Universités Montpellier 1 et 2 – Master Biologie‐Santé Examen écrit 2eme session – Juin 2012 UE Biologie Cellulaire – FMBS103 Durée 2h00 – Aucun document autorisé Les auteurs de ces travaux, Guilluy et al. (Nature Cell Biology, 2011), se proposent d’étudier comment les cellules répondent aux forces mécaniques exercées à leur encontre. Expérimentalement, la mise en jeu de forces de tension est effectuée au moyen de microbilles magnétiques couplées à leur surface par de la fibronectine. Une fois en contact avec les cellules (cellules REF 52) l’application d’un champ magnétique induit la production d’une force de tension réenforçant l’assemblage de complexes adhésifs et le réarrangement du cytosquelette cellulaire provoquant ainsi le développement d’une rigidité (stiffness) cellulaire, appelée encore phénomène de ré‐enforcement. Ce phénomène peut être expérimentalement quantifié en mesurant le déplacement des microbilles induit par la réponse cellulaire à la suite de deux courtes applications successives de champ magnétique. Le déplacement des billes est augmenté lors de l’application du second champ, reflétant le phénomène de ré‐enforcement engendré lors de la première application. RhoAG17A is a nucleotide‐free RhoA mutant. GST‐RBD est utilisé pour retenir puis éluer spécifiquement la fraction de Rho liant le GTP (technique du ‘GST pull‐down’). SU6656 est un inhibiteur des tyrosine‐kinases de la famille Src (Src, Yes, Fyn) U0126 est un inhibiteur de MEK (MAP kinase kinase) 1. Figure 1 (a, b) : RhoA est‐elle activée en réponse à la force de tension exercée ? A quoi sert l’usage d’anticorps anti‐1 intégrine utilisée dans la figure 1a ? Justifiez vos réponses. 2. Figure 1 (c, d) : Quelles GEF parmi les 5 étudiées sont spécifiquement recrutées dans le complexe adhésif sous l’effet de la force de tension ? Qu’est‐ce une GEF ? Que concluez‐vous vis‐à‐vis de l’effet observé précédemment sur RhoA (fig1 a,b). Justifez vos réponses. 3. Figure 1 (e, f) et figure 2 (a‐c): comment ces nouveaux résultats confortent‐ils les conclusions qui découlent des résultats pésentés dans les figures 1(a, b) et 1(c, d) ? justifez vos réponses. 4. Quelles conclusions supplémentaires peuvent être tirées des figures 3 et 4 respectivement pour LARG et GEF‐H1 ? Répondez en proposant, en une phrase, un titre à chacune de ces figures. 5. (question bonus : + 1 point). Quelle autre protéine, étudiée en cours, est également impliquée dans la mécanosensibilité cellulaire ? Figure 1. (a,b) REF52 cells were incubated without or with the function-blocking anti-1 antibody (P4C10) for 30 min and then
with fibronectin-coated magnetic beads. A permanent magnet was used to generate tensional force for different amounts of
time. (a) Active RhoA (RhoA-GTP) was isolated with GST-RBD and analysed by western blotting. (b) Corresponding
densitometric analysis of RhoA-GTP normalized to RhoA levels and expressed relative to the control in the absence of
stimulation by force (error bars represent s.e.m., n=5). (c) REF52 cells were incubated for 30 min with fibronectin-coated beads
and stimulated with tensional force using a permanent magnet for different amounts of time before cell lysis. After magnetic
separation of the adhesion complex fraction, the lysate and the adhesion complex fraction were analysed by western blotting. All
results are representative of at least three independent experiments. (d) REF52 cells were incubated for 30 min with fibronectincoated beads and stimulated with tensional force using a permanent magnet for different amounts of time before cell lysis.
G17A
Active GEFs were sedimented with GST-RhoA
and analysed by western blotting. All results are representative of at least
three independent experiments. (e,f) REF52 cells were transfected for 48 h with control siRNA or siRNA targeting p115, GEFH1, LARG or both GEF-H1 and LARG, and incubated for 30 min with fibronectin-coated beads. (e) After stimulation with
tensional force for 5 min, cells were lysed and active RhoA (RhoA-GTP) was isolated with GST-RBD and analysed by western
blotting. (f) The corresponding densitometric analysis. RhoA-GTP is normalized to RhoA levels and expressed relative to the
control (error bars represent s.e.m., n=4). Figure 2. (a) Typical displacement of a fibronectin-coated bead bound to a REF52 fibroblast during force pulse application. (b)
Change in stiffness during two force pulses applied to fibronectin-coated beads bound to REF52 cells transfected for 48 h with
control siRNA or RhoA siRNA or RhoA siRNA and an siRNA-resistant mutant of RhoA (myc-RhoA) (error bars represent s.e.m.,
n=20; *P <0:01). (c) Change in stiffness during two force pulses applied to fibronectin-coated beads bound to REF52 cells
transfected for 48 h with control siRNA or siRNA targeting p115, GEF-H1, LARG or Ect2 (error bars represent s.e.m., n=20, *P
<0:05).
Figure 3. (a) REF52 cells untreated or treated with SU6656 (2.5 M for 30 min) were incubated with fibronectin-coated beads
and stimulated with tensional forces for different amounts of time. Active LARG and GEF-H1 were sedimented with GSTG17A
and analysed by western blotting. (b) REF52 cells untreated or treated with SU6656 (2.5 M for 30 min) were
RhoA
incubated with fibronectin-coated beads. After stimulation with tensional force for different amounts of time, cells were lysed and
-/active RhoA (RhoA-GTP) was isolated with GST-RBD and analysed by western blotting. (c) SYF cells and SYF cells re+/+
expressing Src, Yes and Fyn (SYF ) or re-expressing Src or Fyn were incubated with fibronectin-coated beads and stimulated
G17A
and analysed by western
with tensional forces for 3 min. Active LARG and GEF-H1 were pulled down with GST-RhoA
-/blotting. (d) Change in stiffness during two force pulses applied to fibronectin-coated beads bound to SYF cells and SYF cells
+/+
re-expressing Src, Yes and Fyn (SYF ) or re-expressing either Src or Fyn (error bars represent s.e.m., *P=0:01; n=20).
Figure 4. (a) REF52 cells untreated or treated with U0126 (5 M for 30 min) were incubated with fibronectin-coated beads and
G17A
stimulated with tensional forces for different amounts of time. Active LARG and GEF-H1 were sedimented with GST-RhoA
and analysed by western blotting. (b) REF52 cells untreated or treated with the FAK inhibitor 14 (5 M for 30 min) were
incubated with fibronectin-coated beads and stimulated with tensional forces for different amounts of time. Active Ras (RasGTP) was sedimented with Raf1-GST. Phosphorylated FAK (Tyr 397), phosphorylated ERK (Thr202 and Tyr204) and total FAK
were analysed by western blotting. (c) REF52 cells untreated or treated with the FAK inhibitor 14 (5 M for 30 min) were
incubated with fibronectin-coated beads and stimulated with tensional forces for different amounts of time. Active LARG and
G17A
and analysed by western blotting. (d) Change in stiffness during two force pulses
GEF-H1 were sedimented with GST-RhoA
applied to fibronectin-coated beads bound to REF52 cells treated with or without U0126 (5 M for 30 min; error bars represent
s.e.m., *P <0:01; n=20).