essential for multicellular organisms the combined effects of … · 2018. 4. 11. · • small or...

Cell-to-Cell Communication

•  Essential for multicellular organisms

•  The combined effects of multiple signals determine cell response

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Cell signaling

•  Signal transduction pathway –  series of steps –  signal from outside the cell –  specific cellular response

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•  Pathway similarities

–  Ancestral signaling molecules evolved in prokaryotes

–  Modified in eukaryotes

Cell signaling

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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

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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

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•  Respond to stimulus

•  Secrete ligands (signaling molecules)

–  Ex. hormones and neurotransmitters

Sensory cells

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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


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?


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•  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


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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

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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

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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

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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

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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)

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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

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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!)

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•  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

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Ligand-gated ion channels

Source: Sadava et al., 2011, Life: The science of biology, 9th edition, Sinauer Associates, Inc.

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•  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

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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

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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

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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

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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

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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)

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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 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

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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

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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

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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

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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

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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

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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+]

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•  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

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Role of secondary messengers


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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?

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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

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Note amplification in a signal transduction cascade


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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

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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

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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

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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?!

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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


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


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.


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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)


H1N1

H5N1 (MB1)

H5N1 (GSB)

H7N7

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essential for multicellular organisms the combined effects of … · 2018. 4. 11. · • small or...

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