A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
Cytoskeleton
A.
B.
C.
D.
Overview
Experimental Methods
Microtubules
Microfilaments
(Updated 4/9/08)
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
A. Overview
1.
2.
3.
4.
Definition
Types of Cytoskeleton Fibers
Dynamic
Polymerization/Depolymerization
Molecular Motors
Alberts: Fig. 16 – 1, Panel 16 – 1, Panel 16 – 2, Fig. 16 –11, Fig 16 – 12, 16 – 8, 16 – 7, 16 – 10, 16 – 13, 16 – 14, 16 – 15,
16 – 16, 16 – 17, 16 – 19, 16 – 56, Table 16 – 1
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
B. Experimental methods
1.
Visualization Approaches
–
–
Light Microscopy
Fluorescence Microscopy
»
–
–
2.
3.
http://www.itg.uiuc.edu/exhibits/gallery/fluorescencegallery.htm
Digital/video Microscopy
Electron Microscopy
Genetic Approaches
Biochemical Approaches
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. Microtubules
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments
1.
2.
3.
4.
5.
6.
7.
Structure
Microtubule-associated proteins
Functions
Microtubule motors
Microtubule organizing centers
Dynamic properties of microtubules
Flagella and cilia
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
C.1. Microtubules: Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments
1.
Structure
–
–
Alberts: Fig 16 – 11
Structure and composition - hollow, tubular; found in
most eukaryotic cells (cilia, spindle, flagella)
»
»
»
–
–
–
Outer diameter - 24 nm
Wall thickness - ~5 nm
May extend across cell length/breadth
Wall composed of globular proteins arranged in
longitudinal rows (protofilaments)
Protofilaments are aligned parallel to tubule long axis
In cross section, consist of 13 protofilaments arrayed
in circular pattern within wall
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C.1. Microtubules: Structure
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments
– Each protofilament is assembled of dimeric building
blocks (one a-tubulin & one b-tubulin; A heterodimer)
organized in linear array along length of protofilament
– Two types of tubulin subunits have similar 3D structure &
fit tightly together
– Protofilaments asymmetric (a-tubulin at one end, btubulin at other); All in single MT have same polarity;
Each assembly unit has 2 nonidentical components
(heterodimer)
– All protofilaments of microtubule have same polarity;
Thus so does full tubule (plus- & minus-end)
– Plus end - fast-growing (b-tubulins on tip); Minus end slow-growing (a-tubulins on tip)
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
C.2. Microtubules: MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments
2.
Microtubule-associated proteins
–
–
–
–
Alberts: Fig 16-40, 16-41
MTs can assemble in vitro from purified tubulin, but
MAPs are found with MTs isolated from cells; most
found only in brain tissue; MAP4 has wider
distribution
Have globular head domain that attaches to MT side
& filamentous tail protruding from MT surface
May interconnect MTs to help form bundles (crossbridges), increase MT stability, alter MT rigidity,
influence MT assembly rate
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 2. Microtubules: MAPs
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments
– MAP activity controlled by addition & removal of
phosphate groups from particular amino acid residues
by protein kinases & phosphatases, respectively;
example - Alzheimer’s disease (AD)
– Abnormally high MAP (tau) phosphorylation
implicated in fatal neurodegenerative diseases like
AD; neurofibrillary tangles in brains made of
hyperphosphorylated tau; may help kill nerve cells
– Excessively phosphorylated tau molecules are unable
to bind to MTs; people with one of these diseases, a
type of dementia called FTDP-17, carry mutations in
tau gene, implicating it as cause
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
C. 3. Microtubules: Functions
4. Microtubule Motors
5. MTOCs
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments
3.
Functions
–

Alberts: Table 16-2; Fig 16-23, 66
Internal skeleton (scaffold) providing structural
support & maintaining organelle position
– Resist compression or bending forces on fiber;
provide mechanical support like girders in `tall
building; prevent distortion of cell by cytoplasmic
contractions
– MT distribution conforms to & helps determine cell
shape: flattened, round cells - radiate from nuclear
area; columnar epithelium - parallel to cell long axis;
like aluminum rods support tent
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
C. 3.
Microtubules: Functions
4. Microtubule Motors
5. MTOCs
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments
– Elongated cell process (axon, axopods of heliozoan
protists) - MTs oriented parallel to each other & axon
or axopod long axis; help move things
– In developing embryo, extend growing central NS
axons to peripheral NS; inhibit (colchicine [CO],
nocodazole [NO]) & outgrowth stops, regresses
(collapses back to rounded cell body)
– Found as core of axopodial processes of heliozoan
protozoa; many MTs arranged in spiral with individual
MTs traversing entire length of process
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
C. 3. Microtubules: Functions
4. Microtubule Motors
5. MTOCs
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments

Plants: play similar role in plants; affect shape
indirectly by influencing cell wall formation;
found in cortex just below membrane during
interphase forming a distinct cortical zone
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
C. 3. Microtubules: Functions
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments


Also have role in maintenance of cell internal
organization (organelle placement) - disrupt MTs
(CO, NO) —> Golgi disperses to cell periphery;
goes back to cell center when inhibitors removed
Move macromolecules & organelles around cell
in directed manner (intracellular motility)
–
–
Halt vesicle transport between compartments if
disrupt MTs so transport dependent on them
Proteins made in neuron cell body move down axon
(neurotransmitters, etc.) in vesicles
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
C. 3. Microtubules: Functions
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
6. Dynamic Properties
– Different materials move at different rates; fastest rate is
5 µm/sec (400 mm/day); vesicles seen attached to MTs
– Structures & materials moving toward neuron terminals
are said to move anterograde
– Other structures, like endocytic vesicles that are formed
at neuron terminals & carry regulatory factors from
target cells, move from synapse to cell body in a
retrograde direction
– Ex.: axons filled with MTs, MFs & IFs; evidence
suggests that both anterograde & retrograde movement
are mediated mostly by MTs; video microscopy shows
vesicles moving along MTs
– Confirmed by EM of axons; molecular motors move
vesicles along the MTs that serve as tracks
7. Flagella and Cilia
D. Microfilaments
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
C. 3. Microtubules: Functions
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments


Motile elements of cilia & flagella (more
later)
Active components of mitotic/meiotic
machinery; move chromosomes
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
C. 4. Microtubules: Motors
3. Functions
4. Microtubule Motors
5. MTOCs
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments
4.
Microtubule motors
–

Alberts: Fig 16-58, 59, 60, 62, 63, 64, 67
Motor proteins: convert chemical energy stored
in ATP into mechanical energy that is used to
move cellular cargo attached to motor
–
–
Types of cellular cargo transported by these
molecular motors include: vesicles, organelles
(mitochondria, lysosomes, chloroplasts),
chromosomes, other cytoskeletal filaments
A single cell may contain dozens of different motor
proteins, each specialized for moving a particular
type of cargo in particular cell region
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 4. Microtubules: Motors
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments
– Collectively, motor proteins are grouped into 3 broad
families: myosins, kinesins, dyneins
» Kinesins & dyneins move along MTs; myosins move along
MFs; None known for ifs
» Motor proteins move unidirectionally along their
cytoskeletal track in a stepwise manner from one binding
site to the next
» As they move along, they undergo a series of
conformational changes (a mechanical cycle)
» Steps of mechanical cycle are coupled to chemical cycle,
which provides energy fueling movement
» Includes motor binding ATP, ATP hydrolysis, product
(ADP & Pi) release & binding of new ATP
» Binding & hydrolysis of 1 ATP moves motor a few nm
along track; Cycles repeated many times
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 4. Microtubules: Motors
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments

Kinesins
– Kinesins move vesicles/organelles from cell body to
synaptic knobs; isolated in 1985 from squid giant
axons; tetramer made of 2 identical heavy chains & 2
identical light chains; smallest & best understood
– Large protein - pair of globular heads generate force
by hydrolyzing ATP & bind MT; each head connected
to a neck, a rodlike stalk & fan-shaped tail that binds
cargo to be hauled
– Diverse superfamily of kinesins - heads similar since
roles similar; tails vary since they haul different
cargoes
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 4. Microtubules: Motors
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments
– In vitro mobility assay - kinesin-coated beads move to
MT "+" end (axon tip); it is a "+" end-directed MT
motor, therefore, kinesin responsible for anterograde
movement
» All MTs of axon are oriented with"-" ends facing cell body &
"+" ends facing synaptic knobs
» Moves through ATP-dependent cross-bridge cycle along
single MT protofilament (rate proportional to [ATP]; up to ~1
µm/sec); at low concentrations, move slowly & see movement
in distinct steps
» Each step is ~8 nm in length, the spacing between tubulin
dimers along protofilament
» Appear to move 2 globular subunits (or 1 dimer at a time);
usually toward membrane & "+" ends
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 4. Microtubules: Motors
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments
– Kinesin possesses 2 motor domains that work by
“hand-over-hand” mechanism; one always firmly
attached to MT
» 2 heads of kinesin behave in coordinated manner, so that they
are always present at different stages in their chemical &
mechanical cycles at a given time
» When one head binds to MT, the interaction induces a
conformational change in adjacent neck region of motor
protein; it swings the other head forward toward binding site
on next dimer
» Force generated by head catalytic activity leads to swinging
movement of neck
» A kinesin molecule walks along a MT, hydrolyzing one ATP
with each step
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 4. Microtubules: Motors
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments

Conventional kinesin (discovered in 1985) is only
one member of a superfamily of related kinesins
– Mammalian genome sequence analysis leads to
estimate that mammals make >50 different kinesins
– Heads of kinesins have related amino acid sequences,
reflecting common evolutionary ancestry & their
similar role in moving along MTs
– In contrast, kinesin tails have diverse sequences,
reflecting variety of cargo different proteins haul
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 4. Microtubules: Motors
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments
– Most kinesins travel toward the "+" end; but one small
subfamily of kinesins (including the Drosophila Ncd
protein) moves toward the MT "-" end
» one would expect that the heads of "+"- & "-"-directed would
have a different structure since the heads contain the catalytic
core of the motor domain
» But the heads are virtually indistinguishable; instead
differences in direction of movement are determined by
differences in the adjacent neck regions of the two proteins
» When the head of a "-" end-directed Ncd molecule is joined to
the neck-stalk portions of a kinesin molecule, the hybrid
protein moves toward the "+" end of a MT track
» Even if the hybrid has a catalytic domain that would normally
move toward the "-" end of a MT, as long as it is joined to the
neck of a "+" end motor, it moves in the "+" direction
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 4. Microtubules: Motors
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments

A third subfamily of kinesinlike proteins is
incapable of movement: kinesins of this
group, like KXKCM1, are thought to
destabilize MTs rather than acting as MT
motors
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 4. Microtubules: Motors
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments

Cytoplasmic Dyneins
– Dyneins - first MT-associated motor found (1963);
responsible for moving cilia & flagella
– Thought to be ubiquitous eukaryotic motor protein;
related protein found in variety of nonneural cells
– Cilia/flagella form of protein was called axonemal
dynein; its new relatives were called cytoplasmic
dynein
– Huge protein (~1.5 million daltons); 2 identical heavy
chains & variety of intermediate & light chains
– Each dynein heavy chain forms large globular head
(~10X larger than a kinesin head) that generates force;
moves along MT toward "-" end
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
C. 4. Microtubules: Motors
3. Functions
4. Microtubule Motors
5. MTOCs
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments

Suggested roles of cytoplasmic dynein
–
–
–
–
Force generating agent for chromosome movement in
mitosis
"-"-directed MT motor for Golgi complex positioning
& movement of vesicles/organelles through
cytoplasm
In nerve cells, cytoplasmic dynein involved in axonal
retrograde organelle movement (toward cell body &
cell center) & anterograde movement of MTs
Fibroblasts & other nonneural cells: may move varied
membranous organelles (endosomes, lysosomes, ERderived vesicles going toward Golgi) from periphery
toward cell center
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 4. Microtubules: Motors
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments
– Cytoplasmic dynein does not interact directly with
membrane-bounded cargo, but requires intervening
multisubunit complex, dynactin that may regulate
dynein activity & help bind it to MT
– Present model may be overly simplistic: kinesin &
cytoplasmic dynein move similar materials in opposite
directions over the same railway network
– Individual organelles may bind kinesin & dynein
simultaneously although only one is active at given
time; myosin may also be present on some of these
organelles
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
C. 5. Microtubules: MTOCS
3. Functions
4. Microtubule Motors
5. MTOCs
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments
5.
Microtubule-organizing centers
–
–
Alberts: Panel 16-1; Fig 16-29, 30, 31, 32, 33
Function of MT in living cell depends on its
location & orientation, thus it is important to
understand why a MT assembles in one place
as opposed to another
controlled by MT-organizing centers
(MTOCs)
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 5. Microtubules : MTOCS
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments

Assembly of MTs from ab-dimers occurs in
2 distinct phases
– Nucleation - slower; small portion of MT
initially formed; occurs in association with
specialized structures in vivo called
microtubule-organizing centers (MTOCs);
centrosome is example
– Elongation - more rapid
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 5. Microtubules : MTOCS
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments

Centrosomes - complex structure with 2 barrelshaped centrioles surrounded by amorphous,
electron dense pericentriolar material (PCM)
– In animal cells, cytoskeleton MTs typically form in
association with centrosome
– Centrioles: cylindrical; ~0.2 nm dia & typically ~twice
as long; usually with 9 evenly spaced fibrils
– Each fibril seen in cross section to be composed of 3
fused MTs (A, [the only complete one]; B & C), A is
attached to centriole center by radial spoke
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 5. Microtubules : MTOCS
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments
– 3 MTs of each triplet arranged in pattern that gives
centriole a characteristic pinwheel appearance
– Centrioles usually in pairs at right angles to each other
near cell center just outside nucleus
– Extraction of isolated centrosomes with 1 M potassium
iodide removes ~90% of PCM protein leaving behind
spaghetti-like scaffold of insoluble fibers
– Centrosomes are sites of convergence of large numbers
of MTs
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 5. Microtubules : MTOCS
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments

MT polymerization & disassembly - treat with
poisons (CO, NO) or cold —> MTs disassemble;
much has been learned about their disassembly &
reassembly in cultured animal cells in this way
– Observe assembly when cells warmed or poisons
removed; fix at various times after & stain with
fluorescent anti-tubulin ABs
– Within a few minutes of inhibition removal, 1 or 2
bright fluorescent spots seen in cytoplasm
– Within 15 - 30 minutes, number of labeled filaments
radiating from these foci rises dramatically
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 5. Microtubules : MTOCS
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments
– In EM: MTs radiate out from centrosome; MTs
don't actually penetrate into centrosome &
contact centrioles, but terminate in dense
pericentriolar material residing at centrosome
periphery
– PCM apparently initiates MT formation;
centrioles not involved in MT nucleation, but
they probably play a role in recruiting
surrounding PCM during centrosome assembly
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
C. 5. Microtubules : MTOCS
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments

Centrosome typically situated near center of cell,
just outside nucleus
– Columnar epithelium - centrosome moves from cell
center to apical region just beneath cortex;
cytoskeletal MTs emanate from site, extending toward
nucleus & basal surface of cell
– Regardless of location, centrosomes are sites of MT
nucleation; polarity is always the same: "-" end at
centrosome, "+" (growing) end at opposite tip
– Thus, even though MTs are nucleated at MTOC, they
are elongated at opposite end of polymer
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 5. Microtubules : MTOCS
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments

Not all MTs associated with centrosome;
– some animal cells (mouse oocytes) lack
centrosomes entirely, but still make spindle
– MTs of axon are not associated with
centrosome, which is located in cell body, but
they may be formed at centrosome, then
released from that MTOC & carried to axon
by motor proteins
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 5. Microtubules : MTOCS
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments

Basal bodies & other MTOCs
– Centrosomes are not the only MTOCs in cells; basal
bodies at base of cilia & flagella serve as origin of
ciliary & flagellar MTs; MTs grow out of them
– Basal body cross-section looks like centriole; in fact,
the two can give rise to one another
– Sperm flagellum arises from basal body derived from
sperm centriole that had been part of meiotic spindle
of spermatocyte from which the sperm arose
– Conversely, sperm basal body typically becomes
centriole during fertilized egg's first mitotic division
of fertilized egg
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 5. Microtubules : MTOCS
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments

Plant MTOC - lack both centrioles &
centrosomes; MTOCs more dispersed than
those of animals
– In plant endosperm cells, the primary MTOC
consists of patches of material situated at outer
surface of nuclear envelope from which
cytoskeletal MTs emerge
– MT nucleation also thought to occur
throughout plant cell cortex
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 5. Microtubules : MTOCS
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments

MT nucleation
– Despite diverse appearances, all MTOCs play
similar roles in all cells
– Control number of MTs that form & their
polarity
– Control the number of protofilaments that
make up their walls
– Control the time & location of MT assembly
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 5. Microtubules : MTOCS
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments
–
–
–
–
All MTOCs share a common protein component, gtubulin (discovered in mid-1980s); it is ~0.005% of
total cell protein while a- & b-tubulins are 2.5% of
total nonneural cell protein
Fluorescent anti-g-tubulin antibodies (ABs) stain all
MTOCs, like centrosome PCM; suggests it is critical
component in MT assembly & nucleation
Microinject anti-g-tubulin AB into living cell —>
blocks MT reassembly after depolymerization by
drugs or cold temperatures
Genetically engineered fungi lacking functional gtubulin gene cannot assemble normal MTs
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 5. Microtubules : MTOCS
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments

Nucleation mechanism revealed by
structure/composition studies of PCM at
centrosome periphery
– Insoluble fibers of PCM are thought to serve as
attachment sites for ring-shaped structures that have
same diameter as MTs (25 nm) & contain g-tubulin
– Ring-shaped structures found when centrosomes were
purified & incubated with gold-labeled anti-g-tubulin
ABs —> cluster in rings/semi-circles at MT minus ends
(ends embedded in PCM)
– Isolate similar ring-shaped complexes (g-TuRCs) from
cell extracts; nucleate MT assembly in vitro
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 5. Microtubules : MTOCS
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments

Model - helical array of 13 g-tubulin subunits
forms open, ring-shaped template on which first
row of ab-tubulin dimers assemble;
– Only a-tubulin of heterodimer can bind to ring of gsubunits, establishing polarity of entire MT

2 other tubulin isoforms d-tubulin & e-tubulin
have also been identified in centrosomes, but their
function has not been determined
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
C. 6. Microtubules : Dynamic
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments
6.
Dynamic properties of microtubules
–
–
–
–
Alberts: Table 16-2; Fig 16-16, 16-17
MTs vary markedly in stability even though similar
morphologically - spindle/cytoskeleton labile; mature
neuron MTs less labile; cilia/flagella very stable;
lability allows cell to respond to stimuli
Cilia/flagella MTs are stabilized by MAP attachment
& by enzymatic modification (e. g. acetylation) of
specific amino acid residues within tubulin subunits
Labile MTs in living cells can be disassembled
without disrupting other cell structures via a number
of treatments
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
C. 6. Microtubules : Dynamic
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments

Treatments that cause MT disassembly;
usually interfere with noncovalent bonds
holding them together
–
–
–
–
Cold temperatures
Hydrostatic pressure
Elevated Ca2+ concentration
Variety of chemicals (often used in
chemotherapy) - CO, vinblastine, vincristine,
NO, podophyllotoxin
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
C. 6. Microtubules : Dynamic
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments

Some treatments (taxol) disrupt MT
dynamic activity & act by doing the
opposite; inhibit disassembly
– Taxol binds MT polymer & thus prevents
disassembly; cell cannot build new MT
structures
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
C. 6. Microtubules : Dynamic
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments


Cytoskeletal MT lability reflects fact that they are
polymers formed by noncovalent association of
dimers; subject to
depolymerization/repolymerization as cell needs
change
Dramatic changes in MT spatial organization may
be achieved by combination of 2 separate
mechanisms
– Rearrangement of existing MTs
– Disassembly of existing MTs & reassembly of new
ones in different cell regions
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
C. 6. Microtubules : Dynamic
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
6. Dynamic Properties
7. Flagella and Cilia

D. Microfilaments
Study of MT dynamics in vitro - suggest that
cytoskeleton can rapidly remodel & respond to
stimuli
– Early studies established that GTP binding to b -subunit
required for MT assembly; GTP hydrolysis not needed
for binding, but it is hydrolyzed soon after dimer
attached to MT end; GDP stays bound
– After dimer is released from MT during disassembly &
enters soluble pool, GDP is replaced by GTP, thus
recharging dimer so that it can add to polymer again
– A GTP molecule is also bound to a-tubulin subunit, but
it is not exchangeable & it is not hydrolyzed after
subunit incorporation
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
C. 6. Microtubules : Dynamic
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
6. Dynamic Properties
7. Flagella and Cilia

D. Microfilaments
Assembly is not energetically inexpensive
since it includes GTP hydrolysis, but it does
allow the cell to control assembly &
disassembly independently
– A dimer being added to MT has a bound GTP;
dimer being released from MT has bound GDP
– GDP- & GTP dimers have different
conformations & participate in different
reactions; the ends of growing & shrinking MTs
have different structures
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
C. 6. Microtubules : Dynamic
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
6. Dynamic Properties
7. Flagella and Cilia

D. Microfilaments
The above facts lead to the following model:
– When a MT is growing, the "+" end is present as an
open sheet to which GTP-dimers are added
– During rapid growth periods, tubulin dimers are added
faster than GTP can be hydrolyzed
– The resultant cap of GTP-dimers on MT at
protofilament ends is thought to favor the addition of
more subunits & hence MT growth
– However, MTs with open ends thought to undergo
spontaneous reaction leading to tube closure
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
C. 6. Microtubules : Dynamic
4. Microtubule Motors
5. MTOCs
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments
– tube closure is accompanied by hydrolysis of
bound GTP, changing tubulin dimer
conformation —> resultant mechanical strain
destabilizes MTs
– Strain is released as protofilaments curl out
from tubule & catastrophically depolymerize
– Disassembly can occur remarkably fast,
especially in vivo, which allows very rapid MT
cytoskeleton disassembly
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
C. 6. Microtubules : Dynamic
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
6. Dynamic Properties
7. Flagella and Cilia

D. Microfilaments
Study of MT dynamics in vivo: dynamic character
of MT cytoskeleton inside cell is best revealed by
microinjecting labeled tubulin into nondividing
cultured cell
– Inject labeled tubulin into nondividing cultured cell —
> labeled subunits rapidly incorporated into preexisting
cytoskeleton MTs, even in absence of any obvious
morphological change
– Watch cell with fluorescent-labeled MTs over time —>
some MTs grow, others shrink; dynamic
– Both growth & shrinkage in vivo occur predominantly
at "+" end of polymer, the end located opposite the
centrosome (or other MTOC)
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
C. 6. Microtubules : Dynamic
4. Microtubule Motors
5. MTOCs
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments
– Single MTs switch randomly & unpredictably
between growing & shrinking (dynamic
instability)
– MTs shrink faster than they grow, so in a matter
of minutes, MTs disappear & are replaced by
new MTs that grow out from centrosome
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
C. 7. Microtubules: Flagella
3. Functions
4. Microtubule Motors
5. MTOCs
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments
7.
Cilia and flagella structure
–


Alberts: Fig 16-80, 81, 82, 83, 84
Entire ciliary or flagellar projection is covered
by membrane continuous with cell membrane
Cilium core (axoneme) contains an array of MTs
that run longitudinally through entire organelle
–
–
Usually 9 peripheral doublet MTs surrounding central
pair of single MTs; known as 9 + 2 pattern or array;
all MTs in array have same polarity ("+" ends at tip,
"-" ends at base)
Doublets - 1 complete (A tubule; 13 subunits) MT; 1
incomplete (B tubule) MT with 10 or 11 subunits
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 7. Microtubules: Flagella
6. Dynamic Properties
7. Flagella and Cilia
D. Microfilaments
– Not all eukaryotes have them; cilia & flagella generally
absent among fungi, nematodes & insects
– Where they do occur, they nearly always show same 9
+ 2 array, a reminder that all living eukaryotes have
evolved from a common ancestor
– Despite high degree of conservation (e. g. 9 + 2 pattern)
some evolutionary departures:
» 9 + 1 array in flatworms
» 9 + 0 array in Asian horseshoe crab, eel, mayfly; some lacking
central elements are motile, some not
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 7. Microtubules: Flagella
6. Dynamic Properties
7. Flagella and Cilia

D. Microfilaments



Central MTs enclosed by projections forming
central sheath; sheath connected to doublet A MTs
by radial spokes; doublets connected by
interdoublet bridge made of elastic protein nexin
Pair of arms (inner & outer) project from A MT in
clockwise direction (when viewed base to tip)
Radial spokes typically in groups of three with
major repeat of 96 nm
Inner & outer dynein arms staggered along A MT
length (outer arms spaced every 24 nm; inner arms
arranged to match unequal spacing of radial
spokes)
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 7. Microtubules: Flagella
6. Dynamic Properties
7. Flagella and Cilia

D. Microfilaments
Cilia/flagellae emerge from basal bodies - 9
peripheral fibers consisting of 3 MTs rather
than 2 (A tube complete, B/C incomplete);
similar in structure to centrioles
– No central MTs as in centrioles; also similar to
centrioles in other ways
– A & B tubules elongate to form cilia/flagella
doublet; if sheared off, regrow from basal body
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 7. Microtubules: Flagella
6. Dynamic Properties
7. Flagella and Cilia

D. Microfilaments

The mechanism of ciliary & flagellar locomotion:
sliding filament model suggested mechanism of
ciliary/flagellar movement was sliding of adjacent
MT doublets relative to one another
In model, dynein arms act as swinging crossbridges that generate forces needed for
ciliary/flagellar movement; dynein arms projecting
from one doublet walk along adjacent doublet wall
—> sliding
A. Overview
B. Experimental Methods
C. Microtubules
1. Structure
2. MAPs
3. Functions
4. Microtubule Motors
5. MTOCs
C. 7. Microtubules: Flagella
6. Dynamic Properties
7. Flagella and Cilia

D. Microfilaments
Sequence of events in ciliary/flagellar sliding
motion
– Dynein arms anchored on a doublet's A MT attach to
binding sites on B MT of adjacent doublet
– Dynein molecules undergo conformational change;
causes A MT doublet to move slightly toward basal end
of attached B MT doublet
– Dynein then releases B tubule of adjacent doublet
– Dynein arms reattach to adjacent doublet's B MT closer
to its base so another cycle can begin
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
1. Structure
2. P/D
3. Myosins
4. Muscle Contraction
D. Microfilaments
5. Nonmuscle Actin
1.
2.
3.
4.
5.
Structure
Polymerization/depolymerization
Myosin
Muscle Contraction
Non-muscle motility
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
1. Structure
2. P/D
D. 1. Microfilaments: Structure
3. Myosins
4. Muscle Contraction
5. Nonmuscle Actin
1.


Structure
Alberts Fig 16-12
Microfilaments:
–
–
–
–
~8 nm diameter
made of globular actin subunits (G-actin)
found in most animal cells, also higher plants
“Microfilament,” “actin filament,” “F-actin
filaments” are synonyms but F-actin often
used for those formed in vitro
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
1. Structure
2. P/D
D. 1. Microfilaments: Structure
3. Myosins
4. Muscle Contraction
5. Nonmuscle Actin

In presence of ATP, G-actin polymerizes to form
stiff filament made of 2 strands of F-actin wound
around each other in a helical configuration
–

Each subunit has polarity & all subunits are pointed
in same direction, so entire MF has polarity
Depending on cell type & activity in which it is
engaged, MFs can be organized into highly
ordered arrays, loose ill-defined networks, or
tightly anchored bundles
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
1. Structure
2. P/D
D. 1. Microfilaments: Structure
3. Myosins
4. Muscle Contraction
5. Nonmuscle Actin

Actin identified more than 50 years ago as one of
major contractile proteins of muscle cells
– Since then found to be major protein in virtually every
eukaryotic cell examined
– Higher plants & animals possess number of actincoding genes whose products are specialized for
different types of motile processes
– Actin structure highly conserved evolutionarily (yeast
actin & rabbit skeletal actin 88% identical); means that
nearly all aminos are crucial to function; actin from
diverse sources can copolymerize
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
1. Structure
2. P/D
D. 1. Microfilaments: Structure
3. Myosins
4. Muscle Contraction
5. Nonmuscle Actin

Actin detected microscopically:
– By electron microscopy, using proteolytically cleaved
myosin head fragments (HMM or S1 fragments)
» HMM & S1 bind actin all along MF —> see polarity in EM;
one end of MF pointed, other end barbed
» Orientation of arrowheads formed by S1-actin complex
provides information as to direction in which MFs are likely to
be moved by myosin motor protein
– By fluorescence microscopy, with fluorescently labeled
S1 or anti-actin ABs
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
1. Structure
D. 2. Microfilaments: P/D
2. P/D
3. Myosins
4. Muscle Contraction
5. Nonmuscle Actin
2.


MF polymerization/depolymerization
Alberts Table 16 – 2, Fig. 16 – 36, 37, 38
Before polymerization, actin monomer binds to
adenosine nucleotide (usually ATP); like GTP in
MTs
–
–

Actin is an ATPase (like tubulin is GTPase); role of
ATP in MF assembly is same as GTP in MT
Some time after incorporation into growing actin
filament, ATP hydrolyzed to ADP
If filaments built at high rate, the end has actinATP cap (hinders disassembly, favors assembly)
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
1. Structure
2. P/D
3. Myosins
4. Muscle Contraction
D. 2. Microfilaments: P/D
5. Nonmuscle Actin

Actin polymerization can be studied in vitro by
labeling or viscosity studies
– In vitro with high concentration of labeled G-actin, both
ends labeled but .....
» One end incorporates monomers at 5 - 10 times higher rate
than the other end
» Decoration with S1 myosin fragment reveals that barbed ("+"
end) of MF is fast-growing end, while the pointed ("-") end is
the slow-growing tip
– In lower concentrations of G-actin:
» Actin-ATP subunits add to "+" end & actin-ADP subunits tend
to leave from "-"
» Can be demonstrated by pulse-chase “treadmilling”
experiments
» Don’t know if treadmilling occurs in vivo
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
1. Structure
2. P/D
3. Myosins
4. Muscle Contraction
D. 2. Microfilaments: P/D
5. Nonmuscle Actin


MFs maintain a dynamic equilibrium between
monomeric & polymeric actin - can be influenced
by a variety of different proteins
Changes in local conditions in particular part of
cell can push equilibrium either toward assembly
or disassembly
– allows cell to reorganize its MFs cytoskeleton by
controlling this equilibrium
– need such reorganization for dynamic processes (cell
locomotion & shape changes, cytokinesis)
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
1. Structure
2. P/D
3. Myosins
4. Muscle Contraction
D. 2. Microfilaments: P/D
5. Nonmuscle Actin

Actin-binding proteins in the cell affect the nucleation and
polymerization rate of microfilaments
– Formin: A dimeric protein that initiates nucleation by capturing
two monomers of actin, then remains associated with the plus end
of a rapidly growing microfolament
– Thymosin: A protein that binds to actin monomers and inhibits
nucleotide exchange or polymerization, keeping much of the
available actin in an unpolymerized state
– Profilin: A protien that competes with thymosin for binding to actin
monomers; it binds opposite the ATP binding site on actin and
promotes polymerization at the plus end of a growing filament
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
1. Structure
2. P/D
3. Myosins
4. Muscle Contraction
D. 2. Microfilaments: P/D
5. Nonmuscle Actin

Inhibitors of microfilament
polymerization/depolymerization used to
study microfilament polymerization (Table
16 - 2
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
1. Structure
2. P/D
D. 3. Microfilaments: Myosins
3. Myosins
4. Muscle Contraction
5. Nonmuscle Actin
Myosins
Alberts fig 16 – 54, 55, 56, 57, 60, 61, 65, 68, 69, 72
Myosin's sole known function is as motor for actin;
3.


–
–
–
Almost all motors known to interact with actin are members of
myosin superfamily
all of them move toward MF plus end (except for myosin VI)
First isolated from mammalian skeletal muscle & then from
wide variety of eukaryotic cells: protists, higher plants,
nonmuscle animal cells, vertebrate cardiac & smooth muscle
tissues
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
1. Structure
2. P/D
D. 3. Microfilaments: Myosins
3. Myosins
4. Muscle Contraction
5. Nonmuscle Actin

Structure of myosins
– All share characteristic motor (head) domain, which has
a site that binds actin filament & one that binds &
hydrolyzes ATP to drive the myosin motor
– While head domains of myosins are similar, tail
domains are highly divergent
– Myosins also contain variety of low molecular weight
(light) chains
– Based on these construction differences divided into 2
large groups - conventional & unconventional
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
1. Structure
2. P/D
D. 3. Microfilaments: Myosins
3. Myosins
4. Muscle Contraction
5. Nonmuscle Actin

Conventional (type II):
– found in various muscle tissues, and also in a variety of
nonmuscle cells (generate tension at focal contacts,
cytokinesis)
– Structure of myosin II molecules: 6 polypeptide chains
(one pair of heavy chains, 2 pairs of light chains);
organized in a way that produces a highly asymmetric
protein with 3 sections
» A pair of globular heads that contain the molecule’s catalytic
site
» A pair of necks, each consisting of a single, uninterrupted ahelix & 2 associated light chains
» A single, long, rod-shaped tail formed by the intertwining of
long a-helical sections of the 2 heavy chains to form an ahelical coiled-coil
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
1. Structure
2. P/D
D. 3. Microfilaments: Myosins
3. Myosins
4. Muscle Contraction
5. Nonmuscle Actin

Immobilize isolated myosin heads (S1 fragments)
on glass cover slip —> cause actin filament
sliding
– Single head domain has all of the machinery needed for
motor activity



The fibrous tail plays a structural role, allowing
the protein to form filaments
Light chain phosphorylation regulates assembly of
myosin II into thick filaments
Tail ends of myosin molecule point toward
filament center; heads point toward ends (bipolar)
– Polarity of filament reverses at its center
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
1. Structure
2. P/D
D. 3. Microfilaments: Myosins
3. Myosins
4. Muscle Contraction
5. Nonmuscle Actin
Skeletal muscle myosin II filaments are
highly stable
 smaller myosin II filaments (most
nonmuscle cells) often display transient
construction (assembling when & where
needed, then disassembling)

A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
1. Structure
2. P/D
D. 3. Microfilaments: Myosins
3. Myosins
4. Muscle Contraction
5. Nonmuscle Actin

Unconventional myosins subdivided into at least
14 different types
– Each type is presumed to have its own specialized
functions
– Several types may be present together in same cell

Some functions of unconventional myosins
– Amoeboid movement & phagocytosis (myosin I)
– Movement of cytoplasmic vesicles & organelles
(myosins I, V, & VI)
– Stereocilia in cochlea hair cells of inner ear (myosin
VIIa)
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
1. Structure
2. P/D
D. 4. Microfilaments: Muscle
3. Myosins
4. Muscle Contraction
5. Nonmuscle Actin
4.


Muscle contraction
Alberts 16 – 73, 74, 75, 76, 61, 77, 78
Skeletal muscle cell structure - highly
unorthodox; cylindrical; 10 - 100 µm thick; up
to 400 mm long
–
–
Skeletal muscle cells are multinucleate (100s), the
result of embryonic fusion of mononucleate
myoblasts (premuscle cells); even myoblasts from
distantly related animals fuse in culture
Because of their properties, these cells are more
appropriately called muscle fibers
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
D. 4. Microfilaments: Muscle
1. Structure
2. P/D
3. Myosins
4. Muscle Contraction
5. Nonmuscle Actin

Muscle fibers may have the most orderly internal
structure of any cell in body
– Muscle fiber is cable made up of hundreds of thinner,
cylindrical strands (myofibrils)
– Each myofibril is repeating linear array of contractile
units (sarcomeres)
– Each sarcomere, in turn, has characteristic banding
pattern that gives muscle fiber a striated look

Myofibrils separated by cytoplasm with
intracellular membranes & mitochondria, lipid
droplets, glycogen granules
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
D. 4. Microfilaments: Muscle
1. Structure
2. P/D
3. Myosins
4. Muscle Contraction
5. Nonmuscle Actin

Banding pattern is result of partial overlap
between thick & thin filaments
– Each sarcomere extends from Z line to Z line (~2.5 µm)
& contains several dark bands & light zones; there is a
pair of light staining I bands at each end of sarcomere
– More densely staining A band is between outer I bands;
lightly staining H zone in A band center
– Densely staining M line lies in center of H zone
– I bands - only thin filaments; H zone - only thick
filaments; A band outside H zone - both overlap
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
D. 4. Microfilaments: Muscle
1. Structure
2. P/D
3. Myosins
4. Muscle Contraction
5. Nonmuscle Actin

Composition & organization of thin filaments
– Thin filaments mostly actin
– In addition to actin, thin filaments also contain two
other proteins: troponin & tropomyosin
» Tropomyosin - elongated, ~40 nm long; fits securely into
grooves between two thin filament actin chains; each rodshaped tropomyosin interacts with 7 actin subunits linearly
along F-actin chain
» Troponin - globular protein complex with 3 subunits - each has
distinct, important functional role; ~40 nm apart on thin
filament, contact both actin & tropomyosin thin filament
components
– Actin filaments of each half sarcomere aligned with
barbed ends linked to Z line by a-actinin
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
D. 4. Microfilaments: Muscle
1. Structure
2. P/D
3. Myosins
4. Muscle Contraction
5. Nonmuscle Actin

Composition & organization of thick filaments
– Thick filaments are several 100 myosins & a small
number of other proteins
– Like filaments formed in vitro, thick filament polarity is
reversed at sarcomere center; filament center is
composed of opposing tail regions of myosin molecules
& is devoid of heads
– Myosin heads project from each thick filament along
remainder of its length due to staggered positions of
myosins making up the body of the filament
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
1. Structure
2. P/D
D. 4. Microfilaments: Muscle
3. Myosins
4. Muscle Contraction
5. Nonmuscle Actin
– Titin (~3 x 106 dalton MW [27,000 aa’s], 1 µm
long) - largest protein yet discovered; originates
at M line & extends along myosin filament past
A band to terminate on Z line
» Highly elastic protein; stretches (bungee cord) as
certain domains within molecule are unfolded
» It is thought to prevent sarcomere from being pulled
apart during muscle stretching
» Also maintains myosin filaments in proper position
in sarcomere center during contraction
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
1. Structure
2. P/D
3. Myosins
4. Muscle Contraction
5. Nonmuscle Actin

D. 4. Microfilaments: Muscle
The Sliding Filament Model of muscle contraction
– muscles contract by shortening at sarcomere level
– combined decrease in sarcomere length accounts for
decrease in length of entire muscle
– Sarcomere banding patterns shift during contraction shows that filaments slide
» H zone & I bands decrease in width during contraction &
eventually disappear; A bands stay same
» During contraction, Z lines move inward & approach A band
outer edges until they touch each other
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
D. 4. Microfilaments: Muscle
1. Structure
2. P/D
3. Myosins
4. Muscle Contraction
5. Nonmuscle Actin

Molecular basis of contraction
– During contraction, each myosin head extends out &
binds tightly to thin filaments forming cross-bridges
– Once bridges form, heads change shape, bending
toward sarcomere center (power stroke)
– Moves actin filament over thick filament (~5 - 15 nm;
controversy exists over these distances) toward
sarcomere center
– Heads hydrolyze ATP & act as levers for thin filament
motion; each myosin cross-bridge mechanical activity
cycle is accompanied by ATPase activity cycle
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
D. 4. Microfilaments: Muscle
1. Structure
2. P/D
3. Myosins
4. Muscle Contraction
5. Nonmuscle Actin

Details of the sliding filament model
– Cycle starts with ATP binding to myosin head, which
induces dissociation of cross-bridge from actin
– ATP binding followed by its hydrolysis before head
contacts actin filament; ADP & Pi products stay bound
at enzyme active site
– Energy released by hydrolysis absorbed by myosin as a
whole, placing the cross-bridge in energized state, like a
stretched spring capable of spontaneous movement
– Energized myosin attaches to actin & releases its bound
Pi
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
D. 4. Microfilaments: Muscle
1. Structure
2. P/D
3. Myosins
4. Muscle Contraction
5. Nonmuscle Actin
– Triggers a large conformational change, driven by
stored free energy
– The shape change shifts actin filament toward
sarcomere center (myosin head power stroke)
– Bound ADP leaves & new ATP attaches, inducing
release of cross-bridge; cycle starts over
– In absence of ATP, cross-bridges stay tightly intact
binding actin (causes rigor mortis after death)

Skeletal muscle contraction is triggered by a
nerve-impulse mediated release of calcium ions
into the sarcomere
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
1. Structure
2. P/D
D. 4. Microfilaments: Muscle
3. Myosins
4. Muscle Contraction
5. Nonmuscle Actin

Regulation of skeletal muscle contraction
– Muscle fibers are organized into motor units;
all fibers of motor unit jointly innervated by
branches of one motor neuron; contract
simultaneously if stimulated by impulse
transmitted along that neuron
» Point where neuron axon terminus & muscle fiber
make contact called neuromuscular junction
» Junction is site of nerve impulse transmission from
axon across synaptic cleft to muscle fiber
» Muscle fiber plasma membrane is also excitable &
capable of conducting an action potential
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
1. Structure
2. P/D
D. 4. Microfilaments: Muscle
3. Myosins
4. Muscle Contraction
5. Nonmuscle Actin
– Unlike a neuron, where an action potential stays
at the cell surface, skeletal muscle cell impulse
is propagated into cell interior along
membranous folds (transverse [T] tubules)
» T tubules terminate in close proximity to a
cytoplasmic membrane system (sarcoplasmic
reticulum [SR]) that forms membranous sleeve
around myofibril (specialized form of smooth ER)
» ~80% of SR membrane integral protein is ATPdriven Ca2+ pump (moves Ca2+ from cytosol into SR
lumen where it is stored until it is needed)
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
1. Structure
2. P/D
3. Myosins
D. 4. Microfilaments: Muscle
4. Muscle Contraction
5. Nonmuscle Actin
– With arrival of action potential at SR via T
tubules, opens Ca2+ channels in SR membrane
are opened
» Ca2+ diffuses out of SR compartment & over short
distance to myofibrils
» [Ca+2] levels go from ~2 x 10-7 M to ~5 x 10-5 M
– In a relaxed sarcomere (low [Ca2+]):
» Tropomyosin blocks myosin-binding sites on actin
molecules
» Tropomyosin’s position in filament groove blocking
sites is controlled by troponin
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
1. Structure
2. P/D
D. 4. Microfilaments: Muscle
3. Myosins
4. Muscle Contraction
5. Nonmuscle Actin
– When [Ca2+] rises:
» Ca2+ binds to troponin C subunits
» This causes a conformational change in troponin
» And this causes adjacent tropomyosin to move ~1.5
nm closer to center of filament’s groove
– The movement of tropomyosin exposes
myosin-binding site on adjacent actins, crossbridges form, contraction occurs
– Each troponin controls position of 1
tropomyosin, which, in turn, controls binding
capacity of 7 adjacent actin monomers
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
1. Structure
2. P/D
D. 4. Microfilaments: Muscle
3. Myosins
4. Muscle Contraction
5. Nonmuscle Actin
– When stimulation from motor neuron stops:
» SR Ca2+ channels close & excess Ca2+ pumped from
cytosol back into SR by the Ca2+ pump
» As [Ca2+] decreases, Ca2+ ions dissociate from
troponin binding sites
» Tropomyosin molecules return to their original
positions and block actin-myosin binding
A. Overview
B. Experimental Methods
C. Microtubules
D. Microfilaments
1. Structure
2. P/D
D. 5. Microfilaments: Nonmuscle
3. Myosins
4. Muscle Contraction
5. Nonmuscle Actin
Some examples of nonmuscle actin-myosin
5.
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Alberts Fig. 16 – 50, 51, 52
Cytokinesis
Phagocytosis
Cytoplasmic streaming (directed bulk flow of
cytoplasm occurring in certain large plant cells)
Vesicle trafficking
Blood platelet activation
Lateral movements of integral proteins within
membranes
Cell substratum interactions, cell locomotion &
axonal outgrowth
Changes in cell shape
Cell projections such as microvilli & stereocilia