Cytoskeleton II – Actin Network Flashcards
Describe the cytoskeleton
- dynamic intracellular network
- drives movement of intracellular organelles and whole cells
- involved in muscle contraction
- organises and provides structural support for the cell
- controls cell shape
Describe actin
- microfilaments
- 7nm
- found in all eukaryotes
Describe tubulin
- microtubules
- 25nm
- found in all eukaryotes
Describe intermediate filaments
- 10nm
- vimentin, cytokeratin, desmin
- found only in animals
Describe the filaments
long unbranched one-dimensional protein polymers
Describe actin microfilaments
- linear polymers of G-actin
- F-actin microfilaments are helical polymers
- left-handed helix with a rotation of 166 degrees per subunit
- 13 monomers per helix repeat of 37 nm
- 7 nm diameter
- flexible ‘ropes’
G-actin
- a single globular protein
- monomeric G-Actin
- binds one ATP
- hydrolyses ATP
- subdomain 2-4 surface binds to subdomain 1-3 surface, resulting in filament polarity
F-actin
filamentous actin
Describe actin filament dynamics
- “in vitro treadmilling”
- end has the highest binding affinity to G-actin
- post-polymerisation into microfilaments, actin monomers hydrolyse their bound ATP; destabilises the filament
- ADP-binding subunit dissociation from - end
What happens in F actin at the - end
- releases G-actin ADP and Pi
- G-actin releases ADP
What happens in F actin at the + end
ATP binds to G-actin, which binds to F-actin
Describe treadmilling
actin filament elongates at + end and shrinks at - end
Describe actin dynamics in vivo
- controlled by actin binding proteins
List the functions of actin binding proteins
- nucleation
- capping
- severing
- sequestering
- bundling
Actin binding proteins determine
rate of filament assembly and stability
Describe the functions of the microfilaments
- strong in tension, weak in compression (better for pulling than pushing)
- linear pathways for organelle movement in plants and fungi
- in animals, form contractile systems together with motor-proteins
- when cross-linked, have a variety of structural roles; can push the growing margins of an animal cell forward
Describe myosin motor proteins
- protein family
- two catalytic ATPase heads walk along actin filament
- motor heads convert chemical energy released by the hydrolysis of ATP into mechanical movement
- motor domain is connected via a neck domain to a tail domain, which interacts with cargo or dimerises
Describe Myosin-II
- 2nm
- C terminus connects to coiled-coil of two light chain alpha-helices
- neck or hinge region connects N terminus
Describe organelle and vesicle transport in plants
- small organelles and vesicles are continuously moved around the cytoplasm
- drag caused by moving organelles causes cytoplasmic streaming
- movement is powered by actin filaments, using myosin motor proteins
- used to overcome diffusion barriers in extremely large vacuolate plant cells
cytoplasmic streaming
the whole cytoplasm cycles round the cell
Describe actin and myosin-II in animals
form contractile arrays
Describe the skeletal muscle - the basics
a contractile machine
Describe muscle fibres in animals
- giant multinucleate syncitial cells
- 50 μm in diameter
- formed by the fusion of myoblasts
- within each fibre are many myofibrils, forming the contractile apparatus
myoblasts
mononucleated precursors
Describe muscle fibre formation
myoblasts make myotubes form myofibrils
Describe sarcomeres
- each myofibril is a highly organised linear array of sarcomeres
- actin-containing thin filaments project with opposite polarities from the two Z-discs
- interdigitating between the thin filaments are thick filaments
Sarcomeres
contractile units
Z-discs
embed the actin plus-ends
What are the sarcomere filaments composed of?
myosin
Describe myosin thick filaments
- spontaneously assembles
- bipolar with a bare central zone
- myosin heads project out sideways in nine radial positions
- three-fold symmetry
- period of 43 nm
Describe nebulin
- giant protein
- acts as a molecular ruler that controls the lengths of the thick and thin filaments
Describe titin
- giant protein
- acts as a molecular ruler that controls the lengths of the thick and thin filaments
- elastic ends hold thick filaments in the centre of the sarcomere
Describe thick and thin filament attachment
- plus end: Z disc and Cap Z
- titin and nebulin
- tropomodulin at - end
- M line
- myosin thick filament
- actin thin filament
- ends with other Z disc
Describe the sliding filament model of contraction
- sliding of the two filament sets, without change in filament lengths creates shortening of the sarcomere
- myosin molecules binding to actin filaments and pulling them towards the centre of the sarcomere
- actin is in tension
Describe the myosin cross-bridge cycle
- myosin head undergoes dramatic changes in conformation depending on its binding
- power stroke
- distance moved by each myosin head is ~6 nm
force generated is 0.7x10-12 N (0.7 piconewtons) - heads are detached most of the time (~95% of the duty cycle)
Describe the ‘power stroke’
Pi release causes strong filament binding and conformational change in neck region
Describe a cross section of insect flight muscle
thick filaments are hexagonally packed with great regularity
Describe myosin cross-bridge binding
- spacing of myosin heads is out of register with spacing of the myosin binding sites on the actin thin filament
- symmetry of myosin heads and surrounding actin binding sites differ
- myosin heads cannot all bind actin at the same time
- ensure some heads are attached at all times to maintain tension
Describe myosin head and binding site spatial discrepancy
– myosin heads repeat distance: 129 nm
– each head moves ca. 6 nm per cycle
– repeat distance of the myosin binding sites on the actin filaments: 37 nm
Describe the asymmetry of myosin heads and actin binding sites
– myosin heads stick out in nine radial positions
– each thick filament it is surrounded by six actin filaments
Describe the co-ordination of actin-myosin interactions
- thin filament contains accessory proteins troponin and tropomyosin
- two calcium ions bind to troponin C
- troponin complex changes shape, induces tropomyosin to roll away from the myosin binding site on the actin microfilament
- myosin can bind to the actin filament and cause contraction
- contraction continues as long as calcium ions are present
Describe how calcium release co-ordinates contraction
- released from stores in the SR
- resting calcium levels in the sarcoplasm are ~10-7 molar
- within the SR, calcium concentration is about ~10-3 molar (10,000 times greater)
- SR membrane is in electrical contact with the plasmamembrane
- arrival of an action potential opens calcium channels in the SR, increasing Ca2+ about 10-fold
SR
- specialised endoplasmic reticulum of the muscle fibre
- sarcoplasmic reticulum
the sarcoplasm
the muscle cytoplasm
Describe a muscle cell
- plasma membrane
- myofibrils
- transverse tubules form invaginations of plasmamembrane
- SR
Describe cytokinesis in animals and fungi
- cytokinesis after mitosis is brought about by a contractile ring of actin and myosin II
- form a ring of mini sarcomere-like structures
What is the contractile ring composed of?
actin and myosin filaments
Describe the distribution of actin and myosin II during cytokinesis in normal Dictyostelium discoideum cells
- actin: spread across cell
- myosin II: centralised
Describe microfilament organization in non-muscle cells
- cytoplasmic bundles of F-actin and myosin-II form stress fibres
Describe stress fibres - the basics
- focal adhesion point
- peri-nuclear cage or PM
- stress fibres are contractile actin arrays
- can be visualised in fibroblast cells with red actin and blue nucleus staining or with deep etching and rotary shadowing
Describe the focal adhesion point of stress fibres
anchored to other cells or extracellular matrix by the PM
Describe the structure of stress fibres
- contain both bipolar actin filaments and myosin II mini- filaments
- contractile
- mini-sarcomeres
Describe actin polymerisation
- can drive rapid cell migration
- e.g. keratocytes from fish scales (~15 μm per minute)
Describe microfilament organisation in crawling cells
Lamellipodium
Describe stress fibres in crawling cells
- contractile bundles
- push the trailing edge
Filopodium in crawling cells
- small, dynamic cell
projections - actin bundle
- senses environmental
signals
Lamellipodium in crawling cells
- large, flattened cell
- extension at the leading edge
- quasi-2D branched actin
meshwork - pulls the cell forward
Describe the crawling cells
- filopodium and microspike
- lamellipodium
- adhesion sites
- lamella
- arcs
- stress fibres
Describe the leading edge of a crawling cell
- lamellipodia contain dense network array of short branched actin filaments
- provide enough rigidity to push on the membrane at the leading edge
- 70 degree intersections
Describe ARPs
- organise branched arrays
- ARP2/3 complex can bind to pre-existing actin filaments at a 70 degree angle
- nucleate formation of new filaments creates a cross-linked meshwork
- analogous to g-Tubulin nucleating MT
ARPs
actin-related proteins
Describe the array treadmilling model for lamellipodium extension
- individual actin filaments become capped at both ends, then remain constant in length, and stationary with respect to the array (and to the substrate over which the cell moves)
- the array as a whole grows forward, polymerising at the front and depolymerising at the rear
- array can thus be described as treadmilling, although the individual filaments are not
What happens at the leading edge of crawling cells?
net filament assembly
What happens behind the leading edge of crawling cells?
net filament disassembly
How is lamellipodium protrusion (new extension growth) at the leading edge mediated
- actin polymerisation without the involvement of myosin II
What do the focal contacts of crawling cells contain?
integrins
Describe the spatial organisation of actin polymerisation
G-proteins
G-proteins
Small, GTP-binding GTPases
Describe some G-proteins
- Rho stimulates stress fibre production
- Rac stimulates extension of lamellipodia
- Cdc-42 stimulates formation of filopodia
- can be viewed under staining
Describe G-proteins and cell control
- G-proteins can bind either GTP or GDP
- slowly cleave GTP to GDP
- when GTP is bound, G-proteins are in an activated state and
can stimulate other proteins - act as molecular switches and control cellular activity
- switching controlled by other factors
switching
GTP binding and release
How are crawling cells activated?
GTP acquisition (fast!)
How are crawling cells inactivated?
GTP hydrolysis into GDP, which is released (slow!)
Describe the interaction among cytoskeletal elements - the basics
- necessary for most cellular processes (e.g. cell division and cell migration)
- requires coordination among cytoskeleton components
Describe some specific interactions among cytoskeletal elements
- static crosslinkers
- motor proteins
- motor protein-binding protein complices
Actin polymerisation is based on …
binding and hydrolysis of ATP
When cross-linked or linked to the substratum actin can…
act under compression to force forward cell membranes in motile cells
Actin can act under tension in contractile systems in single cells and tissues
spatiotemporal control of actin polymerisation is controlled by
small GTPases, that are themselves controlled by diverse spatiotemporal signals from the cell’s internal activities and environment
Actin based cytosplasmic streaming is used to overcome
diffusion barriers in extremely large plant cells
Interaction among cytoskeleton elements ensures
coordinated cellular function
