Contraction, Locomotion (Ch 16), Shelden

Actin-dependent ATP hydrolysis by myosin generates motile force

Molec Biol Cell 5th ed. Supplemental Videos

1

The Myosin ATPase activity cycle

ATP binding releases myosin from actin (rigor is caused by lack of ATP)

ATP hydrolysis to ADP+Pi moves myosin forward

Release of Pi accompanies reattachment of myosin to a new actin subunit

Release of ADP causes force generation and movement

Fig 16-61

2

The Myosin ATPase activity cycle

Molec Biol Cell 5th ed. Supplemental Videos

3

Different Myosins Exhibit Different Motile Behaviors

All myosins except myosin VI move toward the plus

end of actin filaments

Fig 16-57

4

Contractile force is generated in all types of cells

by the action of bipolar myosin II thick filaments

Fig 16-54

Fig 16-55

5

Smooth muscle cells (and non-muscle cells) assemble myosin thick

filaments as needed by regulating myosin light chains

Fig 16-72

6

Myosin thick filaments assemble during smooth muscle cell

contraction and apply force to preassembled actin filaments

Actin filaments are attached to the cell membrane at dense plaques containing alpha

actinin and other actin associated proteins. Fujiwara et al., J. Cell Biol (1983) 96:783-795

Anti alpha actinin

7

Isolated smooth muscle cell contraction

relaxedformation of myosin

thick filaments contraction

Driska et al., Journal of Applied Physiology, Vol. 86,

Issue 1, 427-435, January 1999

8

Formation of skeletal (striated) muscle

Skeletal muscle myotube

Showing nucleiMyotube formation from myoblasts

9

Skeletal muscle cells contain thick filaments

organized into permanent sarcomeres

Fig 16-74

Fig 16-74

I-band A-band

10

Sarcomeres are highly ordered structures

Actin thin filament length is determined by nebulin

The plus ends of actin are attached to Z discs and

capped by CapZ

Minus ends of actin are protected by tropomodulin

Thick filaments are positioned by titin

Fig 16-75Fig 16-76

11

Thick and thin filaments slide past each other

during contraction, but don’t change length.

Fig 16-76

12

Regulation of sarcomere contraction

Nerve impulses cause release of acetylcholine at the junction. Binding of

acetylchline to the muscle cells opens ion changels that lead to depolarization of the

plasma membrane (sarcolema) and transverse tubules

Neuromuscular junctionsFig 16-77

13

Depolarization of the tranverse tubules stimulates release of calcium from the

sarcoplasmic reticulum (a modified ER) into the cytoplasm

Fig 16-77

14

Calcium binds to the troponin complex and causes

attached tropomyosin to move away from actin

Troponin C: binds calcium

Troponin T: attaches

tropomyosin

Troponin I: inhibits myosin

activity by changing

shape

Myosin binding sites on

actin are revealed by

movement of

tropomyosinFig 16-78

15

Smooth and Striated Muscle

Smooth Striated

Actin meshwork anchored in dense

plaques by alpha-actinin. Myosin

not assembled when relaxed.

Individual cells Multicellular syncitia (skeletal)

Actin bundles anchored in Z lines

using alpha-actinin. Myosin

assembled in thick filaments.

Simple cell architecture Myofibrils with M-lines, Z-lines, A bands,

I bands. Transverse tubules and

sarcoplasmic reticulum

Contraction initiated by activation

of myosin light chain kinase, causes

mini-myosin filaments to form

Neurons depolarize sarcolema.

Depolarization enters T-tubule system,

causing calcium release from sarcoplasmic

reticulum. Calcium binds to Troponin

complex, moving tropomyosin out of the

myosin binding site on actin filaments.

Cells

Actin

Organiz

ation

Trigger

16

Movement of cells involves protrusion, attachment, transport of

the cell components and retraction of trailing attachments.

Fig 16-86

17

Amoeboid cell movement

Molec Biol Cell 5th ed. Supplemental Videos

18

Keratocyte movie

Rachael Ream, George Somero & Julie Theriot.

http://cmgm.stanford.edu/theriot/movies.htm#Current

19

Membrane protrusion is driven by actin

polymerization

Lysteria movement (J. Theroit, Ph.D.)

20

Cell Locomotion

Lysteria movement (J. Theroit, Ph.D.)

http://cmgm.stanford.edu/theriot/IFLmBig.jpg

21

Protrusion

Lamellar (and pseudopod)

protrusion of the plasma

membrane is driven by

cyclical actin polymerization

at the cell periphery and

depolymerization in more

interior sites

Cofilin prefers ADP

actin

22

Attachment is mediated by formation

of “focal adhesions”

Fig 19-45

Elements of Focal Adhesions:

1. Transmembrane proteins

(integrins)

2. Regulatory elements

3. Coupling proteins talin and

vinculin

4. Actin

23

Crawling movement of fibroblasts

Copyright Eric Shelden

24

Focal adhesions in a moving

fibroblast

Bhatt et al., Journal of Cell Science 115, 3415-3425 (2002)

25

Forward transport of organelles is mediated by

motor proteins (myosins, dyneins and kinesins)

Wu et al., Cell Biol., Volume 143, Number 7,

December 28, 1998 1899-1918

See also http://micro.magnet.fsu.edu/moviegallery/pondscum/protozoa/amoeba/index.html

26

Fig 16-94

Retraction of trailing cellular attachments is mediated

by myosin II-dependent contraction at the rear of cells

Myosin I (green) and Myosin

II (red) in a motile cell

Da

vid K

necht, U

nive

rsity o

f Conne

cticut

Wild-t

ype a

moeba

Myosi

n II

nul

ls

27

(Video on next slide)

Video