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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
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
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.
The cytoskeleton is a network of filaments that regulates
a cell’s shape, strength and movement [Fig. 1-27]
Actin filaments
Microtubules
Intermediate
filaments
An overview of the cytoskeleton [Fig. 17-2]
Intermediate
Filamentstough ropes
Microtubulesbig hollow
tubes support
cell structures
Actin
Microfilamentshelical polymers
involved in
movement/shape
17_02_01_protein_filament.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
Fig. 21-27: desmosomes connect
epidermal cells
cadherins
Intermediate
filaments
Plasma
membranes
monomer
dimer
Assembly of intermediate filaments
involves coiled coil dimers [Fig. 17-4]
Intermediate filaments strengthen animal cells [Fig. 17-5]
17_05_strengthen_cells.jpg
17_06_filam_categories.jpg
Microtubules (MTs)
17_02_02_protein_filament.jpg
MTs grow from MT organizing centers [Fig. 17-9]
cilia
centrosome
Basal
bodies
Spindle poles
MICROTUBULES
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)
B)
Colchicine: binds free tubulin and
inhibits formation of microtubulues
Taxol: stabilizes microtubules by
preventing loss of subunits
-The growing end of the
microtubule (MT), at the
top, has subunits
arranged with the betatubulin on the outside.
The subunits in the
microtubule all show a
uniform polarity
b
a
tubulin dimer
Growing
end
-Microtubules, like polypeptides 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]
Fig. 16-11 Alberts MBOC-
GTP-bound
tubulin packs
efficiently into
protofilament
GDP-bound
tubulin bind less
strongly to each
otherdepolymerize MT
GTP hydrolyzes to
GDP in MT
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]
17_11_centrosome.jpg
Centrioles are arrays of short MTs and are identical to basal bodies.
GTP cap leads to stability and
growth of MTs
Inhibitor: COLCHICINE
Dynamic instability (loss of
GTP cap) leads to MT shrinking
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]
Aster MTs
Kinetochore MTs
Interpolar MTs
Sister chromatids separate at anaphase [Fig. 19-17]
I.
II.
Each microtubule filament grows and shrinks
17_12_grows_shrinks.jpg
independent of its neighbors [Fig.17-12]
A model of microtubule assembly
[Becker et al. The World of the Cell]
The selective stabilization of MTs can
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
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
Motor proteins transport their cargo
along MTs [Fig. 17-18]
17_18_motor_proteins.jpg
Kinesins move ER outward and Dyneins move Golgi
inward to maintain cell structure [Fig. 17-23]
ER
MT
Golgi
ER
Golgi
MTs
MTs
Nucleus
kinesins
dynein
Kinesin walks along a MT [Fig. 17-22]
17_22_kinesin_moves.jpg
Heads
Kinesin-GFP
moves along
a MT
Moves in a
Series of
8 nm steps
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
Ciliated epithelium in airway [Fig. 17-24]
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.
Flagella propel a sperm cell [Fig. 17-26]
MTs in a cilium or flagellum are arranged
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
The movement of dynein causes bending of flagellum
17_28_dynein_flagell.jpg
Linkers removed
[Fig. 17-28]
Actin Filaments [Fig. 17-2]
17_02_03_protein_filament.jpg
Distribution of actin filaments in different cells
Determines their shape and function [Fig. 17-29]
17_29_Actin_filaments.jpg
Microvilli in
Intestine
(increase
surface area)
Contractile bundles Sheetlike (lamellipodia)
Contractile ring
in cytoplasm
and fingerlike (filipodia)
during cell divn.
protrusions
of a moving cell [important
in cell crawling, endo- and
exo-cytosis
Two F-actin strands wind around each other to form an actin filament [Fig. 17-30]
17_30_protein threads.jpg
Twist-repeating
distance
ATP hydrolysis induce dynamic instability of
actin filaments [Fig.17-31]
Actin
with
Actin with
bound
bound ADP
ATP
minus end
Phalloidin: A cyclic peptide from the death
cap fungus, Amanita phalloides, inhibits
the depolymerization of actin, thereby
stabilizing actin microfilaments
plus end
Cytochalasin D: A fungal metabolite,
Inhibits the polymerization of 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
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)
Actin polymerization pushes cell edge forward,
contraction pulls cell body along [Fig. 17-33]
Actin in amoeboid movement of a fibroblast [Fig. 17-34]
cortex
lamellipodia
filopodia
Filopodium grows by nucleation of actin microfilaments
[Fig. 16-29, ECB 1st ed.]
nucleation
complex at
PM
monomers
added
Growing microfilament
Growing
filopodium
Association of actin and actin related proteins pushes forward lamellipodium
17_36_actin_meshwork.jpg
Roles of actin-dependent motor protein, myosin I [Fig. 17-38]
The head group of myosin I walks towards
the plus end of the actin filament.
17_38_myosin_I.jpg
Myosin I: Move a vesicle relative to an actin filament.
Myosin I: Move an actin filament.
Myosin-II molecules can associate with one another to
form myosin filaments [Fig. 17-40]
17_40_Myosin_II.jpg
[Coiled-coil]
Tails
Bipolar myosin filament
Roles of actin-dependent motor protein, myosin II [Fig. 17-38
17_41_slide_actin.jpg
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 bundles
of actin and myosin
Sarcomeres (contractile units of muscle)
are arrays of actin and myosin [Fig. 17-43]
Z disc: attachment points
For actin filaments
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.
1. The Myosin head
tightly locked onto an
actin filament.
17_45_myosin_walks.jpg
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.
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.
2.
3.
4.
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 deepetch, 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
1. 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