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Review sessions here today, Monday, 6-8PM

Exam Wednesday covers molecular biology through endocytosis

I will upload exam 3 from Gard last fall

5.

ADP

1.Myosin is an “actin-dependent” ATPase that acts as a “molecular motor”

1. No nucleotide. Myosin head is tightly bound to actin (“rigor”)2.

ATP

3.

4.

Pi

Myosin headActin filament

Thick filament

“-” “+”

“-” “+”

2. ATP binding releases myosin from actin

3. ATP hydrolysis “cocks” myosin

4. Pi is released, strengthening binding of myosin to actin

5. Myosin binds actin tightly and undergoes “power stroke” releasing ADP

Myosin heads “walk” towards “barbed” (“plus”) -end of actin filament

17.7-myosin.mov

Muscle contraction involves actin-myosin II sliding

ECB 17-41

Thick filaments are bipolar; myosin heads on two sides ratchet in opposite directions

Both sides ratchet toward + end of actin (myosin is a + end directed motor)

Causes actin filaments to slide in opposite directionsActin filaments don’t slide back because other myosins are bound

Contracted myofibril

Myofibril contraction results from sliding of thin and thick filaments

Thick filaments

Thin filaments

Filaments slide as myosin heads walk toward plus-ends of thin filaments (towards Z-lines)…

ZMZRelaxed myofibril

+ATP+Ca2+

Sarcomere shortens…I-bands shorten…A-bands unchanged…

I-bandA-band

A-band I-band

- +-+

Z Z

Z ZM

ECB 17-44

Filament sliding leads to contraction

Contraction is regulated by toponin and tropomyosin

Tropomyosin filament binds along actin filament

Troponin complex binds to tropomyosinIn the absence of Ca2+, tropomyosin blocks myosin binding siteIn presence of Ca2+, Troponin C binds Ca2+

Conformational change of troponins and tropomyosin uncovers myosin binding site

Myosin walks on actin and myofibril contractsRemoval of Ca2+ restores inhibition

ECB 17-48

Where does Ca2+ come from?

Myofibril contraction is stimulated by release of Ca2+ from the “sarcoplasmic

reticulum

Myofibril

Plasma membrane

T-systemT-tubules formed from invaginations of plasma membrane. The T-system carries “nerve impulse” into muscle fiber…

“Sarcoplasmic reticulum (SR)”

Derivative of ER

SR serves as a Ca2+ reservoir

Signal from neuron causes Ca2+ release thru voltage-gated Ca2+ channels.

Ca2+ stimulates myofibril contraction.

Contraction is terminated by pumping Ca2+ back into the SR…

ECB 17- 46

17.13-muscle_contraction.mov

Calcium release occurs through a voltage-gated channel

ECB 17-47

L19 Non-Muscle Actin

“Non-muscle” actin is abundant in non-muscle cells

Microvilli: “brush border” epithelia of intestine (increased surface area), “hair cells” of inner ear (sound detection)

Filopodia andlamellapodia

Stress fibers andfocal contacts

Contractile ringMicrovilliAdapted from ECB figure 17-29

Stress fibers: adhesion and cell shape (fibroblasts growing in vitro)

Filopodia and lamellipodia: at leading edge of moving cells in vitro and in vivo

Contractile ring: division of the cytoplasm in animal cells

Intracellular transport: myosin-coated organelles move along actin filaments in plants

Cortical actin: just beneath plasma membrane of most eukaryotic cells

Actin filaments in cells are often dynamic

Grow and shorten rapidly (minutes), but not as fast as microtubules

Regulated by 1 - ATP binding to actin

+ end- end

ECB 17-31

ATP bound actin adds at + end

ATP is hydrolyzed and ADP actin destabilizes filament

Filaments with more ADP-actin are less stable and tend to depolymerize

2 - Dynamics also regulated by actin-binding proteins

Modulating the assembly/function of actin:actin-binding proteinsG-actin

F-actin

Nucleating proteins

Monomer-binding proteins

ECB 17- 32

End-capping proteins

Side-binding proteins

Severing proteins

Motors

Bundling proteinsCross-linking proteins

Thymosin 4ARP 2/3 complex

Gelsolin/villin

-ActininFilamin

Tropomyosin

Myosins

CapZ

The surface of a moving cell is very dynamic

Lamellipodia (“ruffles”): sheet-like extensions of the cell’s leading edge

Filopodia (“microspikes”): “finger-like”

Motility is dependent upon actin assembly: inhibited by cytochalasins and latrunculin (fungal toxins that block actin assembly)

How can we visualize actin in cells?

Lamellipodia

Filopodia

01.1-keratocyte_dance.mov

Visualizing actin organization in cells…

Fixed (dead cells)• Electron microscopy… high resolution but limited area1. Fluorescence microscopy

a. Antibodies and immunofluorescence microscopy…b. Fluorescent Phalloidin

• Toxin from the “death angel” mushroom, specifically binds F-actin.

Lamellipodia

Filopodia Filopodia

lamellipodium

Stress fibers

Live cells - Fluorescent actin

F-actin is concentrated in filopodia and lamellipodia…

Branched meshwork of short actin filaments in lamellipodia

Bundles of actin filaments in filopodia

Barbed (+) end of actin filaments oriented towards plasma membrane

Lamellipodia

Filopodia

+

-

Orientation of actin filaments in migrating cell

ECB 17-34

Arrowhead points to + end

Misleading, actually a branched network

What do you guess drives extension of lamellipodia and filopodia?

- +

ARP2/3complex

ARP2

ARP3More proteins

F-actin

Actin filament assembly - Arp 2/3 complex; (all eukaryotes?)

G-actin

VERY SLOW!

Assembly of actin filaments from “pure” subunits is very slow

Rapid elongation

Actin assembly is facilitated by the ARP2/3 complex

Two actin-related proteins (ARPs 2 and 3)

…and several other polypeptides in a macromolecular complex.

ARP2/3 “nucleates” actin filament assembly by providing a template or “seed” that can be elongated by subunit addition.

The ARP2/3 complex caps the minus-end of actin filaments…

ARP2/3 promotes actin assembly at the plasma membrane

ARP2/3 promotes nucleation of actin filaments

Filaments continue to elongated by addition of subunits at their plus ends

Continued elongation drives membrane extension

Bundling and cross-linking proteins bind to and organize actin filaments

G-actin F-actin ARP2/3

End binding protein

Activated ARP2/3 complex

Bundling/cross-linking protein

Subunits add to plus-ends

++

+

Filopodia

Plus-ends (barbed)

May also drive extension of tip-growing cells of non-animals (fungal hyphae, pollen tubes)

Arp 2/3 promotes actin nucleation & branching;

Activated ARP2/3 binds to side of exisiting actin filaments

…and nucleates new filaments from the side (branches)

New, elongating filaments are not yet capped

Network depolymerizes in rear

Branching networks are common in all eukaryotes thus far examined

ECB 17-36

G-actin F-actin ARP2/3Capping protein

Actin filament assembly drives forward membrane extension…

Actin filaments disassemble behind the leading edge…

Actin filament assembly drives extension of lamellipodia

Leading edge of cell membrane

Actin filament assembly drives extension of lamellipodia

G-actin F-actin ARP2/3Capping protein

Actin filament assembly drives forward membrane extension

Actin filaments disassemble behind the leading edge

Actin filament assembly drives extension of lamellipodia

G-actin F-actin ARP2/3Capping protein

Actin filament assembly drives forward membrane extension…

Actin filaments disassemble behind the leading edge…

Net

dis

ass

em

bly

Net

ass

em

bly

Actin assembly also drives movement of intracellular parasites

17.9-listeria_parasites.mov

Actin assembly is regulated by Rho family GTPases

“molecular switches”

Rac activation causes formation of massive lamellipodium

Cdc42 (GTPase family) causes formation of filapodia

ECB 17-39

Modulating the assembly and function of actin: actin-binding proteinsG-actin

F-actin

Nucleating proteins

Monomer-binding proteins

ECB 17- 32

End-capping proteinsSide-binding proteins

Severing proteins

Motors

Bundling proteinsCross-linking proteins

Thymosin 4ARP 2/3 complex

Gelsolin/villin

-ActininFilamin

Tropomyosin

Myosin

CapZ

Bundling vs. cross-linking: -actinin vs filamin

-Actinin (dimer) is rod-shaped with two actin binding sites:– Forms loose parallel bundles of

actin filaments…– Z-line of striated muscle– Stress fibers and focal contacts

Filamin (dimer) is has two actin binding sites on long flexible arms:– Forms cross-linked actin “gels”…– Stress fibers and focal contacts– Smooth muscle

See ECB figure 17-37

Actin filament bundles called “stress fibers” are common in cultured fibroblasts

Organized by actin binding proteins…

Bundling (-actinin)

Crosslinking (filamin)

Type II myosin

Actin cross-linking proteins (filamin)

Actin bundling proteins (-actinin)

Myosin II

Polarity of actin filaments in bundle is not uniform

ECB 17-37

MCB figure 22-10 © Freeman PublishingSee ECB figure 16-30

Stress fibers are lnked to the extracellular matrix at “focal contacts”

Actin cross-linking proteins (filamin)

Actin bundling proteins (-actinin)

Myosin II

Polarity of actin filaments is not uniform

“Focal contacts” aka “focal adhesions”

Integrins link actin filaments to the extracellular matrix in animals cells

Integrins

Extracellular matrix

Plasma membrane

Linker proteins

Actin filaments

Actin filaments in plant cells are also linked to the ECM (cell wall), but the linking molecules are different

There are many other linkages from actin to ECM in animal cells

Modulating the assembly and function of actin: actin-binding proteinsG-actin

F-actin

Nucleating proteins

Monomer-binding proteins

Adapted from ECB figure 16-27

End-capping proteinsSide-binding proteins

Severing proteins

Motors

Bundling proteinsCross-linking proteins

Thymosin 4ARP 2/3 complex

Gelsolin/villin

-ActininFilamin

Tropomyosin

Myosins

CapZ

use energy of ATP hydrolysis to walk along actin filaments…

Non-muscle cells contain multiple myosins

Small bipolar filaments of ~30-40 molecules.

Conventional (Type II)

Single-headed “Type I” myosinMyosin IUnconventional myosin

What roles do different myosins play in cells?

Myosin-I and intracellular transportMain form of transport in many non-animal cells (animal cells primarily use microtubule based motility)

Myosin-I on vesicles moves vesicle toward + end of actin filament

Anchored myosin-I can move filament

Always ratchets towards + end

ECB 17-38

Vesicles, ER, and other organelles move along actin cables in subcortical cytoplasm

Myosin-I powers cytoplasmic streaming in green algae and plants

Cytoplasmic streaming in Elodia…

Powered by myosin-I; speeds up to 100 µm per second!

Myosins and cell motility

In Dictyostelium (cellular slime mold)

Myosin II

Myosin I

•Type II myosin (red) is found in the “tail”

•Type I myosin (green) is found in the leading edge

A model for motility using actin assembly and myosin motors

Actin

Actin network in cortex

Lamellipodium

Focal contact

Actin polymerization extends lamellipodium (myosin-I?)

New focal contact

New focal contact

ContractionMyosin II

Actin assembly

Focal contact and actin disassembly

Cortex under tension

ECB 17-33

Movement of G-actin

Myosin II

Myosin I

MBoC (4) figure 16-93

Movie 1… Movie 2…

Non-muscle cells contain multiple “unconventional” myosins

Adapted from MBoC (4) figure 16-54 © Garland Publishing

17 “subfamilies” of myosins:

See ECB figure 16-32

Specialized for specific functions

Actin Binding Proteins in PlantsG-actin

F-actin

Nucleating proteins

Monomer-binding proteins

ECB 17- 32

End-capping proteins

Side-binding proteins

Severing proteins

Motors

Bundling proteinsCross-linking proteins

ProfilinARP 2/3 complex

Gelsolin/villin

-ActininFilamin

Tropomyosin

Myosin

Cap32

By genome sequencing of Arabidopsis, all major classes are presentFunction of most have not yet been studied

Why tropomyosin in plants?