All skeletal muscles express similar levels of RyR1, but the level or RyR3 expression varies greatly. Anatomically, this correlates with two types of “feet” (= RyR): junctional feet (JF), located in the triad gap of all muscles, and parajunctional feet (PJF), located on the sides of the jSR cisternae and present only in RyR3-expressing muscles. Physiologically, frog fibers (with a high content of RyR3 and PJF) have readily-detected Ca sparks, while adult mouse fibers (with a very low or nil RyR3 and PJF content) do not. Fast fibers in zebrafish tail display abundant PJF. We explored the role of RyR3 by injecting zebrafish oocytes with a splicing blocker morpholino designed to bind to RyR3 pre-mRNA. Tails from several fish at 72 hpf (hours post fertilization) were pooled for western blotting. A pan-RyR antibody (34C) reveals three bands, presumably representing RyR3 and RyR1a and b. The slowest band specifically disappears after morpholino injection. Tails from ∼50 larvae at 72 hpf were collected for enzymatic dissociation of single muscle fibers. Intact 48 and 72 hpf tails and some dissociated fibers were fixed for EM. The PJF/JF ratio in EM images of fast fibers in control larvae at 72 hpf was 0.79±0.14, mean±SD (7 fish, 70 fibers, 310 triads; 3 experiments) and decreased to 0.03±0.03 (10 fish, 100 fibers, 420 triads; 4 experiments) in injected larvae. To measure Ca sparks, dissociated fibers were adhered to coverslips by matrigel, loaded with Fluo-4, and imaged confocally. 0.3 mM caffeine was used to stimulate sparks, which were readily detected in normal cells but almost absent in morpholino-treated cells. These data identify PJF as RyR3 and indicate that RyR3 activity is required for the ready-detection of sparks.

Perni, S., Franzini-Armstrong, C., Hollingworth, S., Baylor, S.M. (2014). Silencing RyR3 Eliminates Parajunctional Feet and Ca Sparks in Zebra Fish Tail Myotomes. BIOPHYSICAL JOURNAL, 106(2), 124A-125A [10.1016/j.bpj.2013.11.740].

Silencing RyR3 Eliminates Parajunctional Feet and Ca Sparks in Zebra Fish Tail Myotomes

Perni, S.;
2014

Abstract

All skeletal muscles express similar levels of RyR1, but the level or RyR3 expression varies greatly. Anatomically, this correlates with two types of “feet” (= RyR): junctional feet (JF), located in the triad gap of all muscles, and parajunctional feet (PJF), located on the sides of the jSR cisternae and present only in RyR3-expressing muscles. Physiologically, frog fibers (with a high content of RyR3 and PJF) have readily-detected Ca sparks, while adult mouse fibers (with a very low or nil RyR3 and PJF content) do not. Fast fibers in zebrafish tail display abundant PJF. We explored the role of RyR3 by injecting zebrafish oocytes with a splicing blocker morpholino designed to bind to RyR3 pre-mRNA. Tails from several fish at 72 hpf (hours post fertilization) were pooled for western blotting. A pan-RyR antibody (34C) reveals three bands, presumably representing RyR3 and RyR1a and b. The slowest band specifically disappears after morpholino injection. Tails from ∼50 larvae at 72 hpf were collected for enzymatic dissociation of single muscle fibers. Intact 48 and 72 hpf tails and some dissociated fibers were fixed for EM. The PJF/JF ratio in EM images of fast fibers in control larvae at 72 hpf was 0.79±0.14, mean±SD (7 fish, 70 fibers, 310 triads; 3 experiments) and decreased to 0.03±0.03 (10 fish, 100 fibers, 420 triads; 4 experiments) in injected larvae. To measure Ca sparks, dissociated fibers were adhered to coverslips by matrigel, loaded with Fluo-4, and imaged confocally. 0.3 mM caffeine was used to stimulate sparks, which were readily detected in normal cells but almost absent in morpholino-treated cells. These data identify PJF as RyR3 and indicate that RyR3 activity is required for the ready-detection of sparks.
Perni, S., Franzini-Armstrong, C., Hollingworth, S., Baylor, S.M. (2014). Silencing RyR3 Eliminates Parajunctional Feet and Ca Sparks in Zebra Fish Tail Myotomes. BIOPHYSICAL JOURNAL, 106(2), 124A-125A [10.1016/j.bpj.2013.11.740].
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11365/1216074