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Eur J Histochem
2014 Dec 05;584:2453. doi: 10.4081/ejh.2014.2453.
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Specific association of growth-associated protein 43 with calcium release units in skeletal muscles of lower vertebrates.
Caprara GA
,
Perni S
,
Morabito C
,
Mariggiò MA
,
Guarnieri S
.
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Growth-associated protein 43 (GAP43), is a strictly conserved protein among vertebrates implicated in neuronal development and neurite branching. Since GAP43 structure contains a calmodulin-binding domain, this protein is able to bind calmodulin and gather it nearby membrane network, thus regulating cytosolic calcium and consequently calcium-dependent intracellular events. Even if for many years GAP43 has been considered a neuronal-specific protein, evidence from different laboratories described its presence in myoblasts, myotubes and adult skeletal muscle fibers. Data from our laboratory showed that GAP43 is localized between calcium release units (CRUs) and mitochondria in mammalian skeletal muscle suggesting that, also in skeletal muscle, this protein can be a key player in calcium/calmodulin homeostasis. However, the previous studies could not clearly distinguish between a mitochondrion- or a triad-related positioning of GAP43. To solve this question, the expression and localization of GAP43 was studied in skeletal muscle of Xenopus and Zebrafish known to have triads located at the level of the Z-lines and mitochondria not closely associated with them. Western blotting and immunostaining experiments revealed the expression of GAP43 also in skeletal muscle of lower vertebrates (like amphibians and fishes), and that the protein is localized closely to the triad junction. Once more, these results and GAP43 structural features, support an involvement of the protein in the dynamic intracellular Ca2+ homeostasis, a common conserved role among the different species.
Figure 1. GAP43 is expressed in skeletal muscle of lower vertebrates. Western blot analysis of skeletal muscle samples deriving from Danio rerio (Zebrafish), Xenopus leavis (Xenopus), Mus musculus (Mouse) and a sample from mouse brain (Brain).
Figure 2. Localization of GAP43 in Zebrafish and Xenopus myotubes has a periodic sarcomeric spacing. Representative immunofluorescence images of muscle cell cultures deriving from Zebrafish (A) and Xenopus (B) samples. Confocal microscopy analysis shows a single striation cross-orientated as indicated by arrowheads (A and B). C) Measurements of distances between striations. Scale bars: A) 20 µm; B) 2 µm.
Figure 3. GAP43 is localized close to CRUs, in adult fibers. Representative immunofluorescence images of double-immunolabeled muscle fibers from Zebrafish (A-C) and Xenopus (D-F) samples. In adult Zebrafish and Xenopus muscle fibers, the staining of CRUs (marked with an anti-RyR antibody, B and E) forms dotted single rows along the Z-lines. Also GAP43 staining produces a single dotted cross-striation (A and D), which appears to co-localize with that of RyR (C and F, merged images). Scale bars: 5 µm.
Figure 4. Fluorescence image profile. Graphs represent fluorescence intensity profiles calculated on images obtained from both Zebrafish (A) and Xenopus (B) muscle fiber samples co-immunostained for RyR and GAP43. The GAP43 fluorescence peak appears close to that of RyR.
Figure 5. Schematic representation of a muscle fiber cross section from fish (A) and frog (B) cut at the Z-line level. At this level, the sarcoplasmic reticulum (black lines inside the fiber perimeter) is engaged in the formation of the triads together with the T-tubules. The images at the bottom C and D represent the projection of a longitudinal optical section of the same fibers (indicated by the green squares). The colored sarcoplasmic reticulum is the portion that is included into the optical section and actually visualized by the observer, the pink color represents RyR (centered in the triad) and the green color represents GAP43 (off-centered from the triad). The overlap between RyR and GAP43 signals is colored in yellow. The particular shape and disposition of the myofibrils in the fish (A) ensure that the sarcoplasmic reticulum delimiting the myofibrils mostly overlaps in the same direction of the laser beam (z).
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