Types of motor proteins
1. Actin-based: myosins
Myosin families: myosin I-XVIII
2. Microtubule based motors
a. Dynein
Flagellar and cytoplasmic dyneins. MW~500kDa
They move towards the minus end of MT
b. Kinesin
Cytoskeletal kinesins
Neurons, cargo transport along the axons
Kinesin family: conventional kinesins + isoforms. MW~110 kDa
They move towards the minus end of MT
Chapter 19
Part II
3. Nucleic acid based
DNA and RNA polymerases
They move along a DNA and produce force
All myosins have head, neck, and tail domains with distinct functions
Myosins are a large superfamily of mechanochemical motor proteins.
All myosin consist of one
or two heavy chains and
several light chains
(generally hae a
regulatory fucntion).
Type I, II V are present in
all eukaryotic cell.
Type I and V, in
cytoskeleton-membrane
interaction (transport of
membrane).
Type VII, XI, XIII are
only in plant.
Type XI, fastest myosin.
Implicated in
cytoplasmic streaming in
green algae and higher
plants.
Myosin I and V, light chain are
calmodulin, a Ca2+ binding
regulatory subunit
Myosin II, has two different light
chains called essential and
regulatory light chains, they are
Ca2+ binding protein, but
differ from calmodulin.
Light chains bound in the neck region
Powers muscle contraction
Myosin are Ca2+ depend, but
different regulatory mechanism.
α-helical sequence form a
rodlike coiled-coil
Chapter 3 motor protein
1
HEAD
NECK
Functions of myosin tail domains.
TAIL
Myosins are associated with
intracellular membrane vesicles or
the cytoplasmic face of the plasma
membrane
Neck
regulation
binds regulatory chains
Helical Tail:
binds cargo:
attachment to vesicles → transport
attachment to filaments →sliding
attachment to itself → formation of filaments
vesicle
membrane
Tail domains of myosins bind the plasma
membrane or the membranes of intracellular
organelles → membrane-related activities
Globular Head
mechano-chemical transduction
force generating domain
ATPase activity
MF(actin) binding site,
ATP hydrolysis results in unidirectional movement
MYOSIN II
Motor proteins
convert chemical energy (derived from ATP hydrolysis)
into mechanical force (movement)
Mysoin heads walk along actin filaments in discrete steps
Sliding-filament assay is used to
detect myosin-powered movement.
ATP add
非連續性
Coverslip
Fixed to myosin
Motor domains of most myosins move along actin
filaments toward the plus ends of the filaments.
This movement is ATP-dependent and is accompanied by
ATP hydrolysis.
An exception is myosin VI, which moves toward the minus
ends of actin filaments.
flash
2
Optical trap determines force & step size generated by a
single myosin molecule.
Can measure the force from myosin
Myosin-bound vesicles are carried along actin filaments.
Vesicle trafficking (myosins I, V, & VI)
– These myosins have high duty ratio (the fraction of time spent
attached to filaments during ATPase cycle).
Cytoplasmic streaming (myosin XI)
Move to +
Myosin II moves in a step of 5-10 nm & generates 3-5 piconewtons (pN) of
force.
Step size of a myosin is correlated to its neck length. Myosin V, with a long
neck, moves in a 36-nm step. However, the correlation is not absolute,
e.g. myosin VI.
麗藻細胞
Large vesicle, is part of the ER
network, contacts the stationary (不增
減) actin filaments and moves along
them by a myosin motor protein
Contractile ring in dividing cells.
Cytokinesis: as division of the cytoplasm.
Organized thick and thin filaments in skeletal muscle slide
past one another during contraction.
Typical skeletal muscle cell
called a myofiber, 1-40
mm in length and 10-50
um in width;
multinucleated (100).
Sarcomere(肌節):
cytoplasm is packed with
a regular repeating array
of filament bundles into a
specialized. And contain
two type: thick (myosin)
and thin (actin) filaments
Contraction: sarcomere
about 70% shorten
(thin filament)
(thick filament)
3
Combine with Figure 3-25.
Myosin + ATP → move to “+” end
Contraction of skeletal muscle is regulated by Ca2+
and actin-binding proteins.
Action potential → neuromuscular junction → opening Ca2+ voltage channel in the
sarcoplasmic reticulum → Ca2+ release →bind to troponin (C subunit) with I and T
subunit → control Tropomysion movement
No Ca2+ → myosin can bind thin filament but TM-TN complex prevent move
Ca2+ → bind → triggered TN-C →conformational change → slight movement of TM
→ expose the myosin-binding site on actin → when Ca2+ high lead contraction
(thin filament; actin filament)
troponin
tropomyosin
4
Thin filament:
(1). Actin (肌動蛋白)
(2). Tropomyosin (原肌球蛋白，旋轉肌球素)
(3). Troponin (肌鈣蛋白，旋轉素) :
a. troponin I (strong affinity for actin)；
b. troponin T (for tropomyosin)；
c. troponin C (for Ca)
Myosin-dependent mechanisms regulate contraction in smooth
muscle and nonmuscle cells.
Calcium-dependent activation of myosin II.
Ca2+ → bind to calmodulin → activate calmodulin → bind to myosin LC kinase
(MLCK) → phosphorylation of mysoin LC → contraction
LC: light chain
5
Signal-induced activation of myosin II by Rho kinase.
In smooth muscle or non-muscle cell, no neurotransmission.
Rho-GTP acts via Rho
Kinase (ROCK) to
activates interaction of
myosin II with actin
filaments, promoting
formation of stress fibers
and contraction of these
stress fibers at the rear of
a moving cell
Muscle cell → contraction regulated by nervous system
Smooth muscle regulated by phosphorylation
Cell functions for myosins
Cell Locomotion
Steps in keratinocyte movement.
lamellipodium: Actin filaments at the leading edge are rapidly cross linked
into bundles and net works in a protruding region.
Filopodia: Slender fingerlike membrane projections.
The head group of the myosin walks toward the plus end of
the actin filament.
6
Cell movement coordinates force generation with cell adhesion
1.
2.
3.
4.
1. Membrane extension.
(a) Actin filaments →
assembled into
branched network
Membrane extension
Cell substrate adhesions
Cell body translocation
Breaking cell attachments
Arp2/3 complex,
formed 70o angle
branch
Profilin promotes ADP/ATP
exchange by G-actin, to
yield the ATP-bound form
competent to polymerize, at
the leading edge of an
advancing cell.
Capping protein adds to
the plus ends of actin
filaments shortly after they
are nucleated by Arp2/3,
keeping actin filaments at
the leading edge short &
highly branched.
Cell locomotion by Controlled Actin Assembly
Focal contacts are made with
extracellular matrix Cause cross
talk between cytoskeletal elements
Actin nucleation is initiated
Web formation occurs Mediated by
Arp2/3 (actin-relatedprotein)
complex
Newly nucleated actin filaments are
attached to the sides of old
filaments at a 70o angle
In a steady state, ends are capped
After ATP hydrolysis, filaments are
depolarized by cofilin
This allows spacing of filament
Result: actin filament network
moves forward, moving cell
(b)Elongation of filament →
generated pushing force; elastic
brownian ratchet model.
The polymerization “motor”
Elastic Brownian ratchet
How can polymerization push ?
Cantilever 懸臂
However, a single filament is too weak…
7
2. Cell-substrate adhesions.
•
•
Prevent the leading lamella (薄板) from retracting (縮進).
Allow the cell to push forward.
3. Cell body translocation.
• Myosin-dependent cortical contraction.
4. Breaking cell attachments.
Measuring the rate of movement in cells that express varying levels of
integrins Æ The fastest migration occurs at an intermediate level of
adhesion.
Ameboid movement entails reversible gel-sol
transitions of actin networks.
Keratinocyte (角質細胞)
actin
•
•
At the front of the cell, actin polymerizes (profilin) to form gel-like
network (filamin, α-actinin).
At the tail of the cell, actin depolymerizes (cofilin, gelsolin) to form the
more fluid endoplasm.
Flash
myosin
Filaments (F actin) are another important
means by which the cytoskeleton is used for trafficking.
It is important for cell shape, location and contraction
G protein
Rho GEF
Rho kinase
偽足
片足
Role of signal transduction pathways in cell locomotion and the organization of
the cytoskeleton
8
An example for actin cytoskeleton
regulation
Actin filaments are rarely single …
Nets and bundles.
Profilin
thymosin
filamin
gelsolin
myosin
Current Opinion in Structural Biology 2002, 12:768–774.
Intermediate Filaments (IF)
Differ in stability, size, and structure from other
cytoskeletal fibers
Intermediate filament assembly
spontaneous assembly
→ No need of chaperone proteins or energy (no hydrolysis of
nucleotides) actin polymerization need energy
the filament has no polarity ≠ from actin or microtubule filaments
Intermediate diameter ~10 nm
Subunits are fibrous
Almost all subunits are incorporated into stable
intermediate filaments
No hydrolysis of ATP or GTP is required for
Intermediate filaments: cytoplasmic and nuclear
non-polar, tough, rope-like, less than 5% in soluble form, no nucleotide
provide protection against mechanical stress,
withstand stretching forces
polymerization
No known polarity of the filament
The formed fibers are not easily soluble
No direct participation in cell motility
IF protein-specific antibodies or cDNA used for cell typing and tumour diagnosis
REGULATION:
phosphorylation by PKC of the N-terminal Ser induces disassembly of IF
(particularly in nuclear lamins during mitosis)
No examples of motor proteins that move along
intermediate filaments
IF associated proteins (IFAPs)
9
INTERMEDIATE FILAMENT PROTEIN MONOMERS:
(cell-type-specific)
A model of intermediate filament construction
Class I. + II. (MW 40 - 70 000)
Cytokeratins
epithelial cells ( > 20 isoforms, skin, hair, nails)
Class III. (MW ~53 000)
Vimentin cells of mesenchymal origin
Desmin muscle
GFAPs astroglial cells (= Glial fibrillary acidic proteins)
Intermediate filaments
are only found in some
Metazoans :vertebrates,
nematodes,molluscs
Parallel
Antiparrel
Not required in
every cell type
Ancesters: nuclear lamins
No polarity!
Class IV. (MW 130 , 100 and 60 000)
Neurofilament proteins in neural cells
“subunit”
Easily bent
Hard to break
Class V. (MW = 65-75 000)
Nuclear lamins
inside surface of the inner nuclear membrane
most dynamic
8 parallel protofilaments
Keratin & lamin filaments in epithelial cells.
Intermediate filaments are
anchored in cell junctions
lamin intermediate
filaments: blue; nucleus
Cytoplasmic keratin
cytosleleton: red
Fig. 19-31
10
Intermediate filaments are resistent to bending or stretching forces
Neurofilaments in a neuronal axon.
Fig. 19-32
IF proteins are classified according to their
distribution in specific tissues.
Keratins: epithelial
Vimentin is the major IF in cells of mesenchymal and neuronal
origin
Glial Fibrillary Acidic Protein forms IF in glial cells and some
Schwann cells
Peripherin is a rare IF, occurring in some types of neurons
Desmin is the predominant IF in skeletal and cardiac muscle
sarcomers and in smooth muscle myofibrils
11
p
core domain & are organized
Parallel dimer
tetramer
similarly into Antiparallel
filaments.
Identity of IF subunits: most likely tetramer.
Homo- and heteropolymers:
– Spacer sequences in the coiled-coil region.
Assembly of IF:
– The N- and C-terminal domains.
– Some mutant IF proteins form hetero-oligomers with
normal proteins but block IF assembly at an intermediate
stage.
Intermediate filament assembly
spontaneous assembly
→ No need of chaperonne proteins or energy (no hydrolysis of nucleotides)
the filament has no polarity
≠ from actin or microtubule filaments
12
Intermediate filaments are
dynamic.
IFAPs cross-link IFs to one another and to other cell
structures (microtubules, actin filaments,
membranes).
Intermediate filament
associated protein (IFAPs):
corss link intermediated
filaments with one another ,
forming a bundle or a network
and with other cell structures,
including the plasma
membrane.
plakin
Fibroblast cell.
Microtubules are red, intermediate filaments-blue, short connecting fibers is green
IF networks form various supportive structure and
are connected to cellular membranes.
• Desmin filaments in muscle.
Some of the proteins of desmosomes and hemidesmosomes
desmosomes : intercellular junctions allowing a strong adhesion
between cells in epithelia, cardiac muscle…
hemidesmosomes :junctions allowing adhesion of cells to the
basal membrane in epithelia such as epidermis, breast…
13
Blistering of the skin caused by mutant keratin genes
Disruption of keratin networks causes blistering.
大皰性表皮鬆解症
Epidermolysis bullosa simplex EBS: the skin blisters in response to
very slight mechanical stress
Normal mouse
Keratin gene mutant
Separation between epidermis and dermis
Truncated keratin (missing both the N- C- domains) Tg mice
Other blistering diseases:
mouth, esophageal lining and cornea of the eye-mutations of different keratins
Fig. 19-37
14
Cytoskeleton associated proteins
Many families of proteins which can bind specifically to actin
A. According to filaments
1. Actin-associated (e.g. myosin)
2. MT- associated (e.g. Tau protein)
3. IF- associated
B. According to the binding site
1. End binding proteins (nucleation, capping, pl. Arp2/3, gelsolin)
2. Side binding proteins (pl. tropomyosin)
C. According to function
1. Cross-linkers
a. Gel formation (pl. filamin, spectrin)
b. Bundling (pl. alpha-aktinin, fimbrin, villin)
2. Polymerization effects
a. Induce depolymerization („severing”, pl. gelsolin)
b. Stabilizing (pl. profilin, tropomiozin)
3. Motor proteins
15