BIOL 200 (Section 921) Lecture # 11 [July 4, 2006] UNIT 8: Cytoskeleton Reading: ECB, 2nd ed. Chap 17. pp 573-606; Questions 17-1, 17-2, 17-12 to 17-23.

BIOL 200 (Section 921)Lecture # 11 [July 4, 2006]

UNIT 8: Cytoskeleton

• Reading:

• ECB, 2nd ed. Chap 17. pp 573-606; Questions 17-1, 17-2, 17-12 to 17-23.

• ECB, 1st ed. Chap 16. pp 513-542; Questions 16-1, 16-2, 16-10 to 16-21.

UNIT 8: Cytoskeleton - Objectives1. Two major roles for cytoskeleton - skeletal support and motility 2. Distinguish between three major cytoskeletal systems - intermediate filaments,

microtubules and actin filaments (microfilaments). 3. Be able to describe how intermediate filaments are assembled from polypeptides

to form a microscopically visible fibre. 4. Know major cell function of intermediate filaments 5. Know structure of microtubules, their process of assembly, the meaning of plus

and minus ends, and the role of MTOCs. 6. Understand dynamic instability and how it may be applied to microtubules and

microtubule containing structures. 7. Understand the role of GTP in the generation and control of dynamic instability of

microtubules. 8. Understand how motor proteins work and how their movement relates to the

polarity of their molecular substrates 9. Be able to describe the structure of flagella and the molecular basis of flagellar

bending 10. assembly of actin filaments, 11. dynamic instability of actin filaments; comparison with that of microtubules. 12. role of actin filaments in formation of the cell cortex, and regulation of cell

structure and movement, 13. myosins and the myosin activity cycle as it relates to muscle.

The cytoskeleton is a network of filaments that regulatesa cell’s shape, strength and movement [Fig. 1-27]

Actin filaments Microtubules Intermediatefilaments

An overview of the cytoskeleton [Fig. 17-2]

Intermediate Filaments-tough ropes

Microtubules-big hollow tubes support cell structures

Actin Microfilaments- helical polymers involved in movement/shape

17_02_01_protein_filament.jpg

17_03_Interm_filaments.jpgIntermediate filaments form a strong, durable network in

the cytoplasm of the cell [Fig. 17-3]

Intermediate keratin filaments (green, fluorescent) from different cells are

connected through the desmosomes [Immunofluorescence micrograph]

A drawing from the electronmicrograph showing the bundlesof intermediate filaments throughthe desmosomes

Fig. 21-27: desmosomes connect epidermal cells

Intermediate filaments

Plasma membranes

cadherins

Assembly of intermediate filaments involves coiled coil dimers [Fig. 17-4]

monomer

dimer

17_05_strengthen_cells.jpgIntermediate filaments strengthen animal cells [Fig. 17-5]

17_06_filam_categories.jpg

17_02_02_protein_filament.jpg

Microtubules (MTs)

MTs grow from MT organizing centers [Fig. 17-9]

centrosome

Spindle poles

Basal bodies

cilia

Structure Long, hollow cylinders made of 13 protofilaments

Diameter 25 nm

Protein Subunit Alpha & Beta Tubulin = globular Proteins

Location in Cell -end: attached to centrosome (or Microtubule organization Center)

+ end is free

Function 1. Chromosome movement during cell division

2. Maintaining cell shape

3. Movement of vesicles

4. Movement of cilia and flagella

5. Positioning organelles within cell

Drug Sensitivity A) Colchicine: binds free tubulin and inhibits formation of microtubulues

B) Taxol: stabilizes microtubules by preventing loss of subunits

MICROTUBULES

-The growing end of the microtubule (MT), at the top, has subunits arranged with the beta-tubulin on the outside. The subunits in the microtubule all show a uniform polarity

tubulin dimer

-Microtubules, like poly-peptides and nucleic acids, grow by addition of subunits at only one end:Growth = plus endNo Growth = minus end.

[Fig. 17-10]

Growingend

Non-growingor fixed end

Fig. 16-11 Alberts MBOC-

GTP-bound tubulin packs efficiently into protofilament

GDP-bound tubulin bind less strongly to each other-depolymerize MT

GTP hydrolyzes to GDP in MT

17_11_centrosome.jpg

The centrosome is the major MT-organizing Center. It contains nucleating sites (rings of of γ-tubulin) which serve

as starting point for growth of MTs [Fig. 17-11]

Centrioles are arrays of short MTs and are identical to basal bodies.

-Tubulin (a G-protein) dimers carrying GTP (red) bind more tightly to one another than tubulin dimers carrying GDP (dark green). -microtubules with freshly added tubulin dimers and GTP keep growing. -when microtubule growth is slow, the subunits in this "GTP cap" will hydrolyze their GTP to GDP before fresh subunits loaded with GTP have time to bind. The GTP cap is then lost-the GDP-carrying subunits are less tightly bound in the polymer and are readily released from the free end, so that the microtubule begins to shrink continuously.

GTP cap leads to stability and growth of MTs

GTP cap

Dynamic instability (loss of GTP cap) leads to MT shrinking

Fig. 17-13Inhibitor: COLCHICINE Inhibitor: TAXOL

Three classes of MTs make up the mitotic spindle at metaphase [Fig. 19-13]

Aster MTs Kinetochore MTs Interpolar MTs

Sister chromatids separate at anaphase [Fig. 19-17]

I. II.

17_12_grows_shrinks.jpg

Each microtubule filament grows and shrinks

independent of its neighbors [Fig.17-12]

A model of microtubule assembly [Becker et al. The World of the Cell]

17_14_polarize_cell.jpgThe selective stabilization of MTs can

polarize a cell [Fig. 17-14]

•A MT can be stabilized by attaching its plus end to a capping protein or cell structure that prevents tubulin depolymerization

•This is how organelles are positioned in cells

Motor proteins [Dynein and Kinesin] transport vesicles along MTs in a nerve cell [Fig. 17-15]

cell body

MT

axon terminal

+-

dynein kinesin

Nerve cell polarity maintained by microtubules

Motor proteins [Dynein and Kinesin] move along MTs using their globular heads [Fig. 17-17]

dynein

dynein

kinesin

kinesin

17_18_motor_proteins.jpgMotor proteins transport their cargo

along MTs [Fig. 17-18]

Kinesins move ER outward and Dyneins move Golgi inward to maintain cell structure [Fig. 17-23]

ER

MTs

kinesins

Golgi

MTs

dynein

ER

Golgi

MT

Nucleus

17_22_kinesin_moves.jpg

Kinesin walks along a MT [Fig. 17-22]

Moves in aSeries of 8 nm stepsHeads

Kinesin-GFPmoves alonga MT

Motor proteins• Two families of motor proteins are involved in

moving vesicles and other membrane-bound organelles along MT tracks

• Both binding sites for tubulin (head) and for their cargo (tail)

• Both use ATP hydrolysis to change conformation and move along MT

• Kinesins move vesicles to plus end of MT away from centrosome [e.g. Kinesins pull ER ouward along MTs]

• Dyneins move vesicles towards minus end of MT, towards the centrosome [e.g. Dyneins pull the Golgi apparatus towards the centre of the cell]

Cilia and Flagella• An array of stabilized

MTs and MT-associated proteins (MAPS)

• Same structure throughout all kingdoms.

• Cilia are short and many. Flagella are long, single or paired.

• Air pollution and cigarette smoking can cause loss of cilia on epithelium of the respiratory tract.

Ciliated epithelium in airway [Fig. 17-24]

Flagella propel a sperm cell [Fig. 17-26]

17_27_9_+_2_array.jpgMTs in a cilium or flagellum are arranged

In a “9 + 2” array [Fig. 17-27]

•9 Doublet MTs and 2 central singlets•Many different MAPs including radial spokes, central sheath element, nexin links, dynein arms•Dynein hydrolyzes ATP and generates a sliding force between MT doublets

17_28_dynein_flagell.jpg

The movement of dynein causes bending of flagellum

[Fig. 17-28]Linkers removed

17_02_03_protein_filament.jpg

Actin Filaments [Fig. 17-2]

17_29_Actin_filaments.jpgDistribution of actin filaments in different cells

Determines their shape and function [Fig. 17-29]

Microvilli in Intestine (increase surface area)

Contractile bundlesin cytoplasm

Sheetlike (lamellipodia)and fingerlike (filipodia)protrusionsof a moving cell [importantin cell crawling, endo- andexo-cytosis

Contractile ring during cell divn.

17_30_protein threads.jpg

Two F-actin strands wind around each other to form an actin filament [Fig. 17-30]

Twist-repeatingdistance

Actin with bound ADP

minus end

Actin with bound ATP

plus end

ATP hydrolysis induce dynamic instability of actin filaments [Fig.17-31]

Cytochalasin D: A fungal metabolite, Inhibits the polymerization of actin microfilaments

Phalloidin: A cyclic peptide from the deathcap fungus, Amanita phalloides, inhibits the depolymerization of actin, thereby stabilizing actin microfilaments

Microfilaments or Actin Filaments

• Distribution: in bundles lying parallel to plasma membrane

• Diameter: 7 mm• Structure: made of a small globular protein known

as G-actin• Polymerizes into filaments known as F-actin• Two F-actin molecules wind around each other to

form a microfilament• Show structural polarity• Show dynamic instability• Associate with actin-binding proteins

17_32_Actin_binding.jpg

Actin-binding proteins regulate the behavior of actin filaments [Fig. 17-32]

(e.g. thymosin and profilin)

(e.g. gelsolin)

Actin polymerization pushes cell edge forward, contraction pulls cell body along [Fig. 17-33]

cortex lamellipodia filopodia

Actin in amoeboid movement of a fibroblast [Fig. 17-34]

Filopodium grows by nucleation of actin microfilaments [Fig. 16-29, ECB 1st ed.]

nucleation complex at PM

Growing filopodium

Growing microfilament

monomers added

17_36_actin_meshwork.jpg

Association of actin and actin related proteins pushes forward lamellipodium

17_38_myosin_I.jpg

Roles of actin-dependent motor protein, myosin I [Fig. 17-38]

Myosin I: Move a vesicle relative to an actin filament.

Myosin I: Move an actin filament.

The head group of myosin I walks towards the plus end of the actin filament.

17_40_Myosin_II.jpgMyosin-II molecules can associate with one another to

form myosin filaments [Fig. 17-40]

[Coiled-coil]

Bipolar myosin filament

Tails

17_41_slide_actin.jpgRoles of actin-dependent motor protein, myosin II [Fig. 17-38

Myosin II: Regulate contraction – move actin filaments relative to each other.

The head group of myosin II walks towards the plus end of the actin filament.

Myofibrils made up of actin and myosin II packed into chains of sarcomeres [Fig. 17-42]

Muscle contraction depends on bundlesof actin and myosin

Sarcomeres (contractile units of muscle) are arrays of actin and myosin [Fig. 17-43]

Z disc: attachment pointsFor actin filaments

17_44_Muscles contract.jpg

Muscles contract by a sliding-filament mechanism [Fig. 17-44]

+ +

The myosin heads walk toward the plus end of the adjacent actin filamentdriving a sliding motion during muscle contraction.

17_45_myosin_walks.jpg

1. The Myosin head tightly locked onto an actin filament.

2. ATP binds to the myosinhead. The Myosin head released from actin.

3. The myosin head displaced by 5 nm. ATP hydrolysis.

4. The myosin head attachesto a new site on actin filament.Pi released. Myosin headregains its originalconformation (power stroke).ADP released.

5. The myosin head is again locked tightly to the actin filament.

Experimental Methodology, Techniques and Approaches for

Studying the Cytoskeleton

1. Modern microscopy techniques

2. Drugs and mutations to disrupt cytoskeletal structures

Modern microscopy techniques to study cytoskeleton

1. Immunofluorescence microscopy: Primary antibodies bind to cytoskeletal proteins. Secondary antibodies labeled with a fluorescent tag bind to the primary antibody. Cytoskeletal proteins glow in the fluorescence microscope. [Fig. A fibroblast stained with fluorescent antibodies against actin filaments].

2. Fluorescence techniques: Fluorescent versions of cytoskeletal proteins are made and introduced into living cells. Flurescence microscopy and video cameras are used to view the proteins as they function in the cell [Fig.Fluorescent tubulin molecules form MTs in fibroblast cells].

3. Computer-enhanced digital videomicroscopy: High resolution images from a video camera attached to a microscope are computer processed to increase contrast and remove background features that obscure the image. [ Several MTs processed to make them visible in detail].

4. Electron microscopy: EM can resolve individual filaments prepared by thin section, quick-freeze deep- etch, or direct-mount techniques. [Bundles of actin filaments in a fibroblast cell prepared by the quick-freeze deep-etch method].

Becker et al. The World of the Cell

Drug Treatments1. Colchicine: An alkaloid from the Autumn

crocus, Colchicum autumnale). Binds to tubulin monomers and prevents polymerization in MTs.

2. Taxol: from the Pacific Yew tree, Taxus brevifolis binds tightly to MTs and stabilizes them. It prevents MTs from dissociating.

3. Cytochalasin D: A fungal metabolite, inhibits the polymerization of actin microfilaments.

4. Phalloidin: A cyclic peptide from the death cap fungus, Amanita phalloides, inhibits the depolymerization of actin, thereby stabilizing actin microfilaments