the number of links connecting the neighbours of node I NETWORK BIOLOGY: UNDERSTANDING THE CELL S FUNCTIONAL ORGANIZATION Albert-László Barabási* & Zoltán N. Oltvai NATURE REVIEWS GENETICS VOLUME 5 FEBRUARY

11 Example of metabolic network: different levels of abstraction NETWORK BIOLOGY: UNDERSTANDING THE CELL S FUNCTIONAL ORGANIZATION Albert-László Barabási* & Zoltán N. Oltvai NATURE REVIEWS GENETICS VOLUME 5 FEBRUARY Example of metabolic network: different levels of abstraction NETWORK BIOLOGY: UNDERSTANDING THE CELL S FUNCTIONAL ORGANIZATION Albert-László Barabási* & Zoltán N. Oltvai NATURE REVIEWS GENETICS VOLUME 5 FEBRUARY

12 Yeast protein interaction network is a hierarchical scalefree network NETWORK BIOLOGY: UNDERSTANDING THE CELL S FUNCTIONAL ORGANIZATION Albert-László Barabási* & Zoltán N. Oltvai NATURE REVIEWS GENETICS VOLUME 5 FEBRUARY Why are protein interactomes scale free? A simple answer. NETWORK BIOLOGY: UNDERSTANDING THE CELL S FUNCTIONAL ORGANIZATION Albert-László Barabási* & Zoltán N. Oltvai NATURE REVIEWS GENETICS VOLUME 5 FEBRUARY

13 Examples of scale free metabolic networks Jeong et al Nature Oct How to obtain a protein interactome? NETWORK BIOLOGY: UNDERSTANDING THE CELL S FUNCTIONAL ORGANIZATION Albert-László Barabási* & Zoltán N. Oltvai NATURE REVIEWS GENETICS VOLUME 5 FEBRUARY

14 The work horse is the Yeast-2-Hybrid system: Normal Transcription Figure 1. Normal Transcription. Normal transcription requires both the DNA-binding domain (BD) and the activation domain (AD) of a transcriptional activator (TA). Figure 2. Yeast two-hybrid transcription. The yeast two-hybrid technique measures protein-protein interactions by measuring transcription of a reporter gene. If protein X and protein Y interact, then their DNA-binding domain and activation domain will combine to form a functional transcriptional activator (TA). The TA will then proceed to transcribe the reporter gene that is paired with its promoter. From: "THE YEAST TWO-HYBRID ASSAY: AN EXERCISE IN EXPERIMENTAL ELOQUENCE" By Solmaz Sobhanifar at "Science Creative Quarterly" 27 Yeast Two Hybrid - construction of plasmid Figure 3. Plasmid construction. The bait and hunter fusion proteins are constructed in the same manner. The bait DNA is isolated and inserted into a plasmid adjacent to the GAL4 BD DNA. When this DNA is transcribed, the bait protein will now contain the GAL4 DNA-binding domain as well. The hunter fusion protein contains the GAL4 AD. From: "THE YEAST TWO-HYBRID ASSAY: AN EXERCISE IN EXPERIMENTAL ELOQUENCE" By Solmaz Sobhanifar at "Science Creative Quarterly" 28

15 Yeast Two Hybrid - bait and hunter Figure 4. Bait and Hunter Plasmids. The yeast two-hybrid assay uses two plasmid constructs: the bait plasmid, which is the protein of interest fused to a GAL4 binding domain, and the hunter plasmid, which is the potential binding partner fused to a GAL4 activation domain. From: "THE YEAST TWO-HYBRID ASSAY: AN EXERCISE IN EXPERIMENTAL ELOQUENCE" By Solmaz Sobhanifar at "Science Creative Quarterly" 29 Yeast Two Hybrid - transfection Figure 5. Transfection. The bait and hunter plasmids are introduced into yeast cells by transfection. In this process, the plasma membrane is disrupted to yield holes, through which the plasmids can enter. Once transfection has occurred, cells containing both plasmids are selected for by growing cells on minimal media. Only cells containing both plasmids have both genes encoding for missing nutrients, and consequently, are the only cells that will survive. From: "THE YEAST TWO-HYBRID ASSAY: AN EXERCISE IN EXPERIMENTAL ELOQUENCE" By Solmaz Sobhanifar at "Science Creative Quarterly" 30

16 Y2H arrays Principle of two-hybrid library and array screens. (a) Typical two-hybrid screens use a library of random DNA or cdna fused to a transcriptional activation domain (AD), expressed in yeast ( preys ; circles denote plasmids). The library clones are mated to a strain of opposite mating type that expresses a protein of interest ( bait, B) as a fusion to a DNA-binding domain (DBD). If bait and prey interact in the resulting diploid cells, they reconstitute a transcription factor, which activates a reporter gene whose expression allows the diploid cell to grow on selective media (here, without histidine). As an alternative to mating, prey libraries can also be transformed into the bait strain in order to express bait and prey in the same cell. In any case, positive clones have to be picked, their DNA isolated and the encoded plasmids sequenced in order to identify interacting proteins. From: Peter Uetz "Two Hybride Arrays" Curr. Opin.Chem. Biol. 2001, 6: 57-62, 31 Y2H arrays From: Peter Uetz "Two Hybride Arrays" Curr. Opin.Chem. Biol. 2001, 6: 57-62, (b) Array screens use defined sets of cloned prey ORFs or fragments there of that are mated systematically to a certain bait strain. Matings and two-hybrid tests can be automated when large sets of preys have to be assayed, as in the case of whole genomes. 32

17 Y2H arrays is a lot of pipetting From: Peter Uetz "Two Hybride Arrays" Curr. Opin.Chem. Biol. 2001, 6: 57-62, 33 Example: yeast interactome Figure 5. An interaction map of the yeast proteome assembled from published interactions. The map contains 1,548 proteins and 2,358 interactions. Proteins are colored according to their functional role as defined by the Yeast Protein Database (YPD); proteins involved in membrane fusion (blue), chromatin structure (gray), cell structure (green), lipid metabolism (yellow), and cytokinesis (red). After Schwikowski et al. (2000). From: Peter Uetz & Andrei Grigoriev "The yeast interactome" 34

18 Example: yeast interactome From: Peter Uetz & Andrei Grigoriev "The yeast interactome" 35 Example: yeast interactome From: Peter Uetz & Andrei Grigoriev "The yeast interactome" 36

19 Yeast interactome is clustered Figure 7. Interactions between functional groups. Numbers in parentheses indicate, first, the number of interactions within a group, and second, the number of proteins in a group. Numbers near connecting lines indicate the number of interactions between proteins of the two connected groups. For example, there are 77 interactions between the 21 proteins involved in membrane fusion and the 141 proteins involved in vesicular transport (upper left corner); 23 protein interactions connect the 21 proteins involved in membrane fusion. Only connections with 15 or more interactions are included here. Note that only proteins with known function are shown (many of these have several functions). The sum of all interactions in this diagram is therefore smaller than the number of all interactions. After Schwikowski et al. (2000). From: Peter Uetz & Andrei Grigoriev "The yeast interactome" 37 Neuronal Networks are a story in itself 38

20 Neuronal Networks simulate learning (and forgetting) 39 Agent world - modelling systems of chemotactic cells AgentCell: a digital single-cell assay for bacterial chemotaxis Thierry Emonet1,!, Charles M. Macal2, Michael J. North2, Charles E. Wickersham1 and Philippe Cluzel1 BIOINFORMATICS Vol. 21 no , pages

21 Agent world - modelling systems of chemotactic cells AgentCell: a digital single-cell assay for bacterial chemotaxis Thierry Emonet1,!, Charles M. Macal2, Michael J. North2, Charles E. Wickersham1 and Philippe Cluzel1 BIOINFORMATICS Vol. 21 no , pages Agent world - modelling systems of chemotactic cells AgentCell: a digital single-cell assay for bacterial chemotaxis Thierry Emonet1,!, Charles M. Macal2, Michael J. North2, Charles E. Wickersham1 and Philippe Cluzel1 BIOINFORMATICS Vol. 21 no , pages

22 Agent world - modelling systems of chemotactic cells AgentCell: a digital single-cell assay for bacterial chemotaxis Thierry Emonet1,!, Charles M. Macal2, Michael J. North2, Charles E. Wickersham1 and Philippe Cluzel1 BIOINFORMATICS Vol. 21 no , pages Building regulatory networks from scratch: the Repressilator We used three transcriptional repressor systems that are not part of any natural biological clock3±5 to build an oscillating network, termed the repressilator, in Escherichia coli. The network periodically induces the synthesis of green!uorescent protein as a readout of its state in individual cells. The resulting oscillations, with typical periods of hours, are slower than the cell-division cycle, so the state of the oscillator has to be transmitted from generation to generation. This arti cial clock displays noisy behaviour, possibly because of stochastic!uctuations of its components. Such `rational network design' may lead both to the engineering of new cellular behaviours and to an improved understanding of naturally occurring networks. A synthetic oscillatory network of transcriptional regulators Michael B. Elowitz & Stanislas Leibler NATURE VOL JANUARY

23 Building regulatory networks from scratch: the Repressilator A synthetic oscillatory network of transcriptional regulators Michael B. Elowitz & Stanislas Leibler NATURE VOL JANUARY Building regulatory networks from scratch: the Repressilator m: mrna p: protein A synthetic oscillatory network of transcriptional regulators Michael B. Elowitz & Stanislas Leibler NATURE VOL JANUARY

24 Building regulatory networks from scratch: the Repressilator A synthetic oscillatory network of transcriptional regulators Michael B. Elowitz & Stanislas Leibler NATURE VOL JANUARY degrees of separation; or its just 19 clicks to any information you want to get Réka Albert, Hawoong Jeong, Albert-László Barabási Diameter of the World-Wide Web NATURE VOL SEPTEMBER