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EXAM 3 REVIEW
CH 15: Signal Transduction and G Protein Coupled Receptor Complexes
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•
Describe the structure, function, ligands and activation of a GPCR and recognize how
downstream signaling components are activated by them.
o Structure:
▪ A receptor that contains 7 membrane-spanning alpha helices
• Same orientation in the membrane (n-terminus outside and cterminus in cytosol)
• Contain 7 transmembrane alphas helical regions (H1-H7)
• 4 extracellular segments (E1-E4)
o act as receptor to interact with ligand (usually E2/E3)
• 4 cytosolic segments (C1-C4)
▪ A receptor-activated heterotrimeric G protein cycling between GTP-active
and GDP-inactive forms
▪ A membrane-bound effector protein
▪ Proteins that desensitize the signaling pathway
o Ligands:
▪ Causes conformational change of receptor that allows it to interact with
the heterotrimeric G protein
▪ Different cell types often have different receptors for the same ligand →
different intracellular signal transduction pathway
▪ Different cell types may have the same receptor for a specific ligand →
different intracellular response
Explain how GTP hydrolysis is involved in signaling.
o Active/ON State (bound GTP before being hydrolyzed)
▪ Switch I and switch II – bound to the GTP terminal  phosphate through
interactions with conserved threonine and glycine residues backbone
amide groups
▪ Switch domain conformation → can bind and activate specific
downstream effector proteins
▪ GEFs catalyze dissociation of bound GDP and replacement by GTP
▪ Accelerated GAPs (GTPase-activating proteins) and RGSs (regulators of
G protein signaling)
▪ G protein on-off transition conformational changes:
•
•
Involves switch I and switch II
Promotes binding to downstream signaling proteins
o Inactive/OFF State (bound GDP after GTP hydrolysis)
▪ Intrinsic GTPase activity → hydrolyzes GTP to GDP (removes GTP 
phosphate)
▪ GTP hydrolysis rate → time G protein remains in the active conformation
▪ Switch I and switch II relaxation into off conformation → inhibits
interaction with downstream effectors
▪ Similar spring-loaded mechanism switches the alpha subunit into
heterotrimeric G proteins between active and inactive conformations by
movement of three switch segments)
•
Recognize and describe the types of targets of GPCRs.
o Endocrine: (epinephrine, insulin)
▪ Signaling molecules synthesized and secreted by signaling cells
▪ Transported through the circulatory system
▪ Affect distant target cells expressing the receptor
o Paracrine: (neurotransmitters, growth factor)
▪ Signaling molecules secreted by a cell
▪ Affect only nearby target cells expressing the receptor
▪ Some may bind to ECM and released only when ECM is degraded
o Autocrine: (growth factors)
▪ Cells respond to signals they themselves secrete
o Membrane Protein Signals:
▪ Signal neighboring cells by direct contact with surface receptors
•
o Second Messengers:
▪ cAMP:
• generated from ATP by adenylyl cyclase
• activates PKA
▪ cGMP:
• generated by guanylyl cyclase
• activates PKG and specific cation channels
▪ IP3 and DAG:
• Both made from PIP2 by phospholipase C
• IP3 opens channels to release Ca2+ from ER
• DAG with Ca2+ activates PKC
2+
▪ Ca :
• Released from intracellular stores or transported into the cell
• Activates calmodulin, specific kinases (PKC), and other regulatory
proteins
Understand the role of the G complex. Recognize the variants of G (Gi, Gt, Gs,
etc.) and know what transduction pathways they are found in.
o Gi:
▪ Inhibitory
▪ Associated with adenylyl cyclase K+ channel to decrease cAMP by
changing membrane potential
▪ Ex) alpha2-adrenergic and muscarinic acetylcholine receptors
o Gt:
▪ Transducin
▪ Associated with cGMP phosphodiesterase to decrease cGMP
▪ Ex) rhodopsin in rod cells
o Gs:
▪ Stimulant
▪ Associated with the effector adenylyl cyclase to increase cAMP by making
cAMP
▪ Ex) beta-adrenergic, glucagon, serotonin, and vasopressin receptors
o Gofl:
▪ Olfactory
▪
Associated with the effector adenylyl cyclase to increase cAMP by making
cAMP
▪ Ex) odorant receptors in nose
o Gq and Go:
▪ Associated with phospholipase C to increase IP3 and DAG
▪ Alphaq → ex) alpha1-adrenergic receptor
▪ Alphao → ex) acetylcholine receptor in endothelial cells
o Process:
▪ Receptor with ligand behaves like a guanine exchange factor → causes G
protein to open up and dump GDP so that GTP binds to alpha subunit and
activate → alpha subunit separates from beta and gamma subunit so alpha
subunit can work on effector protein → stimulate effector protein to
hydrolyze GTP (effector protein acts like GAP) → alpha subunit
disassociates and reassociates with gamma and beta subunits to begin
again
•
Define cAMP, explain how it is made, and describe its function in signal transduction?
o Adenylyl Cyclase Is an Effector that Produces cAMP:
▪ GPCRs activate G proteins that activate or inhibit adenylyl cyclase
generation of cAMP from ATP and are regulated by feedback repression
▪ cAMP activates PKA, which phosphorylates-regulates multiple target
proteins
• metabolism of fats and sugars
• synthesis and secretion of hormones
• muscle contraction
▪ PKA activation can stimulate gene expression
▪ Used to fine-tune cAMP levels
o Regulation of glycogenolysis by cAMP and PKA:
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Recognize the different types of heterotrimeric G proteins and their effectors, as well as
their direct and indirect effects.
o Different G proteins are activated by different GPCRs and regulate different
effector proteins
o Effector protein types:
▪ Membrane-bound ion channels
▪ Membrane-bound enzymes that catalyze formation of one or more second
messenger
o Indirect → influences on membrane potential
o Direct → direct binding
Understand how the GPCR (rhodopsin) works in rods and the downstream signal
transduction pathway involving arrestin and transducin.
o Rhodopsin:
▪ GPCR senses light in rod cells
▪ Located in the flattened membrane disks of the cell outer segment
▪
Has its own special G called Gt (transducin → found in retina)
▪
Light activation of rhodopsin:
• Leads to closing of cGMP-gated cation channels
• Absence of neurotransmitter from rod
Dark adapted rod cells:
• High level of cGMP → keeps cGMP-gated nonselective cation
channels open
• Open channels depolarize the plasma membrane (-30 mV) from
resting potential (-60 to -90 mV)
• Stimulates neurotransmitter release
▪
•
Inhibition of rhodopsin signaling by rhodopsin kinase:
o Several mechanisms act to reset the system
o Feedback repression of overactivated rhodopsin
o Rhodopsin kinase phosphorylates light-activated rhodopsin
but not dark-adapted rhodopsin
▪
▪
▪
▪
•
Rhodopsin phosphorylation is proportional to the
amount of time each rhodopsin spends in the lightactivated form
Greater the light-activated rhodopsin
phosphorylation → greater activation of Gt is
reduced
Arrestin binds to completely phosphorylated
rhodopsin → inhibits activation Gt
Rhodopsin phosphorylation and inactivation by
arrestin within 50 ms
o Transducin and arrestin distribution in dark-adapted and light adapted rod cells
▪ Rod cells adapt to varying levels of ambient light by intracellular
trafficking of arrestin and transducin
▪ Dark:
• Transducin: localized in out segment
• Arrestin: localized in inner segment
▪ Light:
• Transducin: out → inner segment
• Arrestin: inner → out segment
Describe where and how signal amplification can occur by signal transduction.
o Signal transduction pathways amplify the effects of extracellular signals
•
Know how PKA is activated and inactivated and what its potential targets are.
o PKA activated by the binding of cAMP
o PKA Activation of Transcription Factors:
▪ cAMP-PKA regulates gene expression through CREB
• activated PKA catalytic subunits translocate into nucleus
• PKA responsive genes have cAMP response elements (CRE)
• Phosphorylated CRE-binding protein (CREB) binds to CRE
o CREB complex binds to CRE regulatory elements in
promoters of multiple genes
o CREB complex binding stimulates transcription of the
various target genes controlled by CRE
o Suppressing Signaling GPCR/cAMP/PKA:
▪ 1) G alpha binding to effector proteins accelerate GTP hydrolysis → G
alpha/effector dissociation
▪ 2) Hormone levels fall → PDE hydrolyzes cAMP to AMP → PKA off →
signal terminates
▪ 3) Feedback repression: end product inhibits earlier step in the pathway
• High cAMP activates PKA
• High PKA → phosphorylates c-terminus of receptor → ligand still
present but receptor can’t bind G alpha → cannot activate effector
• BARK phosphorylates other residues → beta-arrestin binds
receptor and clathrin/AP2 → receptors endocytosed → some
degraded, some dephosphorylated/resensitized
•
Review the -adrenergic and acetylcholine receptors as GPCR examples.
CH 17: Cytoskeleton and Movement I: Actin Microfilaments
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Describe the subunits and characteristics of microfilaments, how they are made, their
polarity, and what contributes to their branching, breaking, capping, recharging and
recycling.
o Overview:
▪ Smallest component in cytoskeleton → 7-9 nm in diameter
▪ Made by actin binding proteins (ABPs)
▪ Plasma membrane organization (microvilli)
▪ Tracks for ATP-powered motor proteins
▪ Contractile function
o Actin-based Structures: microfilaments can be organized into a variety of
different structures with distinctive activities with a cell/in different types of cells
▪ Tight bundles
▪ Support and organization beneath the plasma membrane (-actin)
▪ Junctions → provide strength to a tissue layer (epithelium)
▪ Leading edge of migrating cells (-actin)
▪ Stress fibers → cell-cell and cell-ECM adhesion (-actin)
▪ Endocytosis and vesicular transport
▪ Cytokinesis and contractility (-actin)
o Subunits:
▪ G-actin:
• The actine monomer
• Structure: 2 approximately equal-sized lobes and four subdomains
(I-IV)
o ATPase fold: Mg+2 and ATP/ADP bind at the bottom of the
cleft and contacts both lobes (cations induce
polymerization)
• G-actin Polymerization to form F-actin:
o Nucleation (lag) Phase: formation of 3 ATP-G-actine
“nucleus/seed” initiates formation of a filament → more
stable than 2 actin associations because of the extra bonds
▪ An addition of short actin filament “nuclei/seeds”
bypasses the slow nucleation phase and elongation
proceeds without any lag period
o Elongation Phase: actin subunits rapidly assemble onto
each end of the filament
o Steady State Phase: G-actin monomers exchange with the
subunits at the filament ends (filament length doesn’t
fluctuate)
▪ F-actin:
• Actin monomers polymerize into a long, helical F-actin polymer
•
Structure: Two helically wound strands with a repeating unit of 28
subunits (14 per strand) → 72 nm per turn
• Polarity: the ATP-binding cleft of every actin subunit is oriented
toward the same end of the filament → the filament end with an
exposed binding cleft is the (-) end and the opposite end is the (+)
end
• Negatively stained (TEM) actin filaments appear as long, flexible,
and twisted strands of beaded actins
o Regulation of Filament Turnover by ABPs
▪ Actin-binding proteins (ABPs) regulate rate of assembly and disassembly
of filaments and availability of G-actin
• Profilin: binds to ADP-G-actin, which opens the cleft and catalyzes
the exchange of ADP for ATP → allows the G-actin monomer end
to assemble onto the filament (+) end (RECYCLE/RECHARGE)
• Cofilin: fragments ADP-actin filament regions, which enhances
overall depolymerization by making more filament (-) ends
• Thymosin-4: buffers ATP-G-actin by sequestering G-actin at high
concentrations and releasing G-actin at low concentrations to
polymerize → provides a buffered reservoir of ATP-G-actin for
polymerization (keeps G-actin concentrations steady)
o Filament Capping Proteins: Capping proteins block assembly and disassembly at
filament ends
▪ (+) end capping proteins:
• Can lead to filament shortening
• CapZ: limits actin assembly and disassembly dynamics to that at
the (-) end → high affinity for the (+) end
• Gelsolin: severs actin filaments by inserting itself between actin
subunits of the helix → blocks the new (+) end
o Some gelsolin family members are activated by a rise in
Ca2+ concentration
▪ (-) end capping protein:
• Can lead to filament lengthening
• Tropomodulin: blocks the end where filament disassembly
normally occurs → stabilizes the filament
o Cell Migration:
▪ Cells migrate during normal development, wound healing, immune
function, and cancer cell metastasis
▪ Requires coordinated activities generated in different parts of a cell:
• The trailing edge of the cell remains attached to the substratum
until the tail eventually detaches and contractile force retracts into
the cell body
•
•
•
The endocytic cycle internalizes membrane and integrins at the
rear of the cell and transports them to the front of the cell for reuse
in making new adhesions
▪ Cells fail to move if they are either too strongly/weakly attached to a
surface
▪ Classes of microfilaments involved in cell migration:
• The network of actin filaments in the leading edge advances the
cell forward
• Contractile fibers in the cell cortex squeeze the cell forward
• Stress fibers ending in focal adhesions pull the bulk of the cell
body up as the rear adhesions are released
o Focal adhesions:
▪ Structure: stress fiber actin filament ends attach
through integrins attached to the underlying
extracellular matrix
▪ Signaling: contain many signaling molecules
important for regulating cell locomotion
• Arp2/3 complexes nucleate the dynamic actin network in the
leading edge
▪ Rho family of small GTPases regulate movement:
• Cells respond to external factors (epidermal growth factor, platelet
derived growth factor, food, chemotaxis, phototaxis)
• Family regulate formation of different actin filament organizations
and myosin II activity to direct cell motility
Define critical concentration (Cc).
o The number of G-actins that you actually need available to be able to start
building a filament → below a certain concentration of G-actin, filaments cannot
form but above a certain concentration, filaments form
Explain/draw how actin treadmilling occurs and works.
o Actine filament assembly-disassembly at each end:
▪ Assembly is almost 10x faster at the (+) end than at the (-) end
▪ ADP-G-actin disassembly is similar at both ends
▪ At steady state, ATP-actin assembly on the (+, lower Cc) end is faster than
actin ATP hydrolysis in the filament → giving rise to a filament with a
short region of ATP-actin (actin being added on) and regions of ADP-Piactin and ADP-actin (actin falling off) toward the (-) end
o Actin filament treadmilling:
▪ At steady state, ATP-G-actin subunits assemble preferentially on the (+)
end, while ADP-G-actin subunits disassemble from the (-) end
▪ 2 actin subunits assemble on the (+) end → over time, more actins
assemble onto the (+) end while actins disassemble from the (-) end → the
2 actine treadmill to the (-) end
▪ Movement of actin in its positive direction
•
•
Define a ‘duty ratio.’
o How long the head of a myosin stays associated with the actin microfilament
Explain/draw how microfilaments started/nucleated.
o Actin Nucleation by Formin: (Helps CHAIN)
▪ Functionally different actin-based structures are nucleated by formins
▪ Formin is activated by Rho-GTP
▪ Formin interacts with profilin to accept ATP-G-actin
▪ Formin assembles long actin filaments
• Muscle cells, stress fibers, filopodia, cytokinesis contractile ring
o Actin Nucleation by the Arp2/3 Complex: (Helps BRANCH)
▪ Functionally different actin-based structures are nucleated by Arp2/3
complexes
▪ Need a chain to start branching off of
▪ Associates to the side of a growing filament and recruits a nucleation
promoting factor, which bridges the divide between active G-actin to bring
in and Arp2/3
▪
•
•
Arp2/3-dependent actin polymerization:
• Branched filament assembly/disassembly
• Moves pathogenic bacteria and endocytic vesicles within cells
• Pushes the leading-edge membrane forward in moving cells
Know where microfilaments are predominantly located in any given cell.
o Plasma membrane
Recognize the toxins that depolymerize actin or prevent depolymerization and how they
accomplish this.
o Toxins affect the dynamics of actin polymerization
▪ Cytochalasin D: (fungal alkaloid) binds to (+) end of F-actin
• actin falls apart
Latrunculin: (sponge toxin) sequesters G-actin resulting in no filament
formation
▪ Jasplakinolide: (sponge toxin) promotes nucleation
▪ Phalloidin: (mushroom toxin) prevents filaments from depolymerizing →
good for visualizing actin filaments
• Filaments stay stuck together
Demonstrate how cells (or pathogens) manipulate microfilaments for various functions.
o Actin polymerization powers intracellular movement
▪ Food-born bacterium → mild gastrointestinal symptoms/fatal to elderly or
immunocompromised
▪ Enters animal cells and divides in the cytoplasm
▪ Hijacks a normal cell motility process for intracellular motility
• ActA mimics an NPF → binds VASP (bind profilin-ATP-actin)
• Enhances Arp2/3-based actin assembly
• Blocks CapZ from (+) growing ends
• Cofilin disassembles the (-) end to replenish from actin supply
• Assembling filaments push the back of the bacterium
▪ Results:
• Bacteria push the host cell plasma membrane → forming a
filopodium
• Protrudes into a neighboring cell and delivers that bacterium to that
cell
• Movement from one cell cytoplasm to another cell cytoplasm
without exiting the cell screen the bacterium from the immune
system
o Leukocyte Phagocytosis and Degradation of a Bacteria
▪ Opsonization: Bacterium is coated by specific antibodies to a cell-surface
protein
▪ Leukocyte surface receptor (Fc) binds bacterium-bound antibodies
▪ Signals the cell to activate Arp2/3 complexes → assemble an actin
filament network that moves the cell membrane around the opsonized
bacterium
▪ Fusion of the membrane projections pinches off the phagosome into the
cytoplasm
▪ Fusion of lysosomes with the phagosome delivers enzymes that degrade
the bacterium
Compare and contrast the different classes of myosin proteins.
o Commonalities:
▪ Myosin superfamily protein structure → common head and specific tail
domains
• Head binds to actin
▪
•
•
• Tails provide regulatory/specific functions
Crossbridge cycle converts ATP hydrolysis energy to mechanical work
actin filaments
▪ Myosin class-specific step sizes processivity support different functions:
• Muscle contraction
• Vesicular transport
• Moving organelles
• Cell migration
▪ Actin-activated ATPase activity
o Myosin Classes:
▪ Myosin I: one heavy chain with a head domain and a neck domain
• only single-headed myosin
• some associate directly with membranes through tail-lipid
interactions
• (+) end-directed motors → move in (+)/growing end direction
• Function in membrane association and endocytosis
▪ Myosin II: two heavy chains – each with a head and a neck domain that
binds two different light chains
• heavy chain long helical tail homodimerizes through a coiled-coli
interaction
• only class that can assemble into bipolar filaments through tail
interactions
• (+) end-directed motors → move in (+)/growing end direction
• Function in skeletal muscle contraction
▪ Myosin V: two head domains and six light chains per neck
• heavy chain helical tail homodimerizes through a coiled-coli
interaction
• end of tails interact with specific receptors on organelles, which
they transport along actin filament tracks
• (+) end-directed motors → move in (+)/growing end direction
• Function in organelle transport
After all this time, are you confident that you know how a sarcomere is structured and
contracts?
o Steps of Muscle Contraction:
▪ Motor neurons synapse with your muscles
▪ They release acetylcholine and it goes through the transverse tubules (Ttubules)
▪ Acetylcholine activates the release of calcium from sarcoplasmic
reticulum
▪ Calcium then goes to the sarcomeres and interacts with troponin and
tropomyosin that turn to open up the binding places on actin so that
myosin heads can bind to it
▪
•
•
o Myosin II light-chain phosphorylation regulates smooth muscle contraction
▪ Light chain phosphorylation regulates myosin II assembly and contractile
interaction with actin filaments
• Relaxed: at low calcium concentrations, the myosin LC is
dephosphorylated by MLC phosphatase → myosin is a folded
blocking head interaction with actin
• Activated: at high calcium concentrations, calcium binds
calmodulin → activates myosin light-chain kinase →
phosphorylates the myosin LC → activates head ATPase activity
and unfolds the tail to assemble into bipolar filaments
• Active myosin II produces force on actin filaments for contraction
o ATP-driven myosin V movement along actin filament
▪ Hand-over-hand processive motor model:
• Fluorescent-labeled myosin V tracked with nm accuracy as myosin
walks toward (+) end of filament
• Myosin head takes 72 nm steps along actin filament
• Trailing head swings forward 72 nm to become the leading head
while the other head remains attached (no dissociation)
• Hydrolysis of 1 ATP drives each head movement
• Myosin V ATPase cycle has a higher duty ratio than myosin II due
to a slower rate of ADP release → myosin V remains in contact
with the actin filament for longer
o A duty ratio for each head > 50% in a two-headed motor
ensures that it maintains contact at all times as it moves
down an actin filament
Describe other new proteins that you learned are involved in maintaining a sarcomere’s
structure.
o Accessory Proteins Found in Skeletal Muscle:
▪ Nebulin: binds actin subunits and determines the length of the thin
filament
▪ Titin: long elastic molecules
• One end attaches to the Z disk and the other end attaches to the M
band
• Interactions with each pole of myosin bipolar filaments centers the
think filaments
• Molecular elasticity prevents catastrophic overstretch of the
sarcomere
• Titin mutation cause cardiomyopathies
• Largest protein ever identified
▪ Tropomyosin:
• Helps keep structure and rotating to make myosin binding sites
available in the presence of calcium
•
Understand how cells move across a substrate using different types of microfilaments,
integrins and foci.
CH 18: Cytoskeleton and Movement II: Microtubules and Intermediate Filaments
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•
•
•
•
Describe the subunits and characteristics of microtubules, how they are made, their
polarity, and what contributes to their recharging and recycling.
o Microtubules:
▪ Made of tubulin and associated with MAPs
• Tubulin dimer:
o -tubulin: GTP is never hydrolyzed and nonexchangeable
o -tubulin: GDP is exchangeable with GTP and can be
hydrolyzed in the site
o -subunit: involved in microtubule assembly (Eukaryotes)
▪ Stiff tubes that are singular or in bundles
▪ Extend long distances (e.g., flagella, mitotic spindle, axons)
▪ Pull/push (no buckling)
▪ Dynamic and flexible
▪ Polarity → utilized by MT-dependent motors in organelle transport
• Structurally Polarized Tube:
o Protofilaments: end-to-end dimers
o Protofilaments pack side by side
o Protofilaments are staggered → -tubulin contacts tubulin in neighboring protofilaments (except at the seam)
o Intrinsic polarity
o Subunits added at the (+) end where -tubulin is exposed
Define critical concentration.
o Amount of subunit needed to build polymer
Explain/draw how microtubule treadmilling occurs.
o Dynamic structure:
▪ Microtubules grow preferentially at the (+)/ end
▪ Can assemble/disassemble rapidly at both ends
▪ Treadmilling: addition at one end and loss at the other (though often
anchored at (-) end)
Describe how dynamic instability and catastrophe and rescue occur. What does GTP have
to do with it?
o Dynamic instability: oscillation between growth and shrinkage (catastrophe vs.
rescue)
▪ Catastrophe → falling apart
▪ Rescuing → building
o Balance depends on the presence of GTP on the (+) end → if it’s been hydrolyzed
to GDP on microtubule that has stopped growing, the microtubule will curl
o GDP--tubulin has structural strain that is contained as long as the GTP--tubulin
cap is in place (or in islands)
Explain what MAPs are – give examples and what they do in microtubule function.
o MAPs stabilize microtubules:
▪
•
•
•
MAP (long arm) and tau (short arm) side-binding proteins stabilize and
space microtubules in neurons
• Microtubule spacing in MAP2-expressing cells is greater than in
tau-expressing cells → enlarges caliber of cell
• Side associations with several monomers along protofilaments
stabilize microtubules and dampen dynamic instability (increase
growth rate or decrease catastrophe)
• MAP/tau phosphorylation can regulate microtubule interactions
▪ (+) end tracking proteins (+TIPs): regulate the properties and functions of
microtubule (+) end
• End-binding 1 (EB1) binds assembling end (GTP--tubulin)
• Other +TIPs hitchhike on EB1
• +TIPs Activities:
o EB1 and XMAP215 (binds free dimers) promote
microtubule growth by enhancing polymerization at the (+)
end
o CLASPs suppress catastrophes
o Other +TIPs link the microtubule (+) end to other cellular
structures, such as ER, F-actin in the cell cortex,
chromosomes during mitosis, plasma membrane
Know which MAPS destabilize microtubules and how they do it. Give an example of
when or where you might need to destabilize a microtubule.
o Kinesin-13:
▪ Enhances the disassembly of either a (+)/(-) microtubule end
▪ ATPase activity dissociates Kinesin-13 from the -tubulin dimer
o Op18/stathmin:
▪ Binds selectively to two dimers in curved protofilaments and enhances
their dissociation from a microtubule end
▪ Enhances GTP hydrolysis
▪ Activity is inhibited by phosphorylation
▪ Contributes to microtubule growth at front of cell
Describe how microtubules are started/nucleated and the novel structures they form.
Describe the organization of an MTOC and where MTOCs are found in a cell.
o Microtubule Organizing Centers (MTOCs)
▪ -tubulin ring complex (-TuRC): MAP that nucleates microtubule
assembly → forms a template for the microtubule (-) end
▪ Centrosome:
• Mother → distal appendages and daughter centrioles each
consisting of 9 linked triplet microtubules
• Pericentriolar material contains -TuRC
▪ Types:
• Spindle poles
•
• Centrosome
• Basal body
• MTOC
Know the various kinesins and how they function (orientation of movement included!).
They are so ki-neat.
o Kinesin Superfamily:
▪ Kinesins form a large superfamily with diverse functions
▪ A conserved motor domain is fused to a variety of class-specific nonmotor
domains
o Kinesin-1:
▪ (+) end-directed microtubule motor involved in organelle transport at cell
periphery
▪ attaches cargo to microtubules and transports them (ATP hydrolysis)
▪ anterograde movement → towards the (+) ends of microtubules
▪ Structure:
• Homodimer of 2 identical heavy chains and associated light chains
• Head motor domain → microtubule and ATP/ADP binding sites
• Flexible linker domain → required for motor activity and connects
head to the coiled-coil stalk
• Two light chains associated with the tail of each heavy chain bind
to receptors on vesicles
▪ Uses ATP to “walk” down a microtubule:
• 1) leading head binds ATP and trailing head binds ADP
• 2) linker region swings forward and docks onto the surface of its
associated head domain → thrusting head forward
• 3) new leading head binds weakly to a site 16 nm toward the
microtubule (+) end
• 4) new leading head releases ADP and binds tightly to microtubule
• 5) new trailing head hydrolyzes ATP to ADP and Pi
• 6) Pi release converts the trailing head into a weak microtubulebinding state with the linker domain released from binding to the
head
o Kinesin-2:
▪ (+) end-directed vesicle transport
▪ Family has 2 closely related nonidentical heavy chains and a third cargobinding subunit
o Kinesin-5:
▪ (+) end-directed motor
▪ 4 heavy chains assembled in a bipolar configuration can slide antiparallel
microtubules past each other
o Kinesin-13:
▪ “motor” domain in the middle of the heavy chain has no motor activity
•
•
▪ Destabilizes microtubule ends for disassembly
o Kinesin-14:
▪ (-) end movement
Know what dynein is and how it functions (orientation of movement included!).
o Kinesins and dyneins cooperate in the transport of organelles throughout the cell;
most organelles have one or more microtubule-based motors associated with
them.
o Cytoplasmic Dynein:
▪ Retrograde transport of organelles toward microtubule (-) ends at the
centrally located MTOC
▪ Dynein heavy chain domains:
• Stem
o Dimerization domain and intermediate chains associate
with the stem region and can link dynein to cargo through
dynactin
• Linker
• 6 AAA ATPase repeats → form a circular structure with a coiledcoil stalk domain containing a microtubule binding site at one end
• Alpha-helical domain that supports stalk
Recognize the structure of cilia and flagella (9+2, nexin, axonemal dynein) – how DO
they MOVE?
o Axoneme:
▪ microtubule bundle in 9+2 arrangement
▪ (+) ends oriented at distal tip
▪ Connects to the basal body (9 triplets)
o Cilium:
▪ Nexin: connects adjacent microtubules to each other → prevents over
sliding of adjacent doublets with respect to each other
▪ Radial spokes project from each outer doublet A tubule toward the central
pair → prevents structural collapse during bending
▪ Axonemal dynein:
• Referred to as “inner-arm” and “out-arm” dyneins
• Project from anchorage on each A tubule toward each adjacent B
tubule
• Produce force for motility
o Movement/Sensation:
▪ Ciliary and flagellar beating pattern are produced by controlled sliding out
outer doublet microtubules
• Activation of the axonemal dynein motors power cilia and flagella
bending
• A tubule axonemal dynein walking toward the adjacent B tubule (-)
end pulls on the adjacent doublet
•
•
Nexin tethering of adjacent doublets converts force generated by
dynein into bending
▪ Intraflagellar Transport
Recognize some characteristics of intermediate filaments – particularly nuclear lamins
and keratins.
o Intermediate Filaments:
▪ More tensile strength
▪ Structural integrity and organization
▪ Not used as tracks for motors
▪ Many types of Ifs
▪ Tissue-dependent expression
▪ No intrinsic polarity
▪ No nucleotide binding
▪ Dynamic but more stable
▪ None present in fungi and plants
▪ 70 genes, 5 families
▪ Subunit dimers
o Major Classes:
▪ Class I: Acidic keratins
• Epithelial cells
• Tissue strength and integrity
▪ Class II: Basic keratins
• Epithelial cells
• Tissue strength and integrity
▪ Class III: Desmin, GFAP, Vimentin
• Muscle, glial cells, mesenchymal cells
• Sarcomere organization and integrity
▪ Class IV: Neurofilaments (NFL, NFM, NFH)
• Neurons
• Axon organization (axon diameter → nerve impulse rate)
▪ Class V: Lamins
• Nucleus
• Nuclear structure and organization
o Helps maintain nuclear integrity
o More movement → less rigidity
o Associated with chromatin in exoplasmic side → facilitate
nuclear transport
o Cyclin Dependent Kinases (CDKs) promote lamin
depolymerization
CH 19: Cell Cycle
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•
•
You should already be familiar with the cell cycle stages as well as what happens during
mitosis. Understand that transition through the cell cycle is tightly regulated at specific
steps and by specific complexes to make sure every step is completed with fidelity before
moving on.
o G1 (growth, RNA/protein synthesis) → S → G2 → M
o The cell cycle is an ordered series of high accuracy events leading to cell division,
controlled by:
▪ the regulation of timing of entry into the division cycle
▪ DNA replication
▪ mitosis
o It is regulated by checkpoint pathways facilitated by:
▪ protein kinases → cyclin-CDK (+/- feedback)
▪ Protein degradation
▪ Checkpoint pathway guarantees each cell cycle step is completed correctly
before the next is initiated
Define the START/restriction point.
o Cells commit to division at the G1
Describe the relationship between Cyclins and CDKS, also CDKIs.
o Cyclins:
▪ Different cyclins present only in the cell cycle stage they promote activate
CDKs at different cell cycle stages
▪ Subject to signal transduction
•
•
•
▪ Activate CDKs and influence specificity
▪ Establish preparation for next stage
o CDKs:
▪ have many different target proteins to help initiate every aspect of each
cell cycle stage
▪ fluctuate during cell cycle via +/- feedback mechanisms
▪ activating and inhibitory phosphorylation of the CDK subunit regulates
activity
• CDK inhibitors (CDKIs) bind directly to the cyclin-CDK complex
Know how the Ubiquitin/Proteasome system is involved in the cell cycle. (Know how
APC functions and the different involvements of APCCdh1 and APCCdc20).
o The ubiquitin-proteasome system limits presence of a cyclin to the appropriate
cell cycle stage
Recognize the phases of the cell cycle and the associated cyclin/CDKs that we focused
on (G1, G1/S and M). You don’t need to memorize the combinations, but you do need to
understand why they are expressed at different times and what the cell commits to in each
phase. Understand that cyclin/CDKs also activate each other.
o Mitosis: (Mitotic CDKs)
▪ CDK1
▪ Cyclin A and B
o S entry into the cell cycle: (G1/S phase CDKs/S phase CDKs)
▪ CDK2
• Free CDK2 → T loop blocks access of protein substrates
• Nonphosphorylated cyclin A-CDK2 → conformational changes
cause the T loop to pull away from the active site so substrate
proteins can bind (phosphotrasnfer reaction)
• Phosphorylated cyclin A-CDK2 → phosphorylation of Thr-160
induces conformational changes I substrate-binding surface which
increases active site affinity for protein substrates
▪ Cyclin E and A
o G1 entry into the cell cycle: (G1 CDKs)
▪ CDK4
▪ CDK6
▪ Cyclin D
What signals control the G1 → S phase transition – internal and external?
o E2F transcription factor and its regulator Rb control G1-S phase transition
▪ Rb inhibits ERF activity
▪ Growth factors (mitogens) stimulate G1 CDK activity
• Mitogens promote growth
• Anti-mitogens inhibit growth which block G1 cyclins, prevent G1CDK accumulation, and promote CDKI expression
▪
▪
▪
▪
•
Cyclin D-CDK4/6 is phosphorylated by Rb
E2F → transcription of early response genes (cyclin E, CDK2, and E2F)
Cyclin E-CDK2 phosphorylates Rb → feedback loop → rapid rise in the
expression/activity of E2F and cyclin E-CDK2)
G1/S phase cyclin E/A-CDK2 CDKs support cell passage through the
restriction point and initiation of DNA replication and centrosome
duplication
What subcellular commitments are made and what must be activated and completed in S
phase (beyond what you basically know about DNA replication, e.g. cohesins). How does
the cell make sure the chromosomes aren’t replicated again?
o S phase:
▪ DNA unwinds at origins of replication
▪ Cdh1 activated APC/C late in anaphase of the previous mitotic cycle
• APC/C ubiquitinates S phase and mitotic cyclins for destruction
•
▪
▪
Reset this to go into S phase again by phosphorylating Cdh1 and
inactivating APC/C
G1/S phase CDKs:
• block S phase inhibitors (CDKIs)
• phosphorylate Cdh1
• turn off machinery that degrades S phase CDKs
• S phase cyclin gene expression and accumulation
• One more blockage to insure that all G1 business is complete
(phosphorylation of Sic1 is a thermometer for S phase CDK levels)
Initiation of Replication:
• Origin recognition complex (ORC) is associated with DNA
replication origins
• 1) Exit from mitosis and early G1: CDK activity is low
o Cdc6 and Cdt1 (MCM loading factors) associate with ORC
and load the MCM replicative helicase complex onto each
DNA replication origin
• 2) Late G1: DDK and S phase CDKs are activated
o Phosphorylation to recruit MCM helicase activators to
unwind DNA
o S phase CDKs prevent reloading of MCM helicases and
also phosphorylate MCM helicases to disengage them from
the DNA when replication is complete and exported from
the nucleus
• 3) DNA polymerases recruited to origins initiate DNA replication
o Cohesin linkage of sister chromatids
▪ Cohesin ring complex:
•
•
Smc1 and Smc3: long coiled domain flanked by a globular ATPase
domain
• Scc1 (Rad21) and Scc3: interact with Smc1/3 ATPase domains
▪ Cohesin load onto DNA and acquisition of cohesive properties:
• G1: Scc2-Scc4 cohesin complexes load cohesins onto
chromosomes
o Cohesins associate with only one chromosome
o Cohesins can dissociate by interacting with the Pds5-WapI
complex
o Interphase dynamicity of cohesin association may be
important for regulating interphase chromatin structure and
gene expression
▪ S phase/G2:
• Cohesin acetyltransferase (CoATs) acetylate Smc3 near replication
fork and encircle the replicated sister chromatids
• Acetylation is accompanied by sororin binding
• Sister chromatids linked along their entire length by cohesins
• Mei-S332/Shugoshin proteins recruit the protein phosphatase 2A
(PP2A) to centromeric regions
▪ Mitosis (prophase and metaphase):
• Pds5-WapI activity + kinases (Polo & aurora B) release cohesins
• Cohesins are retained only in the region of the centromere (PP2A
prevents cohesin phosphorylation and dissociation)
Recognize how mitotic cyclin/CDKs come about and how they are removed before
mitosis even finishes to commit the cell to cytokinesis and reestablishing G1.
o CDK phosphorylation regulates mitotic CDK activity
▪ Centrosome maturation
▪ Nucleus → chromatin condensation
▪ Nuclear envelope breakdown (phosphorylate lamins, nucleoporins,
integral membrane proteins, etc.)
▪ Correct spindle attachment with Polo & Aurora kinases
•
•
Describe how chromosome attachment to microtubules is accomplished correctly and
what important components (Ndc80, aurora kinase, etc.) are involved.
o Chromosome attachment to the mitotic spindle
▪ Sister chromatids must be stably bi-oriented on the mitotic spindle to be
accurately segregated
• Only this arrangement has the correct tension when microtubules
begin pulling
▪ Dynamic microtubules anchored by their (-) ends to each spindle pole
search and capture chromosomes
• Chromosome motor protein propel chromosomes to the (+) end of
microtubules
• Each of the two sister chromatid kinetochore attached to several
microtubules from opposite poles
o CPC regulation of microtubule-kinetochore attachment
▪ Ndc80 complex forms a sleeve linking the kMT (+)
end embedded in the outer kinetochore to the inner
kinetochore, where CPC is linked to the centromere
▪ Chromosomal passenger complex (CPC) regulates
microtubule attachment at kinetochores
▪ Two mechanisms ensure all chromosomes are
correctly bi-oriented before anaphase begins
• Aurora B phosphorylates Ndc80 if it gets
too close to destabilize incorrect attachments
• In correct attachments Ndc80 is pulled away
and dephosphorylated
• Chromosome capture and congression in prometaphase
▪ Sensing mechanisms correct inappropriate attachments
Know what separase and securase are for and how they are regulated.
o Separase:
▪ Protease activity inhibited by CDK phosphorylation and securin binding
▪ released after securin is ubiquitinated and proteolyzes Scc1 and breaks
cohesin tethers
o Securase (Securin):
▪ Ubiquitinated by Cdc20 + APC/C
•
•
Explain why there is a spindle assembly checkpoint pathway.
o Spindle Checkpoint (Cohesin Cleavage):
▪ S phase to M metaphase:
• Sister chromatids tethered by cohesins
• Separase protease activity is inhibited by CDK phosphorylation
and securin binding
▪ Spindle checkpoint controls and then:
• 1) All kinetochores attached
• 2) Microtubules spindle is properly assembled and oriented
• 3) Cdc20 + APC/C ubiquitinylate securin and mitotic cyclins
• 4) Release of separase to proteolyze Scc1 and break cohesin tethers
• 5) Initiation of anaphase and disconnected sister chromatids are
pulled toward opposite spindle poles
o Spindle Assembly Checkpoint Pathway:
▪ Ensures each kinetochore is properly attached to spindle microtubules
before anaphase initiation
▪ Knl1 is an outer kinetochore component that can be phosphorylated
▪ Has multiple checkpoint kinases
▪ Forms mitotic checkpoint complex (MCC)
▪ Blocks APC/C activation of anaphase
Describe the role that motor proteins play in chromosome segregation and preparing the
cell for cytokinesis.
o Chromosome movement and spindle pole separation in anaphase
•
Understand what the Mitotic exit network is and how it is activated.
o Cdc14 phosphatase triggers exit from mitosis
CH 20: Cell in the Context of Tissues
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•
•
Understand Cell-cell vs cell-extracellular matrix (ECM) adhesion and interactions.
o Cell-cell and cell-ECM interactions are critical for:
▪ assembling cells into tissues
▪ controlling cell shape and function
▪ determining the developmental fate of cells and tissues
o Connectivity and communication facilitate information transfer to promote:
▪ Cell survival
▪ Cell proliferation
▪ Cell differentiation
▪ Cell migration
Define CAMs; know the specific types that we discussed (e.g. Cadherins & Integrins; be
able to describe how an E-Cadherin works, how in interacts in the ECM, how it’s
anchored to the cytoplasm).
o Cell-adhesion molecules (CAMs)
▪ Mediate direct cell-cell (homo- or heterophilic) interactions
▪ Adhesion receptors mediate cell-matrix adhesions
▪ Types:
• Collagens: form fibers
• Fibronectin
• Proteoglycan: a unique type of glycoprotein
• Laminin
• Elastic Fibers
• Integrin
o Mediate cell-ECM adhesions (including epithelial-cell
hemidesmosomes)
o Vertebrate integrin combinations: 24 integrin heterodimers
o All integrin evolved from two ancient general subgroups:
▪ Integrins that bind proteins containing the tripeptide
sequence Arg-Gly-Asp RGD motif (e.g.,
fibronectin)
▪ Integrins that bind laminin
Know what types of molecules can be found in the ECM of various environments (multiadhesive matrix proteins).
o Multi-adhesive matrix proteins: Important organizers of the extracellular matrix
▪ Fibronectin and laminin:
• Long, flexible molecules that contain multiple domains
• Bind various types of collagen, other matrix proteins,
polysaccharides, and extracellular signaling molecules as adhesion
receptors
• Interactions with adhesion receptors regulate cell-matrix adhesion
and cell shape and behavior
•
•
•
Understand the various functions/contributions of the ECM to signaling/communication
and anchoring cells together.
o Functions:
▪ Multitude of roles in addition to cell adhesion
▪ Composition imparts characteristics: strength, rigidity, cushioning,
transparency
▪ Positional and signaling information
▪ Reservoir for signaling molecules
▪ Lattice: migration or no movement
▪ Essential for mophogenesis (branching structures)
o Adhesion Receptor Mediated Signaling
▪ Ex) Integrins
• Interact via adapter proteins and signaling molecules with a broad
array of intracellular signaling pathways
• Influence cell survival, gene transcription, cytoskeletal
organization, cell motility, and cell proliferation
Recognize the various types of epithelia and where you might find them.
o Simple columnar epithelia:
▪ Elongated cells
▪ Includes mucus-secreting cells (stomach and cervical tract) and absorptive
cells (small intestine)
▪ Microvilli on apical surface
o Simple squamous epithelia:
▪ Thin cell
▪ Line blood vessels (endothelial cell/endothelium) and many body cavities
o Transitional epithelia:
▪ Several layers of cell with different shapes
▪ Line certain cavities subject to expansion and contraction (e.g., urinary
bladder)
o Stratified squamous (nonkeratinized) epithelia:
▪ Line surfaces such as mouth and vagina
▪ Resist abrasion
▪ Generally prevent material absorption/secretion into or out of lined cavity
o Basal lamina:
▪ Thin, fibrous network of collagen and other ECM components
▪ Connects epithelia to underlying connective tissue
Describe the structure and function of cell-cell and cell-extracellular complexes:
o Anchoring junctions (and their subtypes):
▪ Hold tissues together
▪ Adherens junctions: Joins an actin bundle in one cell to a similar bundle in
a neighboring cell
• E-cadherin clusters mediate initial attachment of cells into sheets
o Exoplasmic:
▪ Cis and trans interactions
▪ Ca2+-dependent
o Cytosolic domains:
▪ Bind directly/indirectly through adapter proteins
(e.g., -catenin) to actin filaments (F-actin) of
cytoskeleton
▪ Participate in intracellular signaling pathways
▪ Depict different sets of adapter proteins that interact
with different adherens junctions
▪ Adapters such as ZO-1 can interact with several
different CAMs
o E-cadherin activity lost during EMT
▪ Focal contacts
▪ Desmosomes: Joins the intermediate filaments in one cell to those in a
neighbor
• Contain two specialized cadherins (desmoglein and desmocollin)
• Pemphigus vulgaris human autoimmune disease:
o Desmoglein auto-antibodies disrupt desmosome adhesion
between epithelial cell, causing blisters of skin and mucous
membranes
▪ Hemidesmosomes: Anchors intermediate filaments in a cell to the basal
lamina
▪ Cadherins and integrins mediate cell-cell and cell-ECM junctions
o Tight junctions (know the proteins involved in tight junctions – occluding and
claudin and how they work)
▪ Hold tissues together
▪ Define epithelia cell polarity and regulate extracellular (paracellular) flow
of water and solutes from one side of the epithelium to the other
▪ Seals neighboring cells together in an epithelial sheet to prevent leakage of
molecules between them
▪ Block transport of many nutrients across the intestinal epithelium between
cells through the paracellular pathway
▪ Maintain cell polarity by restricting diffusion of membrane components
between apical and basolateral membrane regions
▪ Some molecules can move extracellularly through tight junctions
▪ Tight junction permeability to small molecules and ions depends on
composition of the junctional components and the physiological state of
the epithelial cells
▪ Permeability to ions, small molecules, and water varies enormously
among different epithelial tissues
▪ Occludin and claudin are the principal membrane proteins
• Occludin c-terminal cytoplasmic domain binds scaffold proteins
(ZO-1-3 proteins)
•
•
•
Claudin larger extracellular loop contributes to paracellular ion
transport selectivity
• Claudin-15 transmembrane domains four helix bundle permits
paracellular transport of cations
▪ JAM protein:
• single transmembrane domain
• extracellular domain (two immunoglobulin domains)
• tight linkages with adjacent cell JAMs
▪ Enteric pathogens disrupt tight junctions
o Gap Junctions (connexin)
▪ Connect the cytoplasms of adjacent cells for metabolic and electrical
coupling
▪ Allows the passage of small water-soluble ions and molecules
▪ Connexon hemichannels → cylinders of 6 dumbbell-shaped connexin
molecules
• Channel allows small molecules to pass directly between cytosols
of adjacent cells (1.2-2 nm in diameter with polar residues)
• Neurons: ions pass → rapid transmission of electrical signals
• Heart: ions pass → rapid transmission of electrical signals →
cardiac contraction
• Pancreas, smooth muscle: hormones → second messengers →
coordinated neighboring cell response
• Metabolic coupling: one cell transfers nutrients to a neighber
Know what EMT is and what the consequences for cells can be if they lose or disrupt cell
junctions.
o Epithelial-to-mesenchymal transition (EMT)
▪ Snail and slug proteins suppress E-cadherin expression
▪ Associated with conversion of epithelial cells into malignant carcinoma
cells
▪ Normal epithelial MDCK cells grown in culture form sheet of cadherinconnected cells
▪ MDCK cells expressing snail gene undergo EMT and cells dissociate
▪ E-cadherin distribution (dark brown staining) in tissue from a patient with
hereditary diffuse gastric cancer
• E-cadherin at intracellular borders of normal stomach gastric gland
epithelial cells
• No E-cadherin at the borders of invasive carcinoma cells
Understand how the ECM of a basal lamina differs from that of connective tissues.
o Basal Lamina ECM I:
▪ ECM influence signaling pathways (cell growth, proliferation, gene
expression)
•
•
Proteoglycans (perlecan) cross-links ECM components and
cushions cells
• Collagen IV has a 2-D network, structural integrity, mechanical
strength, and resilience
• Multi-adhesive matrix proteins (laminin and fibronectin) has a
fibrous 2-D network that binds receptors (integrins) and ECM
components (collagen)
• Nidogen cross links perlecan, collagen, and laminin
▪ Connects epithelia and other groups of cells to adjacent connective tissue
▪ ECM proteins bind to each other and to cellular receptors
o Connective tissue ECM II:
▪ Composed mostly of ECM produced by fibroblasts
• Glycosaminoglycans (GAGs): highly hydrated polysaccharides
o (ex) hyaluronan
• Proteoglycans: glycoproteins + GAGs
• Fibrillar collagen: 2-D network
• Multi-adhesive matric proteins (fibronectin): cross link adhesion
receptors and ECM components
• Elastin: amorphous core of elastic fibers
o Elastic fibers permit tissues to undergo repeated stretching
and recoiling
o Elastin fibers → tropoelastin molecules covalently cross
linked
▪ Tropoelastins self-associate, extend under stress,
and recoil efficiently
o Elastic fibers → solid core of elastic fibers integrated into a
bundle of microfibrils
Read the brief section in the book on Elastin, Collagen, and Fibrillin.