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Cell-to-Cell Communication
• Essential for multicellular organisms
• The combined effects of multiple signals determine cell response
1
Cell signaling
• Signal transduction pathway – series of steps – signal from outside the cell – specific cellular response
2
• Pathway similarities
– Ancestral signaling molecules evolved in prokaryotes
– Modified in eukaryotes
Cell signaling
3
Fig. 11-3
Individual rod- shaped cells
Spore-forming structure (fruiting body)
Aggregation in process
Fruiting bodies
0.5 mm
1
3
2
Signals in bacteria: • Quorum sensing (regulation of gene expression) • Ex. Biofilm formation
Source: Campbell et al., 2008
4
This species’ ability to "talk" has led to increased incidence of food poisoning in the developed world.
Signal: acyl-homoserine lactone
Assess group density and signal aggregation
Image source: http://www.magma.ca/~pavel/science/Foodbugs.htm
Cell signaling in Salmonella enteritidis
5
• Respond to stimulus
• Secrete ligands (signaling molecules)
– Ex. hormones and neurotransmitters
Sensory cells
6
Local and long distance signaling
Synaptic signals
Direct contact
Source: Solomon, Berg, and Martin, 2011, Biology 9th edition, Brooks/Cole, Cengage Learning, Belmont, CA. 7
Local and long distance signaling
Endocrine
Neuroendocrine
Autocrine
Paracrine Ex. Growth factors Clotting factor
Source: Solomon, Berg, and Martin, 2011, Biology 9th edition, Brooks/Cole, Cengage Learning, Belmont, CA. 8
• Local regulators: messenger molecules that travel only short distances
• Hormones: chemicals used in long-distance signaling
– Plant growth regulators = hormones
• Ex. Ethylene (C2H4), important in fruit ripening
– Synthesis: enzymatic reaction, needs O2, inhibited by CO2 • How do we manipulate this system to our advantage?
Local and long distance signaling
9
• Hormones: chemicals used in long-distance signaling
– Plant growth regulators = hormones, different than animal hormones
• Ex. Green leaf volatiles
– Seem to distinguish among herbivore species
• How do the plants know?
– Attract predators of the herbivores
– Signal nearby plants
Local and long distance signaling
10
The Three Stages of Cell Signaling: A Preview
• Earl W. Sutherland discovered how the hormone epinephrine acts on cells
• Sutherland suggested that cells receiving signals went through three processes: – Reception – Transduction – Response
11
Three Stages of Signal Transduc3on
• Recep%on, transduc%on, and response are common to all signaling systems – although they vary greatly in detail
Outside cell
Signal
Receptor
Cytoplasmic end of
receptor Ac3va3on
Molecules that transfer signal down the pathway
Molecule that brings about
response
Change in cell
Cytoplasm
2
1
3
Figure 9-‐3
Recep3on
Transduc3on
Response
12
Reception: A signal molecule binds to a receptor protein, causing it to change shape
• Specific binding between a signal molecule (ligand) and receptor
• A shape change in a receptor
• Most receptors are plasma membrane proteins
13
Ac3va3on of a Surface Receptor
Outside cell Extracellular signal molecule
Extracellular segment of
receptor
Signal-‐binding site
Transmembrane segment Plasma membrane
Cytoplasmic segment
Site triggering cellular response, in inac3ve state
Inac3ve receptor
Cytoplasm
Figure 9-‐4
Recep3on
Cytoplasmic site is ac3vated
and triggers cellular response
Ac3ve receptor
14
Intracellular Receptors
• In the cytosol or nucleus of target cells
• Small or hydrophobic chemical messengers, can cross the plasma membrane
• Examples:
– steroid hormones (cytoplasmic receptor)
– thyroid hormone (nuclear receptor)
– nitric oxide (NO gas, cytoplasmic receptor)
– ethylene (C2H4, ER membrane)
15
Pathway of Gene Activation by Steroid Hormone Receptors
Figure 9-‐15
Outside cell
Steroid hormone
Cytoplasm
Recep3on Hormone-‐
binding domain Steroid
hormone receptor
Domain for ac3va3ng target genes
Transduc3on DNA-‐binding
domain (ac3ve)
DNA-‐binding site
Response Gene
ac3va3on DNA
Control region of gene
Gene Nucleus
Steroid hormones can pass through lipid bilayer.
Hormone binds to its receptor within cytoplasm.
Hormone-receptor complex can enter nucleus and influence gene expression.
Note intracellular receptor
16
Receptors in the Plasma Membrane
• Three main types of membrane receptors: – Ion channel receptors
– Receptor tyrosine kinases – G protein-coupled receptors
• G protein: guanine nucleotide bonding protein
• http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2012/press.html (find the mistake!)
17
• A ligand-gated ion channel receptor acts as a gate when the receptor changes shape
• When a signal molecule binds as a ligand to the receptor, the gate allows specific ions, such as Na+ or Ca2+, through a channel in the receptor – Ex. neurons
Ligand-gated ion channels
18
Ligand-gated ion channels
Source: Sadava et al., 2011, Life: The science of biology, 9th edition, Sinauer Associates, Inc.
19
• Receptor tyrosine kinases are membrane receptors that attach phosphates to tyrosines
• A receptor tyrosine kinase can trigger multiple signal transduction pathways at once – 90 genes known, 58 proteins
– Abnormal function: some kinds of cancer
Receptor tyrosine kinases
20
Signal molecules bind and two receptors assemble into a
dimer
Outside cell Signal molecules
Signal-‐binding site
RTK (receptor tyrosine kinase)
Protein kinase regions (inac3ve)
Cytoplasm
Plasma membrane
Dimer
Transduc3on and cellular responses
Recep3
on
Signaling protein 1
Signaling protein 2
Transduc3on pathway 1
Transduc3on pathway 2
Tran
sduc3on
Cellular response 1
Cellular response 2 Re
spon
se
Ac3va3on of protein kinases and autophosphoryla3on of the
receptor
Ac3vated protein kinase regions (tyrosines
unphosphorylated in dimer)
Dimer
Receptor autophosphoryla3on
Receptor tyrosine kinase fully ac3ve
(tyrosines phosphorylated in
dimer)
Figure 9-‐7 21
There are other receptor kinases
22
Source: Taiz and Zeiger, 2010, Plant Physiology, 5th edition, Sinauer Associates, Inc.
• A G protein-coupled receptor is a plasma membrane receptor that works with the help of a G protein
• The G protein acts as an on/off switch: If GDP is bound to the G protein, the G protein is inactive
G protein-coupled receptors
23
G-‐Protein–Coupled Receptors Outside cell
Segment binding signal
molecules
Plasma
membrane
Segment binding
G protein
Cytoplasm Figure 9-‐8 24
Figure 9-‐9
Outside cell First messenger signal
G protein–coupled receptor
Recep3
on
Inac3ve G protein
Cytoplasm
Tran
sduc3on
Second messenger
Protein kinase
Respon
se
Cellular response
Response Pathways Ac;vated by G-‐Protein-‐Coupled Receptors
25
G Protein-Coupled Receptors
Source: Raven et al., 2011, Biology, 9th edition, The McGraw-Hill Companies, Inc. 26
Signal transduction cascades
• Signal transduction: multiple steps
• Amplify a signal: A few molecules can produce a large cellular response
• Coordination and regulation of the cellular response
27
Signal transduction cascades
• Signal from receptor to response: relay proteins
• At each step, the signal is transduced into a different form, usually a shape change in a protein
28
Protein phosphorylation and dephosphorylation
• Protein kinases transfer phosphates from ATP to protein = phosphorylation
– >500 different kinases known in humans; control cell growth, movement, and death
– Deregulated kinase activity: certain cancers
– Drugs that inhibit specific kinases?
• Gleevec (certain leukemia and GI tumors) and Iressa (certain lung and breast cancers)
29
Phosphoryla;on Cascade
Figure 9-‐5
Outside cell
Recep3on Ac3ve protein kinase 1
Inac3ve protein kinase 2
Ac3ve protein kinase 2
Target protein
Cellular response
Transduc3on by phosphoryla3on
cascade
Ac3va3on or inac3va3on of
target molecule by phosphoryla3on
Response
Cytoplasm 30
Protein phosphorylation and dephosphorylation
• Protein phosphatases remove the phosphates from proteins = dephosphorylation
– Ex. Involved in brain function and synaptic function
– Ex. Dysregulation linked to cognitive ageing and neurodegeneration
• Molecular switch: phosphorylation/dephosphorylation
31
32
Protein phosphorylation and dephosphorylation
Activated BSU1 dephosphorylates BIN2
Dephosphorylated BIN2 is broken down by the proteasome
Phosphorylated BIN2 is active Active BIN2 phosphorylates BES1/BZR1
Phosphorylated BES1/BZR1 cannot bind DNA, are taken out of the nucleus, and are broken down
Source: Taiz and Zeiger, 2010, Plant Physiology, 5th edition, Sinauer Associates, Inc.
Small molecules and ions as second messengers
• The extracellular signal is “first messenger”
• Second messengers are small, nonprotein, usually water-soluble molecules or ions: readily spread
– Usually short lifetime
– Do not catalyze reactions; bind to and modify enzymes
– Common examples: cAMP and Ca2+
– Pathways initiated by G protein-coupled receptors and receptor tyrosine kinases
33
Adenylyl cyclase
Phospho-‐ diesterase
Pyrophosphate
ATP cAMP (Second messenger)
AMP
Figure 9-‐11 34
cAMP synthesis and break down
• Many signal molecules trigger formation of cAMP
• cAMP usually activates protein kinase A, which phosphorylates various other proteins
• Further regulation: inhibit adenylyl cyclase
• Examples:
– Breaking down glycogen
– Increasing heart rate
cAMP as a second messenger
35
Outside cell
Recep3
on
Ac3ve adenylyl cyclase (effector)
Ac3vated G protein subunit Second
messenger
Tran
sduc3on
Re
spon
se
Cytoplasm
2
1
3
4
Figure 9-‐10
Cellular response
cAMP Receptor-‐Response Pathways
36
Nuclear and cytoplasmic responses
• Ultimate response: regulation of some cellular activity
• Response in cytoplasm or nucleus
• Transcription factors that turn genes on or off in the nucleus: regulate synthesis
• Or regulate the activity of enzymes
37
Calcium ions as secondary messengers
• Calcium ions (Ca2+) act as a second messenger in many pathways
• Calcium is an important second messenger because cells can regulate its concentration
38
EXTRACELLULAR FLUID
Source: Campbell et al., 2008
ATP
Nucleus
Mitochondrion
Ca2+ pump
Plasma membrane
CYTOSOL
Ca2+ pump
Endoplasmic reticulum (ER)
Ca2+ pumpATP
Key
High [Ca2+]Low [Ca2+]
39
• A signal relayed by a signal transduction pathway may trigger an increase in calcium in the cytosol
• Pathways leading to the release of calcium involve inositol triphosphate (IP3) and diacylglycerol (DAG) as additional second messengers
• Ex. root gravitropism
http://plantsinmotion.bio.indiana.edu/plantmotion/movements/tropism/tropisms.html
Calcium ions as secondary messengers
40
Role of secondary messengers
Source: Sadava et al., 2011, Life: The science of biology, 9th edition, Sinauer Associates, Inc.
41
Odorant receptors in sperm
• Activation of hOR17-4 and mOR23 in human and mouse sperm, respectively, mediates distinct flagellar motion patterns and chemotactic behavior in various bioassays
• Activated by small aldehyde molecules
• Mediate calcium signals in sperm
• Adenylyl cyclase and G proteins in sperm
• Downstream steps?
42
Marc Spehr, Katlen Schwane, Jeffrey A. Riffell, Richard K. Zimmer, Hanns Hatt (2006) Odorant receptors and olfactory-like signaling mechanisms in mammalian sperm. Molecular and Cellular Endocrinology 250 128–136
Fig. 2. Proposed model of a bifunctional human odorant receptor. Either expressed in ciliary membranes of nasal OSNs or on the midpiece of mature sperm, hOR17-4 (OR1D2) is activated by the synthetic floral odorant bourgeonal and competitively inhibited by undecanal. In both systems, receptor activation triggers a cAMP-dependent signaling cascade. In sperm, however, the identity of the G protein and mAC isoform involved as well as the nature of downstream signaling components remains elusive. (A–C) Bourgeonal-induced signals in human olfactory epithelium and sperm. (A) Electro-olfactogram recordings from the olfactory mucosa show considerable inhibition of bourgeonal-induced field potentials after brief undecanal exposure. (B and C) Ca2+ signals in individual sperm are blocked by coapplication of both undecanal (B) and SQ22536 (C). 43
Fine-Tuning of the Response
• Multistep pathways have two important benefits: – Amplifying the signal (and thus the response)
– Contributing to the specificity of the response
44
Note amplification in a signal transduction cascade
Source: Sadava et al., 2011, Life: The science of biology, 9th edition, Sinauer Associates, Inc.
45
Termination of the signal
• Inactivation mechanisms are an essential aspect of cell signaling
• When signal molecules leave the receptor, the receptor reverts to its inactive state
– Remove receptors and signal molecules through endocytosis; receptor might be recycled
– Signal is removed/destroyed
– Secondary messengers have to be removed
– Relay molecules have to return to inactive forms too
46
Cross-‐Talk
Outside cell
Ac3vated G protein subunit
Ac3vated G protein subunit
Ac3va3on Ac3va3on
Protein kinases
Cross-‐talk leading to ac3va3on or inhibi3on of
another signal transduc3on pathway
Protein kinases
Cellular response
Cytoplasm
Cellular response
Effector Effector DAG
IP3
Cellular response of the cAMP pathway is modified if
the signal molecules for both pathways bind to the receptors at the same 3me.
Figure 9-‐16 47
The G protein Ras
• Some pathways important in gene regulation link receptor tyrosine kinases to a specific G protein called Ras
48
Receptor Tyrosine Kinases and Gene Regula3on
Outside cell
Plasma membrane
Adapter proteins
Target protein Nucleus Nucleus
Ac3ve
Cytoplasm
Recep3
on
Tran
sduc3on
Re
spon
se
Figure 9-‐13
DNA
Cellular response
Inac3ve Ras Ac3ve Ras
GDP Raf
Mek
ERK
49
The G protein Ras
• Receptor tyrosine kinase activated
• Adapter proteins bind to phosphorylated receptor and bridge to Ras, which is activated by binding GTP
• Activated Ras initiates a phosphorylation cascade in a series of three MAP (mitogen-activated protein) kinases
• The last MAP kinase (ERK) enters the nucleus and phosphorylates proteins that change the expression of certain genes, particularly those involved in cell division
• What if Ras remains active?!
50
Influenza A viruses
Experiment 1: Analyze sample of extract for ERK ac3vity: ERK ac3vity was detected, indica3ng that MAP kinase cascade had been ac3vated.
Cells in culture medium; no U0126
Cell extract of infected cells
What about the influenza virus?
Source: Russell et al., 2014. Biology: The Dynamic Science, 3rd edition, Cengage Learning, Brooks/Cole, Belmont, CA. 51
Influenza A viruses
Analyze sample of extract for ERK ac3vity: Very lible ERK ac3vity was detected, indica3ng that U0126 could block ac3va3on of the MAP kinase cascade by the virus.
Cells growing in culture medium in the presence of
inhibitor U0126
Cell extract of infected cells
What about the influenza virus?
Source: Russell et al., 2014. Biology: The Dynamic Science, 3rd edition, Cengage Learning, Brooks/Cole, Belmont, CA. 52
Source: Pleschka et al. (2001).
Dark stain: ERK activity
ERK2: showing equal presence
No detectable ERK activity
ERK2: showing equal presence
What about the influenza virus?
MOI = multiplicity of infection, ratio of agents to targets. 53
Source: Pleschka et al. (2001). Cells were infected with virus.
Treated with 50 µM U0126 (inhibitor dissolved in DMSO) or DMSO (control) for the indicated times.
After 9 hours samples were analyzed for infectious virions.
Numbers are expressed as percentage of the control.
What about the influenza virus?
54
U0126 is an inhibitor of the central kinase MEK. They measured virus titres in the lungs of infected mice after local aerosolic administration into the trachea. Data are normalized against the control (solvent).
Source: Droebner et al. (2011)
What about the influenza virus?
H1N1
H5N1 (MB1)
H5N1 (GSB)
H7N7
55