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cell communication c o u r s e l a y o u t introduction molecular biology biotechnology bioMEMS bioinformatics bio-modeling cells and e-cells transcription and regulation cell communication neural networks dna computing fractals and patterns the birds and the bees ….. and ants i n t r o d u c t i o n cell communication what is signal transduction? Conversion of a signal from one physical or chemical form into another. In cell biology, it commonly refers to the sequential process initiated by binding of an extracellular signal to a receptor and culminating in one or more specific cellular responses. what is a signal transduction pathway? Chemical signals are converted from one type of signal into another to elicit a molecular response from the organism. All organisms require signaling pathways to live. ABCDEFG Letters represent chemicals or proteins. Arrows represent enzymatic steps. what is a second messenger? An intracellular signaling molecule whose concentration increases (or decreases) in response to binding of an extracellular ligand to a cell-surface receptor. cell signaling How do cells receive and respond to signals from their surroundings? Prokaryotes and unicellular eukaryotes are largely independent and autonomous. In multi-cellular organisms there is a variety of signaling molecules that are secreted or expressed on the cell surface of one cell and bind to receptors expressed by other cells. These molecules integrate and coordinate the functions of the cells that make up the organism. modes of cell-cell signaling Direct cell-cell or cell-matrix Secreted molecules. Endocrine signaling. The signaling molecules are hormones secreted by endocrine cells and carried through the circulation system to act on target cells at distant body sites. Paracrine signaling. The signaling molecules released by one cell act on neighboring target cells (neurotransmitters). Autocrine signaling. Cells respond to signaling molecules that they themselves produce (response of the immune system to foreign antigens and cancer cells). steroid hormones This class of molecules diffuse across the plasma membrane and binds to Receptors in the cytoplasm or nucleus. The y are all synthesized from cholesterol. They include sex steroids (estrogen, progesterone, testosterone) corticosteroids (glucocorticoids and mineralcorticoids) Thyroid hormone, vitamin D3, and retinoic acid have different structure and function but share the same mechanism of action with the other steroids. Steroid Receptor Superfamily. They are transcription factors that function either as activators or repressors of transcription. steroid hormones seven levels of regulation of cell growth pathways are inter-linked Signalling pathway Genetic network STIMULUS Metabolic pathway metabolic pathways 1993 Boehringer Mannheim GmbH - Biochemica overview of cell to cell communication Chemical Autocrine & Paracrine: local signaling Endocrine system: distant, diffuse target Electrical Gap junction: local Nervous system: fast, specific, distant target gap junctions and CAMs Protein channels - connexin Direct flow to neighbor Electrical- ions (charge) Signal chemicals CAMs (cell-adhesion molecules) Need direct surface contact Signal chemical Figure 6-1a, b: Direct and local cell-to-cell communication gap junctions and CAMs Figure 6-1a, b: Direct and local cell-to-cell communication paracrines and autocrines Local communication Signal chemicals diffuse target Example: Cytokines Autocrine–receptor to on same cell Paracrine–neighboring cells Figure 6-1c: Direct and local cell-to-cell communication hormones Signal Chemicals Made in endocrine cells Transported via blood Receptors on target cells long distance communication Figure 6-2a: Long distance cell-to-cell communication neurons and neurohormones Neurons Electrical signal down axon Signal molecule (neurotransmitter) to target cell Neurohormones Chemical and electrical signals down axon Hormone transported via blood to target long distance communication Figure 6-2 b: Long distance cell-to-cell communication neurons and neurohormones long distance communication Figure 6-2b, c: Long distance cell-to-cell communication neurons and neurohormones long distance communication Figure 6-2b, c: Long distance cell-to-cell communication signal pathways Signal molecule (ligand) Receptor Intracellular signal Target protein Response Figure 6-3: Signal pathways receptor locations Cytosolic or Nuclear Lipophilic ligand enters cell Often activates gene Slower response Cell membrane Lipophobic ligand can't enter cell Outer surface receptor Fast response membrane receptor classes Ligand- gated channel Receptor enzymes G-protein-coupled Integrin membrane receptor classes signal transduction Transforms signal energy Protein kinase Second messenger Activate proteins Phosporylation Bind calcium Cell response signal amplification Small signal produces large cell response Amplification enzyme Cascade receptor enzymes Transduction Activation cytoplasmic Side enzyme Example: Tyrosine kinase Figure 6-10: Tyrosine kinase, an example of a receptor-enzyme G-protein-coupled receptors Hundreds of types Main signal transducers Activate enzymes Open ion channels Amplify: adenyl cyclase-cAMP Activates synthesis G-protein-coupled receptors transduction reviewed novel signal molecules Calcium: muscle contraction Channel opening Enzyme activation Vesicle excytosisNitric Oxide (NO) Paracrine: arterioles Activates cAMP Brain neurotransmitter Carbon monoxide (CO) novel signal molecules Calcium as an intracellular messenger quorum sensing quorum sensing the ability of bacteria to sense and respond to environmental stimuli such as pH, temperature, the presence of nutrients, etc has been long recognized as essential for their continued survival it is now apparent that many bacteria can also sense and respond to signals expressed by other bacteria quorum sensing is the regulation of gene expression in response to cell density and is used by Gram positive and Gram negative bacteria to regulate a variety of physiological functions it involves the production and detection of extracellular signaling molecules called autoinducers quorum sensing Tomasz (1965) – Gram-positive Streptococcus pneumoniae produce a “competence factor” that controlled factors for uptake of DNA (natural transformation) Nealson et al. (1970) – luminescence in the marine Gram-negative bacterium Vibrio fischeri controlled by self-produced chemical signal termed autoinducer Eberhard et al. (1981) identified the V. fischeri autoinducer signal to be N-3-oxo-hexanoyl-L-homoserine lactone Engebrecht et al. (1983) cloned the genes for the signal generating enzyme, the signal receptor and the lux genes quorum sensing Vibrio fischeri is a specific bacterial symbiont with the squid Euprymna scolopes and grows in its light organ quorum sensing the squid cultivates a high density of cells in its light organ, thus allowing the autoinducer to accumulate to a threshold concentration at this point, autoinducer combines with the gene product luxR to stimulate the expression of the genes for luciferase, triggering maximal light production studies have shown that hatchling squid fail to enlarge the pouches that become the fully developed organ when raised in sterile seawater quorum sensing In V. fisheri, bioluminsecence only occurs when V. fischeri is at high cell density quorum sensing N-3-oxo-hexanoyl-L-homoserine lactone quorum sensing Fuqua et al. (1994) introduced the term quorum sensing to describe cell-cell signaling in bacteria Early 1990’s – homologs of LuxI were discovered in different bacterial species V. fischeri LuxI-LuxR signaling system becomes the paradigm for bacterial cell-cell communication quorum sensing Vast array of molecules are used as chemical signals – enabling bacteria to talk to each other, and in many cases, to be multilingual Gram-negative bacteria Gram-positive bacteria universal language quorum sensing in Pseudomonas aeruginosa P. aeruginosa uses a hierarchical quorum sensing circuit to regulate expression of virulence factors and biofilm formation quorum sensing in Gram-positive bacteria Gram-positive bacteria utilizes modified oligopeptides as signaling molecules – secreted via an ATP-binding cassette (ABC) transporter complex Detectors for these signals are two-component signal transduction systems quorum sensing in Gram-positive bacteria sensor kinase binding of autoinducer leads to autophosphorylation at conserved histidine residue response regulator -phosphorylation at conserved aspartate by sensor kinase leads to binding of regulator to specific target promoters hybrid quorum sensing circuit in Vibrio harveyi V. harveyi – marine bacterium, but unlike V. fischeri, does not live in symbiotic associations with higher organisms, but is free-living Similar to V. fischeri, V. harveyi uses quorum sensing to control bioluminescence Unlike V. fischeri and other gram-negative bacteria, V. harveyi has evolved a quorum sensing circuit that has characteristics typical of both Gram-negative and Gram-positive systems hybrid quorum sensing circuit in Vibrio harveyi V. harveyi uses acyl-HSL similar to other Gram-negatives but signal detection and relay apparatus consists of twocomponent proteins similar to Gram-positives V. harveyi also responds to AI-2 that is designed for interspecies communication X = transcriptional repressor hybrid quorum sensing circuit in Vibrio harveyi AI-1 AI-2 LuxN and LuxQ – autophosphorylating kinases at low cell densities X = transcriptional repressor Accumulation of autoinducers – LuxN and LuxQ phosphatases draining phosphate from LuxO via LuxU Dephosphorylated LuxO is inactive repressor X not transcribed LuxS and interspecies communication LuxS homologs found in both Gram-negative and Grampositive bacteria; AI-2 production detected in bacteria such as E. coli, Salmonella typhimurium, H. pylori, V. cholerae, S.aureus, B. subtilis using engineered V. harveyi biosensor Biosynthetic pathway, chemical intermediates in AI-2 production, and possibly AI-2 itself, are identical in all AI2 producing bacteria to date – reinforces the proposal of AI-2 as a “universal” language signal processing circuits cell-cell communication circuits Sender cells 0 tetR Receiver cells 0 LuxR luxI VAI VAI aTc aTc pLuxI-Tet-8 pRCV-3 GFP cell-cell communication circuits Sender cells Receiver cells VAI P(tet) VAI + tetR aTc P(Ltet-O1) luxI luxR Lux P(L) Lux P(R) GFP(LVA) 2:4 multiplexer C(4)HSL qsc box C(6)HSL lux box Cell Color 0 0 none 0 1 Green (GFP) 1 0 Red (HcRED) 1 1 Cyan (CFP) significance of multiplexer With a 2:4 mux, the combination of 2 inputs produces 4 different output states / expressed proteins In Eukaryotic cells, these proteins could potentially differentiate the cell into one of four cell types Applications include tissue engineering and more understanding for stem cell fate and determination mux: the sum of three circuits qsc lux A qsc lux B 0 0 0 0 0 0 0 1 green 0 1 0 1 0 0 1 0 red 1 1 0 1 1 0 qsc lux C qsc lux D 0 0 0 0 0 0 0 1 0 0 1 green 1 0 0 1 0 red 1 1 cyan 1 1 cyan + = + case A C6HSL C4HSL luxR GFP lux box RhlR qsc box qsc Lux A 0 0 0 0 1 green 1 0 0 1 1 0 case B C6HSL C4HSL luxR HcRED qsc box RhlR lux box qsc lux B 0 0 0 0 1 0 1 0 red 1 1 0 case C, AND gate cI lux box CFP λP(R) cI qsc box qsc lux C 0 0 0 0 1 0 1 0 0 1 1 cyan case A and B C6HSL C4HSL luxR RhlR GFP lux box qsc box HcRED qsc lux AxorB 0 0 0 0 1 green 1 0 red 1 1 0 design considerations qsc binding site plasmid copy number production of C(x)HSL phenotype tests triple plasmid, regulatory double plasmid, antisensing double plasmid, antisensing + regulatory chromosome, antisensing + regulatory case A -1 0 re gion rrnB T1 P (BLA) AP r LuxR LuxR RBS lux P (L) pRCV-3 4149 bp Inv e rte d Re pe a t QSC box LuxR -1 0 LuxR -3 5 CAP/c AM P Binding S ite lux box LuxICDABE G -1 0 re gion ColE 1 O RI RBSII lux P (R) G FP(LV A) CAP bs rrnB T1 P (LAC) -1 0 re gion -3 5 re gion pASK-102: Single “Parent” Offspring case A -1 0 re gion rrnB T1 P (BLA) AP r LuxR LuxR RBS lux P (L) pASK-102-qsc117 4159 bp Inv e rte d Re pe at LuxR -1 0 LuxR -3 5 CAP/c AM P Binding S ite lux box ColE 1 O RI LuxICDABE G -10 re gion qs c1 1 7 lux box for C4HS L lux P (R) CAP bs RBSII -3 5 re gion -1 0 re gion P (LAC) Plasmid 1 G FP(LV A) rrnB T1 case A -10 region AP r RhlR Ver 2 (8 Mismatch) pASK-103-RhlR-qsc117 4848 bp LuxR ColE1 ORI LuxR RBS lux P(L) Inverted Repeat CAP bs LuxR -10 -35 region LuxR -35 -10 region CAP/cAMP Binding Site P(LAC) lux box rrnB T1 LuxICDABEG -10 region qsc117 lux box for C4HSL lux P(R) RBSII GFP(LVA) Parents: pASK-102-qsc117 (vector), pECP61.5 (insert) Plasmid 2 detecting chemical gradients signal analyte source OO N HO O O O N HO OO N HO OO N HO OO N HO OO O N HO OO O N HO OO O N HO OO O N HO OO O N HO OO O N HO OO O N HO reporter rings analyte source detection GFP [HSL] circuit components OO O N HO OO O N HO OO O N HO O N HO O OO O N HO OO OO OO N HO O N HO P(X) OO LuxR P(R) luxR O N HO LuxR P(lux) X P(Z) Y P(Y) Components 1. Acyl-HSL detect 2. Low threshold 3. High threshold 4. Negating combiner Z1 W P(W) GFP Z2 GFP [HSL] detecting chemical gradients acyl-hSL detection OO O N HO OO O N HO OO O N HO OO OO O N HO OO O OO O N HO N HO O N HO P(X) OO LuxR P(R) luxR O N HO LuxR P(lux) X Z1 P(Z) Y P(Y) W X low threshold Y high threshold P(W) Z2 GFP detecting chemical gradients low threshold detection OO O N HO OO O N HO OO O N HO OO OO O N HO OO O OO O N HO N HO O N HO P(X) OO LuxR P(R) luxR O N HO LuxR P(lux) X Z1 P(Z) Y P(Y) W P(W) Z2 GFP detecting chemical gradients high threshold detection OO O N HO OO O N HO OO O N HO OO OO O N HO OO O OO O N HO N HO O N HO P(X) OO LuxR P(R) luxR O N HO LuxR P(lux) X Z1 P(Z) Y P(Y) W P(W) Z2 GFP detecting chemical gradients protein Z determines range OO O N HO OO O N HO OO O N HO OO OO O N HO OO O OO O N HO N HO O N HO P(X) OO LuxR P(R) luxR O N HO LuxR P(lux) X Z1 P(Z) Y P(Y) W P(W) Z2 GFP detecting chemical gradients negating combiner OO O N HO OO O N HO OO O N HO OO OO O N HO OO O OO O N HO N HO O N HO P(X) OO LuxR P(R) luxR O N HO LuxR P(lux) X Z1 P(Z) Y P(Y) W P(W) Z2 GFP engineering circuit characteristics HSL-mid: the midpoint where GFP has the highest concentration HSL-width: the range where GFP is above 0.3uM HSL-mid 0.3 HSL-width ot h er si g na ls relay signals Signals received at the cell surface either by G-proteinlinked or enzyme-linked receptors are relayed into the cell This is achieved by a combination of small and large intracellular signaling molecules The resulting chain of intracellular signaling events alters a target protein which in turn modifies the behavior of the cell (Fig. 15-1) relay signals The small intracellular mediators are called second messengers (the first messenger being the extracellular signal) e.g. Ca2+ and cyclic AMP, which are water-soluble and diffuse into the cytosol The large intracellular signaling proteins mediators are intracellular They relay the signal by either activating the next signaling protein in the chain or generating small intracellular mediators Different kinds of intracellular signaling proteins along a signaling pathway from the cell surface to the nucleus Relay proteins: pass the message to the next signaling component Adaptor proteins: link one signaling protein to another without themselves participating in the signaling event Bifurcation proteins: spread the signal from one signaling pathway to another Amplifier proteins: usually either enzymes or ion channels that enhance the signal they receive Transducer proteins: convert the signal to a different form e.g. adenyl cyclase Different kinds of intracellular signaling proteins along a signaling pathway from the cell surface to the nucleus Latent gene regulatory proteins: activated at the cell surface by activated receptors & migrate to the nucleus to stimulate gene expression Integrator proteins: receive signals from 2 or more pathways and integrate them before relaying a signal onwards Anchoring proteins: maintain specific signaling proteins at a specific location by tethering them to a membrane Modulator proteins: modify the activity of intracellular signaling proteins & regulate the strength of signaling along the pathway Different kinds of intracellular signaling proteins along a signaling pathway from the cell surface to the nucleus Scaffold proteins: adaptor &/or anchoring proteins that bind multiple signaling proteins together in a functional complex intracellular signaling proteins as molecular switches Many intracellular molecular switches signaling proteins behave like On receipt of a signal, they switch from an inactive to active state until another process turns them off There are two classes of such molecular switches Phosphorylation switches 2. GTP-binding protein switches 1. In both cases, it is the gain or loss of phosphate that determines whether the switch is active or inactive intracellular signaling proteins as molecular switches Switch is turned on by a protein kinase, which adds a phosphate, and turned off by a protein phosphatase, which removes the phosphate group Switch is turned on by exchange of GDP for GTP, and turned off by GTP hydrolysis (ie GTPase activity) phosphorylation cascades ~ 1/3 of the proteins in a cell are phosphorylated at any given time Moreover, many of the signaling proteins controlled by phosphorylation are themselves protein kinases These are organized in phosphorylation cascades One protein kinase , activated by phosphorylation, phosphorylates the next protein kinase in the sequence, and so on, relaying the signal onward protein kinases There are two main types of protein kinase Serine/threonine kinases They phosphorylate proteins on serines and (less often) threonines Tyrosine kinases They phosphorylate proteins on tyrosines signal processing Complex cell behaviors, like cell survival and cell proliferation, are stimulated by specific combinations of signals, rather than one signal acting alone signal processing signal processing Accordingly, the cell has to integrate information coming from separate signals so as to make the appropriate response– e.g. to live or die This depends on integrator proteins, which are analogous to computer microprocessors They require multiple signal inputs to produce an output with the desired biological effect integrator proteins integrator proteins Example of how they work: External signals A and B both activate a different series of protein phosphorylations Each leads to the phosphorylation of protein Y, but at different sites on the protein (Fig. 15-18) integrator proteins integrator protein integrator proteins Example of how they work: Protein Y is activated only when both of these sites are activated, and hence only when signals A and B are simultaneously present For this reason, integrator proteins are sometimes called coincidence detectors integrator proteins Also known as a ‘coincidence detector’ scaffold proteins The complexity of signal response systems, with multiple interacting relay chains of signaling proteins is daunting One strategy the cell uses to achieve specificity involves scaffolding proteins They organize groups of interacting signaling proteins into signaling complexes Because the scaffold guides the interactions between the successive components in such a complex, the signal is relayed with speed In addition, cross-talk between signaling pathways is avoided scaffold proteins G-protein-linked cell-surface signaling G-protein-linked receptors consist of a single polypeptide chain (sometimes called serpentine receptors) G-protein-linked cell-surface signaling Upon binding of a signal molecule, the receptor undergoes a conformational change that enables it to activate trimeric GTP-binding proteins (G- proteins) G-protein-linked cell-surface signaling G-protein-linked cell-surface signaling G-protein-linked cell-surface signaling e.g. adenyl cyclase (makes cyclic AMP, which in turn activates CyclicAMP- dependent Protein Kinase, thus initiating a signaling cascade) G-protein-linked cell-surface signaling
