Cytoskeleton - Actin and IF

Question 1

Describe Actin?

Answer 1

Actin monomer is a globular protein (Mr - 42kDa) that polymerises into filaments
The filament is helical
G-actin = the globular protein
F-actin = the polymerised filament

F-actin is polar due to asymmetrical subunits pointing in the same direction
Minus end (pointed end) = slow growing
Plus end (barbed end) = fast growing
It can be a two stranded right handed helix, pitch - 72 nm
Or alternatively a shallow left handed helix (genetic helix), pitch - 5.9 nm
F- actin is 8 nm wide

It binds ATP - cleft in the middle for this
D-loop is essential for polymerising the actin
3 types of actin: alpha, beta and gamma
It is highly conserved

Question 2

How is energy related to ATP?

Answer 2

Actin can catalyse the hydrolysis of ATP
T-form: bound ATP
Most are in this form due to 10 fold increased ATP concentration
D-form: bound ADP
Most of this energy is stored in the polymer

Question 3

What requires a constant source of ATP hydolysis?

Answer 3

Steady state filament treadmilling - addition of subunits at the + end in the T-form and at the same time losing subunits at the - end in the D-form - resulting in no change of the filament length

Question 4

Describe actin formation?

Answer 4

1. Smaller actin aggregates to create a kinetic barrier to nucleation
2. When polymerization is initiated, this barrier results in a lag phase during which no filaments are formed
3. In the lag phase - some small, unstable aggregates succeed in transitioning to a more stable form
4. Then there’s a phase of rapid filament elongation - subunits are added quickly to the ends of the nucleated filament
5. As the concentration of actin monomers declines, we reach equilibrium - rate of addition of new subunits = the rate of subunit dissociation

Question 5

What are some actin cross-linking proteins to help form higher order structures?

Answer 5

Straight/stiff connections
Fimbrin - excludes myosin, leading to the close packing of actin filaments - parallel bundle
A-actin - allows myosin to enter the bundle, due to loose packing
Villin - has two actin binding sites and helps cross link 20-30 tightly bundled actin filaments in microvilli

Form actin filament webs
Filamin - promotes the formation of loose and highly viscous gel - by clamping 2 actin filaments at right angles
The extend to thin sheet-like membrane projections - helps them crawl across solid surfaces
Loosing filamin is good in melanoma patients, as inability crawl = less likely to metastasise

Spectrin - It cross-links actin filaments into a 3D network, linking the web to the plasma membrane
It provided mechanical support to the membrane and allows red blood cells to spring back to its original shape after moving through a capillary

Question 6

What can actin allow?

Answer 6

The pushing force generated by polymerisation of branched web actin can push the plasma membrane out (protrusion) or propel vesicles/particles through the cell cytoplasm
Cell adhesion and traction allows cells to pull themselves forward

Question 7

What are some actin monomer binding proteins?

Answer 7

They alter the dynamics and organisation through: control of monomer availability

Arp2/3 complex - nucleates assembly to form a web (dendritic structures) at the - end
Formin - nucleates assembly at the + end
Thymosin - binds subunits and prevents assembly (locked state)
Profilin - binds subunits and speeds elongation (competes with thymosin)

Question 8

What are some actin filament binding proteins?

Answer 8

Cofilin - binds ADP-actin filaments and accelerates disassembly - destabilises actin filaments, by causing a twist and therefore mechanical stress = brittle
Gelsolin - severs filaments and binds to the + end (activated by high Ca2+ levels)
Capping protein - prevents assembly/disassembly at + end - slows the rate
Tropomyosin - stabilises fragment, can prevent interaction with other proteins
Tropomodulin - prevents assembly/disassembly at the minus end (only binds after coating with tropomyosin)

Question 9

How can the actin cytoskeleton be rearranged?

Answer 9

Rho protein family:
Signals that trigger global structural rearrangements converge inside the cell close to the monomeric GTPases - that are members of the Rho family
They act as molecular switches - cycling between GTP/GDP bound states
Activation of Rho promotes bundling of actin filaments with myosin filaments into stress fibres
This then forms integrin clusters and focal adhesions

Question 10

How is actin and bacteria related?

Answer 10

Bacteria including Listeria monocytogenes recruits Arp2/3
This nucleates the assembly of actin filaments that generate a force to push the bacterium through the cytoplasm - up to 1µm/sec

Question 11

Describe myosin?

Answer 11

It is a motor protein
Hexameric protein - 2 heavy chains dimerise to form the coiled tail and 2 myosin heads
The head contains the motor domain and the lever
4 light chains bind to the lever, 2 per heavy chain
Tail is about 155 nm long
Head (motor + lever) is about 16nm long
Myosin tails (coiled coil) pack together to form the thick filament
They contain exactly 294 molecules - 1.6 um long

Question 12

How does myosin come together within the filament?

Answer 12

Within the central region myosin uses antiparallel packing (bipolar packing) and as you move further out myosin uses parallel packing
It gives a regular ‘stagger’ between molecules

Question 13

Give an overview of what the sliding filament theory/tilting crossbridge hypothesis is?

Answer 13

This is how myosin interacts with actin in order to produce force
• Thin filament - actin
• Thick filament - myosin
• Crossbridge - connected to the thick filament by myosin (sub-fragment 2)

The thin filament ‘slides’ past the thick filament do to force
Movement of the crossbridge - causes the movement of the actin filament
The myosin is always trying to move towards the barbed end of the filament
It pulls the actin filaments in towards the middle of the muscle sarcomere
This shortens the sarcomere by 0.2 microns (around 10%) in each sarcomere
This leads to dramatic shortening in the overall muscle

All driven by ATP hydrolysis

Question 14

Describe the first part of the sliding filament mechanism?

Answer 14

Tropomyosin prevents the myosin head from attaching to the binding site on actin
Ca2+ ions are released from the sarcoplasmic reticulum - binding to troponin, changing the tertiary structure causing the tropomyosin to pull away from the binding site on the actin
The myosin head attaches to the binding site on the actin forming a crossbridge

Question 15

Describe the second part of the sliding filament mechanism?

Answer 15

ATP binds to a myosin causing myosin’s actin-binding site to open up and release its bound actin
Myosin’s active site closes around the ATP and hydrolysis into ADP+Pi, alters the myosin head into a ‘high energy conformation’
The myosin head binds weakly to another actin monomer
Myosin releases Pi causing its actin-binding site to close
A power stroke follows
ADP is released completing the cycle

Question 16

Give an overview of cardiac muscle disease?

Answer 16

Caused by mutations in sarcomeric proteins (actin, myosin, tropomyosin, troponin, C-protein, titin)
Normally autosomal dominant missense mutations
Mutations in myosin heavy chain cause about 30-40% of cases of HCM
The first symptom tends to be death - asymptomatic

Common in cardiomyopathy loop - Arg403 - R403Q
At least 1/500 carry this mutation - over 1000 mutations have been found

Question 17

Give an overview of skeletal muscle disease?

Answer 17

Mutations in actin/nebulin cause skeletal muscle myopathies
Muscular structure is disrupted
Loss of sarcomeres
Muscle is weak
Death due to effects on respiratory muscle

Actin can polymerise with the nucleus causing difficulty with import/export from the nucleus
Effects include:
Changes to actin polymerisation (mutation in the D-loop)
Effects on actin ATPase
Effects on myosin binding

Question 18

What is actin involved in, within non muscle cells?

Answer 18

Actin cytoskeleton is required for cell-cell adhesion and migration
There are many types of myosin
Class 2 Myosins: all the muscle Myosins (skeletal, cardiac and smooth) and 3 ‘non-muscle’ myosin isoforms (NM2A, NM2B and NM2C)

Examples:
Blood clotting disorders that affect platelets (non-muscle myosin 2A – NM2A)
Deafness and blindness (e.g. Myo7a)

Question 19

Describe activation of platelets - NM2A?

Answer 19

In circulating platelets, most myosin is in 10S shutdown state
When platelets are activated:
Regulatory light chain is phosphorylated
Myosin is activated (6S) – forms filaments

Most of the mutations are in the coiled-coil tail
Mutations could interfere with formation of ‘off state’ and/or interfere with filament formation

Enlarged platelets - from mutation in the coiled coil tail
This could affect filament formation, folding and regulation

Question 20

Describe hearing/deafness - Myo7A?

Answer 20

In the cochlea - there are stereocilia
Stereocilia - contain actin rich bundles (precisely organised)

Different heights connected by tip links
Connected to a single kinocilium (has microtubules)
Myosin 7a is near the lip link (upper end) - needed to make/restore the tip link

Around half the mutations are in the tail
It can result in the disorganisation of the stereocilia - resulting in deafness
Also important in pigment trafficking in the retina

Question 21

Describe intermediate filaments?

Answer 21

Intermediate filaments: polymers of intermediate filament proteins

10nm in diameter
No motors associated with these filaments and non-polar = less dynamic
Strong rope like fibres - forms the nuclear lamina
There subunits are symmetrical and don’t catalyse the hydrolysis of nucleotides e.g. ATP and GTP
They provide mechanical strength by lining the inner face of the nuclear envelope, forming a protective cage for the cell’s DNA; in the cytosol, they can hold epithelial cell sheets together or help nerve cells to extend long and robust axons
They form tougher appendages e.g. Hair and fingernails

Question 22

What are some cell specific intermediate filaments?

Answer 22

Epithelial - keratin
Muscle - desmin
Fibroblasts - vimentin
Neurones - neuronal

Question 23

What are intermediate filaments needed for?

Answer 23

They are required for cell-cell and cell ECM adhesion
Desmosome - where cells attach to each other
Hemi-desmosome/focal adhesion - where cells attach to underlying extracellular matrix (ECM)

Mutations cause a variety of diseases
E.g. Keratin - causes skin blistering diseases - epidermolysis bullosa

Question 24

Describe the formation of intermediate filaments?

Answer 24

There is a central a helical domain within the monomer that forms a parallel coiled-coil dimer with another monomer
A pair of dimers can then associate to form a staggered tetramer (antiparallel)
The tetramers pack tightly together in a lateral form to create the filament - including 8 parallel protofilaments all made up of the tetramers

Question 25

Describe the formation of higher order structures within intermediate filaments?

Answer 25

They are cross linked and bundled into strong arrays
The long bundle of tetrameric subunits - further bundle themselves by self-association

They can be held together by accessory proteins:
Filaggrin - bundles keratin filaments together = outermost layers of skin are very tough
Plectin - a crosslinking protein that: bundles intermediate filaments, links intermediate filaments to microtubule/actin/myosin bundles and helps attach intermediate filaments to adhesive structures at the plasma membrane

Question 26

What are some types of intermediate filament disorders?

Answer 26

Type I and II - epidermolysis; hair/nail defects from keratin mutations
Type III - Desmin-related myopathy: structural disintegration of muscle Z-line and protein build up in muscle fibres; Cardiomyopathy
Type IV - Charcot-Marie-Tooth diseases: types of progressive neurological degeneration
Type V - Lipodystrophy, muscle laminopathies and neurological disorders
Type VI - Autosomal dominant cataract: early onset cataracts

Question 27

Describe nuclear lamins?

Answer 27

A class V intermediate filament
Mutations cause 15 distinct human diseases (laminopathies)
Muscle, metabolic, neuronal diseases and acclerated aging
Most mutations are encoded in type A lamins
Over 600 mutations
Most autosomal dominant - missense mutations

Question 28

What does a lamin A mutation cause?

Answer 28

Progeria
Lamin A mutation - affects the nucleus
Results in premature ageing
Causes of mutation - incorrect splicing of lamina A results in progerin
Progerin accumulates at the nuclear lamina - displacing normal lamin A/C
Impairs signalling pathways, gene expression, cell renewal

Mutation E145K
Blocks normal lamin A localisation in the nuclear envelope
This causes premature senescence