alex's musculosketal

LCRS Alexandra Burke-Smith

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The Molecules of Movement Musc 1 - Professor Michael Ferenczi ([email protected])

1. Appreciate that there are a large number of molecular motors, each with its assigned role.

2. Linear molecular motors are associated with polymeric filaments. Which filaments are myosins and

kinesins associated with?

3. Which molecular motor is that found predominantly in muscle cells?

4. Describe the structure of myosin II.

5. Describe the structure of myosin filaments.

6. Describe the structure of actin filaments.

7. What are sarcomeres? Draw a sarcomere in longitudinal section.

8. Activation of the skeletal muscle cell results from a cascade of events:

9. What causes depolarisation of the muscle cell membrane?

10. How does cell membrane depolarisation propagate

11. along the cell surface?

12. into the core of the muscle cell?

13. Describe how calcium ions are released from intracellular calcium stores (sarcoplasmic reticulum).

Which membrane-bound proteins are involved in this process?

14. Describe the fate of calcium ions during activation of the myofilaments.

15. How is the thin filament affected by calcium? What are the consequences?

16. What is relaxation? What happens to calcium ions during relaxation?

Molecular Motors: Overview

Protein assembles which convert chemical energy into mechanical work

Energy for the biological motors is usually derived directly from the hydrolysis of ATP or from an ionic

gradient (e.g. H+ or Na+)

o Behave like molecular engines

LINEAR MOTORS:

- require protein rails:

o actin filaments for myosins

o microtubules for dynein

o kinesins for NCD

o DNA/RNA for helicases and tropoisomerases

- other linear motors rely on filament polymerisation

o e.g. actin polymerisation in listeria, or microtubule polymerisation

ROTARY MOTORS:

- Require stators:

o E.g. the flagellar motor

o E.g. 2. ATP-hydrolysing F1 portion of the F1F0-ATPase

OSCILLATORY MOTORS:

- Found in cilia and require cross-linked microtubule bundles called AXONEMES

- Powered by dynein

- Cross-links generate oscillation

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Microtubule Polymerisation

EB-1 protein attached to the positive end of microtubules

Polymerise from the centre of the cell to the periphery

Another mechanism by which movement is generated

E.g. how vesicles are moved around the cell

Types of Movement

Intracellular

- Vesicle transport

- Tuning of hair cells in the ear (sensory organs)

- Cytoplasmic streaming

- Anaphase: segregation of chromosomes in mitosis

- Cytokinesis: formation of cleavage furrow

- Unwinding of DNA

Cellular

- Bacterial motility

- Cell locomotion

- Axon growth

- Phagocytosis

- Muscle contraction

- Chemotaxis

Cytokinesis

Spindles contain microtubules and dynein

Formation of a cleavage furrow by the contraction of a circumferential ring of actin and myosin II at the

cell equator

Chemotaxis

Ability of cells to move or orient themselves according to a chemical gradient

o Cells respond to the chemical signal by either moving away or towards the source

Directed movement in response to environment

Anaphase

Spindles contain microtubules and dynein

Involves myosin I and II, as well as other proteins

Chromosomes are segregated to opposite ends of the diving cell during mitosis

Unwinding of/Movement along DNA

X-ray diffraction can be used to assess the structure of a protein, e.g. HUMAN TROPOSIOMERASE 1

HT1 catalyses the transient breaking of DNA strands and movement along the strand

Actin Polymerisation

Another type of molecular motor

Actin: small protein, forms very long filaments


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Listeria moves in infected cells by inducing actin polymerisation

o Makes use of host cell machinery

o The growth of actin filaments forms a COMET TAIL

o Tail generates movement, but does not require self motors

o Unique mechanism as travels through cells, used to travel through placental membrane to infect

foetus

Molecular Motors

Kinesin

Consists of two amino-acid chains, each terminating into a globular ATP-binding “head”, which interacts

with microtubules

Tail of proteins form a coiled-coil; vesicles are held at specialised binding sites at the end of tails

Kinesin moves along microtubules

o Microtubules are polymers of tubulin molecules

o This moves vesicles through cells

o Requires ATP hydrolysis

Dynein

Oscillatory motor found in cilia

CILIA:

o Found lining the respiratory tract

o Also responsible for sperm movement

o Cilium contains an AXONEME (bundle of microtubules)

Between the inner and outer cilium, there are a range of proteins which connect the microtubules:

o ELASTIN proteins – nexin

o MOTOR proteins – dynein

When ATP is hydrolysed, Dynein bends (and vice versa when ATP is synthesised)

o This changes the distance between the inner and outer cilium

o This drives the oscillation

F1-ATPase/ATP Synthase

Found in the inner membrane of mitochondria

Acts as a generator

Driven by the protein gradient between the outer and inner compartments in the mitochondrion

o Result of the electron transport chain


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The flagellum

Rotary motor

Rotation driven by either H+ or Na+

gradient

MS ring does not rotate

Extracellular section allows movement

of the entire cell e.g. bacterium – E. coli

Myosin

The molecular motor found in muscle

cells

Moves along actin filaments

Vast number of different classes identified; phylogenetic tree shows evolutionary formation of the

different classes

o Myosin II conventional

Between classes, length of tails and functional

domains vary

All classes have a motor domain, most have a

light chain binding domain

Myosin II

- 2 amino acid chains coiled around each other

- 2 smaller peptides (light chains) coild around

other chains

- N terminus: globular head (myosin subfragment

1)- actin and ATP binding site

- C terminus: polypeptide tail

- Thick filament structure: tail diameter 2nm

- Backbone diameter: 16.3 nm

- Length of filament: 1.6 micrometer

Myosin V

- Found in brain and involved in

vesicular and mRNA transport

- E.g. melanin vesicle transport into

hair along F-actin filaments

coloured hair

- Binds microtubules

- Found in nerve growth cones

Organs designed for movement: Skeletal

Muscle

Myosin filaments

Skeletal muscle packed with myosin II

Tail diameter: 2 nm


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Thick filament backbone diameter: 16.3 nm

Thick filament and packed myosin heads: 20.3 nm

Length of native thick filaments: 1.6 µm

Form thick filaments by forming tail to tail junctions

o 294 molecules form 1 thick filament

Myosin heads form at either end of the BARE ZONE in opposite orientations

GLOBULAR HEAD:

o Actin binding site—consists of Myosin S1

o ATP-binding site

o 50kDa cleft between binding sites

o Also consists of:

Lever arm

Converter

Pliant region

Switch II

SH1 helix

relay

Actin Filament system- Microfilaments

Made by polymerisation of G-actin

Found in the periphery of cells underlying cell surface

E.g. thin filaments of muscle, core of microvillus in brush border

When it polymerises, hydrolyses ATP- ADP remains in the centre of the filament- involved in cell surface

shape and cell migration

Decorated by myosin globular heads (S1)

Sarcomere

Defined as distance between Z lines

Z-line is source of actin

Thick filament length: 1.6 µm: Containing 294 myosin molecules (588 heads). 3 myosins/crown, spaced at

14.3 nm interval.

Thin filament length: 1.1 µm.

Length regulated by NEBULIN and TROPOMODULIN

TITIN is a large elastic and extensible protein that links the thick filaments to the Z-line, thus ensuring that

the thick filaments remain centred in the sarcomere.

Z-band width: 0.052 µm in Type II fibres, 0.101 µm in Type I fibres.

Bare zone: 0.15 µm wide


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Contraction

Of permeabilised muscle fibre by photolytic release of ATP from caged ATP

STRIATIONS are patterns of repeating sarcomeres

The filaments are stiff and largely inextensible

During contraction, sarcomeres shorten

Muscle shortening is brought about by the interaction of myosin “cross-bridges” interacting with the thing

(actin) filaments, which cause changes in the overlap between the sets of filaments

Myosin cross-bridges act as independent force generators

T-tubule organisation

Each bundle of filament is called a myofibril. There are hundreds of myofibrils in each cell of a skeletal

muscle.

T-tubules are invaginations of the sarcoplasmic membrane

o T= transmembrane

o T-tubules go through muscles at intervals corresponding to the sarcomere length

o Open to extracellular space, therefore have the same ion composition

The sarcoplasmic reticulum is a specialised endoplasmic reticulum, storing calcium.

Mitochondria also power contraction, as it requires ATP

Motor neuron makes close contact with the plasma membrane of muscle fibres

o This is known as a NEUROMUSCULAR JUNCTION

o The combination of a single motor neuron, and its

corresponding muscle fibre is known as a MOTOR UNIT

NEUROMUSCULAR JUNCTION:

o Voltage sensitive receptor (DIHYDROPYRIDINE; DHP

receptor) in the t-tubule interacts with a calcium release

channel in the sarcoplasmic reticulum membrane

(RYANODINE receptor)

Mechanism of Contraction

Action potential propagated along motor neuron to muscle cell

T-tubule is depolarised

o DHP receptor interacts with ryanodine receptor on sarcoplasmic reticulum membrane

o Ryanodine receptor undergoes conformational change intracellular Ca2+ released

Ca2+ binds to TROPONIN-C (actin binding protein on actin filament)

o Conformational change in Troponin causes TROPOMYOSIN to move away from the myosin binding

site

o Myosin can then bind to the actin filament

Tropomyosin

o Long polymer spanning 7 actin monomers

o In relaxed muscle, it prevents the interaction of myosin with actin

o In presence of Ca2+, it moves into the actin groove, so myosin can interact with the myosin

binding sites


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Relaxation

Once the membrane has repolarised, the calcium channels in the sarcoplasmic reticulum close.

The sarcoplasmic Ca-ATPase pumps calcium back into the reticulum, causing a decrease in calcium

concentration in the cytoplasm.

This causes calcium dissociation from troponin, resulting in inactivation of myosin binding sites on actin,

and hence muscle relaxation.

Molecules working together: Force & Movement Musc 2 - Professor Nancy Curtin ([email protected])

1. Appreciate that the myosin cross-bridge cycle is the elementary process resulting in force generation

and shortening by all muscle cells.

2. Draw a diagram of the main steps in the cross-bridge cycle, showing the changes in cross-bridge

structure and how ATP is involved.

3. List 3 key features of the cycle (ATP used in each cycle, one-way sliding, small step size).

4. For the following factors, that control the amount of force produced in vivo, briefly explain the

mechanisms involved.

a. recruitment of motor units

b. frequency of activation (covered earlier)

c. sarcomere length and filament overlap

d. speed and direction of movement

5. List the metabolic sources of ATP and briefly outline the conditions in which each is important.

6. Smooth muscle:

a. Describe its main structural features.

b. Give examples of tissues contain smooth muscle and state the nature of the signal that triggers

contraction.

c. Describe the intracellular events during activation of contraction and during relaxation. Be able

to explain how this is different from the corresponding events in skeletal and cardiac muscle.

7. Cardiac muscle:

a. Describe its main structural features

b. Be aware that the crossbridge cycle is like that in skeletal muscle.

c. Describe the sources of Ca for activation and how this differs from skeletal muscle.

Isometric contraction: force without length change Isovelocity contraction: muscle shortens or is stretched at constant velocity Isotonic contraction: constant force during shortening or lengthening Twitch: force and/or movement in response to a single action potential Tetanus: force and/or movement in response to action potentials in quick succession

The cross-bridge cycle

Steps

1. Attachment

- Ca2+ release

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- Troponin binds to Ca2+

- Tropomyosin displaces from myosin binding site

- Myosin head attaches to actin (along with ADP and Pi)

2. Cross-bridge formation and Pi release

- Pi released from myosin

- ADP remains attached

- Myosin forms cross-bridge with actin; increase binding strength from weak to strong

3. Filament sliding and ADP release

- ADP release causes change in orientation of myosin head

- Myosin filament pulled towards M-line

4. ATP binding

- New ATP molecule binds to myosin head

5. ATP hydrolysis and detachment

- ATP molecule hydrolysed to ADP and Pi (which both remain attached)

- Energy released used to return myosin head to original orientation, and detach from the actin filament

Note: Isometric contraction - In isometric contraction, the myosin head changes orientation, but there is NO FILAMENT SLIDING

- Force is directed towards the M line

Key Features

ATP is used in each cycle to provide the energy

o RIGOR MORTIS occurs if ATP concentration = 0

Direction of filament sliding is one way

o Thin filament moves towards the centre of the sarcomere

o Causes sarcomere shortening

o However the attached bridge does resist a stretch applied to the ends of the muscle

Step size is small

o Sliding produced by one cycle is only 1% of sarcomere length

o Many cycles in succession needed to cause large movements e.g. running

Variation in Force (in vivo)

Recruitment of the Motor Units

Motor Unit: a motor neuron and all the muscle fibres is innervates (has synapses with)

o Functional unit of normal skeletal muscle

o Action potential in the motor neuron triggers an action potential in all the muscle fibres in the

motor unit

Vary the number of motor units that are active and thus vary the force as more muscle fibres are

producing force

Functional anatomy of motor units:

o Motor unit size varies (muscle fibres per motor neurone)

o There is a scattered distribution of motor units within the muscle


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Frequency of stimulation

Force increases with the stimulation frequency

Force summates, but action potentials do not:

o Twitch unfused tetanus force fused tetanus force (increasing force)

o Particularly important for getting high forces In vivo

Mechanism:

o at higher stimulation frequency, more Ca is released into the space around the filaments.

o More troponins bind to Ca,

therefore more crossbridges

attach and produce force

Force depends on filament overlap

Drawing Sarcomeres:

o Align thick (central) filaments

first, then draw thin filaments

o Max force produced when

completely overlapped

o Draw cross bridges attached for each different amount of

overlap

Force depends on velocity and direction of Movement

This behaviour cannot be understood in terms of one cross-bridge. It

is due to a large number of bridges acting together. Force depends on

the:

o proportion of bridges that are attached

o Force each attached bridge produces.

If the velocity is too great, the myosin heads do not have as much time

to form cross-bridges, which may result is less force

Energy Demand and Supply

ATP Supply

Phosphocreatine (PCr) + ADP ↔ ATP + Creatine (involves CREATING KINASE)

o Buffers ATP concentration so that it is constant

o Occurs in every contraction, during recovery reaction is reversed

o Phosphocreatine dedicated to this reaction only, and is stored in the muscle

Glycolysis – does not require oxygen

o Substrate: glycogen and glucose

o Products: lactic acid and/or pyruvate

Oxidation – requires oxygen

o Makes more ATP per carbon atom metabolised than Glycolysis does

o Substrates: glucose, glycogen, fatty acids

o Products: carbon dioxide


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Activity Demand

Activity Fuel supply DURING activity

Sustained, low intensity ATP, PCr, fatty acids (also glycogen & glucose)

Sustained, high intensity ATP, PCr, glycogen, glucose (also fatty acids)

Sprint ATP, PCr

Cardiac Muscle

Structure and function

Small cells

Action potentials spread between cells

Some cell types have SPONTANEOUS action potentials

Intercalated disks with mechanical links between the fibres

Gap junctions, where ionic currents (action potentials) flow between fibres

Action potential and Calcium sources

Differences from skeletal muscle: Timing of ventricular action potential and isometric force

Action potential lasts as long as isometric force

Force is already relaxing during membrane’s refractory period

o Therefore cannot produced fused tetanus (sustained, summated force)

o Designed for pumping

Normally not enough Ca to bind to all troponins, therefore anything that changes Ca changes force

Between action potentials, Voltage-gated Ca channels in sarcoplasmic reticulum closed, therefore Ca is

not bound to Troponin in thin filament. Cross bridges not attached, therefore no force

During the action potential, Ca enters from the SR AND through voltage gated channels in the muscle

membrane during the PLATEAU of the action potential

o Some, but NOT ALL cross-bridges attach

Smooth Muscle

Structure and function

Arranged in sheets forming walls of tubular organs

o blood vessels,

o gastro-intestinal tract

o reproductive tract, etc.

Cells appear smooth (not-striated) in micrographs.

Contain filaments, but they are not arranged in regular arrays.

Contractile mechanism (crossbridge cycle) is similar to skeletal and cardiac muscle

Activation and Relaxation

Different from skeletal and cardiac muscle: involves-

o Intracellular Ca

o Intracellular Cm = CALMODULIN

o Intracellular MIck = MYOSIN LIGHT CHAIN KINASE


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o NO Troponin

Contraction

- Inactive myosin is not phosphorylated so cannot bind to actin

- Action potential depolarisation release of/increase in intracellular Ca

- Ca, Cm and MIck combine to form ACTIVE ENZYME

o Phosphorylates myosin (hydrolysing a molecule of free ATP)

o Myosin binds to actin

Relaxation

- PHOSPHATASE removes phosphate from myosin

- Inactive myosin cannot bind to actin

alex's musculosketal

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