Mechanisms of actin filament assembly
Unbranched filament: formins
Branched filament: Arp2/3
Formins nucleate formation of unbranched actin filaments, such as those in stress fibers.
Formins are found at the plus ends of actin filaments.
Formin is said to be processive(前進的),
because it remains bound to the plus end of an actin filament as actin monomers are added at the plus end.
The continued presence of formin prevents binding of plus-end capping proteins that would inhibit filament growth.
Formins assemble unbranched filament
FH2 domain
Induced actin polymerization
Each formin includes an actin-binding FH2 domain that dimerizes to form a ring-like structure with flexible links.
Models have been proposed involving “stair stepping” (階梯式的步驟) by the dimeric formin to explain its ability to remain at the plus end as actin monomers are added.
Regulation of formins by an intramolecular interaction Second messenger regulation
RBD: Rho binding domain
The Arp2/3 complex nucleates branched filament assembly
WASp: Wiskott-Aldrich syndrome protein
Arp2/3 complex binds at 70
oto the side of actin filaments to nucleate a daughter filament.
Actin-related protein (Arps) about 50 % similarity with actin.
Arp2/3 can stimulates actin assembly.
Arp2/3 combined mother and daughter filaments → created ends of filament elongate and create the force to push the membrane forward
The Arp2/3; active state
The Arp2/3; active state
The actin-related proteins: ARP 2/3
Regulation of the Arp2/3 complex by WASp
Rho is a family of small GTP-binding proteins that regulate the actin
cytoskeleton.
Some members of the Rho family:
Rac activates formation of lamellipodia, in part through activation of WASP.
Cdc42 activates formation of
filopodia, in part through activation of the WASP family protein Scar
(WAVE).
Rho activates formation of focal adhesions & stress fibers, in part through activation of formins.
In each case the active form of the Rho family protein has bound GTP.
Listeria utilizes the power of actin polymeration for intracellular movement
Intracellular movement can be powered by actin
polymerization
ActA →activated
→Arp2/3→nucleate new filament→→
Actin polymerization can generate forces that move certain intracellular bacteria & viruses.
Intracellular movements and changes in cell shape are driven by actin polymerization
Red: surface protein
Intracellular pathogens
Toxin perturb (擾亂) the pool of actin monomers.
Cytochalasin D:
– A fungal alkaloid.
– Binds to (+) end of F-actin & blocking further addition of subunits.
Latrunculin:
– A toxin secreted by sponges.
– Binds to G-actin & inhibits it from adding to a filament.
When these two toxins are added to live cells, actin cytoskeleton disappears and cell locomotion & cytokinesis are inhibited.
Phalloidin:蕈毒素
– Isolated from the "angel of death" mushroom.
– Binds between subunits in F-actin, locks adjacent subunits together
& prevents actin filaments from depolymerizing.
– Fluorescent-labeled phalloidin is commonly used to stain actin filaments for microscopy.
細胞鬆弛素
細胞骨架抑制劑
Organization of actin based cellular structures
Cross-linking proteins organize actin filaments
Adaptor proteins link filaments to membrane
Cross-linking proteins organize actin filaments
TEM of microvillus
Long cross- linking protein such as filamin are flexible
Fimbrin, villin, & α-actinin cross-link actin filaments into
bundles.
Filamin cross-links actin filaments into a three-dimensional gel-like network.
Polymer actin
Adaptor proteins link filaments to membrane
microvilli
Scaffolding protein
EM of RBC Maintain cell surface or shape
Genetic defect in spectin, band 4.1 and ankyrin
球形紅細胞貧血(spherocytic anemia)、
Duchenne muscular dystrophy (DMD) 裘馨氏肌肉萎縮症
X染色體
P21基因缺損(DMD)。
DMD基因會製造一種重要的肌肉萎縮蛋白
(Dystrophin)，此症患者的細胞內完全缺少肌肉 萎縮蛋白，肌纖維膜變得無力脆弱，經年累月伸 展後終於撕裂，肌細胞易死亡。
患者的肌肉隨著年齡的增長會逐漸退化消失。
Affects structure (DGC) involved in linking muscle cell to basal lamina This structure provides strength to cell membrane
disruption leads to weakening of mechanical stability of cell Affects cytosolic adapter protein-Dystrophin
Interacts with dystroglycan (glycoprotein
Muscular dystrophy: connections between the ECM and cytoskeleton are defective
About 1/3300 boys, heat or lung failure
Mutation of dystrophin (a cytoslic protein), bind to dystroglycan
Dystroglycan: α-subunit (peripheral protein) plus β-subunit
(transmembrane protein); the a- subunit also has O-linked
oligosaccharides to bind various basal lamina components
DGC (dystrophin glycoprotein
complex) links extracellular matrix to the cytoskeleton and singling pathways enzyme for muscle’s function
Mutations in components of this pathway (e.g. muscular dystrophy) results in mechanical instability of muscle cells.
Actin Cross-Linking Proteins (Fascin, Villin, Fimbrin, α-Actinin, Spectrin, Dystrophin, Filamin)
Membrane-Microfilament Cross-Linking Proteins (Ponticulin, Ankyrin, Dystrophin)
G-Actin Sequestering Proteins (Thymosin β4, Profilin)
Actin Severing Proteins (Gelsolin)
Actin Capping Proteins (CapZ, Tropomodulin)
Actin Dynamizing Proteins (profilin, cofilin) Structure
Dynamics
How is the polymerization of actin filaments regulated?
Actin Binding Proteins
• Regulation of filament assembly
– [monomer] available for polymerization – Promoting nucleation (rate limiting step) – Regulating nucleotide exchange
• Filament severing – Creates new ends
• End capping
– Prevents addition/loss at specific ends
• Filament branching
– Actin Related Proteins (ARP)
• Cross-linking filaments
– Formation of networks & bundles
1. They can bind to specific filament types 2. They can travel along filaments
3. They hydrolyze ATP 肌凝蛋白
Motor proteins are enzymes that couple the hydrolysis of ATP to a conformational change
17.5 Myosins: actin-base motor protein
1. Actin-based: myosins
Myosin families: myosin I-XVIII
2. Microtubule based motors (next chapter) 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 plus (+) end of MT
3. Nucleic acid based
DNA and RNA polymerases
They move along a DNA and produce force
Types of motor proteins
All myosins have head, neck, and tail domains with distinct functions
Different myosins has different function
Conformational changes in the myosin head couple ATP hydrolysis to movement
Myosin (肌凝蛋白)
Structure of myosin II
All myosins have head, neck, and tail domains with distinct functions
HMM: heavy mero-myosin LMM: light meromyosin Myosine II isolated from skeletal muscle. Myosin II assembles into bipolar
filaments, and head exposed. 2 heavy chain
4 light chain
Mero:不全
Motor proteins
convert chemical energy (derived from ATP hydrolysis) into mechanical force (movement)
HEAD NECK TAIL
Globular Head
mechano-chemical transduction force generating domain
ATPase activity
MF(actin) binding site,
ATP hydrolysis results in unidirectional movement
Helical Tail:
binds cargo:
attachment to vesicles → transport attachment to filaments →sliding attachment to itself → formation of filaments Neck
regulation
binds regulatory chains
MYOSIN II
2 heavy chain
4 light chain
Myosin: the actin motor protein
All myosins have head, neck, and tail domains with distinct functions
XVIII
~40 genes
Functions of myosin tail domains.
membrane
Myosins are associated with
intracellular membrane vesicles or the cytoplasmic face of the plasma membrane
Tail domains of myosins bind the plasma
membrane or the membranes of intracellular organelles → membrane-related activities vesicle
Sliding-filament assay is used to detect myosin-powered movement.
Coverslip
Fixed to myosin ATP add
Mysoin heads walk along actin filaments in discrete steps
非連續性
flash
Myosin superfamily in human
Myosins make up a large family of mechanochemcial motor proteins
S1 head domain from 40 human genome Loss specific induced disease
聾
Myosins have diverse, well-defined, functions
All myosin move to + end of actin, exception is myosin VI Myosin VI: move to – end of actin for endocytosis
8 genes encode heavy chains for myosin I family; 14 genes for myosin II family; and three for myosin V.
only assembly to bipolar filament Class I, II and V are most common.
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.
May be other cargo
1. Binding ATP → disrupting actin-binding site
2. Hydrolysis ATP → head, conformational change → move to new position → rebind
3. Pi release → head
conformation second change
→ move the actin
4. Release ATP → new cycle
flash
Conformational changes in the myosin head couple ATP hydrolysis to
movement
扳起
ATP hydrolysis to movement
The length of the myosin II neck domain determine the rate of movement
Recombinant DNA
↓
Different length neck domain
↓ Analysis
↓ movement
More fast
Optical trap determines force & step size generated by a single myosin molecule.
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.
Can measure the force from myosin
Move to +
The processivity of myosins
5-15nm
The movment of Myosin V
Myosin V has two heads, how to work?
Hand over hand Inchworm model
【昆】尺蠖
Myosin-powered cell movements
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)
The sliding-filament model of contraction in striated muscle
Skeletal muscle is structured by stabilizing and scaffolding proteins
Attach to z-disk, extends to the middle of thick filament; it elastic protein that holds the thick
filament in the middle
Repeat, bind to actin.
Sarcomere(肌節):
+ - + -
+ -
+ -
Contraction of skeletal muscle is regulated by Ca
2+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)
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.
LC: light chain
Ca2+ → bind to calmodulin → activate calmodulin → bind to myosin LC kinase (MLCK) → phosphorylation of mysoin LC → contraction
Ca
+2dependent regulation of smooth muscle contraction
Actin and myosin II form contractile bundles in nonmuscle cells
Hypertrophic cardiomyopathies
心肌肥厚症
Mutation of cardiac myosin heavy chain Heat arrhythmia
Myosin V bound vesicles are carried along actin filament
Myosin V carry many different cargoes in budding yeast
Yeast
Budding SV: secretory vesicle
formin
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)
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
麗藻細胞
Cell migration: signaling and chemotaxis
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.
絲狀偽足 瓣狀偽足 lamellipodium
Filopodia
Cell movement coordinates force generation with cell adhesion
1. Membrane extension 2. Cell substrate adhesions 3. Cell body translocation
4. Breaking cell attachments
Actin-based structures involved in cell locomotion
1. Membrane extension.
Arp2/3 complex, formed 70o angle branch
(a) Actin filaments → assembled into
branched network
(b)Elongation of filament → generated pushing force; elastic brownian ratchet model.
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
The polymerization “motor”
How can polymerization push ?
However, a single filament is too weak…
Cantilever 懸臂
Elastic Brownian ratchet
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.
myosin
Keratinocyte (角質細胞) actin
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.
• 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
The small GTP-binding proteins Cdc42, Rac, and Rho control actin organization
GEF: guanine nucleotide exchange factor GAP: GTPase activating protein
GDI: guanine nucleotide displacement inhibitor
Extracellular signals induce global actin
filament
rearrangements via the Rho protein family – cdc42, Rac,
and Rho.
Contractile stressfilopodia ruffles
Cell migration involves the coordinate regulation of Cdc42, Rac and Rho
絲狀偽足 瓣狀偽足 lamellipodium
Filopodia
The wounded cell monolayer assay can be used to dissect signaling pathways in directed cell movement
