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UCD / Biology / BIOL 104 / What are the five stages of platelet activation?

What are the five stages of platelet activation?

What are the five stages of platelet activation?


School: University of California - Davis
Department: Biology
Course: Cell Biology
Professor: Carrasco garcia and starr
Term: Summer 2015
Cost: 25
Name: Notes: Lectures 15-17
Description: These are detailed notes I created by combining material covered in lectures 15 through 17 with material from chapter 6 of the textbook. Appropriate diagrams are included.
Uploaded: 11/09/2015
11 Pages 94 Views 4 Unlocks

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What are the five stages of platelet activation?

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Lecture 15:10/30/15

Regulation of Actin Dynamics with Drugs OLatrunculin (in sea sponges) : binds to and sequesters free actine

monomers, preventing polymerization

- by lowering C below Ca, Korp takes over and depolymerization occurs

What do you call the contractile unit of a muscle?

Study Soup If you want to learn more check out In what ventricle separates the left & right thalamus?

Tutoos ruby

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time Addition of Latrunaudin Cytochalasin (in a mold): binds to and caps the end of actin filaments, blocking 6 end polymerization If you want to learn more check out Who discovers “priam” and used knowledge of homer’s iliad?

- the end will continue shrinking until [g-actin] = Cc end = 0.6.um.

It is the only class of myosins that move toward the ends of actin filaments, what is it?

Don't forget about the age old question of Who is an an italian artist famous for his etchings of rome and of fictitious and atmospheric "prisons"?

Light Scattering


time Addition of Cytochalasin 3 Phalloidin (a toxin in a toadstool mushroom) binds to sides of

filaments and prevents depolymerization If you want to learn more check out Who is arthur kornberg?



Light Scattering If you want to learn more check out Who founded the mughal empire?
We also discuss several other topics like What are the three basic sales tasks are?

The rest of free action monomers will polymerize into filaments so a higher steady state equilibrium will be reached.

@ Stue


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Addition of Phalloidin




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Regulation of Actin Dynamics by Actin-binding Proteins

Actin-binding proteins control the available pools of free Gactin 0 Thymosin binds and sequesters G-actin (usually in ADP state),

preventing polymerization

- In a typical cell, ~50% of actin is bound to thymosin - provides local flexibility for the cell to control actin dynamics by

maintaining a pool of Gactin to be used when needed - Profilin binds free G-actin and induces the exchange of ADP

for ATP, inducing polymerization

- can be locally activated for local polymerization of actin. -Thymosin and Profilin "fight for control over free Gactin and

polymerization de polymerization (3) Cofilin binds the end of ADP-state actin filaments and induces.


polymerization 6 end



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Actin Filament - (F-actin)


[@=cofilin | = Thymosin 10 = Profilin


ADP-state Lt

o end

ADP state


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Platelet Activation

Actin-binding Proteins in PLATELET ACTIVATION for blood clotting

The rapid shape change that o p latelet Activation, we

occurs in platelet activation

depends entirely Inactivated Platelets

on the in blood

Activated Platelets regulation of action dynamics

at a wound within the cell.

• 5 stages of platelet Activation: Stage 1 Platelets circulating in the blood

Gacting have large pool of ATP-state G-actin bound to profilin

CapZ -Cap protein binds to the 6 end of

filaments, blocking polymerization/ addition of the free G-actin

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Stage 21: wounding causes release of a Ca2+ signal which activates the protein Gelsolin : binds the sides of F-actin and severs it to expose . ends, not capped by Cap Stage 3rapid polymerization is induced as

free G-actin adds to the exposed 6 ends of actin filaments





Stage 4 organization of the actin filaments: -Filamin protein : crosslinks actin filaments

to form a gel - Fimbrin & Villin proteins: bundle actin

filaments to form "spikes characteristic of activated platelets

these proteins are also used for structural support in microvidi $ Stereocilia

(intestines) lear cells) Stage 5 : Activated platelets attach 1 to each other and contraction occurs by the motor myosin to form a clot

at the site of the wound

• Filamin

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Nucleation of Actin Filaments

•The rate-limiting step for polymerization of actin filaments is nucleation cells need a way to bypass the energetically unfavorable delay of nucleation so they can begin polymerization right away model system for studying actin-based motility: Listeria monocytogenes

- intracellular pathoger that causes food poisoning, killing

~500 us individuals per year - due to intracellularity, are protected from the immune system

2) sudys

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- have rocket tails that propel them around inside cells

these tails are actin-bused: the addition of Latrunculin drug

causes cessation of Listeria movement -2 hypotheses for propulsion of Listeria

O actin polymerization occuurs at ( actin polymerization occurs at

the rear tip of the actin cloud, the interface between the bacteric

pushing the entire cloud & head and actin cloud, pushing bacterium forward

the head forward while the cloud

remains site of polymerization site of polymerization

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-To determine the site of polymenzation:

. caged-rhodamine-actin: actin attached to the fluorophore

rhodamine which normally fluoresces red upon excitation; in the presence of the cage fluorescence is inhibited UV light removes the cage so fluorescence may be observed caged- rhodamine-actin was added to cells with Listeria UV light was shown on the middle of the actin cloud The distance x between the Listeria head and the uncaged rhodamine -actin increased, supporting hypothesis a that actin is nucleated and polymerized at the head /cloud interface

unlight and

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increased true site of polymerization - To determine

How bacteria induce actin nudeation $ polymerization:

• Added to test tube : f. Listeria (alive or dead)

• rhodamine-actin

• АТР L-cytosol from human platelets

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bacteria begin

to nucleate, rocket around test


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- Fractionated the platelet cytosol, purified a protein complex

required for actin polymerization = ARP 2/3 complex

- Addition of ARP 2/3 shortens the nucleation phase but

did not eliminate it

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Twith ARP 2/3 A without ARP 2/3


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• Genetic mutant screen identifiedle a transmembrane bacterial

protein ActA (Actin Assembly-inducing Protein) required for actin nucleation

- Addition of both ARP 2 and bacterial ActA was

sufficient to comupletely eliminate the nudeation phase

* ActA is andlagous

to WASP/WAVE protein in mammals for regulation of

actin nucleation




with ARP 2/3 and ActA (no nuestrony

I without ARP


& Acta

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time Lecture 16: 11/2/15

• Actin Dynamics create Forces to move cells - Dynamics at the leading edge of a migrating cell include:

1) signal: telling the cell which direction to migrate 2) nucleation of actin filaments (WASP/WAVE, ARP 2/3) 3) polymerization growth at h ends of F-actin (Profilin)

4) nucleation of branched Factin (ARP 23) cross linkage (Filamin) 5) cap 6 ends of filaments further from leading edge (CapZ). 6) depolymerization of old actin filaments far from leading edge (cofilin)

7) recycling of G-actin monomers to the leading edge Profilin) -The cycling of these dynamics serve to propel cells forward

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Interactions Between Actin and Membranes We will use the example of red blood cells to describe how actin

provides structural support to membranes - why?

- RBCs uniform, cheap to harvest - treatment with hypotonic (low salt) solution caused cells to swell



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and burst so cytoplasm leaks out and you're left with RBC_ghost's containing plasma menibrane and scaffold proteins

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• By doing SDS-PAGE analysis on RBC ghosts, spectrin proteins were discovered: long filancentous proteins that interact with transmembrane

Proteins and actin to form the scaffold of RBCS -spectrin-related family of proteins (contain spectrin repeats & interact with

0 Dystrophin: attaches actin to the ECM in skeletal muscle cells

at the plasma membrane, interacts with DGC(Dystrophin. Glycoprotein Complex) which spans the membrane to attach the ECM dystrophin mutations result in muscular dystrophy, where muscle

flexion results in "rips" in the plasma membrane ( Nesprin-1 (called ANC-1 in c. edegans): a KASH protein on the ONM

that connects cytoplasmic actin to the lamin scaffold of the nucleus thru SUN proteins in the INM.

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Myosin Motors and Force Generation

Myosins: a family of motors that hydrolyze ATP to move along actin filament

•2 classes

Conventional Myosin = Myosin-II -important in cytokinesis & muscle contraction - consists of 6 proteins a heavy chains with ATPase heads that bind.

actin filaments, and necks that each interact with a light chains and are connected to the long coiled-coil tails

light chains


neary chains

bind actin

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- Myosin-I



actin filaments towards



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2) Unconventional Myosins contain at least 17 classes of Myosin

- Myosin-I: single head motor binds membranes & actin filaments,

is regulated by Cat


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- Myosin- has a motor heads connected to coiled tails by

long necks so it can "walk" head-over-head towards h ends of actin filaments

tails bind Rab proteins allowing attachment to vesicles so myosin can carry cargo along actin filaments

v sou

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bind actin, "walk"

head over head (AP

bind Rab proteins

and vesicles carrying cargo

0 00

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• Ex) myosin-VA interacts with Rab27 to move melanosomes

(Pigments), mutations in Myosin IA result in albinism because pigments are unable to spread out in cells

Ex) another mutation in myosin-IITA causes Usher Syndrome a deafness due to disorganized stereocilia in ear hair cells


- Myosin-II is the only class of myosins that move toward the

o ends of actin filaments

associates with clathrin -coated pits at the plasma membrane during endocytosis to move vesides toward the interior of cells in the o direction of actin filaments.


The Myosin Power Stroke slides actin filaments ~5 nm.

- Attached, no ATP - rigor state myosin head

- Myosin is released when bound to ATP




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Hydrolysis of ATP → ADP+P; causes conformational change so myosin I head becomes "cocked"

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Tight binding of the cocked myosin head to the action filament



Power stroke: the Pi is released, reversing myosin's conformational change and

Sliding the actin filament vśnm The myosin head will replace ADP with ATP release the filament, and begin

the cycle again

~5 nm

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muscle contraction

• The contractile unit of a muscle is called a sarcomere

• Sarcomeres consist of thick filaments of myosin-I proteins and thin filaments of actin stabilized by Capī at their @ends forming Z-bands

- from 2-band to Z-band is one sarcomere

•Force is generated by the sliding of thin & thick filaments pasteach other

Tropomyosin protein along the sides of thin filaments blocks myosin- I from binding actin by hiding myosin-II binding sites

- when Ca 2+ ions are released from the sarcoplasmic reticulum (ER of the muscle) they signal contraction by binding the protein troponin which moves tropomyosin to expose myosin-I binding

sites on the thin filament - myosin I can then bind actin to slide thin and thick

filaments past each other, shortening the sarcomere

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myosin (thick) filament Actin (thin) filament



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Lecture 17:11/4/15

• Functions of Microtubules 1) Support for internal cell structure and organelle placement

-Ex) Golgis reside at o ends of Mis, Els reside at 2 ends 2) Transport of organelles and of resicles between organelles -Ex) the addition of colchicine to cells resulted in Golgis scattered

throughout the cell due to disorganized MTS 3) motile elements of cilia & flagella 4) Formation of the MT spindle for the movement of chromosomes

during mitosis and meiosis Ex) The organization of mits in mitosis vs. in interphase is

completely different and the Switch is fast: MTs must be dynamic Interphase




• Properties of Microtubules -exist in all eukaryotic cells - make up 10-20% of the protein in your brain -dynamic structures at steddy state there is continual assembly

and disassembly -each MT is composed of 13 protofilaments arranged in a tube

by noncovalent side-to-side interactions

building blocks of proto filaments are heterodinners of 2-tubulin

and B-tubulin 8-tubulin faces the end of MTs ; a- tubulin faces the end Both & and ß-tubulin get Energy from GTP hydrolysis: 2-tubulin is always in the GTP-state while stubulin hydrolyzes GTP and

switches from GTP-state to GDP-state and back protofilament [000000000


aßaß upda

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-Polyncerization of MTs in vitro

nucleation rate - limiting, even more difficult in Mts than

actin because 13 protofilaments must be built; centrosomes soare centers of MT nucleation in the cell

growth lelongation GTP-state tubulin has a KoN >> GDP-state tubulin,

growth is a race between addition of GTP-tubulin / which forms a cap stabilizing the 2 end) and hydrolysis of GTP-tubulin (if it catches up, the 2 end is GDP-state and depolymerization is avored

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• Regulation of Microtubule Dynamics with Drugs

Colchicine (in the meadow saffron plant): binds and sequesters free 4/B-tubulin dimers, lowering their concentration to LC

and causing depolymerization @ Taxol (in yew trees) : stabilizes MTs by binding their sides and

preventing depolymerization you an important chemotherapy drug used to block cell division

• Microtubule Nucleation and the Centrosame - Experiment Cells were treated with colchicine to depolymerize all MTs, then

colchicine was washed out and MT recovery was observed using GFP

5 mins

15 mins

+ Colchicine

after wash

after wash


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Result: All MTs originated from a MTOC: Microtubule Organizing center -The MTOC in rapidly dividing animal cells is the Centrosome

Each centrosome consists of a centrioles and peri-centrio lar material

• Each centriole is a bundle of 9 triplet Microtubules arranged in a cylinder

AB Centrosome

A has 13 protofilaments, Bec have <13 (10) protofilannents en centrioles sit at a right (900) angle to each other

peni-centriolar material poorly defined tog of

proteins surrounding the centrides

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Peri-centriolar material contains 8-TuRe (8 tubulin ring complex), the scaffold for MT nucleation with the same diameter as a MT

.8-TURC caps the o end of MTs stabilizing them and preventing

polymerization /depolymerization at that end thus o ends of Mis remain in the peri-centriolar material and the B ends extend out


* Note: not all MTOC's are centrosomes (plants and animals such as

flatworms do not have centrosomes for their MT nudeation)

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• Dynamic Instability of Microtubules -At steady state, total length of all MTs remains constant but an

individual MT alternates between growing $ shrinking at its o end -Experiment | Added to test tube:

C. fluorescently labeled tubulin 3. purified centrosomes l GTP

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•Result when allowed to reach steady state and observed under a

microscope, the end of mis was constantly growing or shrinking -The growing /shrinking state of a single MT is dependent on

the GTP cap: if it remains in GTP-state, growth will continue; if the rate of hydrolysis passes the rate of new a/B-tubulin addition,

the GDP-state tip will shrink -catastrophe: the point in time where a MT Switches from growing to

shrinking (GDP-state cap) - rescue the point in time where a MT switches from shrinking to

Growing (GTP-state cap)


growth rate slope at time 1 shrink rate - slope at tince 2


(man utsuar Im

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time (min)

• frequency of catastrophes = the number of catastrophe events observed

crudy over the time the MT was growing frequency of rescues - the number of rescue events observed over

the time the MT was shrinking - MAPs (microtubule-associated proteins) bind MTs regulate dynamic instability

MAP2: binds the side of MTs, increasing the frequency of rescue events and decreasing time spent shrinking

time (min)

^ with maps


(mm) utbust the

without Map2

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