Cytoskeleton System
A,Conception of Cytoskeleton (Narrow sense)
A complex network of interconnected microfilaments,
microtubules and intermediate filaments that extends
throughout the cytosol.
Chapter 10
Microbubules Microfilamemts Intermediate filaments
1,Introduction
Figure 10-2,The three types of protein filaments that form the
cytoskeleton.
B,Techniques for studying the cytoskeleton
? Fluorescent microscopy and Electron microscopy,
Immunofluorescence,fluorescently-labeled antibody
Fluorescence,microinject into living cells
Video microscopy,in vitro motility assays
Electron,Triton X-100,Metal replica
? Drugs and mutations (about functions)
? Biochemical analysis(in vitro)
C,The self-assembly and dynamic structure of cytoskeletal
filaments
?Each type of cytoskeletal filament is constructed
from smaller protein subunits.
?The cytoskeleton is a network of three filamentous
structures.
?The cytoskeleton is a dynamic strucrure with many
roles,
2,Microfilament,MF
A,MFs are made of actin and involved in cell motility.
?Using ATP,G-actin polymerizes to form MF(F-actin)
Figure 16-51 The trapping of ADP in an actin filament.
B,MF assembly and disassembly
?Characteristics:
(1) Within a MF,all the actin monomers are oriented
in the same direction,so MF has a polarity
Myosin is
molecular
motor for
actins.
(2) In vitro,(Polymerization) both ends of the MF grow,
but the plus end faster than the minus,
Because actin monomers tend to add to a filament’s plus
end and leave from its minus end----,Tread-milling”
(3) Dynamic equilibrium between the G-actin and
polymeric forms,which is regulated by ATP hydrolysis
and G-actin concentration.
(4) Dynamic equilibrium is required for the cell
functions,Some MFs are temporary and others
permanent.
(5)The nucleation of actin filaments at the PM is frequently
regulated by external signals,allowing the cell to change its
shape and stiffness rapidly in response to changes in its
external environment,
This nucleation is catalyzed by a complex of proteins that
includes two actin-related proteins,or ARPs(Arp2 and Arp3).
Actin arrays in a cell.
Figure 16-55 Lamellipodia and microspikes at the leading
edge of a human fibroblast migrating in culture,The arrow in
this scanning electron micrograph shows the direction of cell movement,As the
cell moves forward,lamellipodia and microspikes that fail to attach to the tissue
culture dish sweep backward over its dorsal surface - a movement known as
ruffling,(Courtesy of Julian Heath.)
C,Specific drugs affect polymer dynamics
Cytochalasins:
Prevent the addition of new monomers to existing
MFs,which eventually depolymerize.
Phalloidin:
A cyclic peptide from the death cap fungus,blocks
the depolymerization of MF
Those drugs disrupt the monomer-polymer
equilibrium,so are poisonous to cells
Figure 16-52 The effect of cytochalasin on the leading edge of
the growth cone of a nerve cell in culture,A living growth cone is
viewed by Nomarski differential-interference-contrast microscopy both before (A)
and after (B) treatment with cytochalasin,The cell in (B) has then been stained
with rhodamine phalloidin to reveal the actin filaments (C),Note how the region
behind the leading edge of the cytochalasin-treated growth cone is devoid of
actin filaments,Cytochalasin B (D),(A,B,and C,courtesy of Paul Forscher.)
D,Actin-binding proteins
The structures and functions of cytoskeleton
are mainly controlled by its binding proteins
(1) Monomer-sequestering proteins
Bind with actin monomers and prevent them from
polymerizing.
thymosin and ( profilin) Promoting the assembly
of MF
Figure 16-53 Two possible mechanisms by which an actin-
monomer-binding protein could inhibit actin
polymerization,It is thought that thymosin inhibits actin
polymerization in one of these ways,
(2) MF-binding proteins
?Actin filaments are likewise strongly affected by the
binding of accessory proteins along their sides.
?Actin filaments in most cells are stabilized by the
binding of tropomyosin,an elongated protein,Which
can prevent the filament from interacting with other
proteins.
?Another important actin filament binding protein,
cofilin,present in all eucaryotic cells,which
destabilized actin filaments(also called actin
depolymerizing factor),
?Cofilin binds along the length of the actin filament,forcing
the filament to twist a little more tightly,
?In addition,cofilin binding cause a large increase in the rate
of actin filament treadmilling.
The modular structures of four actin-cross-linking proteins
The formation of two types
of actin filament bundles:
?Contractile bundle
mediated by ?-actinin
?parallel bundle mediated
by fimbrin.
?Gel-like network Actin filaments are often nucleated at the plasma membrane,The highest
density of actin filaments is at the cell
periphery forming cell cortex.
Filamin cross-links actin filaments into a
three-dimensional network with the physical
properties of a gel.
Loss of filamin causes abnormal cell motility
E,Functions of MFs
(1) Maintain cell’s shape and enforce PM
Figure 10-75 A model for how integrins in the plasma membrane connect
intracellular actin filaments to the extracellular matrix at a focal
contact,The formation of a focal contact occurs when the binding of matrix
glycoproteins (such as fibronectin) on the outside of the cell causes the integrin
molecules to cluster at the contact site,as illustrated schematically in (A),A
possible arrangement of some of the intracellular attachment proteins that
mediate the linkage between an integrin and actin filaments is shown in (B),
(2) Cell migration (Fibroblast et al)
?Platelet activation is a controlled sequence of actin filament
severing,uncapping,elongation,recapping,and cross-linking.
(3) Microvillus,Support the projecting
membrane of intestinal epithelial cells
Figure 16-77 Freeze-etch electron micrograph of an intestinal epithelial cell,
showing the terminal web beneath the apical plasma membrane,Bundles
of actin filaments forming the core of microvilli extend into the terminal web,
where they are linked together by a complex set of cytoskeletal proteins that
includes spectrin and myosin-II,Beneath the terminal web is a layer of
intermediate filaments,(From N,Hirokawa et al.,J,Cell Biol,91:399-409)
(4) Stress fibers
Composed of actin filaments and myosin-II
Stress FibersFocal contacts Focal contacts
IFs
Response to tension
(5) Contractile ring,For cytokinesis
(6) Muscle contraction
?Organization of skeletal muscle tissue
?Sarcomere
Figure 16-84 Electron micrographs of an insect flight muscle viewed in
cross-section,The myosin and actin filaments are packed together with
almost crystalline regularity,(From J,Auber,J,de Microsc,8:197-232)
?Proteins play important roles in muscle
contraction
Myosin,The actin motor portein
ATPase
Binding
sites
Myosin II--Dimer
Mainly in muscle
cells
Thick filamemts
Light-chain phosphorylation and the regulation of
the assembly of myosin II into thick filaments
Tropomyosin,Tm and Tropnin,Tn
Ropelike molecule
Regulate MF to bind
to the head of myosin
Complex,Ca2+-subunit
Control the position of
Tm on the surface of MF
?Thick and thin filaments sliding model
?Excitation-contraction
coupling process
Action potential
Ca2+ rise in cytosol
Tn
Tm
Sliding
3.Microtubule,MT Tubulin heterodimersare the protein building
blocks of MTsA,Structures:
Arrangement of protofilaments in singlet,
double,and triplet MTs
Singlet Double Triplet
A
B
A
B
CIn cilia and flagella
In centrioles and basal bodies
B,MTs assemble from microtubule-organizing
centers (MTOCs)
(1) Interphase,Centrosome
Dynamic instability
(2) Dividing cell,
Mitotic spindle
Dynamic instability
(3) Ciliated cell,Basal body
Stability
Basal body structure
C,Characteristics of MT assembly
Dynamic
instability due to
the structural
differences
between a growing
and a shrinking
microtubule end.
?GTP cap;
?Catastrophe,
accidental loss of
GTP cap;
?Rescue,regain
of GTP cap
? Why the centrosome can act as MTOC
Structure
No centrioles
in Plant and
fungi
? Experiments supporting that centrosome is
the MTOC
Treat cell with
colcemid
Cytosolic MTs depoly,except
those in centrosome
Remove
colcemid Tublin repoly
Expla I,MTOC nucleate
poly of tubulins
Expla II,MTOC gather
MTs in cytosol
centrosome + Tubulins MT
+ Tubulins No
A
B
?MT are nucleated by a protein complex
containing ?-tubulin
The centrosome is the major MTOC of animal cells
? Drugs affect the assembly of MTs
(1) Colchicine
Binding to tubulin dimers,prevent MTs
polymerization
(2) Taxol
Binding to MTs,stabilize MTs
These compounds are called antimitotic drugs,and have
application in medical practice as anticancer drugs
? Microtuble-associated proteins (MAPs)
MAPs modulate MT structure,assembly,
and function
Katanin like proteins MAPs
Tau,In axon,cause MTs to form tight bundles
MAP2,In dendrites,cause MTs to form looser bundles
MAP1B,In both axons and dendrites to form crossbridge
between microtubules
Control
organization
MAPs
MAP1A,MAP1B,MAP1C
MAP2,MAP2c
MAP3,MAP4
Tau
The importance of MAPs for neurite formation
Organization of MT bundles by MAPs,
Spacing of MTs depends on MAPs
Insect cell expressing
MAP2
Insect cell expressing
tau
From J,Chen et al,1992,Nature 360,674
The effects of proteins that bind to MT ends
(A)The transition
between Mt
growth and Mt
shrinking is
controlled in cells
by special
proteins.
(B)Capping
proteins help to
localize Mt in
budding yeast
cell.
5,Functions of MTs
1,Maintain cell shape
Fig,10-31 Microtubule dynamics in a living cell,A fibroblast was injected
with tubulin that had been covalently linked to rhodamine,so that approximately
1 tubulin subunit in 10 in the cell was labeled with a fluorescent dye,Note,for
example,that microtubule #1 first grows and then shrinks rapidly,whereas
microtubule #4 grows continuously,(P.J,Sammak et al.,Nature 332,724-736)
细胞内物质运输
Motor Protein
1,Kinesin Family
2,Dynein Family
染色体运动
Intermediate filaments,IFs
IFs are the most abundant and stable
components of the cytoskeleton
Figure 10-13 The domain organization of intermediate filament
protein monomers,Most intermediate filament proteins share a
similar rod domain that is usually about 310 amino acids long and
forms an extended alpha helix,The amino-terminal and carboxyl-
terminal domains are non-alpha-helical and vary greatly in size
and sequence in different intermediate filaments.
Figure 10-14,A current model of intermediate filament
construction.
Figure 10 –16,Electron micrographs of two types of intermediate
filaments in cells of the nervous system,(A) Freeze-etch image of
neurofilaments in a nerve cell axon,showing the extensive cross-linking
through protein cross-bridges, (B) Freeze-etch image of glial filaments in glial
cells illustrating that these filaments are smooth and have few cross-bridges,(C)
Conventional electron micrograph of a cross-section of an axon showing the
regular side-to-side spacing of the neurofilaments,which greatly outnumber the
microtubules,(A and B,courtesy of Nobutaka Hirokawa; C,courtesy of John
Hopkins.)
3,Function of IFs,Confer mechanical
strength on tissues
Disruption of keratin networks causes blistering
Figure 10-18,The nuclear lamina,(A) Schematic drawing
showing the nuclear lamina in cross-section in the region of a
nuclear pore,The lamina is associated with both the chromatin
and the inner nuclear membrane,(B) Electron micrograph of a
portion of the nuclear lamina in a frog oocyte prepared by freeze-
drying and metal shadowing,(C) Electron micrograph of metal-
shadowed isolated lamin dimers (marked L),They have an overall
form similar to muscle myosin (marked M),with a rodlike tail and
two globular heads,but they are much smaller molecules,(B and
C,courtesy of Ueli Aebi.)
Summary,Cytoskeletal functions
Summary of cytoskeleton
1,Three types of cytoskeletal filaments are common to many
eucaryotic cells and are fundamental to the spatial
organization of these cells.
2,The set of accessory proteins is essential for the controlled
assembly of the cytoskeletal filaments(includes the motor
proteins,myosins,dynein and kinesin)
3,Cytoskeletal systems are dynamic and adaptable.
Nucleation is rate-limiting step in the formation of a
cytoskeletal polymer.
2,Regulation of the dynamic behavior and assembly of the
cytoskeletal filaments allows eucaryotic cells to build an
enormous range of structures from the three basic filaments
systems.
A,Conception of Cytoskeleton (Narrow sense)
A complex network of interconnected microfilaments,
microtubules and intermediate filaments that extends
throughout the cytosol.
Chapter 10
Microbubules Microfilamemts Intermediate filaments
1,Introduction
Figure 10-2,The three types of protein filaments that form the
cytoskeleton.
B,Techniques for studying the cytoskeleton
? Fluorescent microscopy and Electron microscopy,
Immunofluorescence,fluorescently-labeled antibody
Fluorescence,microinject into living cells
Video microscopy,in vitro motility assays
Electron,Triton X-100,Metal replica
? Drugs and mutations (about functions)
? Biochemical analysis(in vitro)
C,The self-assembly and dynamic structure of cytoskeletal
filaments
?Each type of cytoskeletal filament is constructed
from smaller protein subunits.
?The cytoskeleton is a network of three filamentous
structures.
?The cytoskeleton is a dynamic strucrure with many
roles,
2,Microfilament,MF
A,MFs are made of actin and involved in cell motility.
?Using ATP,G-actin polymerizes to form MF(F-actin)
Figure 16-51 The trapping of ADP in an actin filament.
B,MF assembly and disassembly
?Characteristics:
(1) Within a MF,all the actin monomers are oriented
in the same direction,so MF has a polarity
Myosin is
molecular
motor for
actins.
(2) In vitro,(Polymerization) both ends of the MF grow,
but the plus end faster than the minus,
Because actin monomers tend to add to a filament’s plus
end and leave from its minus end----,Tread-milling”
(3) Dynamic equilibrium between the G-actin and
polymeric forms,which is regulated by ATP hydrolysis
and G-actin concentration.
(4) Dynamic equilibrium is required for the cell
functions,Some MFs are temporary and others
permanent.
(5)The nucleation of actin filaments at the PM is frequently
regulated by external signals,allowing the cell to change its
shape and stiffness rapidly in response to changes in its
external environment,
This nucleation is catalyzed by a complex of proteins that
includes two actin-related proteins,or ARPs(Arp2 and Arp3).
Actin arrays in a cell.
Figure 16-55 Lamellipodia and microspikes at the leading
edge of a human fibroblast migrating in culture,The arrow in
this scanning electron micrograph shows the direction of cell movement,As the
cell moves forward,lamellipodia and microspikes that fail to attach to the tissue
culture dish sweep backward over its dorsal surface - a movement known as
ruffling,(Courtesy of Julian Heath.)
C,Specific drugs affect polymer dynamics
Cytochalasins:
Prevent the addition of new monomers to existing
MFs,which eventually depolymerize.
Phalloidin:
A cyclic peptide from the death cap fungus,blocks
the depolymerization of MF
Those drugs disrupt the monomer-polymer
equilibrium,so are poisonous to cells
Figure 16-52 The effect of cytochalasin on the leading edge of
the growth cone of a nerve cell in culture,A living growth cone is
viewed by Nomarski differential-interference-contrast microscopy both before (A)
and after (B) treatment with cytochalasin,The cell in (B) has then been stained
with rhodamine phalloidin to reveal the actin filaments (C),Note how the region
behind the leading edge of the cytochalasin-treated growth cone is devoid of
actin filaments,Cytochalasin B (D),(A,B,and C,courtesy of Paul Forscher.)
D,Actin-binding proteins
The structures and functions of cytoskeleton
are mainly controlled by its binding proteins
(1) Monomer-sequestering proteins
Bind with actin monomers and prevent them from
polymerizing.
thymosin and ( profilin) Promoting the assembly
of MF
Figure 16-53 Two possible mechanisms by which an actin-
monomer-binding protein could inhibit actin
polymerization,It is thought that thymosin inhibits actin
polymerization in one of these ways,
(2) MF-binding proteins
?Actin filaments are likewise strongly affected by the
binding of accessory proteins along their sides.
?Actin filaments in most cells are stabilized by the
binding of tropomyosin,an elongated protein,Which
can prevent the filament from interacting with other
proteins.
?Another important actin filament binding protein,
cofilin,present in all eucaryotic cells,which
destabilized actin filaments(also called actin
depolymerizing factor),
?Cofilin binds along the length of the actin filament,forcing
the filament to twist a little more tightly,
?In addition,cofilin binding cause a large increase in the rate
of actin filament treadmilling.
The modular structures of four actin-cross-linking proteins
The formation of two types
of actin filament bundles:
?Contractile bundle
mediated by ?-actinin
?parallel bundle mediated
by fimbrin.
?Gel-like network Actin filaments are often nucleated at the plasma membrane,The highest
density of actin filaments is at the cell
periphery forming cell cortex.
Filamin cross-links actin filaments into a
three-dimensional network with the physical
properties of a gel.
Loss of filamin causes abnormal cell motility
E,Functions of MFs
(1) Maintain cell’s shape and enforce PM
Figure 10-75 A model for how integrins in the plasma membrane connect
intracellular actin filaments to the extracellular matrix at a focal
contact,The formation of a focal contact occurs when the binding of matrix
glycoproteins (such as fibronectin) on the outside of the cell causes the integrin
molecules to cluster at the contact site,as illustrated schematically in (A),A
possible arrangement of some of the intracellular attachment proteins that
mediate the linkage between an integrin and actin filaments is shown in (B),
(2) Cell migration (Fibroblast et al)
?Platelet activation is a controlled sequence of actin filament
severing,uncapping,elongation,recapping,and cross-linking.
(3) Microvillus,Support the projecting
membrane of intestinal epithelial cells
Figure 16-77 Freeze-etch electron micrograph of an intestinal epithelial cell,
showing the terminal web beneath the apical plasma membrane,Bundles
of actin filaments forming the core of microvilli extend into the terminal web,
where they are linked together by a complex set of cytoskeletal proteins that
includes spectrin and myosin-II,Beneath the terminal web is a layer of
intermediate filaments,(From N,Hirokawa et al.,J,Cell Biol,91:399-409)
(4) Stress fibers
Composed of actin filaments and myosin-II
Stress FibersFocal contacts Focal contacts
IFs
Response to tension
(5) Contractile ring,For cytokinesis
(6) Muscle contraction
?Organization of skeletal muscle tissue
?Sarcomere
Figure 16-84 Electron micrographs of an insect flight muscle viewed in
cross-section,The myosin and actin filaments are packed together with
almost crystalline regularity,(From J,Auber,J,de Microsc,8:197-232)
?Proteins play important roles in muscle
contraction
Myosin,The actin motor portein
ATPase
Binding
sites
Myosin II--Dimer
Mainly in muscle
cells
Thick filamemts
Light-chain phosphorylation and the regulation of
the assembly of myosin II into thick filaments
Tropomyosin,Tm and Tropnin,Tn
Ropelike molecule
Regulate MF to bind
to the head of myosin
Complex,Ca2+-subunit
Control the position of
Tm on the surface of MF
?Thick and thin filaments sliding model
?Excitation-contraction
coupling process
Action potential
Ca2+ rise in cytosol
Tn
Tm
Sliding
3.Microtubule,MT Tubulin heterodimersare the protein building
blocks of MTsA,Structures:
Arrangement of protofilaments in singlet,
double,and triplet MTs
Singlet Double Triplet
A
B
A
B
CIn cilia and flagella
In centrioles and basal bodies
B,MTs assemble from microtubule-organizing
centers (MTOCs)
(1) Interphase,Centrosome
Dynamic instability
(2) Dividing cell,
Mitotic spindle
Dynamic instability
(3) Ciliated cell,Basal body
Stability
Basal body structure
C,Characteristics of MT assembly
Dynamic
instability due to
the structural
differences
between a growing
and a shrinking
microtubule end.
?GTP cap;
?Catastrophe,
accidental loss of
GTP cap;
?Rescue,regain
of GTP cap
? Why the centrosome can act as MTOC
Structure
No centrioles
in Plant and
fungi
? Experiments supporting that centrosome is
the MTOC
Treat cell with
colcemid
Cytosolic MTs depoly,except
those in centrosome
Remove
colcemid Tublin repoly
Expla I,MTOC nucleate
poly of tubulins
Expla II,MTOC gather
MTs in cytosol
centrosome + Tubulins MT
+ Tubulins No
A
B
?MT are nucleated by a protein complex
containing ?-tubulin
The centrosome is the major MTOC of animal cells
? Drugs affect the assembly of MTs
(1) Colchicine
Binding to tubulin dimers,prevent MTs
polymerization
(2) Taxol
Binding to MTs,stabilize MTs
These compounds are called antimitotic drugs,and have
application in medical practice as anticancer drugs
? Microtuble-associated proteins (MAPs)
MAPs modulate MT structure,assembly,
and function
Katanin like proteins MAPs
Tau,In axon,cause MTs to form tight bundles
MAP2,In dendrites,cause MTs to form looser bundles
MAP1B,In both axons and dendrites to form crossbridge
between microtubules
Control
organization
MAPs
MAP1A,MAP1B,MAP1C
MAP2,MAP2c
MAP3,MAP4
Tau
The importance of MAPs for neurite formation
Organization of MT bundles by MAPs,
Spacing of MTs depends on MAPs
Insect cell expressing
MAP2
Insect cell expressing
tau
From J,Chen et al,1992,Nature 360,674
The effects of proteins that bind to MT ends
(A)The transition
between Mt
growth and Mt
shrinking is
controlled in cells
by special
proteins.
(B)Capping
proteins help to
localize Mt in
budding yeast
cell.
5,Functions of MTs
1,Maintain cell shape
Fig,10-31 Microtubule dynamics in a living cell,A fibroblast was injected
with tubulin that had been covalently linked to rhodamine,so that approximately
1 tubulin subunit in 10 in the cell was labeled with a fluorescent dye,Note,for
example,that microtubule #1 first grows and then shrinks rapidly,whereas
microtubule #4 grows continuously,(P.J,Sammak et al.,Nature 332,724-736)
细胞内物质运输
Motor Protein
1,Kinesin Family
2,Dynein Family
染色体运动
Intermediate filaments,IFs
IFs are the most abundant and stable
components of the cytoskeleton
Figure 10-13 The domain organization of intermediate filament
protein monomers,Most intermediate filament proteins share a
similar rod domain that is usually about 310 amino acids long and
forms an extended alpha helix,The amino-terminal and carboxyl-
terminal domains are non-alpha-helical and vary greatly in size
and sequence in different intermediate filaments.
Figure 10-14,A current model of intermediate filament
construction.
Figure 10 –16,Electron micrographs of two types of intermediate
filaments in cells of the nervous system,(A) Freeze-etch image of
neurofilaments in a nerve cell axon,showing the extensive cross-linking
through protein cross-bridges, (B) Freeze-etch image of glial filaments in glial
cells illustrating that these filaments are smooth and have few cross-bridges,(C)
Conventional electron micrograph of a cross-section of an axon showing the
regular side-to-side spacing of the neurofilaments,which greatly outnumber the
microtubules,(A and B,courtesy of Nobutaka Hirokawa; C,courtesy of John
Hopkins.)
3,Function of IFs,Confer mechanical
strength on tissues
Disruption of keratin networks causes blistering
Figure 10-18,The nuclear lamina,(A) Schematic drawing
showing the nuclear lamina in cross-section in the region of a
nuclear pore,The lamina is associated with both the chromatin
and the inner nuclear membrane,(B) Electron micrograph of a
portion of the nuclear lamina in a frog oocyte prepared by freeze-
drying and metal shadowing,(C) Electron micrograph of metal-
shadowed isolated lamin dimers (marked L),They have an overall
form similar to muscle myosin (marked M),with a rodlike tail and
two globular heads,but they are much smaller molecules,(B and
C,courtesy of Ueli Aebi.)
Summary,Cytoskeletal functions
Summary of cytoskeleton
1,Three types of cytoskeletal filaments are common to many
eucaryotic cells and are fundamental to the spatial
organization of these cells.
2,The set of accessory proteins is essential for the controlled
assembly of the cytoskeletal filaments(includes the motor
proteins,myosins,dynein and kinesin)
3,Cytoskeletal systems are dynamic and adaptable.
Nucleation is rate-limiting step in the formation of a
cytoskeletal polymer.
2,Regulation of the dynamic behavior and assembly of the
cytoskeletal filaments allows eucaryotic cells to build an
enormous range of structures from the three basic filaments
systems.
