Beruflich Dokumente
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CHAPTER 13
The Cytoskeleton
AB: microfilaments AB: microtubules
AB: overlay
AB: intermediate
filaments
Hepatocyte
Keys
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Introduction
Introduction
Properties of cytoskeletal components
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Protein localization
using fluorescent
antibodies: centrin, a
protein found in the
al
g alce l
l
sfla gell
a Dynamic changes in
microtubule length in an
epithelial cell using a
rhodamine-tagged tubulin
2013 John Wiley & Sons, Inc. All rights reserved.
Cy5
In vivo snapshot
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Determining the
mechanical properties of
single cytoskeletal
filaments. A single
intermediate filament
subjected to mechanical
forces by the tip of an
atomic force microscope.
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(13.3) Microtubules
Structure and
Composition
Microtubules are hollow,
cylindrical structures.
The microtubule is a set
of globular proteins
arranged in longitudinal
rows called
protofilaments.
Microtubules contain 13
protofilaments.
Each protofilament is
assembled from dimers Electron micrograph
of - and -tubulin Electron micrograph cross section through a
subunits assembled into of negatively stained microtubule revealing
tubules with plus and microtubules the 13 subunits
minus ends.
Microtubules
The structure of microtubules
Structure and
Composition
Microtubules are hollow,
cylindrical structures.
The microtubule is a set
of globular proteins
arranged in longitudinal
rows called
protofilaments.
Microtubules contain 13
protofilaments.
Each protofilament is
assembled from dimers
of - and -tubulin
subunits assembled into
tubules with plus and Ribbon model: 3D Diagram of a
minus ends. structure of the - longitudinal section
tubulin heterodimer of a microtubule
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Microtubules
Microtubule-associated proteins
Microtubule-Associated
Proteins (MAPs)
MAPs comprise a
heterogeneous group of
proteins.
MAPs attach to the surface of
microtubules to increase their
stability and promote their
assembly.
MAPs are regulated by
phosphorylation of specific
Schematic diagram of a brain amino acid residues.
MAP2 molecule bound to the
surface of a microtubule.
Microtubules
Location of microtubules
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Microtubules
Structure support and organizers
Microtubules help
maintain cell shape
by influencing
formation of the cell
wall in wheat.
Microtubules (A) and
cell wall cellulose (B)
are co-aligned
Microtubules
Axonal transport
Movement of vesicles down
the length of an axon along
tracks of microtubules
Microtubule and
intermediate filament
organization in an axon
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Microtubules
Axonal transport
Movement of vesicles down
the length of an axon along
tracks of microtubules
EM of an axon (cultured
nerve cell) showing
parallel microtubules that
function as tracks for
axonal transport
Microtubules
Axonal transport
Cell body
Autophagic Retrograde
vesicle movement Video micrographs
show the
progression of a
membranous
organelle along a
axon branched axon
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Microtubules
Microtubules
Kinesin
Kinesins
Kinesinmember of a
superfamily called KLPs
(kinesin-like proteins).
A kinesin is a tetramer Schematic illustration
of two identical heavy of kinesin-mediated
chains and two identical and dynein-mediated
light chains. transport of vesicles
Each kinesin includes a
pair of globular heads
(motor domain),
connected to a rod-like
stalk.
Kinesin is a plus end- Force-generating
directed microtubular heads bind to the
motor based on its microtubule; tail
movement. binds to the cargo
being transported
2013 John Wiley & Sons, Inc. All rights reserved.
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Microtubules
Kinesin GFP head
hand-over-hand model
microtubule
Kinesins
They move along a single protofilament of a microtubule at a velocity
proportional to the ATP concentration.
Movement is processive, motor protein moves along an individual
microtubule for a long distance without falling off.
KLPsmov eca rgot owa rdt hec ell
splasmame mbr a ne
.
Microtubules
Kinesin-mediated organelle transport
microtubule AB microtubule AB
Alteration in the
phenotype of a cell
lacking a member of the
kinesin superfamily.
Control KO Control cells from the
extraembryonic tissues
of a normal 9.5 day
Mito AB Mito AB
mouse embryo or
embryo that lacked both
copies of gene encoding
the kinesin-like protein
KIF5B.
Control KO
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Microtubules
Cytoplasmic dynein
Cytoplasmic Dynein
Dynein responsible for the
movement of cilia and
flagella.
Structure of a cytoplasmic
Cytoplasmic dynein Huge
dynein molecule
protein with a globular,
force-generating head.
Schematic
It is a minus end-directed diagram of two
microtubular motor. vesicles moving
Requires an adaptor in opposite
(dynactin) to interact with directions along
membrane-bounded cargo. the same
microtubule
Microtubules
Cytoplasmic dynein
Cytoplasmic Dynein
Dynein responsible for the
movement of cilia and
flagella.
Cytoplasmic dynein Huge
protein with a globular,
force-generating head.
It is a minus end-directed
microtubular motor.
Requires an adaptor
(dynactin) to interact with
membrane-bounded cargo.
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Microtubules
The centrosome
Microtubule-Organizing
Schematic diagram of
Centers (MTOCs)
a centrosome shows
MTOCs specialized the paired centrioles,
structures for the surrounding PCM, and
nucleation of microtubules. microtubules forming
Centrosome structures from the PCM
responsible for initiating
microtubules in animal cells.
It contains two barrel-
shaped centrioles
surrounded by
pericentriolar material
(PCM).
Centrioles are usually
EM cross section of a
found in pairs.
centriole showing its
pinwheel arrangement
Microtubules
The centrosome
Centrosomes
Responsible for initiation and organization of the microtubular cytoskeleton.
Microtubules terminate in the PCM.
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Microtubules
The centrosome
Fluorescence micrograph of a
fibroblast exposed to colcemid with The growth of microtubules occurs by
30 min recovery before fluorescent addition of subunits at the plus end of
anti-tubulin antibody treatment the polymer away from the centrosome
Microtubules
The centrosome
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Microtubules
Microtubules
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Microtubules
Microtubules
Assembly and stability
EM: microtubules
polymerized in vitro.
Middle microtubule
contains 11
protofilaments.
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Microtubules
Assembly and stability
The Underlying Basis of
Microtubule Dynamics
Insight into factors
that influence
microtubule
assembly and
disassembly came
from studies in vitro. Structural
cap model
GTP is required for of dynamic
microtubule instability
assembly.
Hydrolysis of GTP
leads to a
replacement of
bound GDP by new
GT Pt o recha rge
the tubulin dimer.
Microtubules
Assembly and stability
Dynamic instability
Growing and shrinking microtubules can coexist in the same region of a cell.
A given microtubule can switch back and forth between growing and
shortening phases.
It is an inherent property of the plus end of the microtubule.
Proteins called +TIPS regulate the rate of growth and shrinkage.
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Microtubules
Assembly and stability
Dynamic instability
Growing and shrinking microtubules can coexist in the same region of a cell.
A given microtubule can switch back and forth between growing and
shortening phases.
It is an inherent property of the plus end of the microtubule.
Proteins called +TIPS regulate the rate of growth and shrinkage.
Microtubules
Cilia and flagella
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Microtubules
Cilia and flagella
Eukaryotic flagella: The unicellular alga with Forward movement from asymmetric
labeled flagellar membrane protein (green) waveform resembling the breast stroke
Microtubules
The structure of the axoneme
Cilia and flagella
The structure of cilia and
flagella contains a central core
Cross section
(axoneme) consisting of
through a sperm
microtubules in a 9 + 2
axoneme
arrangement.
The basic structure of the
axoneme includes a central
sheath, connected to the A
tubules of peripheral doublets
by radial spokes.
The doublets are Schematic
interconnected to one another diagram of
by an interdoublet bridge. a protist
axoneme
A longitudinal view of the
axoneme shows continuous
nature of microtubules
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Microtubules
Longitudinal view of an axoneme
Cilia and flagella
The structure of cilia and
flagella contains a central core
(axoneme) consisting of EM: median
microtubules in a 9 + 2 longitudinal
arrangement. section of a
The basic structure of the cilium
axoneme includes a central
sheath, connected to the A
tubules of peripheral doublets
by radial spokes.
The doublets are
interconnected to one another Schematic
by an interdoublet bridge. diagram of a
longitudinal
A longitudinal view of the
section of a
axoneme shows continuous
flagellar doublet
nature of microtubules
Microtubules
Basal bodies and axonemes
EM: longitudinal section
through the basal
bodies of several cilia
Structural relationship
between basal body MTs
and the axoneme of a
cilium or flagellum
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Microtubules
Intraflagellar transport
Cilia and flagella
EM: longitudinal
Cilia and flagella emerge section of a
from basal bodies. flagellum with
The growth of an two rows of
axoneme occurs at the protein particles
plus ends of microtubules. (arrowheads)
Intraflagellar transport between the
(IFT) is the process outer doublet
responsible for microtubules
assembling and and plasma
maintaining flagella. membrane.
IFT depends on the Inset shows
activity of both plus end- cargo moved by
and minus end-directed a direction
microtubules. specific motor
protein
Microtubules
Ciliary dynein
Sea urchin sperm (w/o cilia membrane) after
ATP
A sea urchin sperm
reactivated with
ATP following
demembranation
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Microtubules
Structure and function of ciliary dynein
Microtubules
Structure and function of ciliary dynein
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Microtubules
Sliding-microtubule mechanism
Microtubules
Sliding-microtubule mechanism
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Microtubules
Sliding-microtubule mechanism
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Intermediate filaments
(IFs)heterogeneous group
of proteins, divided into five
major classes.
IFs classes IIV are used in
the construction of
filaments; type V (lamins)
are present in the inner
lining of the nucleus.
IFs radiate through the
cytoplasm of a wide variety
of animal cells and are
often interconnected to
other cytoskeletal filaments
by thin, wispy cross-bridges.
Intermediate Filaments
Intermediate filaments
(IFs)heterogeneous group
of proteins, divided into five
major classes.
IFs classes IIV are used in
the construction of
filaments; type V (lamins)
are present in the inner
lining of the nucleus.
IFs radiate through the
cytoplasm of a wide variety
of animal cells and are
often interconnected to
other cytoskeletal filaments Cytoskeletal elements are connected to one
by thin, wispy cross-bridges. another by protein cross-bridges (plectin).
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Intermediate Filaments
Assembly and Disassembly
Assembly:
Basic building block is
a rod-like tetramer
formed by tow
antiparallel dimers.
Both the tetramer and
the IF lack polarity.
IFs are less sensitive to
chemical agents than
other types of
cytoskeletal elements.
Assembly and
disassembly of IFs are
controlled by
phosphorylation and
dephosphorylation.
Models of IF assembly and architecture
Intermediate Filaments
Assembly and Disassembly
Assembly:
Basic building block is Localization of
a rod-like tetramer injected
formed by tow biotinylated
antiparallel dimers. keratin during
Both the tetramer and a 20-minute
the IF lack polarity. label period
IFs are less sensitive to
chemical agents than
other types of Distribution of
cytoskeletal elements. intermediate
Assembly and filaments in the
disassembly of IFs are cell as revealed
controlled by by anti-keratin
phosphorylation and antibodies
dephosphorylation.
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Intermediate Filaments
Types and Functions
(13.5) Microfilaments
Microfilaments are
composed of actin and are
involved in cell motility.
Using ATP, actin
polymerizes to form actin
filame nts(F-a cti
n).
The two ends of an actin
filament have different
structural characteristics
and dynamic properties.
One of the micro-filaments
appears pointed, and the
other appears barbed.
Orientation of the
EM: Determining the
arrowheads formed by
actin provides information Actin filament structure: location and polarity of
about direction of the model and EM showing its actin filaments with the
microfilament movement. double-helical architecture S1 subunit of myosin.
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Microfilaments
Assembly and Disassembly
EM: short actin filament
labeled with S1 myosin
then used to nucleate
actin polymerization.
Addition occurs much
more rapidly at the
barbed (plus) end than
at the pointed (minus)
Microfilaments
Drug disruption
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Microfilaments
Myosin as a molecular motor
Microfilaments
Structure of myosin II
Conventional (Type II)
Myosins
They generate force in
muscles and some non-
muscle cells.
Each myosin II is composed
of two heavy chains, two
light chains, and two
globular heads (catalytic
sites).
All of the machinery
required for motor activity
is contained in a single head.
The tail portion plays a
structural role allowing the Electron micrograph and schematic drawing of
protein to form filaments. a myosin II molecule with one pair of heavy
chains (blue) and two pairs of light chains
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Microfilaments
Structure of myosin II
Conventional (Type II)
Myosins
They generate force in
muscles and some non-
muscle cells.
Each myosin II is composed Myosin heads bound to a silicone-coated
of two heavy chains, two coverslip are incubated with actin filaments.
light chains, and two
globular heads (catalytic
sites).
All of the machinery Arrowheads show the
required for motor activity
actin filaments sliding
is contained in a single head.
over the myosin
The tail portion plays a
heads during the brief
structural role allowing the
protein to form filaments. period between two
exposures (1.5 sec)
Microfilaments
Structure of myosin II
Conventional (Type II)
Myosins
They generate force in
muscles and some non-
muscle cells.
Each myosin II is composed
of two heavy chains, two
light chains, and two
globular heads (catalytic
sites).
All of the machinery
required for motor activity
is contained in a single head.
Diagram of the staggered arrangement
The tail portion plays a
of the individual myosin molecules in a
structural role allowing the
protein to form filaments. myosin II filament and EM of a bipolar
myosin filament formed in vitro
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Microfilaments
Unconventional myosins
Microfilaments
Unconventional myosins
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Microfilaments
Unconventional myosins
Microfilaments
Unconventional myosins and hair cells
Drawing of a hair
cell of the cochlea
Transmission EM
cross section of
a stereocilium
Hair cells are named for the bundle of stiff, hair-like stereocilia that project
from the apical surface of the cell into the fluid-filled cavity of the inner ear.
Displacement of the stereocilia by mechanical stimuli leads to the generation
of nerve impulses that we perceive as sound.
Each stereocilium is supported by a bundle of parallel actin filaments
2013 John Wiley & Sons, Inc. All rights reserved.
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Microfilaments
Unconventional myosins and hair cells
Left: Myosin VIIa (green) in hair cell of Left: SEM from normal mouse cochlea hair
bullfrog. Right: Myosin XVa (green) at cells. Right: SEM from knockout mouse for
stereocilia tips in a rat auditory hair cell. myosin VIIa KO, which suffers from deafness.
Hair cells are named for the bundle of stiff, hair-like stereocilia that project
from the apical surface of the cell into the fluid-filled cavity of the inner ear.
Displacement of the stereocilia by mechanical stimuli leads to the generation
of nerve impulses that we perceive as sound.
Each stereocilium is supported by a bundle of parallel actin filaments
2013 John Wiley & Sons, Inc. All rights reserved.
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Muscle Contractility
The sarcomere
Sarcomere
Each myofibril showing the
consists of a overlapping
repeating array of array of thin
sarcomeres. actin-containing
(orange) and
Each sarcomere has
thick myosin-
a banding pattern
containing
that gives muscle
(purple)
fiber a striated
filaments
appearance.
Banding pattern: EM: cross section
Thin filaments through an insect
(I and A bands) flight muscle showing
Thick filaments the hexagonal
(H and A bands) arrangement of the
thin filaments around
each thick filament
Muscle Contractility
The sarcomere
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Muscle Contractility
The sarcomere
Muscle Contractility
The sarcomere
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Muscle Contractility
Molecular basis of contraction
A model of
the power
stroke of
the myosin
motor
Neck moves through a rotation of domain
approximately 70o producing a
movement of actin filament of 10 nm.
Muscle Contractility
Actinomyosin contractile cycle
The Energetics of Filament
Sliding
Energy is provided by
ATPase activity in the
myosin head.
Activated myosin attaches
to actin initiating the power
stroke.
Release of bound ADP is
followed by binding of
another ATP.
Absence of ATP prevents
dissociation of cross-
bridges causing rigor mortis.
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Muscle Contractility
Actinomyosin contractile cycle
Two cycles mechanical (interaction of
myosin with actin) and chemical
(hydrolysis of ATP) begin in
Step 1: binding of ATP to a cleft in the
myosin head, causing the detachment of
the head from the actin filament.
Step 2: hydrolysis of ATP to Pi and ADP
which energizes the head and fosters
Step 3: causing the head to bind weakly
to the actin filament
Step 4: the release of Pi which causes a
tighter attachment of the myosin head
to the thin filament and the power
stroke that moves the thin filament
toward the center of the sarcomere.
Step 5: the release of the ADP (step 5)
which sets the stage for another cycle .
Model of the actin-myosin contractile cycle
Muscle Contractility
The functional anatomy of a muscle fiber
Excitation-Contraction Coupling
Contact between nerve and muscle is called the neuromuscular junction.
The linking of the nerve impulse to the shortening of the sarcomere is referred
to as excitation-contraction coupling.
Action potential in muscles is propagated into the cell interior by transverse (T)
tubules.
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Muscle Contractility
The role of tropomyosin in muscle contraction
Excitation-contraction coupling
T tubules terminate near the sarcoplasmic reticulum (SR), which stores Ca2+.
In a relaxed sarcomere, Ca2+ levels are low.
An action potential opens calcium channels in the SR, releasing Ca2+.
Binding of Ca2+ to troponin causes a conformational changes, shifting
tropomyosin and exposing the myosin binding site.
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Non-muscle motility
The roles of actin-binding proteins
1) Nucleating proteins
provide a template for
adding actin monomers.
(Arp2/3 complex; formin)
branched versus
unbranched filaments
2) Monomer-sequestering
proteins bind to actin-
ATP monomers and
prevent them from
polymerizing. (thymosin
b4)
3) End-blocking (capping)
proteins regulate the
length of actin filaments
(capZ; tropomodulin).
4) Monomer-polymerizing
proteins promote the
growth of actin
filaments. (profilin)
Non-muscle motility
The roles of actin-binding proteins
5) Actin filament depolymerizing
proteins bind actin-ADP
subunits for rapid turnover of
actin filaments. Example:
cofilin
6) Cross-linking proteins alter
the three-dimensional
organization of actin filaments.
Examples: filamin (X-link),
villin, fimbrin (bundling)
7) Filament-severing proteins
shorten filaments and
decrease cytoplasmic viscosity.
Example: gelsolin
8) Membrane-binding proteins
link contractile proteins to
plasma membrane. Examples:
vinculin, spectrin (dystrophin)
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Non-muscle motility
The roles of actin-binding proteins
Non-muscle motility
Polymerization as a force generator
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Non-muscle motility
Cell locomotion
The repetitive
sequence of
activities that
occurs as a
cell crawls
over the
Scanning electron micrograph of substratum
a mouse fibroblast crawling over
the surface of a culture dish
Non-muscle motility
Cell locomotion
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Non-muscle motility
Motility using a lamellipodium
The leading edge of
this motile fibroblast
is fattened against
the substratum and
spread out into a veil-
like lamellipodium.
Non-muscle motility
Major steps in formation of a lamellipodium
Stimulus at the cell
surface (step1) leads to
Arp2/3 complex
activation of by WASP
family (step2). Activated
complexes initiate
nucleation of actin
polymerization-
Neutrophil responding formation of actin
to a chemoattractant filaments (step 3). Once
from pipette (right) filaments have formed,
complexes attach to
Stimulus at the plasma membrane triggers localized filament side (step 4) and
polymerization of actin, causes polarization of the cell and initiate side branches
movement toward stimulus. (step 5); 70o angle
Wiskott-Aldrich syndrome: Disorder with crippled immune relative to the existing
system because white blood cells lack a functional WASP filaments. The complexes
protein and fail to respond to chemotactic signals push the PM outward
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Non-muscle motility
Lamellipodial extension
Non-muscle motility
Lamellipodial extension
Migrating cell with traction
forces at highest levels (red) at
lamellipodial extension (arrow)
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Non-muscle motility
Actin and myosin in lamellipodial movement
Fluorescence micrographs
(fish keratocyte) moving
over a culture dish by
means of a broad,
flattened lamellipodium.
Actin and myosin
distribution is shown in
micrograph and drawing.
Keratocytes have been a favored system for studying locomotion because their
rapid gliding movement depends on the formation of a very broad, thin
lamellipodium.
Leading edge is filled with actin but devoid of myosin II.
Roleofmy osi
nII tog ene ratec ontrac t
il
efor cesnee dedt o
pul
l
ther
estofcell
behind the leading edge.
Non-muscle motility
Axonal outgrowth
The bulk of the axon shows little
evidence of motile activity. Video image of
The tip of the axon (growth cone) a live growth
is highly motile and shows several cone shows rod-
types of locomotor protrusions: like microspikes
(arrows) and
Microspikes- point outward to
filopodia
the edge of lamellipodium.
(arrowheads)
Filopodia - elongations that
extend and retract during
motile activity. Fluorescence
Growth cone explores its micrograph
environment and elongates its (neuron growth
axon. cone) shows actin
Lamellipodia and filopodia of filaments (green)
growth cone respond to presence and microtubules
of physical and chemical stimuli. (orange)
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Non-muscle motility
Axonal outgrowth
The bulk of the axon shows little
evidence of motile activity.
The tip of the axon (growth cone)
is highly motile and shows several
types of locomotor protrusions:
Microspikes- point outward to
the edge of lamellipodium.
Filopodia - elongations that
extend and retract during
motile activity.
Growth cone explores its
environment and elongates its Directed movements of a growth
axon. cone. Video image of a live growth
Lamellipodia and filopodia of cone (Xenopus neuron) turning
growth cone respond to presence toward a diffusible protein (netrin-1)
of physical and chemical stimuli. released from a pipette (arrow).
Non-muscle motility
Axonal outgrowth
The bulk of the axon shows little
evidence of motile activity.
The tip of the axon (growth cone)
is highly motile and shows several
types of locomotor protrusions:
Microspikes- point outward to
the edge of lamellipodium.
Filopodia - elongations that
extend and retract during
motile activity.
Growth cone explores its
environment and elongates its Growth cone (green) at the tip of a
axon. motor axon has made contact by
means of its filopodia with a target
Lamellipodia and filopodia of
cell that is expressing the neuronal
growth cone respond to presence
guidance factor ephrin (red).
of physical and chemical stimuli.
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Non-muscle motility
Cell shape changes during embryonic development
Ectodermal cells
elongate and form a
neural plate as
microtubules
become oriented
pa ra l
l
eltot hec e l
ls
axis.
Change in cell shape
produced by
contraction of
microfilaments.
Curvature of the
neural tube causes
outer edges to
contact one another
forming a tube Changes in cell shape: a layer of flattened ectodermal
which gives rise to cells at the mid-dorsal region of the embryo roll into a
nervous system. neural tube. Initial cell elongation likely due to
microtubules while rolling of the plate is actin-mediated.
2013 John Wiley & Sons, Inc. All rights reserved.
Non-muscle motility
Cell shape changes during embryonic development
Ectodermal cells
elongate and form a
neural plate as
microtubules
become oriented
pa ra l
l
eltot hec e l
ls
axis.
Change in cell shape
produced by
contraction of
microfilaments.
Curvature of the
neural tube causes
outer edges to
Scanning electron micrograph of the dorsal
contact one another
forming a tube surface of a chick embryo as its neural
which gives rise to plate is being folded to form a tube.
nervous system.
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Synopsis
The cytoskeleton is composed of three distinct types of fibrous structures:
microtubules, intermediate filaments, and microfilaments (actin
filaments), which participate in a number of cellular activities.
Microtubules are hollow, tubular structures 25 nm in diameter that are
assembled from the protein tubulin and, in addition to the cytoskeleton,
form part of the mitotic spindle, centrioles, and the core of cilia and
flagella.
Three families of motor proteins have been identified and characterized:
kinesins and dyneins, which move along microtubules, and myosins,
which move along microfilaments.
In most cases, kinesins and cytoplasmic dynein move materials along
microtubules in opposite directions.
The nucleation of microtubules in vivo occurs in association with a
variety of MTOCs (microtubule organizing centers).
Synopsis
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Synopsis
48