1. cell communication

2.  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 course layout

3. introduction

4. cell communication

5. 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.

6. 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.  Letters represent chemicals or proteins. Arrows represent enzymatic steps. ABCDEFGABCDEFG

7. 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.

8. 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.

9. 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).

10. 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.

11. steroid hormones

12. seven levels of regulation of cell growth

13. pathways are inter-linked Signalling pathway Genetic network Metabolic pathway STIMULUS

14. metabolic pathways 1993 Boehringer Mannheim GmbH - Biochemica

15. 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

16. gap junctions and CAMs Figure 6-1a, b: Direct and local cell-to-cell communication  Protein channels - connexin  Direct flow to neighbor  Electrical- ions (charge)  Signal chemicals  CAMs (cell-adhesion molecules)  Need direct surface contact  Signal chemical

17. gap junctions and CAMs Figure 6-1a, b: Direct and local cell-to-cell communication

18. paracrines and autocrines Figure 6-1c: Direct and local cell-to-cell communication  Local communication  Signal chemicals diffuse to target  Example: Cytokines  Autocrine – receptor on same cell  Paracrine – neighboring cells

19. hormones Figure 6-2a: Long distance cell-to-cell communication  Signal Chemicals  Made in endocrine cells  Transported via blood  Receptors on target cells long distance communication

20. 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 Figure 6-2 b: Long distance cell-to-cell communication long distance communication

21. Figure 6-2b, c: Long distance cell-to-cell communication long distance communication neurons and neurohormones

22. Figure 6-2b, c: Long distance cell-to-cell communication long distance communication neurons and neurohormones

23. signal pathways  Signal molecule (ligand)  Receptor  Intracellular signal  Target protein  Response Figure 6-3: Signal pathways

24. 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

25. membrane receptor classes  Ligand- gated channel  Receptor enzymes  G-protein-coupled  Integrin

26. membrane receptor classes

27. signal transduction  Transforms signal energy  Protein kinase  Second messenger  Activate proteins  Phosporylation  Bind calcium  Cell response

28. signal amplification  Small signal produces large cell response  Amplification enzyme  Cascade

29. receptor enzymes Figure 6-10: Tyrosine kinase, an example of a receptor-enzyme  Transduction  Activation cytoplasmic  Side enzyme  Example: Tyrosine kinase

30. G-protein-coupled receptors  Hundreds of types  Main signal transducers  Activate enzymes  Open ion channels  Amplify:  adenyl cyclase-cAMP  Activates synthesis

31. G-protein-coupled receptors

32. transduction reviewed

33. novel signal molecules  Calcium: muscle contraction  Channel opening  Enzyme activation  Vesicle excytosisNitric Oxide (NO)  Paracrine: arterioles  Activates cAMP  Brain neurotransmitter  Carbon monoxide (CO)

34. novel signal molecules Calcium as an intracellular messenger

35. quorum sensing

36.  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

37. 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

38.  Vibrio fischeri is a specific bacterial symbiont with the squid Euprymna scolopes and grows in its light organ quorum sensing

39.  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

40. In V. fisheri, bioluminsecence only occurs when V. fischeri is at high cell density quorum sensing

41. N -3-oxo-hexanoyl-L-homoserine lactone quorum sensing

42.  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

43. Gram-negative bacteria Gram-positive bacteria universal language  Vast array of molecules are used as chemical signals – enabling bacteria to talk to each other, and in many cases, to be multilingual quorum sensing

44. quorum sensing in Pseudomonas aeruginosa  P. aeruginosa uses a hierarchical quorum sensing circuit to regulate expression of virulence factors and biofilm formation

45. 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

46. 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

47. 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

48. X = transcriptional repressor 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 two- component proteins similar to Gram-positives  V. harveyi also responds to AI-2 that is designed for interspecies communication

49. AI-1AI-2 LuxN and LuxQ – autophosphorylating kinases at low cell densities Accumulation of autoinducers – LuxN and LuxQ  phosphatases draining phosphate from LuxO via LuxU Dephosphorylated LuxO is inactive  repressor X not transcribed X = transcriptional repressor hybrid quorum sensing circuit in Vibrio harveyi

50. LuxS and interspecies communication  LuxS homologs found in both Gram-negative and Gram- positive 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 AI- 2 producing bacteria to date – reinforces the proposal of AI-2 as a “ universal ” language

51. signal processing circuits

52. Receiver cells pLuxI-Tet-8pRCV-3 aTc luxI  VAI VAI LuxR GFP tetR aTc 0 0 Sender cells cell-cell communication circuits

53. VAI Receiver cellsSender cells tetR P(tet) luxI P(Ltet-O1) aTc GFP(LVA) Lux P(R) luxR Lux P(L) + cell-cell communication circuits

54. C(4)HSL qsc box C(6)HSL lux box Cell Color 00none 01Green (GFP) 10Red (HcRED) 11Cyan (CFP) 2:4 multiplexer

55. 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

56. qscluxA 000 01green 100 110 qscluxB 000 010 10red 110 qscluxC 000 010 100 11cyan qscluxD 000 01green 10red 11cyan ++ = mux: the sum of three circuits

57. lux box qsc box GFP luxR RhlR C4HSL C6HSL qscLuxA 000 01green 100 110 case A

58. lux box qsc box HcRED luxR RhlR C4HSL C6HSL qscluxB 000 010 10red 110 case B

59. λ P(R) CFP cI lux box qsc box qscluxC 000 010 100 11cyan case C, AND gate

60. lux box qsc box HcRED luxRRhlR C4HSLC6HSL qscluxAxorB 000 01green 10red 110 GFP case A and B

61.  qsc binding site  plasmid copy number  production of C(x)HSL design considerations

62. phenotype tests  triple plasmid, regulatory  double plasmid, antisensing  double plasmid, antisensing + regulatory  chromosome, antisensing + regulatory

63. pASK-102: Single “Parent” Offspring QSC box case A

64. Plasmid 1 case A

65. Parents: pASK-102-qsc117 (vector), pECP61.5 (insert) Plasmid 2 case A

66. OO O O N H O OO O O N H OO O O N H O O O O N H O O O O N H OO O O N H detecting chemical gradients analyte source detection analyte source reporter rings O O O N H OO O N H OO O N H OO O N H OO O O N H OO O N H signal

67. Components 1.Acyl-HSL detect 2.Low threshold 3.High threshold 4.Negating combiner circuit components

68. Y  high threshold X  low threshold acyl-hSL detection detecting chemical gradients

69. low threshold detection detecting chemical gradients

70. high threshold detection detecting chemical gradients

71. protein Z determines range detecting chemical gradients

72. negating combiner detecting chemical gradients

73. HSL-width HSL-mid 0.3 engineering circuit characteristics  HSL-mid: the midpoint where GFP has the highest concentration  HSL-width: the range where GFP is above 0.3uM

74. other signals

75. relay signals  Signals received at the cell surface either by G-protein- linked 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)

76.  The small intracellular mediators are called second messengers (the first messenger being the extracellular signal)  e.g. Ca 2+ and cyclic AMP, which are water-soluble and diffuse into the cytosol  The large intracellular mediators are intracellular signaling proteins  They relay the signal by either activating the next signaling protein in the chain or generating small intracellular mediators relay signals

77. 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 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 Bifurcation proteins: spread the signal from one signaling pathway to another Different kinds of intracellular signaling proteins along a signaling pathway from the cell surface to the nucleus

78. Integrator proteins: receive signals from 2 or more pathways and integrate them before relaying a signal onwards Latent gene regulatory proteins: activated at the cell surface by activated receptors & migrate to the nucleus to stimulate gene expression Modulator proteins: modify the activity of intracellular signaling proteins & regulate the strength of signaling along the pathway Anchoring proteins: maintain specific signaling proteins at a specific location by tethering them to a membrane Different kinds of intracellular signaling proteins along a signaling pathway from the cell surface to the nucleus

79. Scaffold proteins: adaptor &/or anchoring proteins that bind multiple signaling proteins together in a functional complex Different kinds of intracellular signaling proteins along a signaling pathway from the cell surface to the nucleus

80. intracellular signaling proteins as molecular switches  Many intracellular signaling proteins behave like molecular switches  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 1. Phosphorylation switches 2. GTP-binding protein switches  In both cases, it is the gain or loss of phosphate that determines whether the switch is active or inactive

81. 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) intracellular signaling proteins as molecular switches

82. 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

83. 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

84. signal processing  Complex cell behaviors, like cell survival and cell proliferation, are stimulated by specific combinations of signals, rather than one signal acting alone

85. signal processing

86.  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 signal processing

87. integrator proteins

88. 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)

89. integrator protein integrator proteins

90. 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

91. Also known as a ‘coincidence detector’ integrator proteins

92. 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

93. scaffold proteins

94. G-protein-linked cell-surface signaling G-protein-linked receptors consist of a single polypeptide chain (sometimes called serpentine receptors)

95.  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

98. e.g. adenyl cyclase (makes cyclic AMP, which in turn activates Cyclic- AMP- dependent Protein Kinase, thus initiating a signaling cascade) G-protein-linked cell-surface signaling