Xcell-Center

Uncategorized

Phosphorylation of eIF2α Is a Translational Control Mechanism Regulating Muscle Stem Cell Quiescence and Self-Renewal

http://www.cell.com/cell-stem-cell/abstract/S1934-5909(15)00458-0?rss=yes by

 

Figure 2

Satellite Cells Unable to Phosphorylate eIF2α Enter the Myogenic Program In Vivo

(A) A serine to alanine switch at position 51 (S51A) prevents eIF2α phosphorylation. Mice homozygous for this allele are not viable and are rescued by a transgene with wild-type eIF2α under the control of CMV enhancer and chicken β-actin promoter (actb). The wild-type eIF2α is flanked by two loxP sites and positioned upstream of a GFP reporter (green). Crossing this line with a Pax7CreERT2/+ allele, followed by tmx administration, permits the conditional expression of homozygous eIF2α S51A and GFP (green) in Pax7 satellite cells.

(B) Immunoblotting for P-eIF2α and total eIF2α (eIF2α) of cell lysates from newly isolated GFP+ cells from muscle of Pax3GFP/+ (WT) and tmx-treated Pax7CreERT2/+, tg(actb-eIF2afl-GFP), eIF2aS51A/S51A (S51A) animals. The tmx regime and day of analysis are shown. Relative levels of P-eIF2α, normalized to total eIF2α, are indicated, with representative immunoblots.

(C) Immunostaining for Pax7 (green) and p54/RCK (RCK, red) on isolated EDL myofibers from tmx-treated WT (Pax7+/+; no Cre) and S51A (eIF2αS51A/S51A) mice. Lower panels show merged images with DAPI.

(D) Numbers of p54/RCK+ granules per Pax7-positive satellite cell in (C).

(E) Immunostaining for Pax7 (red) and GFP (green), combined with detection of OPP (far red) on EDL myofibers from tmx-treated WT (Pax7+/+; no Cre) and S51A (eIF2αS51A/S51A) mice. EDL myofibers were also cultured for 6 hr (right panels) or in the presence of cycloheximide (CHX, left panels). Upper panels show merged images with DAPI, overlayed on brightfield to show myofibers.

(F) Rates of protein synthesis, reported by total cell OPP fluorescence in (E).

(G) Immunostaining for Pax7 (red) and GFP (green) on transverse sections of TA muscle after 5 daily doses of tmx (indicated). Right panels show merged images with DAPI, which are overlayed on brightfield images of transverse fiber sections. Asterisk indicates a Pax7+GFP+ satellite cell. Arrows indicate position of GFP+ cells between muscle fibers.

(H) Fraction of Pax7+ nuclei that show immunofluorescence for GFP indicated in (G).

(I) Immunostaining MyoD (red) and GFP (green) on transverse sections of TA muscle after tmx treatment. Right panels are merged images with DAPI, overlayed on brightfield to show myofibers.

(J) Fraction of GFP+ cells that are MyoD+ in (I).

(K) Immunoblotting against Myf5 and β-tubulin from cell lysates of newly isolated GFP+ cells from muscle of Pax3GFP/+ (WT) and tmx-treated Pax7CreERT2/+, tg(actb-eIF2afl-GFP), eIF2aS51A/S51A (S51A) animals. Relative levels of Myf5 normalized to β-tubulin are indicated, with representative immunoblots shown.

(L) Immunostaining Pax7 (green) and Laminin (Lam, red) on transverse sections of TA muscle after tmx treatment. Right panels show merged images with DAPI. Arrows indicate position of satellite cells outside basal lamina.

(M) Fraction of Pax7+ nuclei outside the basal lamina of myofibers after tmx treatment indicated in (L).

(N) Representative images of EdU+ satellite cells isolated from tmx-treated Pax7CreERT2/+, tg(actb-eIF2afl-GFP), eIF2α+/+, or eIF2αS51A/S51A and deposited on slides by cytospin.

(O) Fraction of EdU+ satellite cells isolated from tmx-treated mice, as indicated in (N).

Scale bars represent 50 μm except in (C) and (N) where they represent 10 μm and 20 μm, respectively. All values indicate mean (n ≥ 3) ± SEM. p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; nd, not detected. See also Figure S1 .