Module 632

Lecture 10 JCS

Unconventional myosins and cell

biology

MODULE - 632 Lecture 8 Muscle Contraction

Lecture outcomes:

  At the end of this lecture a student will be aware that :

  1)   there are many different types of motility within cells

2) many movements require directed polymerisation of the actin cytoskeleton as well as cell growth (axons)

3)   most movements are driven by molecular motors – myosin (actin -based) and kinesin/dyneins (microtubular based).

• 5)   there is a large family of myosins in eukaryotes 

• 6)   myosins are important for cellular transport, and changes in cell shape

• 7) myosins are important in how we hear

• 8) mutations in myosins can cause human genetic diseases – deafness, cardiomyopathy, sk. muscle myopathy.

Cell motility is driven by different motors:

• Cytoskeleton polymerisation/depolymerisation– Actin filaments

• Pseudopodia, lamellipodia, phagocytosis, acrosomal process extension – listeria hijacks this system, pollen tubes.

– Tubulin (forms microtubules)

• Molecular motors:– Actin-based motors

• MYOSIN FAMILY– Microtubule-based motors

• KINESIN family• DYNEIN family.

Fibroblast Fish keratocyte

ACTIN FILAMENT DYNAMICS

Plus end - barbed end

Minus end - pointed end

Acrosomal process of sea urchin sperm:

Many transport functions require molecular motors:

e.g.Actin-based Myosin family

MT-based Kinesin & Dynein families

Many cell motilities require several different motors or motors + filament

dynamics!

Locomotion of cells: Actin filament polymerisation Actin filament “stress fibres” Focal Adhesions Myosin I Myosin II

Axon growth - Neuronal vesicle transport:

MYOSIN V

kinesindynein

microtubule

Other myosins found here include Myo VI, Is,

VIIIX

IXIIV

I

XIX

VIII

III

VII

VI

II

Most myosins have been identified using the “Walker” P- motif (J.E. Walker). The sequence is GESGAGKT = Gly-Glu-Ser-Gly-Ala-Gly-Lys-Thr

The myosin family :

From Mermall et al. (1998) Science 279, 527-533

Cellular functions of the myosin family

Plant cells also exhibit vesicular transport: (called cytoplasmic streaming)

But driven by myosin!

Especially the giant cells of the pond algae, Nitella and Chara

Up to60m.sec-1

100m

Direction determined by actin filament polarity: All myosins, except myosin VI, move towards the plus (+) or barbed end of actin.

Note:The + end of actin is defined at the most rapidly polymerising end. It is also called the barbed end because of the arrow-head pattern when decorated with myosin S1 heads. The + end usually points towards the cell peripheryThe other end is called the minus (–) or pointed end

- e.g. most cytoskeletal myosins walk away from the nucleus

- fits with the muscle thick filaments moving along the thin filaments towards

the Z-line (where the thin filament F-actin barbed ends are)

Velocity is determined by the myosin isotype.

Myosin motor polarity:

Many unconventional myosins are associated

with hearing.

Myosin-I, II, III, VI, VIIa

Slides courtesy of Chris Batters (NIMR Mill Hill)

The Human EarSound enters the ear canal and causes the tympanic membrane to vibrate. This vibration is changed from a low force large movement to a high force small movement by the three bones in the inner ear. The stapes uses this higher force to vibrate the oval window, which separates the air filled outer ear to the fluid filled inner ear. The vibration causes the fluid in the ear to accelerate around the cochlea deflecting arrays of stereocilia. When they are deflected mechanosensitive ion channels open and action potentials are created allowing us to hear (Very similar action in the vestibular organ, allowing us to balance).

Single hair cell – note stereocilia

Stereocilia

Variety of myosins in hair cells –

From website of Dr Sutherland MacIver – Univ. Edinburgh

Stereocilia

Outer hair cells Inner hair cells

StereociliaStereocilia

Tip-Link

Hair Cell

Jeffrey R Holt, PNAS 2000.

Transmission electron micrograph (x 50,000) of the tip of the stereocilia and its adjacent, taller neighbour.

Scanning electron micrograph (x 2,500) hair bundles in the bullfrog saccule.

“Tip Link” tensioning motor

D.Corey

Gillespie and Walker Nature, 2001. 413:194-202

Myo1c Physiological significance

The stereocilia are linked together by tip links ‘Cadherin 23’ (May 2004, Nature). The tip links allow the coordinated opening of the ion channels ‘very important so that the action potential threshold is reached’.

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The tension in the tip link is maintained by an array of unconventional myosin-I (myo1c)

Slow adaptation within the ear

Calcium marker showing the entry of calcium into the stereocilia upon deflection

An array of 20-100 myo1c molecules maintains the mechanosensitive ion channel in a ‘just off’ position by climbing up the stereocilia or slipping down.

Myosin II mutations: Human familial hypertrophic cardiomyopathy (HCM) mutants (“Sudden death syndrome”). Human cardiac myosin is encoded by two genes (V1 and V3 isoforms) which are expressed at low levels in skeletal muscle.

HCM mutations occur in at least 9 different cardiac sarcomeric protein genes including myosin heavy chain, tropomyosin, actin, MyBPC, troponin genes.

Myosin VII mutations: Usher’s deaf/blind syndrome (myosin VII)

Myosin V mutations: Dilute mutation (in mice) mild phenotype = altered coat colour : severe phenotype = convulsions and early death.

Myosin mutations and disease:

Familial hypertrophic cardiomyopathy – FHC

‘Sudden death syndrome’

Most mutations which cause this are in the myosin heavy chain – others occur in troponins, TM, actin, MyBPC etc.

Skeletal myopathies –

Nemaline myopathies

Nema = rod/thread (Gk)

Mutations identified in:

& tropomyosin

troponin

nebulin

-skeletal actin

-tropomyosin mutant

Nebulin mutants most common – large gene – not many sequenced.

Actin mutants (ACTA1) >105 mutants. Most dominant.

1

2a

2x

Actin (ACTA1) gene mutants

Classical nemaline

CFTD –congenital fibre type disproportion