The Builders of the Junction: Roles of Junctophilin1 and Junctophilin2 in the Assembly of the Sarcoplasmic Reticulum–Plasma Membrane Junctions in Striated Muscle

Contraction of striated muscle is triggered by a massive release of calcium from the sarcoplasmic reticulum (SR) into the cytoplasm. This intracellular calcium release is initiated by membrane depolarization, which is sensed by voltage-gated calcium channels CaV1.1 (in skeletal muscle) and CaV1.2 (in cardiac muscle) in the plasma membrane (PM), which in turn activate the calcium-releasing channel ryanodine receptor (RyR) embedded in the SR membrane. This cross-communication between channels in the PM and in the SR happens at specialized regions, the SR-PM junctions, where these two compartments come in close proximity. Junctophilin1 and Junctophilin2 are responsible for the formation and stabilization of SR-PM junctions in striated muscle and actively participate in the recruitment of the two essential players in intracellular calcium release, CaV and RyR. This short review focuses on the roles of junctophilins1 and 2 in the formation and organization of SR-PM junctions in skeletal and cardiac muscle and on the functional consequences of the absence or malfunction of these proteins in striated muscle in light of recently published data and recent advancements in protein structure prediction

Neuronal junctophilins recruit specific Cav and RyR isoforms to ER-PM junctions and functionally alter Cav2.1 and Cav2.2

Junctions between the endoplasmic reticulum and plasma membrane that are induced by the neuronal junctophilins are of demonstrated importance, but their molecular architecture is still poorly understood and challenging to address in neurons. This is due to the small size of the junctions and the multiple isoforms of candidate junctional proteins in different brain areas. Using colocalization of tagged proteins expressed in tsA201 cells, and electrophysiology, we compared the interactions of JPH3 and JPH4 with different calcium channels. We found that JPH3 and JPH4 caused junctional accumulation of all the tested high-voltage-activated CaV isoforms, but not a low-voltage-activated CaV. Also, JPH3 and JPH4 noticeably modify CaV2.1 and CaV2.2 inactivation rate. RyR3 moderately colocalized at junctions with JPH4, whereas RyR1 and RyR2 did not. By contrast, RyR1 and RyR3 strongly colocalized with JPH3, and RyR2 moderately. Likely contributing to this difference, JPH3 binds to cytoplasmic domain constructs of RyR1 and RyR3, but not of RyR2

Junctophilins 1, 2, and 3 all support voltage-induced Ca2+ release despite considerable divergence

In skeletal muscle, depolarization of the plasma membrane (PM) causes conformational changes of the calcium channel CaV1.1 that then activate RYR1 to release calcium from the SR. Being independent of extracellular calcium entry, this process is termed voltage-induced calcium release. In skeletal muscle, junctophilins (JPHs) 1 and 2 form the SR–PM junctions at which voltageinduced calcium release occurs. Previous work demonstrated that JPH2 is able to recapitulate voltage-induced calcium release when expressed in HEK293 cells together with CaV1.1, β1a, Stac3, and RYR1. However, it is unknown whether JPH1 and the more distantly related neuronal JPH3 and JPH4 might also function in this manner, a question of interest because different JPH isoforms diverge in their interactions with RYR1. Here, we show that, like JPH2, JPH1 and JPH3, coexpressed with CaV1.1, β1a, Stac3, and RYR1 in HEK293 cells, cause colocalization of CaV1.1 and RYR1 at ER–PM junctions. Furthermore, potassium depolarization elicited cytoplasmic calcium transients in cells in which WT CaV1.1 was replaced with the calcium impermeant mutant CaV1.1(N617D), indicating that JPH1, JPH2, and JPH3 can all support voltage-induced calcium release, despite sequence divergence and differences in interaction with RYR1. Conversely, JPH4-induced ER–PM junctions contain CaV1.1 but not RYR1, and cells expressing JPH4 are unable to produce depolarization-induced calcium transients. Thus, JPHs seem to act primarily to form ER–PM junctions and to recruit the necessary signaling proteins to these junctions but appear not to be directly involved in the functional interactions between these proteins

De novo reconstitution reveals the proteins required for skeletal muscle voltage-induced Ca2+ release

Skeletal muscle contraction is triggered by Ca2+ release from the sarcoplasmic reticulum (SR) in response to plasma membrane (PM) excitation. In vertebrates, this depends on activation of the RyR1 Ca2+ pore in the SR, under control of conformational changes of CaV1.1, located ∼12 nm away in the PM. Over the last ∼30 y, gene knockouts have revealed that CaV1.1/RyR1 coupling requires additional proteins, but leave open the possibility that currently untested proteins are also necessary. Here, we demonstrate the reconstitution of conformational coupling in tsA201 cells by expression of CaV1.1, β1a, Stac3, RyR1, and junctophilin2. As in muscle, depolarization evokes Ca2+ transients independent of external Ca2+ entry and having amplitude with a saturating dependence on voltage. Moreover, freeze-fracture electron microscopy indicates that the five identified proteins are sufficient to establish physical links between CaV1.1 and RyR1. Thus, these proteins constitute the key elements essential for excitation-contraction coupling in skeletal muscle

Novel Details of Calsequestrin Gel Conformation in Situ

Calsequestrin (CASQ) is the major component of the sarcoplasmic reticulum (SR) lumen in skeletal and cardiac muscles. This calcium-binding protein localizes to the junctional SR (jSR) cisternae, where it is responsible for the storage of large amounts of Ca2+, whereas it is usually absent, at least in its polymerized form, in the free SR. The retention of CASQ inside the jSR is due partly to its association with other jSR proteins, such as junctin and triadin, and partly to its ability to polymerize, in a high Ca2+ environment, into an intricate gel that holds the protein in place. In this work, we shed some light on the still poorly described in situ structure of polymerized CASQ using detailed EM images from thin sections, with and without tilting, and from deep-etched rotary-shadowed replicas. The latter directly illustrate the fundamental network nature of polymerized CASQ, revealing repeated nodal points connecting short segments of the linear polymer. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A

Prolonged antimicrobial activity of PMMA bone cement with embedded gentamicin-releasing silica nanocarriers

Antibiotic laden bone cements are regularly employed to prevent infections after joint replacement surgeries. We have developed silica nanocarriers loaded with gentamicin as a drug delivery system to be dispersed in poly methyl-methacrylate (PMMA) bone cement for controlling and extending the release of the antibiotic from bone cements, thus proving a prolonged antimicrobial activity. Layer-by-layer self-assembly was used to deposit gentamicin between alginate layers and two different poly β-amino esters on the silica nanoparticles. The release of gentamicin from PMMA bone cement containing silica nanocarriers continued for about 30 days compared to 6 days when the same amount of antibiotic was added as a pure powder (as in commercial formulations); moreover, the medium containing the released antimicrobial drug was capable of preventing the growth of numerous bacteria species responsible for prosthetic joint infections (both catalogue strains and clinical isolates) for longer periods of time than in the case of commercial formulations, thus confirming the extended antimicrobial properties of the drug once released from the carrier. No detrimental effects toward human osteoblasts were also observed; moreover, bone cement material characteristics such as curing time, water uptake, and mechanical properties were unaffected when the silica nanocarriers were added

Efficacy of a novel light-activated antimicrobial coating for disinfecting hospital surfaces.

Silicone polymers containing the light-activated antimicrobial agent methylene blue with or without gold nanoparticles were evaluated for their ability to reduce the microbial load on surfaces in a clinical environment. When irradiated with white light, polymers containing nanogold were more effective in this respect than those containing only methylene blue

Efficacy of a novel light-activated antimicrobial coating for disinfecting hospital surfaces.

Silicone polymers containing the light-activated antimicrobial agent methylene blue with or without gold nanoparticles were evaluated for their ability to reduce the microbial load on surfaces in a clinical environment. When irradiated with white light, polymers containing nanogold were more effective in this respect than those containing only methylene blue

Highly extensible skeletal muscle in snakes

Many snakes swallow large prey whole, and this process requires large displacements of the unfused tips of the mandibles and passive stretching of the soft tissues connecting them. Under these conditions, the intermandibular muscles are highly stretched but subsequently recover normal function. In the highly stretched condition we observed in snakes, sarcomere length (SL) increased 210% its resting value (SL0), and actin and myosin filaments no longer overlapped. Myofibrils fell out of register and triad alignment was disrupted. Following passive recovery, SLs returned to 82% SL0, creating a region of double-overlapping actin filaments. Recovery required recoil of intracellular titin filaments, elastic cytoskeletal components for realigning myofibrils, and muscle activation. Stretch of whole muscles exceeded that of sarcomeres as a result of extension of folded terminal tendon fibrils, stretching of extracellular elastin and independent slippage of muscle fibers. Snake intermandibular muscles thus provide a unique model of how basic components of vertebrate skeletal muscle can be modified to permit extreme extensibility. © 2014. Published by The Company of Biologists Ltd

Detachment of Listeria innocua and Pantoea agglomerans from cylinders of agar and potato tissue under conditions of Couette flow

Cylinders of raw potato or agar were contacted with suspensions of Listeria innocua and Pantoea agglomerans and then used as replacement rotors in a rheometer in order to investigate detachment under the influence of known shear forces. These shear forces were functions solely of the rotational speed of the rotor and the fluid (glycerol) in which the cylinders were caused to rotate. With this system surface shear forces ranging from 1.3 to 125 Pa could be generated corresponding to rotational speeds of 12.5 to 775 rpm. Under these conditions detachment phenomena were quite rapid with in most cases complete detachment being achieved over timescales of the order of 30 s. In general, lower shear forces were required to detach L. innocua from both agar and potato. For agar cylinders an applied shear force of only 1.3 Pa was sufficient to achieve 98 % detachment of L. innocua after 20 s. By contrast, relatively high shear forces were required to detach P. agglomerans particularly from potato; under an applied shear force of 2.8 Pa only 9.5 % detachment was achieved after 30 s. The results obtained at the highest shear forces studied here (125 Pa) with potato cylinders were suggestive of mass transfer into glycerol of one or more constituents present in potatoes that caused detached cells to aggregate causing an apparent decrease in percentage detachment. The data obtained could be used as a basis for the rational design of washing processes for fresh ready to eat food products