cell communication

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

introduction

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.

Letters represent chemicals or proteins. Arrows represent enzymatic steps.

ABCDEFG

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 se

creted 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

Geneticnetwork

Metabolic pathway

STIMULUS

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

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

gap junctions and CAMs

Figure 6-1a, b: Direct and local cell-to-cell communication

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

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

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

Figure 6-2b, c: Long distance cell-to-cell communication

long distance communication

neurons and neurohormones

Figure 6-2b, c: Long distance cell-to-cell communication

long distance communication

neurons and neurohormones

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

Figure 6-10: Tyrosine kinase, an example of a receptor-enzyme

Transduction Activation cytoplasmic

Side enzyme Example: Tyrosine kinase

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

Vibrio fischeri is a specific bacterial symbiont with the squid Euprymna scolopes and grows in its light organ

quorum sensing

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

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

quorum sensing

N-3-oxo-hexanoyl-L-homoserinelactone

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

Gram-negativebacteria

Gram-positivebacteria

universallanguage

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

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 kinasebinding 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

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

AI-1 AI-2LuxN and LuxQ – autophosphorylating kinases at low cell densities

Accumulation of autoinducers – LuxN and LuxQ phosphatasesdraining phosphate from LuxO via LuxUDephosphorylated LuxO is inactive repressor X not transcribedX = transcriptional repressor

hybrid quorum sensing circuit in Vibrio harveyi

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

signal processing circuits

Receiver cells

pLuxI-Tet-8 pRCV-3

aTc

luxI VAI

VAI

LuxRGFP

tetR

aTc

00

Sender cells

cell-cell communication circuits

VAI VAI

Receiver cellsSender cells

tetRP(tet)

luxIP(Ltet-O1)

aTc

GFP(LVA)Lux P(R)luxR Lux P(L)

+

cell-cell communication circuits

C(4)HSLqsc box

C(6)HSLlux box

Cell Color

0 0 none

0 1 Green (GFP)

1 0 Red(HcRED)

1 1 Cyan(CFP)

2:4 multiplexer

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

qsc lux A

0 0 0

0 1 green

1 0 0

1 1 0

qsc lux B

0 0 0

0 1 0

1 0 red

1 1 0

qsc lux C

0 0 0

0 1 0

1 0 0

1 1 cyan

qsc lux D

0 0 0

0 1 green

1 0 red

1 1 cyan

+ +

=

mux: the sum of three circuits

luxbox

qscbox

GFP

luxR

RhlR

C4HSL

C6HSL

qsc Lux A

0 0 0

0 1 green

1 0 0

1 1 0

case A

luxbox

qscbox

HcRED

luxR

RhlR

C4HSL

C6HSL

qsc lux B

0 0 0

0 1 0

1 0 red

1 1 0

case B

λP(R)CFP

cI

cI

luxbox

qscbox

qsc lux C

0 0 0

0 1 0

1 0 0

1 1 cyan

case C, AND gate

luxbox

qscbox

HcRED

luxR RhlR

C4HSLC6HSL

qsc lux AxorB

0 0 0

0 1 green

1 0 red

1 1 0

GFP

case A and B

qsc binding site plasmid copy number production of C(x)HSL

design considerations

phenotype tests

triple plasmid, regulatory double plasmid, antisensing double plasmid, antisensing + regulatory chromosome, antisensing + regulatory

pRCV-34149 bp

AP r

GFP(LVA)

LuxR

CAP bs

CAP/cAMP Binding Site

P(BLA)

P(LAC)

lux P(L)

lux P(R)

lux box

RBSII

LuxR RBS

ColE1 ORI

Inverted Repeat

rrnB T1

rrnB T1

-10 region

-10 region

LuxR -10

LuxICDABEG -10 region

-35 region

LuxR -35

pASK-102: Single “Parent” Offspring

QSC box

case A

pASK-102-qsc1174159 bp

AP r

GFP(LVA)

LuxR

CAP bs

CAP/cAMP Binding Site

P(BLA)

P(LAC)

lux P(L)

lux P(R)

lux box

qsc117 lux box for C4HSL

LuxR RBS

RBSII

ColE1 ORI

Inverted Repeat

rrnB T1

rrnB T1

-10 region

-10 region

LuxR -10

LuxICDABEG -10 region

-35 region

LuxR -35

Plasmid 1

case A

pASK-103-RhlR-qsc1174848 bp

AP r

GFP(LVA)

LuxR

RhlR Ver 2 (8 Mismatch)

CAP bs

CAP/cAMP Binding Site

P(LAC)

lux P(L)

lux P(R)

lux box

qsc117 lux box for C4HSL

LuxR RBS

RBSII

ColE1 ORI

Inverted Repeat

rrnB T1

-10 region

-10 region

LuxR -10

LuxICDABEG -10 region

-35 region LuxR -35

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

case A

OO OONH

O

OO OONH

OO OONH

OO OONH

OO OONH

OO OONH

detecting chemical gradients

analyte source detection

analytesource

reporter rings

OOONH

OOONH

OOONH

OOONH

OO OONH

OOONH

signal

Components1. Acyl-HSL detect2. Low threshold3. High threshold4. Negating combiner

LuxRO O

O

ONHLuxR

O OO

ONH O O

O

ONHO O

O

ONH

O OO

ONH

P(lux) X Y

Z2P(W)

GFPP(Z)

Z1P(X)

WP(Y)

O OO

ONH

O OO

ONH

O OO

ONH

luxRP(R)

circuit components

Y high threshold

X low threshold

acyl-hSL detection

LuxRO O

O

ONHLuxR

O OO

ONH O O

O

ONHO O

O

ONH

O OO

ONH

P(lux) X Y

Z2P(W)

GFPP(Z)

Z1P(X)

WP(Y)

O OO

ONH

O OO

ONH

O OO

ONH

luxRP(R)

detecting chemical gradients

low threshold detection

LuxRO O

O

ONHLuxR

O OO

ONH O O

O

ONHO O

O

ONH

O OO

ONH

P(lux) X Y

Z2P(W)

GFPP(Z)

Z1P(X)

WP(Y)

O OO

ONH

O OO

ONH

O OO

ONH

luxRP(R)

detecting chemical gradients

high threshold detection

LuxRO O

O

ONHLuxR

O OO

ONH O O

O

ONHO O

O

ONH

O OO

ONH

P(lux) X Y

Z2P(W)

GFPP(Z)

Z1P(X)

WP(Y)

O OO

ONH

O OO

ONH

O OO

ONH

luxRP(R)

detecting chemical gradients

protein Z determines range

LuxRO O

O

ONHLuxR

O OO

ONH O O

O

ONHO O

O

ONH

O OO

ONH

P(lux) X Y

Z2P(W)

GFPP(Z)

Z1P(X)

WP(Y)

O OO

ONH

O OO

ONH

O OO

ONH

luxRP(R)

detecting chemical gradients

negating combiner

LuxRO O

O

ONHLuxR

O OO

ONH O O

O

ONHO O

O

ONH

O OO

ONH

P(lux) X Y

Z2P(W)

GFPP(Z)

Z1P(X)

WP(Y)

O OO

ONH

O OO

ONH

O OO

ONH

luxRP(R)

detecting chemical gradients

HSL-width

HSL-mid0.3

engineering circuit characteristics

HSL-mid: the midpoint where GFP has the highest concentration

HSL-width: the range where GFP is above 0.3uM

other signals

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)

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 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

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

Integrator proteins: receive signals from 2 or more pathways and integrate thembefore 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

Scaffold proteins: adaptor &/or anchoring proteins that bind multiple signaling proteins together in afunctional complex

Different kinds of intracellular signaling proteins along a signaling pathway from the cell surface to the nucleus

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 switches1. Phosphorylation switches2. GTP-binding protein switches

In both cases, it is the gain or loss of phosphate that determines whether the switch is active or inactive

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

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, phosphoryla

tes 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 kinasesThey 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

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

integrator proteins

integrator proteins

Example of how they work: External signals A and B both activate a different series of prot

ein phosphorylations Each leads to the phosphorylation of protein Y, but at different

sites on the protein (Fig. 15-18)

integrator protein

integrator proteins

Example of how they work: Protein Y is activated only when both of these sites are activat

ed, 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’

integrator proteins

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)

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 Cyclic-AMP- dependent Protein Kinase, thus initiating a signaling cascade)

G-protein-linked cell-surface signaling

G-protein-linked cell-surface signaling