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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 - Objectives.

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Biol 200 section 921 lecture 11 july 4 2006
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 objectives
UNIT 8: Cytoskeleton - Objectives

  • Two major roles for cytoskeleton - skeletal support and motility

  • Distinguish between three major cytoskeletal systems - intermediate filaments, microtubules and actin filaments (microfilaments).

  • Be able to describe how intermediate filaments are assembled from polypeptides to form a microscopically visible fibre.

  • Know major cell function of intermediate filaments

  • Know structure of microtubules, their process of assembly, the meaning of plus and minus ends, and the role of MTOCs.

  • Understand dynamic instability and how it may be applied to microtubules and microtubule containing structures.

  • Understand the role of GTP in the generation and control of dynamic instability of microtubules.

  • Understand how motor proteins work and how their movement relates to the polarity of their molecular substrates

  • Be able to describe the structure of flagella and the molecular basis of flagellar bending

  • assembly of actin filaments,

  • dynamic instability of actin filaments; comparison with that of microtubules.

  • role of actin filaments in formation of the cell cortex, and regulation of cell structure and movement,

  • myosins and the myosin activity cycle as it relates to muscle.


Biol 200 section 921 lecture 11 july 4 2006

The cytoskeleton is a network of filaments that regulates

a cell’s shape, strength and movement [Fig. 1-27]

Intermediate

filaments

Actin filaments

Microtubules


Biol 200 section 921 lecture 11 july 4 2006

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

Actin Microfilaments- helical polymers involved in movement/shape

Microtubules-big hollow tubes support cell structures

Intermediate Filaments-tough ropes



17 03 interm filaments jpg

Intermediate filaments form a strong, durable network in

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

17_03_Interm_filaments.jpg

Intermediate keratin filaments (green,

fluorescent) from different cells are

connected through the desmosomes

[Immunofluorescence micrograph]

A drawing from the electron

micrograph showing the bundles

of intermediate filaments through

the desmosomes


Biol 200 section 921 lecture 11 july 4 2006

Fig. 21-27: desmosomes connect epidermal cells

cadherins

Intermediate filaments

Plasma membranes


Biol 200 section 921 lecture 11 july 4 2006

monomer

dimer

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




17 02 02 protein filament jpg

Microtubules (MTs)

17_02_02_protein_filament.jpg


Biol 200 section 921 lecture 11 july 4 2006

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

cilia

centrosome

Basal bodies

Spindle poles



Biol 200 section 921 lecture 11 july 4 2006

-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

b

a

tubulin dimer

Growing

end

  • Microtubules, like poly-

  • peptides and nucleic acids, grow by addition of

  • subunits at only one end:

  • Growth = plus end

  • No Growth = minus end.

Non-growing

or fixed end

[Fig. 17-10]


Biol 200 section 921 lecture 11 july 4 2006

Fig. 16-11 Alberts MBOC- subunits arranged with the beta-tubulin on the outside. The subunits in the microtubule all show a uniform

GTP-bound tubulin packs efficiently into protofilament

GTP hydrolyzes to GDP in MT

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


17 11 centrosome jpg

The subunits arranged with the beta-tubulin on the outside. The subunits in the microtubule all show a uniform centrosomeis 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]

17_11_centrosome.jpg

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


Biol 200 section 921 lecture 11 july 4 2006

GTP cap leads to stability and growth of MTs subunits arranged with the beta-tubulin on the outside. The subunits in the microtubule all show a uniform

Dynamic instability (loss of

GTP cap) leads to MT shrinking

Inhibitor: COLCHICINE

GTP cap

Inhibitor: TAXOL

Fig. 17-13

  • 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.


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

Aster MTs

Kinetochore MTs

Interpolar MTs



17 12 grows shrinks jpg

Each microtubule filament grows and shrinks metaphase [Fig. 19-13]

independent of its neighbors [Fig.17-12]

17_12_grows_shrinks.jpg


Biol 200 section 921 lecture 11 july 4 2006

A model of microtubule assembly metaphase [Fig. 19-13]

[Becker et al. The World of the Cell]


17 14 polarize cell jpg

The selective stabilization of MTs can metaphase [Fig. 19-13]

polarize a cell [Fig. 17-14]

17_14_polarize_cell.jpg

  • 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


Biol 200 section 921 lecture 11 july 4 2006

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

cell body

axon terminal

MT

-

+

dynein

kinesin

Nerve cell polarity maintained by microtubules


Biol 200 section 921 lecture 11 july 4 2006

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

dynein

kinesin

dynein

kinesin


17 18 motor proteins jpg

Motor proteins transport their cargo their globular heads [Fig. 17-17]

along MTs [Fig. 17-18]

17_18_motor_proteins.jpg


Biol 200 section 921 lecture 11 july 4 2006

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

ER

MT

ER

Golgi

Golgi

Nucleus

MTs

MTs

kinesins

dynein


17 22 kinesin moves jpg

Kinesin walks along a MT [Fig. 17-22] maintain cell structure [Fig. 17-23]

17_22_kinesin_moves.jpg

Moves in a

Series of

8 nm steps

Heads

Kinesin-GFP

moves along

a MT


Motor proteins
Motor proteins maintain cell structure [Fig. 17-23]

  • 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
Cilia and Flagella maintain cell structure [Fig. 17-23]

  • 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 jpg

MTs in a cilium or flagellum are arranged maintain cell structure [Fig. 17-23]

In a “9 + 2” array [Fig. 17-27]

17_27_9_+_2_array.jpg

  • 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 maintain cell structure [Fig. 17-23]

17_28_dynein_flagell.jpg

Linkers removed

[Fig. 17-28]


17 02 03 protein filament jpg

Actin Filaments [Fig. 17-2] maintain cell structure [Fig. 17-23]

17_02_03_protein_filament.jpg


17 29 actin filaments jpg

Distribution of actin filaments in different cells maintain cell structure [Fig. 17-23]

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

17_29_Actin_filaments.jpg

Microvilli in

Intestine (increase surface area)

Contractile bundles

in cytoplasm

Sheetlike (lamellipodia)

and fingerlike (filipodia)

protrusions

of a moving cell [important

in cell crawling, endo- and

exo-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]

17_30_protein threads.jpg

Twist-repeating

distance


Biol 200 section 921 lecture 11 july 4 2006

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

Actin with bound ATP

Actin with bound ADP

plus end

minus end

Phalloidin: A cyclic peptide from the death

cap fungus, Amanita phalloides, inhibits

the depolymerization of actin, thereby

stabilizing actin microfilaments

Cytochalasin D: A fungal metabolite,

Inhibits the polymerization of actin

microfilaments


Microfilaments or actin filaments
Microfilaments or Actin Filaments [Fig.17-31]

  • 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]

17_32_Actin_binding.jpg

(e.g. thymosin and profilin)

(e.g. gelsolin)



Biol 200 section 921 lecture 11 july 4 2006

Actin in amoeboid movement of a fibroblast [Fig. 17-34] pulls cell body along [Fig. 17-33]

cortex

filopodia

lamellipodia


Biol 200 section 921 lecture 11 july 4 2006

Filopodium grows by nucleation of actin microfilaments pulls cell body along [Fig. 17-33] [Fig. 16-29, ECB 1st ed.]

nucleation complex at PM

Growing filopodium

monomers added

Growing microfilament


17 36 actin meshwork jpg

Association of actin and actin related proteins pushes forward lamellipodium

17_36_actin_meshwork.jpg


17 38 myosin i jpg

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

17_38_myosin_I.jpg

The head group of myosin I walks towards

the plus end of the actin filament.

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

Myosin I: Move an actin filament.


17 40 myosin ii jpg

Myosin-II molecules can associate with one another to 17-38]

form myosin filaments [Fig. 17-40]

17_40_Myosin_II.jpg

[Coiled-coil]

Tails

Bipolar myosin filament


17 41 slide actin jpg
17_41_slide_actin.jpg 17-38]

Roles 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.


Biol 200 section 921 lecture 11 july 4 2006

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

Muscle contraction depends on bundles

of actin and myosin


Biol 200 section 921 lecture 11 july 4 2006

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

Z disc: attachment points

For actin filaments


17 44 muscles contract jpg

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

17_44_Muscles contract.jpg

+

+

The myosin heads walk toward the plus end of the adjacent actin filament

driving a sliding motion during muscle contraction.


17 45 myosin walks jpg

1. The Myosin head 17-44]

tightly locked onto an

actin filament.

2. ATP binds to the myosin

head. The Myosin head released from actin.

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

4. The myosin head attaches

to a new site on actin filament.

Pi released. Myosin head

regains its original

conformation (power stroke).

ADP released.

5. The myosin head is

again locked tightly to

the actin filament.

17_45_myosin_walks.jpg


Experimental methodology techniques and approaches for studying the cytoskeleton

Experimental Methodology, Techniques and Approaches for Studying the Cytoskeleton

Modern microscopy techniques

Drugs and mutations to disrupt cytoskeletal structures


Modern microscopy techniques to study cytoskeleton
Modern microscopy techniques to study cytoskeleton Studying the Cytoskeleton

  • 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].

  • 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].

  • 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].

  • 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 treatments
Drug Treatments Studying the Cytoskeleton

  • Colchicine: An alkaloid fromthe Autumn crocus, Colchicum autumnale).Binds to tubulin monomers and prevents polymerization in MTs.

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

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

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