1. Announcements Review sessions here today, Monday, 6-8PM Exam Wednesday covers molecular biology through endocytosis I will upload exam 3 from Gard last fall

2. 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 head Actin 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

3. 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 directions Actin filaments don’t slide back because other myosins are bound

4. 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)… ZMZ Relaxed myofibril +ATP +Ca 2+ Sarcomere shortens… I-bands shorten… A-bands unchanged… I-bandA-band I-band - + - + ZZ ZZM ECB 17-44

5. Filament sliding leads to contraction

6. Contraction is regulated by toponin and tropomyosin Tropomyosin filament binds along actin filament Troponin complex binds to tropomyosin In the absence of Ca 2+, tropomyosin blocks myosin binding site In presence of Ca 2+, Troponin C binds Ca 2+ Conformational change of troponins and tropomyosin uncovers myosin binding site Myosin walks on actin and myofibril contracts Removal of Ca 2+ restores inhibition ECB 17-48 Where does Ca 2+ come from?

7. Myofibril contraction is stimulated by release of Ca 2+ from the “sarcoplasmic reticulum MyofibrilPlasma membraneT-system T-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 Ca 2+ reservoir Signal from neuron causes Ca 2+ release thru voltage-gated Ca 2+ channels. Ca 2+ stimulates myofibril contraction. Contraction is terminated by pumping Ca 2+ back into the SR… ECB 17- 46 17.13-muscle_contraction.mov

8. Calcium release occurs through a voltage- gated channel ECB 17-47

9. L19 Non-Muscle Actin

10. “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 and lamellapodia Stress fibers and focal contacts Contractile ring Microvilli Adapted 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

11. 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

12. Modulating the assembly/function of actin:actin-binding proteins G-actin F-actin Nucleating proteins Monomer- binding proteins ECB 17- 32 End-capping proteins Side-binding proteins Severing proteins Motors Bundling proteins Cross-linking proteins Thymosin  4 ARP 2/3 complex Gelsolin/villin  -Actinin Filamin Tropomyosin Myosins CapZ

13. 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

14. Visualizing actin organization in cells… Fixed (dead cells) Electron microscopy… high resolution but limited area 1.Fluorescence microscopy a.Antibodies and immunofluorescence microscopy… b.Fluorescent Phalloidin Toxin from the “death angel” mushroom, specifically binds F-actin. Lamellipodia Filopodia lamellipodium Stress fibers Live cells - Fluorescent actin

15. 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 + -

16. 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?

17. -+ ARP2/3 complex ARP2 ARP3 More 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…

18. 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)

19. 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

20. G-actinF-actinARP2/3 Capping 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

21. Actin filament assembly drives extension of lamellipodia G-actinF-actinARP2/3 Capping protein Actin filament assembly drives forward membrane extension Actin filaments disassemble behind the leading edge

22. Actin filament assembly drives extension of lamellipodia G-actinF-actinARP2/3 Capping protein Actin filament assembly drives forward membrane extension… Actin filaments disassemble behind the leading edge… Net disassembly Net assembly Actin assembly also drives movement of intracellular parasites 17.9-listeria_parasites.mov

23. 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

24. Modulating the assembly and function of actin: actin-binding proteins G-actin F-actin Nucleating proteins Monomer- binding proteins ECB 17- 32 End-capping proteins Side-binding proteins Severing proteins Motors Bundling proteins Cross-linking proteins Thymosin  4 ARP 2/3 complex Gelsolin/villin  -Actinin Filamin Tropomyosin Myosin CapZ

25. 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

26. 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

27. MCB figure 22-10 © Freeman Publishing See 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”

28. 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

29. Modulating the assembly and function of actin: actin-binding proteins G-actin F-actin Nucleating proteins Monomer- binding proteins Adapted from ECB figure 16-27 End-capping proteins Side-binding proteins Severing proteins Motors Bundling proteins Cross-linking proteins Thymosin  4 ARP 2/3 complex Gelsolin/villin  -Actinin Filamin Tropomyosin Myosins CapZ use energy of ATP hydrolysis to walk along actin filaments…

30. Non-muscle cells contain multiple myosins Small bipolar filaments of ~30-40 molecules. Conventional (Type II) Single-headed “Type I” myosin Myosin I Unconventional myosin What roles do different myosins play in cells?

31. Myosin-I and intracellular transport Main 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

32. 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!

33. 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

34. 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 Contraction Myosin 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…

35. 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

36. Actin Binding Proteins in Plants G-actin F-actin Nucleating proteins Monomer- binding proteins ECB 17- 32 End-capping proteins Side-binding proteins Severing proteins Motors Bundling proteins Cross-linking proteins Profilin ARP 2/3 complex Gelsolin/villin  -Actinin Filamin Tropomyosin Myosin Cap32 By genome sequencing of Arabidopsis, all major classes are present Function of most have not yet been studied Why tropomyosin in plants?