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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 Minus end - pointed end Plus end - barbed 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 Other myosins found here include Myo VI, Is, dynein kinesin microtubule The myosin family : II VII XI XI I IV VI VI I II XI I VII X I I 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 Cellular functions of the myosin family From Mermall et al. (1998) Science 279, 527-533 Plant cells also exhibit vesicular transport: (called cytoplasmic streaming) But driven by myosin! Especially the giant cells of the pond algae, Nitella and Chara 100mm Up to 60mm.sec-1 Myosin motor polarity: 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 periphery The 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. Many unconventional myosins are associated with hearing. Myosin-I, II, III, VI, VIIa Slides courtesy of Chris Batters (NIMR Mill Hill) The Human Ear Sound 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). Stereocilia Single hair cell – note stereocilia Stereocilia Outer hair cells Inner hair cells Variety of myosins in hair cells – From website of Dr Sutherland MacIver – Univ. Edinburgh Stereocilia Scanning electron micrograph (x 2,500) hair bundles in the bullfrog saccule. Transmission electron micrograph (x 50,000) of the tip of the stereocilia and its adjacent, taller neighbour. Hair Cell Tip-Link Jeffrey R Holt, PNAS 2000. Myo1c Physiological significance “Tip Link” tensioning motor Gillespie and Walker Nature, 2001. 413:194-202 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’. ******************************************* The tension in the tip link is maintained by an array of unconventional myosin-I (myo1c) D.Corey 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 mutations and disease: 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. 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 Nebulin mutants most common – large gene – not many sequenced. Actin mutants (ACTA1) >105 mutants. Most dominant. -tropomyosin mutant 1 Actin (ACTA1) gene mutants 2a 2x CFTD –congenital fibre type disproportion Classical nemaline