Mercredi 21 novembre 2007 Le cytosquelette

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1 Mercredi 21 novembre 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

2 IV - Fonctions du cytosquelette dans la cellule

3 Coordination des éléments du cytosquelette
Mercredi 21 novembre 2007 Coordination des éléments du cytosquelette Forme de la cellule Déplacement de la cellule Division pendant la mitose (cf. infra) #IVp969

4 Plan La levure comme modèle d'étude
Locomotion des cellules (filipodes, lamellipodes et pseudopodes) Action du cytosquelette sur l'environnement Action de l'environnement sur le cytosquelette Cas particulier : le neurone

5 I - La levure comme modèle d'étude
Mercredi 21 novembre 2007 I - La levure comme modèle d'étude Levure Saccharomyces cerevisiae #1p969

6 Asymétrie de Saccharomyces cerevisiae
Mercredi 21 novembre 2007 Asymétrie de Saccharomyces cerevisiae Grosse cellule mère + petite cellule fille après division Due à l'orientation des faisceaux d'actine Très peu de microtubules  polarité supportée par l'actine Deux types d'assemblage de filaments d'actine câbles Patchs associés au cortex (lieux d'endo- et exo-cytose) #1p969

7 Polarité de Saccharomyces cerevisiae
Mercredi 21 novembre 2007 Polarité de Saccharomyces cerevisiae De la cellule mère naît un bourgeon À l'extrémité du bourgeon nombreux câbles d'actine #1p969

8 Polarité des patchs et câbles d'actine au cours du cycle cellulaire
Actine-phalloïdine* : cables + patches Mercredi 21 novembre 2007 Cellule mère avant la formation du bourgeon Fig 16-81 #1p969 Apparition de nouveaux patches Aymétrie des patches et des cables Naissance du bourgeon Les patches sont principalement dans le bourgeon

9 Mercredi 21 novembre 2007 J. Cell Biol., Volume 142, Number 6, September 21, (Tatiana S. Karpova, James G. McNally, Samuel L. Moltz, and John A. Cooper) Actin in eukaryotic cells is found in different pools, with filaments being organized into a variety of supramolecular assemblies. To investigate the assembly and functional relationships between different parts of the actin cytoskeleton in one cell, we studied the morphology and dynamics of cables and patches in yeast. The fine structure of actin cables and the manner in which cables disassemble support a model in which cables are composed of a number of overlapping actin filaments. No evidence for intrinsic polarity of cables was found. To investigate to what extent different parts of the actin cytoskeleton depend on each other, we looked for relationships between cables and patches. Patches and cables were often associated, and their polarized distributions were highly correlated. Therefore, patches and cables do appear to depend on each other for assembly and function. Many cell types show rearrangements of the actin cytoskeleton, which can occur via assembly or movement of actin filaments. In our studies, dramatic changes in actin polarization did not include changes in filamentous actin. In addition, the concentration of actin patches was relatively constant as cells grew. Therefore, cells do not have bursts of activity in which new parts of the actin cytoskeleton are created. Fig16-81 p970

10 Mercredi 21 novembre 2007 Fig. 1.   Rotating images of 3-D reconstructions of the actin cytoskeleton. (A) A small-budded cell. (B) A budding zygote. Lines point to features of interest. 1, patches that overlap at certain angles. 2, an elongated patch that appears round at a certain angle of view. 3, a cable of uneven thickness along its length. 4, round patches at the ends of thin cables. 5, an elongated patch at the end of a thick cable. 6, a cable in the bud ending on a patch. 7, a cable end that is free but appears to overlap with a patch at certain angles of view. 8, a cable, both ends of which are not associated with a patch. These frames are sequential 2-D projections of rotating images of a vegetative cell and of a zygote with a step size of 24°. Angles of view are marked. The full data set in each case contains 180 frames with sequential projections at 2° angles. The movies are available at the authors' Web site, . Cells were stained with rhodamine-phalloidin. Deconvolution was with the EM method. Bar, 10 µm. J. Cell Biol., Volume 142, Number 6, September 21, (Tatiana S. Karpova, James G. McNally, Samuel L. Moltz, and John A. Cooper) Fig. 1.   Rotating images of 3-D reconstructions of the actin cytoskeleton. (A) A small-budded cell. (B) A budding zygote. Lines point to features of interest. 1, patches that overlap at certain angles. 2, an elongated patch that appears round at a certain angle of view. 3, a cable of uneven thickness along its length. 4, round patches at the ends of thin cables. 5, an elongated patch at the end of a thick cable. 6, a cable in the bud ending on a patch. 7, a cable end that is free but appears to overlap with a patch at certain angles of view. 8, a cable, both ends of which are not associated with a patch. These frames are sequential 2-D projections of rotating images of a vegetative cell and of a zygote with a step size of 24°. Angles of view are marked. The full data set in each case contains 180 frames with sequential projections at 2° angles. The movies are available at the authors' Web site, . Cells were stained with rhodamine-phalloidin. Deconvolution was with the EM method. Bar, 10 µm.

11 Mercredi 21 novembre 2007 J. Cell Biol., Volume 142, Number 6, September 21, (Tatiana S. Karpova, James G. McNally, Samuel L. Moltz, and John A. Cooper) Fig. 2.   Filamentous actin disassembly in cells treated with latrunculin. Time points as labeled on the figure. Cells were stained with rhodamine-phalloidin. Bar, 5 µm. Fig. 2.   Filamentous actin disassembly in cells treated with latrunculin. Time points as labeled on the figure. Cells were stained with rhodamine-phalloidin. Bar, 5 µm.

12 Mercredi 21 novembre 2007 Fig. 3.   Stereopairs of the actin cytoskeleton in sequential stages of the cell cycle (A-D). Arrows indicate cells with thin cables in a random meshwork. Cells were stained with rhodamine-phalloidin. Deconvolution was with the LLS method (A and D) or the EM method (B and C). Bar, 10 µm. J. Cell Biol., Volume 142, Number 6, September 21, (Tatiana S. Karpova, James G. McNally, Samuel L. Moltz, and John A. Cooper) Fig. 3.   Stereopairs of the actin cytoskeleton in sequential stages of the cell cycle (A-D). Arrows indicate cells with thin cables in a random meshwork. Cells were stained with rhodamine-phalloidin. Deconvolution was with the LLS method (A and D) or the EM method (B and C). Bar, 10 µm.

13 Mercredi 21 novembre 2007 Fig. 4.   Optical sectioning of a large-budded cell with a meshwork of thin cables. The distance (µm) of sequential planes along the z-axis is indicated. This cell is also shown as a stereopair in Fig. 3 D. The cell was stained with rhodamine-phalloidin. Deconvolution was with the PWLS method. Bar, 10 µm. J. Cell Biol., Volume 142, Number 6, September 21, (Tatiana S. Karpova, James G. McNally, Samuel L. Moltz, and John A. Cooper) Fig. 4.   Optical sectioning of a large-budded cell with a meshwork of thin cables. The distance (µm) of sequential planes along the z-axis is indicated. This cell is also shown as a stereopair in Fig. 3 D. The cell was stained with rhodamine-phalloidin. Deconvolution was with the PWLS method. Bar, 10 µm.

14 Mercredi 21 novembre 2007 Fig. 5.   Cables after heat shock. (A) Control cells, no heat shock applied. (B) Cells after 5 min of heat shock. (C) Cells after 30 min of heat shock. Single-focal plane images of cells stained with rhodamine-phalloidin are shown. Bar, 10 µm. J. Cell Biol., Volume 142, Number 6, September 21, (Tatiana S. Karpova, James G. McNally, Samuel L. Moltz, and John A. Cooper) Fig. 5.   Cables after heat shock. (A) Control cells, no heat shock applied. (B) Cells after 5 min of heat shock. (C) Cells after 30 min of heat shock. Single-focal plane images of cells stained with rhodamine-phalloidin are shown. Bar, 10 µm.

15 *cells with partially delocalized patches, in which cables are thinner and less well oriented than the cables in wild-type cells Mercredi 21 novembre 2007 (A) The actin cytoskeleton in cap2 mutant : single-focal plane images of cells at different stages Fig. 6.   The actin cytoskeleton in cap2 (A) and tpm1 (B and C) mutants. Single-focal plane images of cells at different stages of the cell cycle are presented in A and B. Asterisks mark the cells with partially delocalized patches, in which cables are thinner and less well oriented than the cables in wild-type cells. C shows a stereopair of a tpm1 cell. A rotating movie of the data set for C, with 180 frames at sequential projections of 2° angles, is available at the authors' Web site, . Cells were stained with rhodamine-phalloidin. Deconvolution was with the EM method (C). Bar, 10 µm J. Cell Biol., Volume 142, Number 6, September 21, (Tatiana S. Karpova, James G. McNally, Samuel L. Moltz, and John A. Cooper) (B) The actin cytoskeleton in tpm1 mutant : single-focal plane images of cells at different stages Fig. 6.   The actin cytoskeleton in cap2 (A) and tpm1 (B and C) mutants. Single-focal plane images of cells at different stages of the cell cycle are presented in A and B. Asterisks mark the cells with partially delocalized patches, in which cables are thinner and less well oriented than the cables in wild-type cells. C shows a stereopair of a tpm1 cell. A rotating movie of the data set for C, with 180 frames at sequential projections of 2° angles, is available at the authors' Web site, . Cells were stained with rhodamine-phalloidin. Deconvolution was with the EM method (C). Bar, 10 µm (C) The actin cytoskeleton in tpm1 mutant : stereopair of a tpm1 cell Cells were stained with rhodamine-phalloidin.

16 The actin cytoskeleton of the bee1/las17 mutant.
Mercredi 21 novembre 2007 Fig. 7.   The actin cytoskeleton of the bee1/las17 mutant. (A) Single-focal plane images of four cells at different stages of the cell cycle with a completely random cable meshwork. (B) A stereopair of another such cell. Movie 3 shows rotating images of this cell, with 180 frames at sequential projections of 2° angles. Movies are available at . (C) Single- focal plane images of two cells with partial orientation of cables. Cells were stained with rhodamine-phalloidin. Deconvolution was with the EM method. Bar, 10 µm. (A) Single-focal plane images of four cells at different stages of the cell cycle with a completely random cable meshwork. J. Cell Biol., Volume 142, Number 6, September 21, (Tatiana S. Karpova, James G. McNally, Samuel L. Moltz, and John A. Cooper) (B) A stereopair of another such cell. Movie 3 shows rotating images of this cell, with 180 frames at sequential projections of 2° angles. Fig. 7.   The actin cytoskeleton of the bee1/las17 mutant. (A) Single-focal plane images of four cells at different stages of the cell cycle with a completely random cable meshwork. (B) A stereopair of another such cell. Movie 3 shows rotating images of this cell, with 180 frames at sequential projections of 2° angles. Movies are available at . (C) Single- focal plane images of two cells with partial orientation of cables. Cells were stained with rhodamine-phalloidin. Deconvolution was with the EM method. Bar, 10 µm. C) Single- focal plane images of two cells with partial orientation of cables. Cells were stained with rhodamine-phalloidin. Bar, 10 µm.

17 Mercredi 21 novembre 2007 Fig. 8.   Diagram summarizing results testing for correlations between patch and cable rearrangement Diagram summarizing results testing for correlations between patch and cable rearrangement J. Cell Biol., Volume 142, Number 6, September 21, (Tatiana S. Karpova, James G. McNally, Samuel L. Moltz, and John A. Cooper) Fig. 8.   Diagram summarizing results testing for correlations between patch and cable rearrangement

18 Mercredi 21 novembre 2007 Fig. 9.   The random meshwork of thin cables in large-budded cells. A-C show three examples. D shows three small-budded polarized cells for comparison. Single-focal plane images of cells stained with rhodamine-phalloidin are shown. Bar, 5 µm. A-C show three examples J. Cell Biol., Volume 142, Number 6, September 21, (Tatiana S. Karpova, James G. McNally, Samuel L. Moltz, and John A. Cooper) D shows three small-budded polarized cells for comparison Fig. 9.   The random meshwork of thin cables in large-budded cells. A-C show three examples. D shows three small-budded polarized cells for comparison. Single-focal plane images of cells stained with rhodamine-phalloidin are shown. Bar, 5 µm. The random meshwork of thin cables in large-budded cells. Single-focal plane images of cells stained with rhodamine-phalloidin are shown. Bar, 5 µm.

19 (A) Cells with mating projections after 1 h of -factor treatment
(B) Cells with mating projections after 3 h of -factor treatment Mercredi 21 novembre 2007 Fig. 10.   Cables in zygote formation and development. (A) Cells with mating projections after 1 h of -factor treatment. (B) Cells with mating projections after 3 h of -factor treatment. (C- E) Sequential stages of zygote formation and development. Single-focal plane images of cells stained with rhodamine-phalloidin are shown. Bar, 10 µm. (C- E) Sequential stages of zygote formation and development J. Cell Biol., Volume 142, Number 6, September 21, (Tatiana S. Karpova, James G. McNally, Samuel L. Moltz, and John A. Cooper) Cables in zygote formation and development. Single-focal plane images of cells stained with rhodamine-phalloidin are shown. Bar, 10 µm. Fig. 10.   Cables in zygote formation and development. (A) Cells with mating projections after 1 h of -factor treatment. (B) Cells with mating projections after 3 h of -factor treatment. (C- E) Sequential stages of zygote formation and development. Single-focal plane images of cells stained with rhodamine-phalloidin are shown. Bar, 10 µm.

20 Levure Saccharomyces cerevisiae
Mercredi 21 novembre 2007 Levure Saccharomyces cerevisiae Forme haploïde Prolifère Si conditions difficiles  doivent s'accoupler pour devenir diploïde et sporuler Facteurs d'accouplement a et  (récepteurs correspondants) Forme diploïde Seule peut sporuler si conditions difficiles #1p969

21 Facteurs d'accouplement (a ou  )
Mercredi 21 novembre 2007 Facteurs d'accouplement (a ou  ) Se lient à la surface de la cellule (haploïde) Entraîne la polarisation de la cellule  Modification de la forme de la cellule en "shmoo"  Préparation à l'accouplement #1p969

22 Mercredi 21 novembre 2007 Saccharomyces cerevisiae devenant polarisé après traitement par le facteur d'accouplement a ou  Fig 16-82 #1p970

23 Voie de signalisation de la réponse de la levure au facteur d'accouplement
Mercredi 21 novembre 2007 GTPase Fig 16-83 #1p970

24 Transmission au réseau de microtubules
Polymérisation de l'actine  COMT (inclus dans la membrane nucléaire chez S. cerevisiae) se place sur le côté du noyau près de l'extrémité du shmoo  Alignement du noyau lui permettant de fusionner avec son partenaire

25 Mercredi 21 novembre 2007 Effets de Rac, Rho et Cdc42 sur l'organisation de l'actine dans le fibroblaste Fig16-50 #1p970

26 Mercredi 21 novembre 2007 Même mécanisme pour la croissance du bourgeon qui deviendra une cellule fille Construction d'un nouveau bourgeon juste à côté du bourgeon précédent Intervention de Cdc 42 pour la transmission du message au cytosquelette #1p970

27 Localisation des molécules dans la cellule
Mercredi 21 novembre 2007 Localisation des molécules dans la cellule ARN Protéines #2p971

28 Exemple de la protéine de régulation de gène Ash1
Mercredi 21 novembre 2007 Exemple de la protéine de régulation de gène Ash1 Le messager de Ash1 et sa protéine sont localisés exclusivement dans le bourgeon en croissance et ne se retrouveront donc que dans la cellule fille Nécessité d'une des deux myosines V pour cette distribution asymétrique de l'ARNm de Ash1 Nécessité d'au moins 6 autres produits de gène pour cette polarité (tropomyosine, profiline, actine, protéines intermédiaires entre ARN et myosine V,[She2 & She3]) #2p971

29 Mercredi 21 novembre 2007 Localisation d'ARNm à l'extrémité du bourgeonnement de la levure (répartition asymétrique de l'ARNm de la protéine ash1) ARNm de ash1 Fig 16-84 #2p971

30 Mercredi 21 novembre 2007 Autres applications du rôle du cytosquelette dans la polarité de la cellule Embryon de drosophile : axe antéro-postérieur ARN localisés à l'extrémité postérieure uniquement grâce au… … transport des ARN le long de microtubules #2p971

31 II – Locomotion des cellules (filipodes, lamellipodes et pseudopodes)

32 Presque toutes les cellules sont capables de migrer sur un support
Mercredi 21 novembre 2007 Presque toutes les cellules sont capables de migrer sur un support Amibe Exception : le spermatozoïde Migrations chez l'embryon Cellules de la crête neurale  cellules pigmentaires de la peau, cellules ganglionnaires, Macrophages, neutrophiles, ostéoclastes, fibroblastes, entérocytes de la villosité intestinale, cellules cancéreuses, … #3p972

33 Mécanismes de la locomotion de la cellule
Mercredi 21 novembre 2007 Mécanismes de la locomotion de la cellule A – Expansion = protrusion de structures riches en actine B - Attachement C - Traction #3p972

34 Fig 16-85 Forces déployées au cours du déplacement de la cellule
Mercredi 21 novembre 2007 Forces déployées au cours du déplacement de la cellule Parfois impression de mouvement continu Parfois impression de mouvement irrégulier Fig 16-85 #3p972

35 Forces déployées au cours du déplacement de la cellule
Mercredi 21 novembre 2007 Forces déployées au cours du déplacement de la cellule Protrusion du bord avant par polymérisation de l'actine Fixation du lamellipode au support  force d'étirement sur le cortex d'actine  contraction à l'arrière pour soulager la tension Création de nouveaux contacts focaux à l'avant Désassemblage de contacts focaux à l'arrière  Déplacement de la cellule sur le support #3p972

36 A - Rôle de l'actine dans la création d'expansions membranaires
Mercredi 21 novembre 2007 A - Rôle de l'actine dans la création d'expansions membranaires Filopodes (= microspikes = spicules) Lamellipodes (les plus étudiés) Pseudopodes #4p973

37 Les différentes formes de "bord avant"
Mercredi 21 novembre 2007 Les différentes formes de "bord avant" Filipodes, lamellipodes, pseudopodes La structure est fonction de la cellule Rempli de faisceau d'actine  pas d'organites 1, 2 ou 3 –D (cf supra) #4p973

38 Filopodes Cônes de croissance des neurones Fibroblastes
Mercredi 21 novembre 2007 Filopodes Cônes de croissance des neurones Fibroblastes Ressemblent à des microvillosités mais plus longs, plus fins et plus dynamiques 1-D #4p973

39 Mercredi 21 novembre 2007 Lamellipodes Cellules épithéliales de l'épiderme de poisson ou grenouille = keratocyte Fibroblastes Certains neurones Damier orthogonal de filaments d'actine parallèle au support Les plus faciles à étudier 2-D #4p973

40 Pseudopodes Amibes et neutrophiles Gel de filaments d'actine 3-D
Mercredi 21 novembre 2007 Pseudopodes Amibes et neutrophiles Gel de filaments d'actine 3-D #4p973

41 Mercredi 21 novembre 2007 Les "kératocytes" Cellules de l'épiderme des poissons et de la grenouille Riches en filament de kératine Peuvent se déplacer très rapidement pour réparer une blessure (jusqu'à 30 µ/minute) Aspect particulier en culture Grand lamellipode Petit corps cellulaire contenant le noyau et non adhérent au support #4p973

42 Fig 16-86 Kératocytes en migration (épiderme de poisson) #4p973
Mercredi 21 novembre 2007 Culture toutes les 15 sec (15 m/sec) Fig 16-86 Petit corps cellulaire contenant le noyau et non adhérent au support #4p973 Actine Microtubules Filaments intermédiaires MEB

43 Comportement de fragment de lamellipode séparé par microchirurgie
A Continue à migrer avec la même organisation Actine  réseau dans le bord avant Myosine II  bande à l'arrière B S'arrête de migrer et devient rond Actine  répartie en périphérie Myosine II  spots dans tout le cytoplasme Mercredi 21 novembre 2007 Fig 16-87 #4p973

44 Mercredi 21 novembre 2007 Verkovski,AB1999p11 : Cytoskeletal organization of (a–d) a keratocyte fragment undergoing locomotion and (e–g) a stationary keratocyte fragment. The distribution of myosin II (red), as revealed by antibody staining, and that of actin (cyan), as revealed by staining with tetramethylrhodamine–phalloidin, is shown (a,e). (b–d,f,g) Electron microscopy of platinum replicas. (b) An overview and (c,d) regions of the moving fragment reveal a crisscross actin network at the leading edge (c) and a bundle at the trailing edge (d). A cluster of myosin II minifilaments revealed after the extraction of actin from the cytoskeleton of a similar fragment is shown in the inset in (d) at an approximate position that it would occupy in the intact cytoskeleton. (f) An overview and (g) a region of a stationary fragment reveals a narrow actin brush (indicated with brackets) at the edge of the fragment. (h) Triple staining for actin (cyan), myosin (red) and tubulin (yellow) in several fragments reveals the presence of few or no microtubules in the fragments. Bars represent 2 mm in (a,b,h) and 200 nm in (c). Verkovski,AB1999p11 Verkovski,AB1999p11 : Cytoskeletal organization of (a–d) a keratocyte fragment undergoing locomotion and (e–g) a stationary keratocyte fragment. The distribution of myosin II (red), as revealed by antibody staining, and that of actin (cyan), as revealed by staining with tetramethylrhodamine–phalloidin, is shown (a,e). (b–d,f,g) Electron microscopy of platinum replicas. (b) An overview and (c,d) regions of the moving fragment reveal a crisscross actin network at the leading edge (c) and a bundle at the trailing edge (d). A cluster of myosin II minifilaments revealed after the extraction of actin from the cytoskeleton of a similar fragment is shown in the inset in (d) at an approximate position that it would occupy in the intact cytoskeleton. (f) An overview and (g) a region of a stationary fragment reveals a narrow actin brush (indicated with brackets) at the edge of the fragment. (h) Triple staining for actin (cyan), myosin (red) and tubulin (yellow) in several fragments reveals the presence of few or no microtubules in the fragments. Bars represent 2 mm in (a,b,h) and 200 nm in (c).

45 ARP Actine + ARP Actine Nucléation des filaments d'actine et formation du réseau par le complexe ARP dans un lamellipode de kératocyte Actine (rouge) + ARP (vert)  jaune sur le bord avant tapis roulant s'assemblant en avant et se désassemblant en arrière (C) FG de (B) embranchement à 70° du complexe ARP Mercredi 21 novembre 2007 Fig 16-88

46 Comportement du réseau d'actine
Mercredi 21 novembre 2007 Comportement du réseau d'actine Étude dans le lamellipode du kératocyte Les filaments d'actine restent fixes par rapport au support au fur et à mesure que le kératocyte se déplace Extrémité Extrémité + : distale vers l'avant Extrémité – : fixée au complexe ARP Impression de tapis roulant de l'ensemble du bord avant #4p973-4

47 Rôle de la cofiline (Actine Depolymeristion Factor)
Mercredi 21 novembre 2007 Rôle de la cofiline (Actine Depolymeristion Factor) Le nucléation a lieu au bord avant et pousse la membrane plasmique La dépolymérisation a lieu en arrière Cofiline se lie préférentiellement à la forme D (ADP) de l'actine  le bord avant (riche en T-actine) résiste à la dépolymérisation par cofiline Le filament vieillit  hydrolyse de l'ATP  désassemblage du filament par la cofiline #4p974

48 Actine + cofiline = Actine-Cofiline(en jaune)
Cofiline (=actine depolymerizing factor) dans les lamellipodes de kératocyte Mercredi 21 novembre 2007 Actine Cofiline Fig 16-89 #4p974 Actine + cofiline = Actine-Cofiline(en jaune)

49 Fig 16-90 Protrusion du réseau d'actine au bord avant #4p974
Nucléation médiée par les complexes ARP du bord avant Mercredi 21 novembre 2007 Fig 16-90 #4p974 Protrusion du réseau d'actine au bord avant

50 Kératocyte Mercredi 21 novembre 2007 Fibroblaste Svitkina,TM1999p1009 Fig. 1.   Multiple branching of actin filaments in lamellipodia. EM of lamellipodia of Xenopus keratocytes (a-g) and fibroblasts (h-o) showing overviews of the leading edge (a and h) and enlargements of the boxed regions (b-g and i-o). Many examples of filaments with tightly spaced multiple branches (cyan) can be visualized in lamellipodia despite the high overall density of the actin network. Bar, 0.5 µm. Fig. 1.   Multiple branching of actin filaments in lamellipodia. EM of lamellipodia of Xenopus keratocytes (a-g) and fibroblasts (h-o) showing overviews of the leading edge (a and h) and enlargements of the boxed regions (b-g and i-o). Many examples of filaments with tightly spaced multiple branches (cyan) can be visualized in lamellipodia despite the high overall density of the actin network. Bar, 0.5 µm.

51 Mercredi 21 novembre 2007 Embranchements de filaments d'actine Svitkina,TM1999p1009 Fig. 2.   Improved visualization of actin filament branching in lamellipodia. EM of keratocyte or fibroblast lamellipodial actin network after CD treatment (a and b, 0.2 µM for 30 min or 0.5 µM for 10 min), 1 min recovery from serum starvation of a mouse fibroblast (c), LA treatment (0.2 µM for 10 min), or unprotected extraction (d). All examples demonstrate frequent branching of actin filaments. Bars, 0.1 µm; b and d are shown at the same magnification as c Fig. 2.   Improved visualization of actin filament branching in lamellipodia. EM of keratocyte or fibroblast lamellipodial actin network after CD treatment (a and b, 0.2 µM for 30 min or 0.5 µM for 10 min), 1 min recovery from serum starvation of a mouse fibroblast (c), LA treatment (0.2 µM for 10 min), or unprotected extraction (d). All examples demonstrate frequent branching of actin filaments. Bars, 0.1 µm; b and d are shown at the same magnification as c

52 Mercredi 21 novembre 2007 Svitkina,TM1999p1009 Keratocyte Xenopus Lamellipode riche en ARP Fig. 3.   Localization of Arp2/3 complex in lamellipodia. (a-c and e-g) Fluorescence microscopy of Xenopus keratocyte (a-c) or fibroblast (e-g). Staining with p21 antibody (green) and TRITC-phalloidin (red) shows ARP2/3 complex highly enriched in lamellipodia. Boxed region in g is enlarged in insets; it shows several filopodia lacking and only one filopodium containing Arp2/3 complex. (d and h) Immuno-EM of lamellipodia of Xenopus keratocyte (d) or fibroblast (h) stained with p21 primary antibody and 10-nm gold-conjugated secondary antibody after glutaraldehyde fixation and SDS treatment of detergent-extracted cells. Gold particles are highlighted in yellow. Bars: (a and e) 10 µm; (d and h) 0.1 µm Fig. 3.   Localization of Arp2/3 complex in lamellipodia. (a-c and e-g) Fluorescence microscopy of Xenopus keratocyte (a-c) or fibroblast (e-g). Staining with p21 antibody (green) and TRITC-phalloidin (red) shows ARP2/3 complex highly enriched in lamellipodia. Boxed region in g is enlarged in insets; it shows several filopodia lacking and only one filopodium containing Arp2/3 complex. (d and h) Immuno-EM of lamellipodia of Xenopus keratocyte (d) or fibroblast (h) stained with p21 primary antibody and 10-nm gold-conjugated secondary antibody after glutaraldehyde fixation and SDS treatment of detergent-extracted cells. Gold particles are highlighted in yellow. Bars: (a and e) 10 µm; (d and h) 0.1 µm Lamellipode riche en ARP fibroblaste

53 Mercredi 21 novembre 2007 Svitkina,TM1999p1009 Fig. 4.   Localization of Arp2/3 complex at actin filament branching points. Xenopus keratocytes and fibroblasts were treated with CD (0.2 µM for 30 min or 0.5 µM for 10 min), extracted in the presence of phalloidin, fixed with glutaraldehyde, treated with 33% methanol, and immunostained with p21 antibody followed by 10-nm gold-conjugated secondary antibody. Gold particles are highlighted in yellow. Bar, 50 nm Fig. 4.   Localization of Arp2/3 complex at actin filament branching points. Xenopus keratocytes and fibroblasts were treated with CD (0.2 µM for 30 min or 0.5 µM for 10 min), extracted in the presence of phalloidin, fixed with glutaraldehyde, treated with 33% methanol, and immunostained with p21 antibody followed by 10-nm gold-conjugated secondary antibody. Gold particles are highlighted in yellow. Bar, 50 nm

54 Mercredi 21 novembre 2007 Svitkina,TM1999p1009 Fig. 5.   Localization of cross-linking proteins in fibroblast cytoskeleton. (a-c) Fluorescence microscopy and corresponding intensity profiles (a'-c') of Xenopus (a and c) or human 356 (b) fibroblast lamellipodia double stained with TRITC-phalloidin (red) and either p21 (a and a'), ABP-280 (b and b'), or -actinin (c and c') antibodies (green). The protein/actin ratio at the leading edge of the lamellipodium is high for Arp2/3 complex (a and a'), medium for ABP-280 (b and b'), and low for -actinin (c and c') compared with internal actin structures. (d-i) Immuno-EM of the cell edge (d-f) or interior (g-i) of CD-treated Xenopus (d, f, g, and i) or human 356 (e and h) fibroblasts stained with p21 (d and g), ABP-280 (e and h), or -actinin (f and i) primary antibody and 10-nm (d, e, g, and h) or 18-nm (f and i) gold-conjugated secondary antibody. Gold particles (yellow) reveal Arp2/3 complex at Y-junctions at cell edge and ABP-280 and -actinin at filament crossovers in the cell interior. Bars: (a-c) 1 µm; (d-i) 0.1 µm Fig. 5.   Localization of cross-linking proteins in fibroblast cytoskeleton. (a-c) Fluorescence microscopy and corresponding intensity profiles (a'-c') of Xenopus (a and c) or human 356 (b) fibroblast lamellipodia double stained with TRITC-phalloidin (red) and either p21 (a and a'), ABP-280 (b and b'), or -actinin (c and c') antibodies (green). The protein/actin ratio at the leading edge of the lamellipodium is high for Arp2/3 complex (a and a'), medium for ABP-280 (b and b'), and low for -actinin (c and c') compared with internal actin structures. (d-i) Immuno-EM of the cell edge (d-f) or interior (g-i) of CD-treated Xenopus (d, f, g, and i) or human 356 (e and h) fibroblasts stained with p21 (d and g), ABP-280 (e and h), or -actinin (f and i) primary antibody and 10-nm (d, e, g, and h) or 18-nm (f and i) gold-conjugated secondary antibody. Gold particles (yellow) reveal Arp2/3 complex at Y-junctions at cell edge and ABP-280 and -actinin at filament crossovers in the cell interior. Bars: (a-c) 1 µm; (d-i) 0.1 µm

55 Mercredi 21 novembre 2007 Svitkina,TM1999p1009 Fig. 6.   Structural differentiation of actin network in lamellipodium. EM of Xenopus keratocytes (a-c) or Xenopus fibroblasts (d-f) after regular extraction in the presence of PEG and phalloidin (a and d) or after unprotected extraction without PEG and phalloidin (b, c, e, and f). Boxed areas from b and e are enlarged in c and f, respectively. Actin network at lamellipodial rear disassembled in the course of unprotected extraction, whereas front zone remained as dense as in control cells. Bar, 1 µm Fig. 6.   Structural differentiation of actin network in lamellipodium. EM of Xenopus keratocytes (a-c) or Xenopus fibroblasts (d-f) after regular extraction in the presence of PEG and phalloidin (a and d) or after unprotected extraction without PEG and phalloidin (b, c, e, and f). Boxed areas from b and e are enlarged in c and f, respectively. Actin network at lamellipodial rear disassembled in the course of unprotected extraction, whereas front zone remained as dense as in control cells. Bar, 1 µm

56 Mercredi 21 novembre 2007 Svitkina,TM1999p1009 Fig. 7.   Differential response of lamellipodial actin network to LA. (A) Phase-contrast sequence of a locomoting Xenopus keratocyte. After addition of 0.1 µm LA at 9 min time point, the cell continued to translocate, retaining the crescent-like shape. (B) Plot showing rate of front edge protrusion versus time of the cell shown in A. LA addition (arrow) decreased rate of protrusion from ~4 µm/min before LA application to 1 µm/min at the end of the sequence. (C) Fluorescence microscopy of the boxed region of the cell shown in A, which was lysed at 34 min time point, fixed, and stained with TRITC-phalloidin. Actin-staining reveals narrow bright lamellipodium at the leading edge, separated by the wide actin-depleted zone from the internal actin structures. (D and E) EM of a Xenopus keratocyte (D) or Xenopus fibroblast (E) lamellipodium treated with LA (D, 0.1 µM for 30 min; E, 0.25 µM for 10 min) reveals actin depletion from the lamellipodial rear. Bars: (A) 10 µm; (D and E) 1 µm Fig. 7.   Differential response of lamellipodial actin network to LA. (A) Phase-contrast sequence of a locomoting Xenopus keratocyte. After addition of 0.1 µm LA at 9 min time point, the cell continued to translocate, retaining the crescent-like shape. (B) Plot showing rate of front edge protrusion versus time of the cell shown in A. LA addition (arrow) decreased rate of protrusion from ~4 µm/min before LA application to 1 µm/min at the end of the sequence. (C) Fluorescence microscopy of the boxed region of the cell shown in A, which was lysed at 34 min time point, fixed, and stained with TRITC-phalloidin. Actin-staining reveals narrow bright lamellipodium at the leading edge, separated by the wide actin-depleted zone from the internal actin structures. (D and E) EM of a Xenopus keratocyte (D) or Xenopus fibroblast (E) lamellipodium treated with LA (D, 0.1 µM for 30 min; E, 0.25 µM for 10 min) reveals actin depletion from the lamellipodial rear. Bars: (A) 10 µm; (D and E) 1 µm

57 Mercredi 21 novembre 2007 Svitkina,TM1999p1009 Fig. 8.   Localization of XAC in Xenopus keratocytes. (a-e) Fluorescence microscopy of a whole cell (a-c) and the intensity profile (d) of the enlarged lamellipodium (e) from the boxed region in c, double stained with XAC antibody (green) and TRITC-phalloidin (red). XAC in lamellipodium is excluded from the narrow zone at the extreme leading edge. (f) Phase-contrast sequence of a locomoting cell; time is shown in min. (g-i) Immuno-EM with XAC antibody of the cell shown in f, which was lysed and processed at 8 min time point. (g) Cell overview. (h) Intermediate magnification of the boxed region from g showing distribution of gold particles (yellow) in lamellipodia. (i) High magnification of the boxed region from d showing leading edge. XAC is excluded from the extreme front of the lamellipodium (h and i). Bars: (a and f) 10 µm; (h) 10 µm Fig. 8.   Localization of Xenopus ADF/Cofilin (XAC) in Xenopus keratocytes. (a-e) Fluorescence microscopy of a whole cell (a-c) and the intensity profile (d) of the enlarged lamellipodium (e) from the boxed region in c, double stained with XAC antibody (green) and TRITC-phalloidin (red). XAC in lamellipodium is excluded from the narrow zone at the extreme leading edge. (f) Phase-contrast sequence of a locomoting cell; time is shown in min. (g-i) Immuno-EM with XAC antibody of the cell shown in f, which was lysed and processed at 8 min time point. (g) Cell overview. (h) Intermediate magnification of the boxed region from g showing distribution of gold particles (yellow) in lamellipodia. (i) High magnification of the boxed region from d showing leading edge. XAC is excluded from the extreme front of the lamellipodium (h and i). Bars: (a and f) 10 µm; (h) 10 µm

58 Mercredi 21 novembre 2007 Fig. 9.   Localization of XAC to posterior regions of depolymerization-resistant actin brush. Electron (a and c) and fluorescence (b) microscopy of lamellipodia of Xenopus keratocytes after unprotected extraction (a) or LA treatment (b, 0.15 µM for 30 min; c, 0.25 µM for 10 min) and subsequent staining with XAC antibody (a and c) or double staining with TRITC-phalloidin and XAC antibody (b). Svitkina,TM1999p1009 Fig. 9.   Localization of XAC to posterior regions of depolymerization-resistant actin brush. Electron (a and c) and fluorescence (b) microscopy of lamellipodia of Xenopus keratocytes after unprotected extraction (a) or LA treatment (b, 0.15 µM for 30 min; c, 0.25 µM for 10 min) and subsequent staining with XAC antibody (a and c) or double staining with TRITC-phalloidin and XAC antibody (b).

59 Mercredi 21 novembre 2007 Svitkina,TM1999p1009 Fig. 10.   Localization of XAC in Xenopus fibroblasts. (a-e) Fluorescence microscopy of a cell fragment (a-c) and the intensity profile (d) of the enlarged lamellipodium (e) from the boxed region in c, double stained with XAC antibody (green) and TRITC-phalloidin (red). XAC is distributed throughout the entire lamellipodium. (f and g) Phase-contrast sequence showing protruding lamellipodium of a motile cell (f) and the overview of the same cell (g) at 80 s time point. (h and i) Immuno-EM with XAC antibody of the cell shown in g. (h) Cell overview. Nucleus and surrounding regions look bright because of high content of cellular proteins, which creates high electron density (brightness in inverted contrast). Immunogold-labeling in these central regions is very low. (i) High magnification of the protruding region pointed to by arrow in h. Gold particles (yellow) revealing XAC localization are found at the extreme leading edge, as well as throughout the lamellipodia Fig. 10.   Localization of XAC in Xenopus fibroblasts. (a-e) Fluorescence microscopy of a cell fragment (a-c) and the intensity profile (d) of the enlarged lamellipodium (e) from the boxed region in c, double stained with XAC antibody (green) and TRITC-phalloidin (red). XAC is distributed throughout the entire lamellipodium. (f and g) Phase-contrast sequence showing protruding lamellipodium of a motile cell (f) and the overview of the same cell (g) at 80 s time point. (h and i) Immuno-EM with XAC antibody of the cell shown in g. (h) Cell overview. Nucleus and surrounding regions look bright because of high content of cellular proteins, which creates high electron density (brightness in inverted contrast). Immunogold-labeling in these central regions is very low. (i) High magnification of the protruding region pointed to by arrow in h. Gold particles (yellow) revealing XAC localization are found at the extreme leading edge, as well as throughout the lamellipodia

60 Mercredi 21 novembre 2007 Svitkina,TM1999p1009 Fig. 11.   Two treadmilling models for actin turnover in lamellipodia. Left, treadmilling of individual filaments suggests that each actin filament in the actin network simultaneously assembles subunits at its barbed end and releases subunits from the pointed end, thus continuously reproducing itself by treadmilling mechanism. Treadmilling of individual filaments collectively results in the treadmilling of the lamellipodial network. Right, treadmilling of the dendritic array suggests frequent formation of new filaments by de novo nucleation, which occurs within the narrow zone at the leading edge, the actin brush. Newborn filaments become immediately incorporated into the actin array as branches of pre-existing filaments. Within the actin brush, filaments are protected from depolymerization at pointed ends. Nucleation, cross-linking, and pointed end capping are proposed to be mediated by the Arp2/3 complex. Many barbed ends are predicted to be capped to prevent exponential increase in filament mass. Release of the Arp2/3 complex from Y-junctions behind the actin brush, followed by ADF/cofilin-mediated dissociation of actin subunits from pointed ends, may be a major pathway for actin array disassembly. By this model, an individual filament in the dendritic array does not treadmill, but rather first grows at the barbed end and later shrinks at the pointed end. However, the actin filament array as a whole treadmills, reproducing itself at the cell front and dismantling itself at the lamellipodial rear. Growing barbed ends are shaded in gray Fig. 11.   Two treadmilling models for actin turnover in lamellipodia. Left, treadmilling of individual filaments suggests that each actin filament in the actin network simultaneously assembles subunits at its barbed end and releases subunits from the pointed end, thus continuously reproducing itself by treadmilling mechanism. Treadmilling of individual filaments collectively results in the treadmilling of the lamellipodial network. Right, treadmilling of the dendritic array suggests frequent formation of new filaments by de novo nucleation, which occurs within the narrow zone at the leading edge, the actin brush. Newborn filaments become immediately incorporated into the actin array as branches of pre-existing filaments. Within the actin brush, filaments are protected from depolymerization at pointed ends. Nucleation, cross-linking, and pointed end capping are proposed to be mediated by the Arp2/3 complex. Many barbed ends are predicted to be capped to prevent exponential increase in filament mass. Release of the Arp2/3 complex from Y-junctions behind the actin brush, followed by ADF/cofilin-mediated dissociation of actin subunits from pointed ends, may be a major pathway for actin array disassembly. By this model, an individual filament in the dendritic array does not treadmill, but rather first grows at the barbed end and later shrinks at the pointed end. However, the actin filament array as a whole treadmills, reproducing itself at the cell front and dismantling itself at the lamellipodial rear. Growing barbed ends are shaded in gray

61 Modification de l'orientation des filaments d'actine
Mercredi 21 novembre 2007 Modification de l'orientation des filaments d'actine Intervention de filaments bipolaires de myosine II Les filaments d'actine deviennent parallèles au bord avant Évite la formation d'une protrusion #4p974

62 Les cellules se déplacent par B - adhésion et C - traction
Mercredi 21 novembre 2007 Les cellules se déplacent par B - adhésion et C - traction Rôle très important de l’environnement sur la locomotion donc de l’adhésion Adhésion est inversement proportionnel à migration kératocyte : faiblement adhérent lamellipode facile à créer neurone de Aplysia : très adhérent  gros lamellipode ne permettant pas la migration mais même cycle pour l'actine  l’actine se retrouve en arrière de la cellule #5p975

63 Mercredi 21 novembre 2007 Mouvement rétrograde du réseau d'actine dans un lamellipode (neurones de la limace Aplysia) (A) Culture sur un support collant Organites et MT localisé à l’arrière Réseau de filaments d’actine dans le lamellipode (B) Traitement par la cytochalasine (capping des +) le réseau de filaments d’actine dans le lamellipode se retire (C) id (B)actine – phalloïdine Fig 16-91 #5p975

64 Répartition de l’actine
Mercredi 21 novembre 2007 Répartition de l’actine Si support très collant  actine concentrée en arrière de la cellule (eg neurone de Aplysia) Si support peu adhérent  actine dans les lamellipodes (eg kératocyte) En général entre ces deux extrêmes  la répartition de l’actine dépend de l’environnement #5p975

65 Sites d’attachement Se forment en avant du lamellipode
Mercredi 21 novembre 2007 Sites d’attachement Se forment en avant du lamellipode Identiques aux contacts focaux Si absence de fixation du lamellipode, bascule sur le bord supérieur de la cellule et forme des ondulations #5p975

66 Mercredi 21 novembre 2007 Mouvement de « vagues » vers l’arrière en cas d’échec d’attachement du lamellipode Fig 16-92 #5p975 Lamellipodes et ondulations au bord avant d’un fibroblaste

67 Rôle des contacts focaux
Mercredi 21 novembre 2007 Rôle des contacts focaux Sites d’ancrage sur le support pour tirer le corps cellulaire en avant Rôle de la myosine II comme moteur Moteurs … fonctionnement mal connu ... #5p976

68 Mercredi 21 novembre 2007 Localisation de la myosine I et II dans l'amibe Dictyostelium Myosine I localisée dans le bord avant du pseudopode Myosine II localisée dans le cortex postérieur riche en actine Myosine I Fig 16-93 #5p976 Myosine II 5m

69 Rôle des myosines Myosine I  pour projeter le bord avant
Mercredi 21 novembre 2007 Rôle des myosines Myosine I  pour projeter le bord avant Myosine II  pour faire avancer le corps cellulaire Amibe Dictyostelium déficiente en myosine II Peut pousser des pseudopodes Mais ramène lentement le reste du corps cellulaire… comment ?? affaiblissement des contacts focaux à la suite de l'étirement ? #5p976-7

70 III - Action du cytosquelette sur l'environnement
Mercredi 21 novembre 2007 III - Action du cytosquelette sur l'environnement #6p977

71 Forces de traction exercées par les cellules sur le support : plissement du support de silicone
Mercredi 21 novembre 2007 Fig 16-94 #5p977

72 Mercredi 21 novembre 2007 Explants de deux cœurs de poulet sur un gel de collagène au bout de 4 jours modification de la matrice Fig 16-95 #5p977

73 IV - Action de l'environnement sur le cytosquelette
Polarisation = préalable indispensable à la locomotion de la cellule  Polarisation  organisation pluri cellulaire Réponse à des modifications de l'environnement physique ou chimique  chimiotactisme

74 Mercredi 21 novembre 2007 Chimiotactisme "Mouvement d'une cellule dans une direction donnée contrôlée par un gradient d'une substance chimique diffusible" eg : mouvement du neutrophile vers une source bactérienne Des récepteurs à la surface des neutrophiles peuvent détecter (différence de 1% de chaque côté de la cellule) de très faibles concentrations de peptides N- formylés (N-formyl-méthionine) #6p977

75 Fig 16-96 #6p978 Polarisation et chimiotactisme du neutrophile
Mercredi 21 novembre 2007 Libération par la pipette de formyl-Met-Leu-Phe  apparition d'un lamellipode vers la source du chémoattractant Agrandissement du lamellipode et polarisation du cytosquelette. Localisation de la myosine II à l’arrière de la cellule Déplacement de la cellule vers le peptide Fig 16-96 #6p978 Polarisation et chimiotactisme du neutrophile

76 Interprétation moléculaire
Mercredi 21 novembre 2007 Interprétation moléculaire Fixation du ligand à un récepteur Diffusible (chimiotactisme) Non diffusible (molécules fixées sur la matrice) Activation d’une GTPase de la famille Rho Polymérisation d’actine Création d’une expansion Migration de la cellule dans cette direction #6p978

77 Rôle des microtubules #6p978
Mercredi 21 novembre 2007 La polymérisation d’actine dans le bord avant  Activation de moteurs qui tirent sur le centrosome via les microtubules  Position du centrosome Qui nuclée de nouveaux microtubules dont les extrémités + vont dans le bord avant  Modification de l’adhésion locale  Activation de Rac GTPase  Augmentation de la polymérisation d’actine dans le bord avant  Allongement de ce bord avant  Feed back positif  Renforcement de la polarité de la cellule  Actine agit sur les MT qui agissent en retour sur l’actine #6p978

78 Mercredi 21 novembre 2007 Autres exemples Mort d’une cellule cible par les lymphocytes T cytotoxiques Reconnaissance de l’antigène par des récepteurs de la surface des lymphocytes T Activation des GTPases de la famille Rho  Polarisation de l’actine corticale sous la zone de contact  Réorientation du centrosome vers la zone de contact  Repositionnement du Golgi sous la zone de contact #6p978

79 Mercredi 21 novembre 2007 Modifications du cytosquelette d'un lymphocyte T cytotoxique à la suite d'un contact avec une cible (modification du cytosquelette des deux cellules) Fig 16-97(A) #6p978

80 Fig 16-97(B) Cellule T et cible colorées par un AC anti-microtubule
Dans la cellule T irradiation des microtubules dirigés vers le point de contact entre les deux cellules Dans la cellule cible le réseau de microtubules n’est pas polarisé Mercredi 21 novembre 2007 Cellule T Fig 16-97(B) #6p978 Cellule cible 10 m

81 V – Cas particulier : le neurone
Mercredi 21 novembre 2007 V – Cas particulier : le neurone Les grosses différences entre pro et eucaryote sont des conséquences du cytosquelette Pro : transport par diffusion Eu : transport par moteurs  cellules plus grosses et plus complexes Le plus gros (long) et le plus complexe : le neurone #7p979

82 Le neurone Cellule embryonnaire  Migration +++
Mercredi 21 novembre 2007 Le neurone Cellule embryonnaire  Migration +++ Apparition de longs prolongements (= neurites) Axones Dendrites Remplis de microtubules Dia déplacée#7p979

83 Axone Tous les microtubules sont orientés selon la même direction
Mercredi 21 novembre 2007 Axone Tous les microtubules sont orientés selon la même direction Extrémité - vers le corps cellulaire Extrémité + vers l’extrémité de l’axone Un microtubule  quelques microns : un microtubule ne traverse pas tout l’axone d’un bout à l’autre : il y a chevauchement  Autoroute pour le transport des protéines et des vésicules ( vésicules synaptiques) #7p979

84 Dendrites Beaucoup plus courts que l'axone Reçoivent les signaux
Les microtubules sont parallèles mais de polarité mixte Se forment comme les axones

85 Vésicules synaptiques
Mercredi 21 novembre 2007 Vésicules synaptiques Utilité à l’extrémité des axones Construites dans le corps cellulaire et les dendrites où sont les ribosomes Très grande longueur (jusqu’à 1 mètre) #7p979

86 Transport axonal antérograde
Mercredi 21 novembre 2007 Transport axonal antérograde Concerne les mitochondries, les protéines, les vésicules synaptiques Se fait le long des microtubules Par des moteurs moléculaires de la famille des kinésines Jusqu'à un mètre par jour Sinon 8 ans pour une mitochondrie ! Nombreux types de moteur  spécificité du transport #7p979

87 Transport axonal rétrograde
Mercredi 21 novembre 2007 Transport axonal rétrograde Concerne le retour des vieux organites Se fait le long des mêmes microtubules Par des moteurs moléculaires de la famille des dynéines cytoplasmiques (moteur qui se déplace dans le sens moins) #7p979

88 Organisation des microtubules dans les fibroblastes et les neurones
Mercredi 21 novembre 2007 Les MT ont une polarité mixte Tous les MT ont la même polarité Fig 16-98 #7p979

89 L'organisation des neurones dépend du cytosquelette
Mercredi 21 novembre 2007 L'organisation des neurones dépend du cytosquelette Actine, filaments intermédiaires, microtubules Actine Localisée sous le cortex de l'axone Moteurs de myosine V Filaments intermédiaires = neurofilaments Rôle de structure Attachés aux microtubules et aux filaments d'actine par des protéines #7p979-80

90 Développement du neurone (= neurogenèse) (1/4)
Mercredi 21 novembre 2007 Développement du neurone (= neurogenèse) (1/4) Repose sur la mobilité liée à l'actine Apparition d'un cône de croissance : structure mobile riche en actine #7p980

91 Fig 16-99 Croissance du cône de croissance neuronal #7p980 Neurite
Mercredi 21 novembre 2007 Deux cônes à l'extrémité d'un neurite Fig 16-99 Nombreux filopodes et un gros lamellipode #7p980 Neurite Aspect raide du neurite dû à la traction de l'extrémité Cône de croissance d'un neurone sensoriel à la surface interne de l'épiderme

92 Développement du neurone (= neurogenèse) (2/4)
Mercredi 21 novembre 2007 Développement du neurone (= neurogenèse) (2/4) Les cônes de croissance produisent des filopodes voire des lamellipodes Ces filopodes sont stabilisés exclusivement par l'environnement Nétrine protéine soluble qui attire ou repousse les cônes de croissance Marqueurs de surface sur la matrice extra cellulaire ou les cellules  #7p980

93 Développement du neurone (= neurogenèse) (3/4)
Mercredi 21 novembre 2007 Développement du neurone (= neurogenèse) (3/4) Formation rapide de contacts focaux  Collapsus du réseau d'actine des cônes non stabilisés Combinaison complexe de signaux positifs et négatifs, solubles et insolubles #7p980

94 Développement du neurone (= neurogenèse) (4/4)
Mercredi 21 novembre 2007 Développement du neurone (= neurogenèse) (4/4) Les microtubules suivent avec leur instabilité dynamique Stabilisé par les signaux d'adhésion (contacts focaux) #7p980

95 Mercredi 21 novembre 2007 Fig 21-97 #7p981

96 Fig 16-100 Complexité de l'architecture d'un neurone (rétine de singe)
Mercredi 21 novembre 2007 Complexité de l'architecture d'un neurone (rétine de singe) Fig #7p981

97 Mercredi 21 novembre 2007 Conclusion Le cytosquelette est à la base de la construction du neurone, donc du système nerveux #7p981

98 Mercredi 21 novembre 2007 Au,Y2004p3016 Au,Y2004p3016