Mercredi 24 octobre 2007 Le cytosquelette I - Les principes communs aux trois types de filaments, assemblage, désassemblage II - Les protéines associées et leur rôle III - Les moteurs moléculaires IV - Fonctions du cytosquelette dans la cellule

III - Moteurs moléculaires Mercredi 24 octobre 2007 III - Moteurs moléculaires p949

Généralités Protéines associées au cytosquelette Mercredi 24 octobre 2007 Généralités Protéines associées au cytosquelette Se déplacent sur un filament polarisé Cycle d'hydrolyse de l'ATP Des douzaines p949

Mercredi 24 octobre 2007 Spécificités Type de filaments pour se déplacer : microfilaments d'actine ou microtubules La direction de déplacement La charge organite à membrane : Golgi, vésicules, … autre filament : contraction musculaire, cil, flagelle, mitose, … p949

Analogies Avec : Car ADN polymérase ARN polymérase Hélicase Ribosomes Mercredi 24 octobre 2007 Analogies Avec : ADN polymérase ARN polymérase Hélicase Ribosomes Car utilisent de l'énergie chimique se déplacent sur une voie linéaire dans une direction qui dépend de la polarité du support p949

Fonctionnement d'un moteur Mercredi 24 octobre 2007 Fonctionnement d'un moteur Une tête et une queue Tête = moteur Hydrolyse du nucléotide coordonnée avec changement de conformation Cycle d'attachement / détachement liaison au filament changement de conformation détachement du filament reprise de la conformation de départ nouvelle liaison au filament comme une "marche à pied" Fonction de la tête : identité de la voie direction du mouvement Fonction de la queue : identité de la charge fonction biologique du moteur p949

Plan Les trois groupes de moteurs Mode d’action Contraction musculaire Mercredi 24 octobre 2007 Plan Les trois groupes de moteurs Myosines (marche sur actine) Kinésines (marche sur tubuline) Dynéines (marche sur tubuline) Mode d’action Comment créer un mouvement ? Transport des organites limités par une membrane Régulation de l'activité des moteurs Contraction musculaire Mouvement des cils et flagelles p949

I – A - Moteurs à microfilaments d'actine Mercredi 24 octobre 2007 I – A - Moteurs à microfilaments d'actine Myosine Premier moteur à être identifié dans le muscle #1p949

Myosine II Six chaînes Deux chaînes lourdes Mercredi 24 octobre 2007 Myosine II Six chaînes Deux chaînes lourdes Deux fois deux chaînes légères #1p949

Chaînes lourdes Tête globulaire (N terminale) Mercredi 24 octobre 2007 Chaînes lourdes Tête globulaire (N terminale) Queues longues enroulées l'une dans l'autre Établissement de liaisons queue-queue Fabrication de filaments épais #1p949

Mercredi 24 octobre 2007 Chaînes légères Deux paires #1p949

Fig 16-51 A - Molécule de myosine II Mercredi 24 octobre 2007 A - Molécule de myosine II deux chaînes lourdes quatre chaînes légères (2 fois 2) B - Molécules de myosine en microscopie électronique ombrées au platine Fig 16-51 #1p949

Filament épais Enroulement de deux queues Mercredi 24 octobre 2007 Filament épais Enroulement de deux queues Assemblage de dimères tête bêche Plusieurs centaines de molécules = Un filament épais #1p949

Fig 16-52 Filament épais de myosine II Zone nue sans têtes Mercredi 24 octobre 2007 Filament épais de myosine II Zone nue sans têtes Explique la théorie du glissement de la contraction musculaire Fig 16-52 #1p949

Myosin molecules and thick filaments Mercredi 24 octobre 2007 Fig 1 Myosin molecules and thick filaments. (a) Striated muscle sarcomere showing overlapping arrays of thick and thin filaments. Thick filaments are connected at the M-line and thin filaments at the Z-line. The sarcomere shortens (bringing about contraction of muscle as a whole) by sliding of the thin filament arrays towards each other, increasing their overlap with thick filaments. (b) Central portion of the thick filament model showing the helical array (red dashed line) of pairs of myosin heads on the filament surface (each pair is represented by a yellow sphere). The green backbone contains myosin tails. The model represents a filament with fourfold rotational symmetry. The axial distance between adjacent levels of heads is 14.5 nm and, in this model, the helical repeat (43.5 nm) is three times the 14.5 nm spacing. (c) Schematic representation of the assembly of myosin molecules in a bipolar thick filament. The head ends of the molecules point away from the filament center in each half. Tails have antiparallel and parallel overlaps in the central bare zone (free of heads), but only parallel overlaps in the distal portions of the filament. (d) Myosin molecule. The tail (S2 and LMM) is a coiledcoil formed from the C-terminal halves of each heavy chain (red and green). Heads (S1) comprise the motor domain (MD) and light chain domain (LCD), which contains the essential light chain (ELC, blue) and regulatory light chain (RLC, yellow). Curr Opin Struct Biol. 2006,16(2):204-12. Structure and function of myosin filaments. Craig R, Woodhead JL. Fig 1 Myosin molecules and thick filaments. (a) Striated muscle sarcomere showing overlapping arrays of thick and thin filaments. Thick filaments are connected at the M-line and thin filaments at the Z-line. The sarcomere shortens (bringing about contraction of muscle as a whole) by sliding of the thin filament arrays towards each other, increasing their overlap with thick filaments. (b) Central portion of the thick filament model showing the helical array (red dashed line) of pairs of myosin heads on the filament surface (each pair is represented by a yellow sphere). The green backbone contains myosin tails. The model represents a filament with fourfold rotational symmetry. The axial distance between adjacent levels of heads is 14.5 nm and, in this model, the helical repeat (43.5 nm) is three times the 14.5 nm spacing. (c) Schematic representation of the assembly of myosin molecules in a bipolar thick filament. The head ends of the molecules point away from the filament center in each half. Tails have antiparallel and parallel overlaps in the central bare zone (free of heads), but only parallel overlaps in the distal portions of the filament. (d) Myosin molecule. The tail (S2 and LMM) is a coiledcoil formed from the C-terminal halves of each heavy chain (red and green). Heads (S1) comprise the motor domain (MD) and light chain domain (LCD), which contains the essential light chain (ELC, blue) and regulatory light chain (RLC, yellow).

Organisation des têtes dans les filaments du muscle strié Mercredi 24 octobre 2007 Organisation des têtes dans les filaments du muscle strié Figure 2. Head organization in striated muscle filaments. (a) Negatively stained thick filament from tarantula muscle (protein white). Helical tracks of heads are apparent by viewing along the filament axis at a glancing angle. (b) Helical 3D reconstruction of tarantula thick filament (5 nm resolution) based on negative-stain images. Data set from surface rendered using UCSF Chimera . (c) Single-particle 3D reconstruction of tarantula thick filament (2.5 nm resolution) based on cryo-EM images . The axial distance between adjacent levels of heads is 14.5 nm and the helical repeat is 43.5 nm. The ‘tilted-J’ (blue dots) represents a pair of myosin heads. The larger diameter of this reconstruction compared with (b), which is at the same scale, is due to shrinkage with negative staining (the heads also appear to partially collapse onto the backbone, resulting in loss of information and lower resolution in negative stain). (d) Reconstruction in (c) fitted with the atomic model of smooth muscle myosin (PDB code 1i84) at two 14.5 nm levels . In both the atomic model (based on crystallographic studies) and the filament, one head of each pair (green) has its actin-binding site blocked by binding to the converter region (located in the motor domain) and essential light chain of the other (red). Interactions also occur between heads from different levels. S2, whose position was uncertain in the atomic model, has been modeled as an α-helical coiled-coil (the α-helices have the same colors as the corresponding heads from which they arise), filling a rod of density in the reconstruction that runs from the head–head junction down towards the filament backbone. S2 appears to interact with blocked heads from both its own and the next 14.5 nm level. These multiple interactions could prevent the heads from hydrolyzing ATP and interacting with actin, thus switching the filament off. Curr Opin Struct Biol. 2006,16(2):204-12. Structure and function of myosin filaments. Craig R, Woodhead JL. Figure 2. Head organization in striated muscle filaments. (a) Negatively stained thick filament from tarantula muscle (protein white). Helical tracks of heads are apparent by viewing along the filament axis at a glancing angle. (b) Helical 3D reconstruction of tarantula thick filament (5 nm resolution) based on negative-stain images. Data set from surface rendered using UCSF Chimera . (c) Single-particle 3D reconstruction of tarantula thick filament (2.5 nm resolution) based on cryo-EM images . The axial distance between adjacent levels of heads is 14.5 nm and the helical repeat is 43.5 nm. The ‘tilted-J’ (blue dots) represents a pair of myosin heads. The larger diameter of this reconstruction compared with (b), which is at the same scale, is due to shrinkage with negative staining (the heads also appear to partially collapse onto the backbone, resulting in loss of information and lower resolution in negative stain). (d) Reconstruction in (c) fitted with the atomic model of smooth muscle myosin (PDB code 1i84) at two 14.5 nm levels . In both the atomic model (based on crystallographic studies) and the filament, one head of each pair (green) has its actin-binding site blocked by binding to the converter region (located in the motor domain) and essential light chain of the other (red). Interactions also occur between heads from different levels. S2, whose position was uncertain in the atomic model, has been modeled as an α-helical coiled-coil (the α-helices have the same colors as the corresponding heads from which they arise), filling a rod of density in the reconstruction that runs from the head–head junction down towards the filament backbone. S2 appears to interact with blocked heads from both its own and the next 14.5 nm level. These multiple interactions could prevent the heads from hydrolyzing ATP and interacting with actin, thus switching the filament off.

Structure des filaments épais du muscle strié Mercredi 24 octobre 2007 Structure des filaments épais du muscle strié Figure 3. Backbone structure of striated muscle thick filaments (transverse views, shown for a filament with fourfold rotational symmetry). (a) Molecular crystal model. Each circle represents one myosin tail). (b) Subfilament model. Each subfilament is 4 nm in diameter and contains three myosin tails.. (c) Projected density of a 43.5 nm repeat of tarantula thick filament reconstruction (protein white). Twelve subunits, 4 nm in diameter and of high density (red circles), representing the thick filament backbone, run parallel to the filament axis — as predicted by the subfilament model for a filament with the tarantula symmetry. Note that (c) is shown at a smaller scale than (a,b); lower density material at higher radius represents heads. Curr Opin Struct Biol. 2006,16(2):204-12. Structure and function of myosin filaments. Craig R, Woodhead JL. Figure 3. Backbone structure of striated muscle thick filaments (transverse views, shown for a filament with fourfold rotational symmetry). (a) Molecular crystal model. Each circle represents one myosin tail). (b) Subfilament model. Each subfilament is 4 nm in diameter and contains three myosin tails.. (c) Projected density of a 43.5 nm repeat of tarantula thick filament reconstruction (protein white). Twelve subunits, 4 nm in diameter and of high density (red circles), representing the thick filament backbone, run parallel to the filament axis — as predicted by the subfilament model for a filament with the tarantula symmetry. Note that (c) is shown at a smaller scale than (a,b); lower density material at higher radius represents heads.

Mercredi 24 octobre 2007 Protéines des filaments épais du muscle strié des vertébrés en dehors de la myosine Curr Opin Struct Biol. 2006,16(2):204-12. Structure and function of myosin filaments. Craig R, Woodhead JL. Figure 4. Non-myosin proteins in vertebrate striated muscle thick filaments. Schematic diagram of a sarcomere (thin filaments omitted) showing a thick filament (green backbone, myosin heads omitted) and myosin-associated proteins. Titin molecules (blue) run from the M-line (where they overlap with molecules from the opposite half of the sarcomere), along the thick filament, then through the I-band (the zone between the thick filament and the Z-line), where they have an elastic domain, and ending at the Z-line, where they would overlap with titin from the next sarcomere. Myosin-binding proteins (MyBP, yellow), primarily MyBP-C, occupy 11 sites 43 nm apart in the proximal portions of each half thick filament. Figure 4. Non-myosin proteins in vertebrate striated muscle thick filaments. Schematic diagram of a sarcomere (thin filaments omitted) showing a thick filament (green backbone, myosin heads omitted) and myosin-associated proteins. Titin molecules (blue) run from the M-line (where they overlap with molecules from the opposite half of the sarcomere), along the thick filament, then through the I-band (the zone between the thick filament and the Z-line), where they have an elastic domain, and ending at the Z-line, where they would overlap with titin from the next sarcomere. Myosin-binding proteins (MyBP, yellow), primarily MyBP-C, occupy 11 sites 43 nm apart in the proximal portions of each half thick filament

Schéma d’un filament épais à polarité latérale (muscle lisse) Mercredi 24 octobre 2007 Schéma d’un filament épais à polarité latérale (muscle lisse) Curr Opin Struct Biol. 2006,16(2):204-12. Structure and function of myosin filaments. Craig R, Woodhead JL. Figure 5. Schematic diagram of a side-polar thick filament. In the side-polar mode of assembly, found in smooth muscle thick filaments, antiparallel tail interactions occur along the entire length of the filament (c.f. bipolar filaments), so that opposite sides, rather than opposite ends, of the filament have opposite polarity. Functionally, the filament is still bipolar and able to pull actin filaments in opposite directions from opposite ends. The double arrows indicate how bipolar myosin dimers could easily associate with and dissociate from the filament ends, consistent with assembly-disassembly processes thought to occur in some smooth muscles in different physiological states. Figure 5. Schematic diagram of a side-polar thick filament. In the side-polar mode of assembly, found in smooth muscle thick filaments, antiparallel tail interactions occur along the entire length of the filament (c.f. bipolar filaments), so that opposite sides, rather than opposite ends, of the filament have opposite polarity. Functionally, the filament is still bipolar and able to pull actin filaments in opposite directions from opposite ends. The double arrows indicate how bipolar myosin dimers could easily associate with and dissociate from the filament ends, consistent with assembly-disassembly processes thought to occur in some smooth muscles in different physiological states.

Mercredi 24 octobre 2007 Domaines de queues de myosine impliqués dans l’assemblage des filaments épais Curr Opin Struct Biol. 2006,16(2):204-12. Structure and function of myosin filaments. Craig R, Woodhead JL. Figure 6. Myosin tail domains involved in thick filament assembly. Schematic representation of the myosin tail sequence showing the locations of various regions thought to be involved in the correct assembly of thick filaments (composite diagram based on different myosins; not all sites are involved in all myosins). These include a negative charge cluster, N1 (red), and two positive charge clusters, P1 (blue) and P2 (purple); an assembly competence domain or ACD (yellow) [55 and 58••]; the non-helical tailpiece or NHT (green); and four skip residues (dark blue). Smooth muscle and non-muscle myosins lack the indicated skip residue. Figure 6. Myosin tail domains involved in thick filament assembly. Schematic representation of the myosin tail sequence showing the locations of various regions thought to be involved in the correct assembly of thick filaments (composite diagram based on different myosins; not all sites are involved in all myosins). These include a negative charge cluster, N1 (red), and two positive charge clusters, P1 (blue) and P2 (purple); an assembly competence domain or ACD (yellow) [55 and 58••]; the non-helical tailpiece or NHT (green); and four skip residues (dark blue). Smooth muscle and non-muscle myosins lack the indicated skip residue.

Théorie du glissement Muscle squelettique Cœur et muscle lisse Mercredi 24 octobre 2007 Théorie du glissement Muscle squelettique Orientations opposées des têtes de myosine dans le filament épais Permet le glissement de filaments d’actine d’orientation opposée Cœur et muscle lisse Même disposition mais gènes différents #1p949-950

Digestions enzymatiques Mercredi 24 octobre 2007 Digestions enzymatiques Chymotrypsine et papaïne Séparation de la tête appelée S1 S1 est responsable du glissement #1p950

Démonstration de l’activité motrice des têtes de myosine Mercredi 24 octobre 2007 Démonstration de l’activité motrice des têtes de myosine 2 filaments d'actine marqués en vert et en rouge Fig 16-53 Filament d'actine marqué Vue toutes les 0,6 sec Vitesse  4 m/sec S1 #1p950

Les myosines La myosine n'est pas que musculaire Mercredi 24 octobre 2007 La myosine n'est pas que musculaire Dans l’amibe Acanthamoaba castellanii, myosine même moteur queue différente monomère  myosine I  Myosine musculaire renommée myosine II (deux têtes) Actuellement ordre de découverte ( XVIII) Puis nombreuses autres myosines moteur reconnaissable en -N queue variable en -C fonctions inconnues pour la plupart #1p950

Fig 16-54(A) Arbre de la superfamille des myosines Mercredi 24 octobre 2007 Arbre de la superfamille des myosines Myosine I et II les plus répandus Myosine XIV Toxoplasma et Plasmodium Fig 16-54(A) #1p950

Structure des chaînes lourdes de myosine Mercredi 24 octobre 2007 que chez les plantes que chez les vertébrés Fig 16-54(B) #1p950 que chez les plantes

Chaînes lourdes de myosine Mercredi 24 octobre 2007 Chaînes lourdes de myosine Forment dimères ou monomères (I, IX, XIV) Myosine VI : marche vers le – (la seule) de l ’actine (insertion dans le domaine de la tête) #1p950

Chaînes lourdes de myosine Mercredi 24 octobre 2007 Chaînes lourdes de myosine Saccharomyces cerevisiae : deux myosines I, une myosine II, deux myosines V Caenorhabditis elegans : 15 gènes de myosine (7 classes) Homme : au moins 40 gènes #1p951

Rôle des myosines Myosine II Myosine I Myosine V Myosine VII #1p952 Mercredi 24 octobre 2007 Myosine II Contraction musculaire et non musculaire Cytodiérèse Migration cellulaire Myosine I second site de liaison à l’actine ou site de liaison à une membrane organisation intra cellulaire expansions cellulaires Myosine V transport des vésicules et des organites Myosine VII oreille interne #1p952

I – B & C - Moteurs à microtubules Mercredi 24 octobre 2007 I – B & C - Moteurs à microtubules Kinésines (superfamille) la kinésine les kinesin-related proteins (KRPs) au moins 10 familles de KRPs Dynéines #2p952

B - La kinésine Moteur protéique Se déplace sur les microtubules Mercredi 24 octobre 2007 Moteur protéique Se déplace sur les microtubules Axone géant de calmar (transport d’organites à membrane) Transport dans le sens + Semblable à la myosine II deux chaînes lourdes et deux chaînes légères dimérisation Saccharomyces cerevisiae : 6 kinésines Caenorhabditis elegans : 16 kinésines Homme :  40 kinésines #2p952

Fig 16-55 Kinésines et Kinesin-related proteins Mercredi 24 octobre 2007 Kinésines et Kinesin-related proteins Kinésine conventionnelle moteur N-terminal queue C-terminale : transport d'organites à membrane Fig 16-55 #2p952

Fig 16-55 Kinésines et Kinesin-related proteins #2p952 KIFC2 Mercredi 24 octobre 2007 Kinésines et Kinesin-related proteins KIFC2 moteur C-terminal comprend protéine Ncd de la drosophile protéine KAR3 de la levure se dirige dans le sens - Fig 16-55 #2p952

Fig 16-55 Kinésines et Kinesin-related proteins #2p952 KIF2 Mercredi 24 octobre 2007 Kinésines et Kinesin-related proteins KIF2 moteur au milieu de la chaîne lourde pas d'activité moteur classique se lie aux extrémités des microtubules pour augmenter leur instabilité dynamique  nom de catastrophine Fig 16-55 #2p952

Fig 16-55 Kinésines et Kinesin-related proteins KIF IB #2p952 Mercredi 24 octobre 2007 Kinésines et Kinesin-related proteins KIF IB agit comme monomère déplace les vésicules le long des microtubules Fig 16-55 #2p952

BimC (KRP) BimC forme des tétramères Mercredi 24 octobre 2007 BimC (KRP) BimC forme des tétramères s'associent par les queues  moteur bipolaire  fait glisser les microtubules les uns sur les autres #2p952

#2p952 http://www.proweb.org/kinesin/KinesinTree.html Mercredi 24 octobre 2007 #2p952

Microtubule protofilament with the bound motor domain of Neurospora crassa conventional kinesin Mercredi 24 octobre 2007 Microtubule protofilament with the bound motor domain of Neurospora crassa conventional kinesin. Model based on Song et al. (2001). In the microtubule, two alpha-tubulin subunits (alphaT1 and alphaT2) and a beta-tubulin (betaT1) are shown (structure adapted from Lowe et al., 2001). Kinesin would move along the microtubule towards the top of the figure. N. crassa kinesin and the microtubule interact predominantly through helices H11 and H12 (yellow coils) and the carboxy-terminal tail (thin red strand, C) of betaT1, and the region of kinesin that includes the structural elements in dark blue (alpha4-L12-alpha5 and beta5a-L8). Other interactions may include the carboxy-terminal tail (pink, C) of alphaT2 and kinesin’s neck linker/neck region (grey), as well as alphaT1 and kinesin’s L11. The top of the kinesin core shows the neck domain in two conformations: a random coil found in NcKin (protein backbone in grey), and the ordered conformation of alpha7 from rat brain kinesin (RnKin; Sack et al. 1997) in red. The group of yellow spheres represents GTP and GDP in tubulin and ADP in NcKin. The drug taxol (red spheres) is shown bound to betaT1 Microtubule protofilament with the bound motor domain of Neurospora crassa conventional kinesin. Model based on Song et al. (2001). In the microtubule, two alpha-tubulin subunits (alphaT1 and alphaT2) and a beta-tubulin (betaT1) are shown (structure adapted from Lowe et al., 2001). Kinesin would move along the microtubule towards the top of the figure. N. crassa kinesin and the microtubule interact predominantly through helices H11 and H12 (yellow coils) and the carboxy-terminal tail (thin red strand, C) of betaT1, and the region of kinesin that includes the structural elements in dark blue (alpha4-L12-alpha5 and beta5a-L8). Other interactions may include the carboxy-terminal tail (pink, C) of alphaT2 and kinesin’s neck linker/neck region (grey), as well as alphaT1 and kinesin’s L11. The top of the kinesin core shows the neck domain in two conformations: a random coil found in NcKin (protein backbone in grey), and the ordered conformation of alpha7 from rat brain kinesin (RnKin; Sack et al. 1997) in red. The group of yellow spheres represents GTP and GDP in tubulin and ADP in NcKin. The drug taxol (red spheres) is shown bound to betaT1

C - Les dynéines Moteur protéique Se déplacent sur les microtubules Mercredi 24 octobre 2007 Moteur protéique Se déplacent sur les microtubules Transport dans le sens - Sans rapport avec la super famille des kinésines Deux ou trois chaînes lourdes associées à de nombreuses chaînes légères Deux catégories ancienne : cytoplasmique plus récente : axonémale #2p953

Fig 16-56 Homodimère Deux têtes moteurs Euk +++ Vésicules, Golgi 25 nm Homodimère Deux têtes moteurs Euk +++ Vésicules, Golgi Hétéro et homodimères Deux ou trois têtes moteur Mouvements rapides (cils et flagelles)

Dynéines / kinésines Dynéines Direction – Très gros Très rapides Mercredi 24 octobre 2007 Dynéines / kinésines Dynéines Direction – Très gros Très rapides 14 m/sec Kinésines Direction + Plus petites Plus lentes Les plus rapides : 2-3 m/sec #2p953

Analogie myosine kinésine Mercredi 24 octobre 2007 Analogie myosine kinésine Moteur myosine > moteur kinésine (850 vs 350  ) Myosine marche sur actine / kinésine sur MT Pas de séquences d' en commun Mais moteurs presque identiques en 3-D Les différences viennent des boucles qui s'échappent du noyau central site de liaison à l'actine (pour la myosine) site de liaison aux microtubules (pour la kinésine) #3p953

Mercredi 24 octobre 2007 Analogie myosine kinésine Fig 16-57 #3p953

Rôle du noyau central dans la génération de force Mercredi 24 octobre 2007 Rôle du noyau central dans la génération de force Analogie avec le site de liaison nucléotidique des petites GTPases de la superfamille Ras conformations différentes : GTP-lié actif / GDP-lié inactif #3p954

Mercredi 24 octobre 2007 Amos,LA1997 En haut à gauche : Kinesin and ncd bind to the same site on tubulin. Tubulin sheets decorated with motor domains and then negatively stained show an 8 nm repeating polar pattern (much magnified in [a], compared with in [b–e]). The pattern is shown as it appears when microtubule plus ends are oriented towards the top of the page. (a) shows an image of tubulin decorated with ncd that was obtained by computer analysis of many sheets. Solid contours represent higher protein density; and dashed contours include more stain and thus less protein. Tubulin sheets decorated with kinesin heads give an extremely similar image (not shown). (b–e) show individual decorated sheets with visible ends (top of each part of the figure). Protein appears white here; surrounding stain is black. (b,d) show unprocessed images; (c,e) show the sheets after computer enhancement of the periodic signal and filtering out of noise. Whether the decoration is with kinesin or ncd, the pattern stops at the same stage at the end of the sheet, suggesting that kinesin and ncd bind in the same way to a- and b-tubulin. En haut à droite : Various polymers of tubulin. All the tubulin polymers that have been used in electron microscopic studies of tubulin–motor-domain complexes consist of polar arrangements of longitudinal protofilaments. Each protofilament is a linear arrangement of ab-tubulin heterodimers (each being shown here as a light monomer above a dark monomer). Most natural microtubules have 13 protofilaments but those assembled in vitro vary in protofilament number. (a) A tubulin sheet, or opened-out microtubule. Purified tubulin assembles mainly into the so-called B-lattice, in which heterodimers in adjacent protofilaments line up at a shallow angle. (b) A reassembled microtubule with 10 protofilaments. If the heterodimers lie on a single shallow helix, as shown here (see the laterally exposed dimers at the top and bottom of the tube), the subunit is helically symmetrical; except near the broken ends, every dimer has an identical set of neighbours. (c) A 14-protofilament microtubule exhibiting a ‘seam’ where lateral contacts are between light and dark subunits rather than between like subunits. (Note also the ends of 1.5 dimer helices at the top and bottom of the tube.) Reassembled 13- or 14-protofilament microtubules must always have at least one such seam; those with any other number of protofilaments may also have seams. (d) Natural 16-protofilament microtubules are apparently always seamless and, therefore, helically symmetrical, with two shallow dimer helices. When there are 13 protofilaments in a microtubule (not shown) they are exactly longitudinal, but other numbers of protofilaments twist slowly around their tubes. En bas à gauche : Comparison of different 3-D images of tubulin–motor-protein complexes. (a) Set of 3-D images obtained by combining tilted views of negatively stained tubulin sheets. The left-hand protofilament is undecorated; the two monomers of a tubulin heterodimer are labelled (T). The middle and right-hand protofilaments are decorated with monomeric kinesin (K) and ncd (N), respectively. (b) Merged 3-D images of frozen, hydrated 16-protofilament microtubules. Part of an undecorated microtubule is shown at the bottom, with tubulin monomers labelled ‘T’; successive helical segments are decorated with monomeric kinesin (K), dimeric kinesin (attached head, K1; tethered head, K2) and dimeric ncd (attached head, N1; tethered head, N2). (c,d) Images of frozen, hydrated 10-protofilament microtubules The models have been inverted so that microtubule polarity is consistent with (a) and (b). (c) shows an undecorated microtubule with tubulin monomers labelled ‘T’; (d) shows a microtubule decorated with monomeric kinesin (K). (e) Merged 3-D images of negatively stained 16-protofilament microtubules. Successive helical turns show microtubules decorated with monomeric kinesin under different nucleotide conditions. White lines on the right-hand side indicate the change in tilt of a projecting feature, from roughly horizontal (ADP-bound state) to 45° (nucleotide-free [no nucl] and AMP.PNP-occupied states). En bas à droite : Structural interactions between tubulin (grey circles) and motor domains (white shapes). On the left is an enlargement of part of the decorated tubulin sheet on the right. a, a-tubulin; b, b-tubulin. Tubulin dimers appear to be oriented so that the b-tubulin monomer (shown here as a light grey subunit) is located towards the microtubule plus end [44,45·]. Image reconstructions (see Fig. 3) show that both kinesin and ncd motor domains contact two tubulin subunits; this is consistent with findings by cross-linking and blot overlay that both motors interact with both tubulin species. As there are no extra, undecorated tubulin monomers at the ends of the decorated tubulin sheets (see Fig. 1), the two tubulin monomers associated with one motor head domain are most probably a stable heterodimer. The underlying shapes of the two sorts of monomer in Figure 1a, as compared with undecorated a- and b-tubulin, also support this positioning of the motor domains relative to tubulin dimers. The points at which double heads of kinesin or ncd are joined (see Fig. 3b) indicate the approximate locations of the amino (N) and carboxyl (C) termini of the motor domains. Amos,LA1997 Kinesin and ncd bind to the same site on tubulin. Tubulin sheets decorated with motor domains and then negatively stained show an 8 nm repeating polar pattern (much magnified in [a], compared with in [b–e]). The pattern is shown as it appears when microtubule plus ends are oriented towards the top of the page. (a) shows an image of tubulin decorated with ncd that was obtained by computer analysis of many sheets. Solid contours represent higher protein density; and dashed contours include more stain and thus less protein. Tubulin sheets decorated with kinesin heads give an extremely similar image (not shown). (b–e) show individual decorated sheets with visible ends (top of each part of the figure). Protein appears white here; surrounding stain is black. (b,d) show unprocessed images; (c,e) show the sheets after computer enhancement of the periodic signal and filtering out of noise. Whether the decoration is with kinesin or ncd, the pattern stops at the same stage at the end of the sheet, suggesting that kinesin and ncd bind in the same way to a- and b-tubulin. Various polymers of tubulin. All the tubulin polymers that have been used in electron microscopic studies of tubulin–motor-domain complexes consist of polar arrangements of longitudinal protofilaments. Each protofilament is a linear arrangement of ab-tubulin heterodimers (each being shown here as a light monomer above a dark monomer). Most natural microtubules have 13 protofilaments but those assembled in vitro vary in protofilament number. (a) A tubulin sheet, or opened-out microtubule. Purified tubulin assembles mainly into the so-called B-lattice, in which heterodimers in adjacent protofilaments line up at a shallow angle. (b) A reassembled microtubule with 10 protofilaments. If the heterodimers lie on a single shallow helix, as shown here (see the laterally exposed dimers at the top and bottom of the tube), the subunit is helically symmetrical; except near the broken ends, every dimer has an identical set of neighbours. (c) A 14-protofilament microtubule exhibiting a ‘seam’ where lateral contacts are between light and dark subunits rather than between like subunits. (Note also the ends of 1.5 dimer helices at the top and bottom of the tube.) Reassembled 13- or 14-protofilament microtubules must always have at least one such seam; those with any other number of protofilaments may also have seams. (d) Natural 16-protofilament microtubules are apparently always seamless and, therefore, helically symmetrical, with two shallow dimer helices. When there are 13 protofilaments in a microtubule (not shown) they are exactly longitudinal, but other numbers of protofilaments twist slowly around their tubes. Comparison of different 3-D images of tubulin–motor-protein complexes. (a) Set of 3-D images obtained by combining tilted views of negatively stained tubulin sheets. The left-hand protofilament is undecorated; the two monomers of a tubulin heterodimer are labelled (T). The middle and right-hand protofilaments are decorated with monomeric kinesin (K) and ncd (N), respectively. (b) Merged 3-D images of frozen, hydrated 16-protofilament microtubules. Part of an undecorated microtubule is shown at the bottom, with tubulin monomers labelled ‘T’; successive helical segments are decorated with monomeric kinesin (K), dimeric kinesin (attached head, K1; tethered head, K2) and dimeric ncd (attached head, N1; tethered head, N2). (c,d) Images of frozen, hydrated 10-protofilament microtubules The models have been inverted so that microtubule polarity is consistent with (a) and (b). (c) shows an undecorated microtubule with tubulin monomers labelled ‘T’; (d) shows a microtubule decorated with monomeric kinesin (K). (e) Merged 3-D images of negatively stained 16-protofilament microtubules. Successive helical turns show microtubules decorated with monomeric kinesin under different nucleotide conditions. White lines on the right-hand side indicate the change in tilt of a projecting feature, from roughly horizontal (ADP-bound state) to 45° (nucleotide-free [no nucl] and AMP.PNP-occupied states). Structural interactions between tubulin (grey circles) and motor domains (white shapes). On the left is an enlargement of part of the decorated tubulin sheet on the right. a, a-tubulin; b, b-tubulin. Tubulin dimers appear to be oriented so that the b-tubulin monomer (shown here as a light grey subunit) is located towards the microtubule plus end [44,45·]. Image reconstructions (see Fig. 3) show that both kinesin and ncd motor domains contact two tubulin subunits; this is consistent with findings by cross-linking and blot overlay that both motors interact with both tubulin species. As there are no extra, undecorated tubulin monomers at the ends of the decorated tubulin sheets (see Fig. 1), the two tubulin monomers associated with one motor head domain are most probably a stable heterodimer. The underlying shapes of the two sorts of monomer in Figure 1a, as compared with undecorated a- and b-tubulin, also support this positioning of the motor domains relative to tubulin dimers. The points at which double heads of kinesin or ncd are joined (see Fig. 3b) indicate the approximate locations of the amino (N) and carboxyl (C) termini of the motor domains.

Mercredi 24 octobre 2007 En haut à gauche Comparison of crystal structures. Corresponding views of (a) myosin and (b) kinesin, looking down on the nucleotide (shown in solid black). The structure of ncd (not shown) is almost identical to that of kinesin. The homology in the structural elements that support the nucleotide-binding pocket is less apparent than in views at right angles to this, but the myosin domains that move relative to one another are more distinct. The distal domain (residues 457–762) appears to rotate around a hinge near residue 457 (marked by asterisk). SH1 and SH2 indicate two cysteines that can be cross-linked in solution; their large separation in all the available crystal structures of myosin suggests that only part of the full conformational change has been shown. Insertion 1, consisting of loop L8 with strands b5a and b5b, and Insertion 2, consisting of loop L12, are predicted to make contact with tubulin (see Fig. 3c). The crystal structure of kinesin lacks a short segment (residues 325–332) at its C terminus (shown by arrow head); the structures of this segment and the subsequent stretch of a helix are therefore hypothetical. En haut à droite Fitting the kinesin crystal structure to 3D electron microscope images. (a) Part of a model combining views of a microtubule decorated with monomeric kinesin motor domains in three different nucleotide states (AMP.PNP, no nucleotide [nucl], ADP. The individual 3D models are calculated from electron microscope images of negatively stained specimens. A prominent spike, on the left of each motor domain in this view, appears to rotate towards the microtubule plus end (oriented towards the top of the page) when Mg.ADP is lost from its nucleotide-binding site. Binding of AMP.PNP, a nonhydrolyzable analogue of ATP, does not cause a reverse movement. The vertical spacing of the motor domains corresponds to the 8 nm length of tubulin heterodimers. (b) A view of the kinesin motor domain at about 90° to that in Figure 2b, rotated so that Insertions 1 and 2 (see Figs 1b,2b) are on the reverse side. In this orientation, both the N and C termini of the traceable regions of the polypeptide are towards the top of the page; there are six unseen residues at the N terminus and 24 at the C terminus. (c) A model obtained by combining several 3D electron microscope images of frozen-hydrated microtubules decorated with motor domains. The longitudinal ‘protofilaments’ of the microtubule show a 4 nm periodicity corresponding to the tubulin monomer spacing. Attached to some sites on the protofilaments are images of monomeric kinesin (K), dimeric kinesin (attached domain, K1; tethered domain, K2) and dimeric ncd (N1 and N2). Superimposed on images of two kinesin monomers are miniaturised views of the kinesin crystal structure (one oriented as in Fig. 3b, the other rotated 135°). The side views on the left (based on Fig. 2b) show loops L8 (incorporating b5a and b5b) and L12 binding separately to tubulin; the hypothetical dimer structure illustrates the proposal that the two strands of the coiled coil may separate to allow the tethered motor domain to find the next available binding site. En bas à droite Functional and structural relationships between myosin and kinesin motors. Cyclic transition of binding states between motor and track. Motor domains (K, kinesin; M, myosin) cycle between weakly bound and strongly bound conformations, depending on whether nucleotide (solid black circle) is present or not in the active site. Both families of motor are strongly bound when the site is empty, but the transition steps between weak and strong binding differ (see Table 1). A kinesin domain remains strongly attached to tubulin whilst cleaving Pi from ATP and releasing it; following detachment kinesin becomes strongly bound again by releasing ADP. Myosin is immediately detached from actin on binding ATP and reattaches strongly by releasing Pi. Kinesin only releases ADP readily when interacting with microtubules; myosin only releases Pi readily when interacting with actin filaments (see Table 2). (b) Homology in the arrangement of sequence segments. Schematic comparison of the amino acid sequences of the S1 fragment of myosin and the motor domains of kinesin and ncd. a-helical segments are shown in black, b-sheet strands are shown in grey and the putative coiled-coil extensions from kinesin and ncd motor domains are shown as cross-hatched segments; the latter segments were absent from the proteins that were crystallized but were present in dimeric motors studied by electron microscopy. The scale at the top shows amino acids in the myosin sequence numbered from the N terminus. The 25, 50 and 20 kDa fragments obtained by proteolysis are also marked. A1 (405–415), A2 (529–558) and A3 (626–647) label points in the myosin sequence that are believed to contact actin. ELC and RLC refer to the essential and regulatory light chains of myosin II, which are associated with the long a-helical lever arm or ‘regulatory domain’ (762–842). Insertion 1 in the myosin sequence seems to be functionally equivalent to loop L8 of kinesin or ncd; insertion 2 seems to be equivalent to loop L12. Small loops at N1–N4 are responsible for binding nucleotide to kinesin or ncd; N2 and N3 are also known as ‘Switch 1’ and ‘Switch 2’ [7··]. AMP.PNP is a nonhydrolysable analogue of ATP. En bas à gauche Motility schemes for kinesin and ncd. (a) The processive behaviour of kinesin may possibly be explained by melting of part of the coiled-coil structure through which the motor domains (heads) are paired [8,9]. Thermal motion will then allow the tethered head to search for the nearest available binding site (stage I). One of the tubulin-binding loops binds first (stage II). After release of Mg.ADP and attachment of the second loop, the leading head is strongly attached (stage III). Because of the conformational change associated with ADP loss, the lead head exerts a tension on the rear head; the effect of tension is highly directional, causing the rear head (but not the leading head) to detach (stage IV) and it is pulled forwards to begin the next cycle. (b) In order to explain the different directions of kinesin and ncd movement, we suggest that initial attachment is via different loops (stage II), before settling down into the strongly attached state (stage III). In addition, on the one hand, when a kinesin head is fully attached, it needs to be pulled off much more easily by its partner pulling from the plus direction than by tension from the minus direction. On the other hand, ncd needs to resist tension from the plus direction when pulling a load towards the minus end. Amos,LiA1997 Amos,LA1997 En haut à gauche Comparison of crystal structures. Corresponding views of (a) myosin and (b) kinesin, looking down on the nucleotide (shown in solid black). The structure of ncd (not shown) is almost identical to that of kinesin. The homology in the structural elements that support the nucleotide-binding pocket is less apparent than in views at right angles to this, but the myosin domains that move relative to one another are more distinct. The distal domain (residues 457–762) appears to rotate around a hinge near residue 457 (marked by asterisk). SH1 and SH2 indicate two cysteines that can be cross-linked in solution; their large separation in all the available crystal structures of myosin suggests that only part of the full conformational change has been shown. Insertion 1, consisting of loop L8 with strands b5a and b5b, and Insertion 2, consisting of loop L12, are predicted to make contact with tubulin (see Fig. 3c). The crystal structure of kinesin lacks a short segment (residues 325–332) at its C terminus (shown by arrow head); the structures of this segment and the subsequent stretch of a helix are therefore hypothetical. En haut à droite Fitting the kinesin crystal structure to 3D electron microscope images. (a) Part of a model combining views of a microtubule decorated with monomeric kinesin motor domains in three different nucleotide states (AMP.PNP, no nucleotide [nucl], ADP. The individual 3D models are calculated from electron microscope images of negatively stained specimens. A prominent spike, on the left of each motor domain in this view, appears to rotate towards the microtubule plus end (oriented towards the top of the page) when Mg.ADP is lost from its nucleotide-binding site. Binding of AMP.PNP, a nonhydrolyzable analogue of ATP, does not cause a reverse movement. The vertical spacing of the motor domains corresponds to the 8 nm length of tubulin heterodimers. (b) A view of the kinesin motor domain at about 90° to that in Figure 2b, rotated so that Insertions 1 and 2 (see Figs 1b,2b) are on the reverse side. In this orientation, both the N and C termini of the traceable regions of the polypeptide are towards the top of the page; there are six unseen residues at the N terminus and 24 at the C terminus. (c) A model obtained by combining several 3D electron microscope images of frozen-hydrated microtubules decorated with motor domains. The longitudinal ‘protofilaments’ of the microtubule show a 4 nm periodicity corresponding to the tubulin monomer spacing. Attached to some sites on the protofilaments are images of monomeric kinesin (K), dimeric kinesin (attached domain, K1; tethered domain, K2) and dimeric ncd (N1 and N2). Superimposed on images of two kinesin monomers are miniaturised views of the kinesin crystal structure (one oriented as in Fig. 3b, the other rotated 135°). The side views on the left (based on Fig. 2b) show loops L8 (incorporating b5a and b5b) and L12 binding separately to tubulin; the hypothetical dimer structure illustrates the proposal that the two strands of the coiled coil may separate to allow the tethered motor domain to find the next available binding site. En bas à droite Functional and structural relationships between myosin and kinesin motors. Cyclic transition of binding states between motor and track. Motor domains (K, kinesin; M, myosin) cycle between weakly bound and strongly bound conformations, depending on whether nucleotide (solid black circle) is present or not in the active site. Both families of motor are strongly bound when the site is empty, but the transition steps between weak and strong binding differ (see Table 1). A kinesin domain remains strongly attached to tubulin whilst cleaving Pi from ATP and releasing it; following detachment kinesin becomes strongly bound again by releasing ADP. Myosin is immediately detached from actin on binding ATP and reattaches strongly by releasing Pi. Kinesin only releases ADP readily when interacting with microtubules; myosin only releases Pi readily when interacting with actin filaments (see Table 2). (b) Homology in the arrangement of sequence segments. Schematic comparison of the amino acid sequences of the S1 fragment of myosin and the motor domains of kinesin and ncd. a-helical segments are shown in black, b-sheet strands are shown in grey and the putative coiled-coil extensions from kinesin and ncd motor domains are shown as cross-hatched segments; the latter segments were absent from the proteins that were crystallized but were present in dimeric motors studied by electron microscopy. The scale at the top shows amino acids in the myosin sequence numbered from the N terminus. The 25, 50 and 20 kDa fragments obtained by proteolysis are also marked. A1 (405–415), A2 (529–558) and A3 (626–647) label points in the myosin sequence that are believed to contact actin. ELC and RLC refer to the essential and regulatory light chains of myosin II, which are associated with the long a-helical lever arm or ‘regulatory domain’ (762–842). Insertion 1 in the myosin sequence seems to be functionally equivalent to loop L8 of kinesin or ncd; insertion 2 seems to be equivalent to loop L12. Small loops at N1–N4 are responsible for binding nucleotide to kinesin or ncd; N2 and N3 are also known as ‘Switch 1’ and ‘Switch 2’ [7··]. AMP.PNP is a nonhydrolysable analogue of ATP. En bas à gauche Motility schemes for kinesin and ncd. (a) The processive behaviour of kinesin may possibly be explained by melting of part of the coiled-coil structure through which the motor domains (heads) are paired [8,9]. Thermal motion will then allow the tethered head to search for the nearest available binding site (stage I). One of the tubulin-binding loops binds first (stage II). After release of Mg.ADP and attachment of the second loop, the leading head is strongly attached (stage III). Because of the conformational change associated with ADP loss, the lead head exerts a tension on the rear head; the effect of tension is highly directional, causing the rear head (but not the leading head) to detach (stage IV) and it is pulled forwards to begin the next cycle. (b) In order to explain the different directions of kinesin and ncd movement, we suggest that initial attachment is via different loops (stage II), before settling down into the strongly attached state (stage III). In addition, on the one hand, when a kinesin head is fully attached, it needs to be pulled off much more easily by its partner pulling from the plus direction than by tension from the minus direction. On the other hand, ncd needs to resist tension from the plus direction when pulling a load towards the minus end.