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9. Cell Communication

Cells talk to each other. Lets look at two examples beforeconsidering how this works

1. Chemotaxis individual Dictyostelium amoebae movingtowards a chemoattractant chemical

2. Quorum sensing individual bacteria that communicatesignals that can regulate light production or the ability totake up DNA molecules.

How does the lymphocyte find thebacterium and how does this makethe lymphocyte move?

Signals received by the frontend of the cell (these are thesoil ameba Dictyostelium)induce actin filaments (white)to assemble and push the cellforward.

Here bacteria are dark until they sense that there are enough bacteria in theflask to form a quorum sufficient to trigger them to luminesce.

Jessica Marshall PNAS 2013;110:2690

2013 by National Academy of Sciences

To learn more about this, watch the video by Bonnie Bassler, who is making importantdiscoveries about quorum sensing https://www.youtube.com/watch?v=S3BOcUrJ5Vg or read the short article by Jessica Marshall that Ill post on line.


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9. Cell Communication

How do individual cells find each otherand how do they known when to formanother structure?

How does a mammalian sperm find andfertilize an egg and why doesnt it try tofertilize every other cell along a femaleoviduct?

How do cells in our body communicatewith one another during embryonicdevelopment?

How do cells in an adult know how torespond to changes in a body?

How do cells in our body know when to

divide and when they should not divide?

First, consider how pneumonococcus bacteria(Streptococcus pneumoniae) can take up DNA that helpsthem express genes to make them drug-resistant.


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a. Low cell density

At low population density,

the concentration of thesignaling peptide is toolow to stimulate DNAuptake by bacteria.

Receptor

Bacterialcell

Signalingmolecule

b. High cell density

At high population density,the concentration of thesignaling peptide is highenough to stimulate theDNA-uptake response.

DNAmolecule

The signaling cellreleases signalingmolecules.

Signalingmolecule

Receptor

The responding cell hasreceptor proteins thatbind to the signalingmolecule.

Basic cell signaling requires a (1) signaling molecule (ligand), (2) a receptor,and (3) molecules inside the cell that transduce the signal and make the cellrespond to the signal.


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The synthesis of cell-surface hormone receptors is regulated by the expression of genes that code for these receptors.Assume that a receptor gene is expressed and is being translated by a ribosome. Describe the pathway by which thatreceptor protein is eventually displayed on the surface of the plasma membrane in an orientation that will allow the

receptor to bind an extracellular hormone.

If all cells in a body are exposed to every hormone released into the bloodstream, why do some cells respond to aspecific hormone while other cells ignore the hormone?

All of the genes in a cell are simply parts of a sequence of nucleotides in a DNA molecule. Can cells incorporate DNAfrom other intact or broken cells? Explain your answer and provide examples that support your answer. Yourexamples may include examples from previous portions of this course.

Study questions 9.1

We are familiar with cell signaling that allows cells in our bodies to communicate with other cells. Do free-living singlecells, such as bacteria or amoeba, signal each other or did signaling evolve when multicellular organisms developed?Explain your answer.

The signal binds to areceptor, which isthen activated.

Receptor activation Signal transduction

Response Termination

Plasma membrane

Extracellularfluid

Cytoplasm

The signal istransmitted to theinterior of the cell bya signal transductionpathway.

The cellresponds, forexample, byactivating anenzyme orturning ontranscription ofa gene.

The responseis terminatedso that newsignals canbe received.

a. Endocrine signaling

Signal travels throughthe circulatory system.

Signalingmolecule

Receptor


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b. Paracrine signaling

Muscle cell

Nerve cell

Plasmamembrane Acetylcholine

OpenClosed Binding site

Na+

Acetylcholinebinding opension channels,allowing Na+toflow into themuscle cell.

c.Autocrine signaling


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Direct contact

signaling

What can signaling do?

Regulate cell growth ex PDGF (platelet derived growth factor)

Regulate metabolism ex sugar uptake induced by insulin

Regulate embryonic development

Communicate signals from nerve cells to target cells

Unclottedblood

Clottedblood

Plasma SerumFibroblast

Cultured with serum

Cultured with plasma


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Cultured with platelet proteins

Cultured with plasma

There are a number of different mechanisms by which cells release and receive signals. Give an example of each ofthe following or describe the mechanisms by which they work:

Study questions 9.2

endocrine signalingparacrine signalingdirect contact between animal cells

direct contact between plant cells

One difficulty of stimulating mammalian cells to grow in culture is that even if you provide them with the foodthey need, they do not produce a greater number of cells. Describe an experiment in which growth factorsthat stimulate cell growth were identified.

You have a population of cells that respond to growth factors and will reproduceat a reasonable pace, shown in this figure.

Now, suppose that you obtain a population of these cells that will not reproduce,even though you add the growth factors and plenty of food.

Assuming that the growth factor stimulates cells by binding to an extracellularreceptor, propose a reason why the non-reproducing cells are no longerresponsive to the growth factors.

a. Cell-surface receptorLigand-binding site

Activated receptor

Polar signalingmolecule

Transmembranedomain

Cytoplasmicdomain

Extracellulardomain

Plasmamembrane

Nucleus

DNA

Polar signalingmolecules cannotcross the plasma

membrane andrely on cell-surface receptors.


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b. Intracellularreceptor

Activated receptorsignal

complex

Small, nonpolarsignaling molecule

Small nonpolarsignaling moleculescan freely passthrough the plasmamembrane andactivate cytoplasmicreceptors.

Intracellular receptors

Here is the structure of testosterone.Why can it cross the plasmamembrane?

Cell signaling requires a signal molecule (ligand) and a receptor. Types of ligands that function as hormones inhumans include steroids and peptides.

a. What chemical properties distinguish steroid and peptide hormones?b. Where are the receptors located for steroid and for peptide hormones?

Most steroid hormones can diffuse across the plasma membrane.a. Why might one cell in your body be responsive to a steroid hormone while another cell is not responsive?b. Sketch a typical animal cell and describe the mechanism by which a steroid hormone can activate gene

expression. Draw the plasma membrane and any other organelles that are appropriate for your answer.

Examine the structure of testosterone shown in the lecture slides. Explain why this would be expected to cross amembrane rather than bind to a receptor on the external surface of a cell.

Compare the pathways by which extracellular receptor is synthesized and positioned on the plasma membrane withthe pathway required for synthesis of the st eroid hormone receptor.

Suppose the steroid receptor mRNA had a signal sequence to target the protein to the ER. Describe what you think

might happen to that receptor and explain your answer.

Study questions 9.3


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b. G protein-coupled receptor

Response

Signalingpathways

Inactive Active

GTPGDP

Ligand

Receptor G protein

c. Receptorkinase

Response

Phosphategroup

b. Ligand-gated ionchannel

Response

Ions


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Some more detailed mechanisms

Important point Well consider how a signaling molecule

(a ligand) can bind to and activate a receptor protein

but

its important to consider how a cell can stop beingactivated by a signal, so think of this as we go throughsome examples.

When the !subunit is boundto GDP, the three subunits are

joined together and the Gprotein is inactive.

When the G protein is boundby an activated receptor, theGDP bound to the !subunit isreplaced by GTP, whichactivates the subunit to bind to

target proteins.

Activated!subunit binds toand activates target protein.

Secondmessengers

Response

! " #

! " #

!"#

GDP

GDP

GTP

GTP

Activeeffector

LigandReceptor

Inactiveeffector

G protein

Adrenaline

Receptor

! " #

GDP

Inactiveadenylylcyclase

Adrenalinereceptor

Activatedadenylylcyclase

!" #

GTP

Active

G protein

ATP

cAMP

ActiveInactive

Proteinkinase A

Activated protein kinase Aphosphorylates proteins in theheart muscle, causing heartrate to increase.


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AdrenalineActivatedreceptor

Amplification

Amplification

Amplification

Activatedadenylyl

cyclase

Each activated receptor activates multiple G proteinswhich in turn activate adenylyl cyclase enzymes.

Active!subunitof G protein

!

GTP

!

GTP

!

GTPATP

cAMP

ATPcAMP

ATPcAMP

Each adenylyl cyclase enzymeproduces large amounts of the secondmessenger cAMP, which activatesmany molecules of protein kinase A.

Each protein kinase Aenzyme phosphorylates andactivates multiple proteintargets.

ATP

ADP

Inactivated proteinkinase A

Activated proteinkinase A

One molecule ofepinephrine binding to aG-protein coupledepinephrine receptorcan stimulate the1,000,000 glycogenphosphorylase enzymesto release 100,000,000glucose molecules fromglycogen.

Occurs on plasma membrane

Occurs in the cytoplasm

Activatedadenylylcyclase

!" #GTP ATP

cAMP

Active

InactiveProteinkinase A

PhosphataseInactive Active

Phosphategroup

Phosphatases removephosphate groups fromproteins, causing them tobecome inactive.

cAMP AMP

Phosphodiesterase

! " #

GDP

The signal moleculeadrenaline detachesfrom the receptor aftera certain amount of

time, inactivating thereceptor so that it canno longer bind to andactivate the G protein.

Within a very short time, anactivated G protein deactivatesitself by converting GTP to GDP. Enzymes in the cytosol specifically

degrade cAMP which stops thephosphorylation and activation oftarget proteins by PKA.

How cAMP signaling is turned off


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Many signaling activities in cells depend on G proteins. What distinguishes G-proteins from any other proteinassociated with signaling in a cell?

G proteins can be activated when a signal ligand binds a receptor but they also are inactivated after a short time in thecytoplasm (the time period depends on the G protein). How are G proteins activated and how are they inactivated?

Phosphodiesterase is an important enzyme associated with adenylyl cyclase signaling.What does this enzyme do in a cell?Choose an example of signaling associated with cAMP and describe what could happen if the cell had a mutation

that produced an inactive phosphodiesterase.

Study questions 9.4

Receptor tyrosine kinases are important for many signaling activities some of which are associated with mammaliangrowth and cell division. When the receptor binds a signaling molecule, the receptors dimerize (two receptors bind to eachother) and activate a tyrosine kinase.

a. What kind of molecule is a kinase?b. What does a protein kinase do?c. What is tyrosine?

What is the difference in function between a protein kinase and a protein phosphatase? Why are these enzymesimportant in cells give one example from this lecture.

One molecule of epinephrine binding to a G-protein coupled epinephrine receptor can stimulate the 1,000,000glycogen phosphorylase enzymes to release 100,000,000 glucose molecules from glycogen. What do glucoseand glycogen have in common and why might a cell have any reason to release glucose from glycogen (i.e.,

what use is glucose to a cell?)

Signalingmolecules

Inacti ve receptor D imer iza tion Act ive recepto r Acti ve receptor

Phosphategroup

Cytoplasmicsignalingproteins

Each member of thereceptor pairattaches phosphategroups to the othermember.

The attachedphosphate groupsprovide binding sitesfor intracellularsignaling proteins.

Signalingmolecule

Inactive receptor

Activated Rassignaling protein

Active receptor

GTP


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GTP

Inactivekinase 1

Activekinase 1

Activekinase 2

Activekinase 3

ATP

ATP

ADP

ADP

A small amount of signalreceived by receptorkinase is amplified whenthe signal is passedfrom kinase to kinase aseach is phosphorylated.

Transcription regulators

Nucleus

Changesin gene

expression

Muscle cell

Nerve cell

Plasmamembrane Acetylcholine

OpenClosed Binding site

Na+

Acetylcholinebinding opension channels,allowing Na+toflow into themuscle cell.

Study questions 9.5

Some signaling systems activate transcription.

Where, in a eukaryotic cell, does transcription occur?What is a product of transcription?What would a cell do with this product of transcription?Why would it be useful for a cell to regulate transcription?

Botulism toxin contains several proteins, one of which can enter a nerve cell and prevent fusion of a synapticvesicle with the plasma membrane. One of these is t he Botox that doctors inject into patients to preventwrinkles. Why does Botox treatment prevent wrinkling?


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A gated ion channel is theCFTR (cystic fibrosisconductance regulator).

This channel transportschloride ions out of a cell

and, by increasing chlorideconcentration, water isdrawn out of the intestinalcells by osmosis.

This helps keep our lungsmoist and reduces thestickiness of mucus.

Cystic fibrosisis causedby defects in the chloridechannel thatcan preventchloride release and theaccumulation of stickymucus that cannot beremoved by the cilia liningthe lungs.

CFTR

G-proteinreceptor

GTP

GDP

Signalmolecule

Adenylylcyclase

ATP

cAMP

Activate Proteinkinase A

Phosphorylateand activateCFTR

Chloride ionsleave cell

Important:

Chloride is only releasedwhen the signal moleculeactivates the G-proteinreceptor.

When the receptor is nolonger activated, the G-protein hydrolyzes GTP andthe GDP-G protein nolonger activates adenylylcyclase, which stopsproduction of cAMP

CFTR

G-proteinreceptor

GTP

GDP

Adenylylcyclase

ATP

cAMP

Phospho-diesterase

PkA nolongeractive

AMP

CFTRchannelcloses

The CFTR channeltransports chloride ions outof a cell and, by increasingchloride concentration,water is drawn out of theintestinal cells and tissuespaces by osmosis.

Cholera toxin binds to the

G-protein receptor, andpermanentlyactivates theG-protein.

CFTR

G-proteinreceptor

GTP

GDP

Adenylylcyclase

ATP

cAMP





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So the key to G-proteinsignaling is thereversibility of the signal.

CFTR

G-proteinreceptor

GTP

GDP

Adenylylcyclase

ATP

cAMP




A Worsening Haitian TragedyBy THE EDITORIAL BOARD, New York Times March 17, 2013The aid group Doctors Without Borders said last Tuesdaythat the cholera crisis in Haiti was getting worse, for the most unnecessaryand appalling of reasons: a lack of money and basic medical supplies.The disease has killed 8,000 people and sickened 649,000 since October 2010. International efforts to defeat the epidemic include a 10-

year, $2.2 billion plan for major investments in clean water, sanitation and medical infrastructure. But that is a project for the future, onethat isnt even funded yet. Doctors Without Borders says people are dying now, needlessly, because attention and money are runningout. Aid groups are leaving. Staff members at some treatment centers havent been paid in months, equipment is wearing out, andsanitary precautions are being abandoned. The death rate has reached an intolerably high 4 percent in some places, the group said.

And the rainy season is about to make things much more difficult.

The dreadful backdrop to this emergency is an abdication of responsibility by organizations that have pledged to help Haiti, particularlythe United Nations. The U.N. said last monththat it would not pay financial compensation for the epidemics victims, claiming immunity.This is despite overwhelming evidence that the U.N. introduced the disease, which was unknown in Haiti until it suddenly appeared neara base where U.N. peacekeepers had let sewage spill into a river.

Though the U.N. has done much good in Haiti since the 2010 earthquake, its handling of cholera is looking like a fiasco. While it insiststhat it has no legal liability for cholera victims, it must not duck its moral obligations. That means mobilizing doctors and money to save

lives now, and making sure the eradication plan gets all the money and support it needs.

Its record so far is dubious. A U.N. appeal last year for $24 million for cholera programs ended the year only 32 percent financed, and in

December, the U.N. said it would contribute $23.5 million to the new 10-year plan about 1 percent of what is needed.

Florida

CubaHaiti

A recent cholera example

New York TimesMarch 22, 2013Cholera in Haiti

To the Editor:A Worsening Haitian Tragedy (editorial, March 18) points out the sad reality that cholera isnow endemic in Haiti. But it gives the impression that most aid organizations are leaving the

country at a time when thousands are dying from a preventable disease.While some aid groups have indeed left the country or are scaling back programs, othershave made fighting the epidemic their top priority.

Since the cholera outbreak began in 2010, AmeriCares alone has delivered enoughmedicines to treat about 150,000 patients, and we continue to expand our Haitian staff toensure that we can continue stocking cholera treatment centers all across the country withmedicines and supplies.

Going forward, N.G.O.s in Haiti must come together and intensify focus on preventionactivities that offer the best hope for preventing needless deaths, unnecessaryhospitalizations and unquantifiable suffering. Better coordination and leadership will bringabout more lasting change than any amount of recrimination and finger pointing.

No one should have to die from a disease that can

be avoided with soap and water, and the collective

will to make them available.

CURT WELLINGPresident and Chief Exec., A meriCaresStamford, Conn., March 19, 2013


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So CFTR channels open because

A signal

binds to G-protein receptors that

Activate G-proteins by exchanging GDP for GTP whichBinds to and activates adenylyl cyclase which

Converts ATP to cyclic AMP which

Binds to and activates protein kinase A which

Phosphorylates and opens chloride channels and

Chloride ions draws water from tissue spaces and

Hydrates mucus

So CFTR channels close

When no more water release is needed, the signal releasesfrom the G-protein receptor

G proteins hydrolyze GTP and become inactive GDP-G proteins

Adenylyl cyclase stops working and cAMP is converted to AMPby phosphodiesterases

Without cAMP, the protein kinase is no longer active

Without active PkA the CFTR channel closes.

The cholera toxin modifies the G protein, whichkeeps the G protein in the GTP-G protein stage

Adenylyl cyclase keeps working and produces cAMP is

cAMP stimulates and keeps the protein kinase active

This keeps the CFTR channels open

Water is drawn from tissues and into the intestine

Which results in diarrhea, water loss, and possibledeath


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Cholera toxin, released from Vibrio choleraebacteria, induces diarrhea and dehydration. Infected individuals diebecause they are dehydrated, due to water loss from the bloodstream. Water is lost because chloride channels thatbind and are activated by cyclic AMPrelease chloride ions into the intestine. This increase in salt (chloride andsodium ions) draws water (by osmosis) out of t he bloodstream and into the intestine, resulting in diarrhea. Choleratoxin acts on the G-protein and prevents GTP hydrolysis.

a. Sketch the process by which the binding of a bacterial protein opens chloride channels. (Hint chloridechannels are ligand-gated ion channels).

b. Cholera toxin blocks GTP hydrolysis by t he G protein. Why does it induce diarrhea?c. Based on your knowledge of osmosis, how can osmosis induce movement of water from tissue into the

intestine?d. How can infection of a human with Vibrio cholera cause diarrhea?d. Without proposing antibiotics, to kill bacteria, or miracle drugs, to block the action of cholera toxin, propose a

simple and inexpensive method that might be (is) used to treat patients infected with Vibrio choleraebacteria.

Study questions 9.6

Receptor tyrosine kinases are important for many signaling activities some of which are associated with mammaliangrowth and cell division. When the receptor binds a signaling molecule, the receptors dimerize (two receptors bind to eachother) and activate a tyrosine kinase.

a. What kind of molecule is a kinase?b. What does a protein kinase do?

c. What is tyrosine?

Compare the functions of protein kinases and protein phosphatases. Give one example from this lecture toillustrate their importance in cells..

One of the key properties of signal transduction pathways is the ability of each signaling molecule in the pathway to beturned on or turned off.

Consider each of the following pathways and identify,for each step in the pathway,

(a) the components that are turned on,(b) the mechanism or molecule that activates each molecule,(c) the mechanism or molecule that inactivates the activated molecule(d) what would happen to the process if any one of the steps were not able to be inactivated.

Pathway:

1. A pathway in which a ligand stimulates a G-protein coupled receptor to open a CFTR (cystic fibrosischannel protein) channel to allow chloride ions to diffuse out of a cell. Your answer should include the

mechanism by which the CFTR channel is opened (within the limits of what we discussed in lecture), themechanism by which the CFTR channel would be closed, and what would happen if GTP could not behydrolyzed by G-proteins associated with this process. (This question and answer should be similar to the first

question in this set)

2. A pathway by which the binding of a single epinephrine molecule to a cell-surface G-protein receptor caninduce the release of 100,000,000 glucose molecules from stored glycogen molecules. Your answer shouldinclude the steps by which the production of glucose from glycogen occurs and what happens if epinephrineis no longer bound to the receptor.

Study questions 9.7

Types of G proteins

There are two major classes of G proteins

Monomeric G proteins

Heterotrimeric G proteins

All are inactivewith GDP bound and activewith GTP bound

What is a difference between a monomeric and a

heterotrimeric G protein?


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Heterotrimeric G proteins

Composed of 3 protein

subunits - !, ", #

These are coupled to G-

protein receptors we have

discussed in class

When the receptor is activated, GTP is bound to G!and theG!dissociates from G"and G#.

The G!and G"#then can target different target molecules

Monomeric G proteins (small G proteins)

More than 100 different small G proteins that can be groupedin 5 classes:

Ras Rho Rab Ran Arf

Have a variety of functions in membrane trafficking and otheraspects of cell regulation

Importance: mutations in Ras alone are found in ~25% of allhuman tumors. Ras is particularly important in signaling

pathways that stimulate cell growth or inhibit cell death

(apoptosis).


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Scaffold proteins can bring several signalingcomponents together.

Scaffold proteins are found in a number of cells, With regard to signaling, what might be a function of ascaffolding protein?

Study questions 9.8

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