BRAF activates PAX3 to control muscle precursor cell migration during forelimb muscle development
- Max-Planck-Institute for Heart and Lung Research, Germany
Figures
Figure 1 with 1 supplement
BRAF mediates muscle precursor cell migration independent of MEK/ERK signaling.
(A) Immunofluorescence staining of migrating muscle cells (C2C12) after knock down of the hepatocyte growth factor (HGF) receptor (siMet), Braf (siBraf) and Pax3 (siPax3) or after transfection of …
Figure 1—figure supplement 1
BRAF and PAX3 stimulate limb muscle precursor cell migration.
Microscopic images of chicken somitic explants after infection with retroviral vectors expressing Braf, Pax3 or human alkaline phosphatase (AP). BRAF and PAX3 both stimulated migration of cells out …
Figure 2
Inactivation of Braf in limb muscle precursor cells.
(A) Strategy for generation of Braf floxed mice (Brafnfl). Breeding with MeuCre mice yielded Braffl mice, in which the neomycin cassette was removed but exon three is flanked by loxP-sites. (B) Braff…
Figure 3 with 1 supplement
BRAF is required for limb muscle precursor cell migration during mouse embryogenesis.
(A) Pax3 whole mount in situ hybridization of WT, germ line Brafdel/del and Pax3-Cre//Brafdel/del mutant embryos at E10.5. (B) Transverse sections of WT and Pax3-Cre//Brafdel/del mutant embryos at …
Figure 3—figure supplement 1
Inactivation of Braf in Pax3 expressing cells impairs limb muscle precursor cell but not endothelial cell migration during mouse embryogenesis.
(A, B, E, F) Immunofluorescence staining of E10.5 Pax3-Cre (A, B) and Pax3-Cre//Brafdel/del embryos (E, F) for BRAF (red). White arrows in (A, B) indicate the presence of BRAF in the dermomyotome. (C…
Figure 4 with 1 supplement
BRAF directly interacts with PAX3 in migrating muscle precursor cells.
(A) PAX3, GST-PAX3 and PAX3 were immunoprecipitated from protein extracts of E10.5 wild type embryos or from C2C12 muscle cells after transfection with HA-Pax3 or CA Braf (V600E). …
Figure 4—figure supplement 1
Selected lists of BRAF interaction partners identified by mass spectrometry analysis.
Mass spectrometry was performed after in-gel digestion of Coomassie-stained bands from polyacrylamide gels. (A) Proteins identified after GST-PAX3 pull down of protein extracts from WT mouse …
Figure 5 with 1 supplement
A fraction of BRAF co-localizes with PAX3 in nuclei of muscle cells.
(A) Western blot analysis of cytoplasmic (C) and nuclear fractions (N) of C2C12 cells transfected with CA Braf (V600E), WT Braf, HA-Pax3 or HA-Pax3-GFP. n = 3. Successful fractionation was monitored …
Figure 5—figure supplement 1
Intracellular localization of BRAF in C2C12 cells.
(A) Immunofluorescence images of C2C12 cells in a Boyden micro chemotaxis chamber. Cultures were either not stimulated (Con) or treated with HGF after transfection with CA Braf (V600E). Note: …
Figure 6
Nuclear translocation of BRAF and migration of muscle cells depend on intact endosomal trafficking.
(A) Western blot analysis of isolated subcellular fractions of C2C12 cells after transfection of CA Braf V600E and knockdown of Eea1 or Erk1/2. n = 3. Knockdown of Eea1 prevented accumulation of …
Figure 7
Quantitative mass spectrometry analysis of PAX3 phosphorylation sites.
(A) Map of PAX3 phosphorylation sites determined by mass spectrometry. Sites shown in green have been published before (phosphosite.org) and sites displayed in black were newly identified. The red …
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Figure 7—source data 1
Source data for mass spectrometry analysis.
- https://doi.org/10.7554/eLife.18351.013
Figure 8
Phosphorylation of PAX3 at Ser205 is critical for stimulation of muscle cell migration.
(A) Exchange of serine by alanine in PAX3 at Ser205 reduces C2C12 myoblast migration after transfection with CA Braf (V600E). Cultures were either transfected with a control vector (-), with …
Figure 9
Schematic model of the MET-BRAF-PAX3 feedback loop enabling migration of limb muscle precursor cells.
(A) Activation and subsequent internalization of MET leads to activation of BRAF and transport via endosomal trafficking to a perinuclear position. (B) After translocation of BRAF into the nucleus …
