Effects of Germline VHL Deficiency on Growth, Metabolism, and Mitochondria.

Mutations in VHL, which encodes von Hippel-Lindau tumor suppressor (VHL), are associated with divergent diseases. We describe a patient with marked erythrocytosis and prominent mitochondrial alterations associated with a severe germline VHL deficiency due to homozygosity for a novel synonymous mutation (c.222C→A, p.V74V). The condition is characterized by early systemic onset and differs from Chuvash polycythemia (c.598C→T) in that it is associated with a strongly reduced growth rate, persistent hypoglycemia, and limited exercise capacity. We report changes in gene expression that reprogram carbohydrate and lipid metabolism, impair muscle mitochondrial respiratory function, and uncouple oxygen consumption from ATP production. Moreover, we identified unusual intermitochondrial connecting ducts. Our findings add unexpected information on the importance of the VHL-hypoxia-inducible factor (HIF) axis to human phenotypes. (Funded by Associazione Italiana Ricerca sul Cancro and others.).

Congenital myopathy results from misregulation of a muscle Ca2+ channel by mutant Stac3.

Skeletal muscle contractions are initiated by an increase in Ca2+ released during excitation-contraction (EC) coupling, and defects in EC coupling are associated with human myopathies. EC coupling requires communication between voltage-sensing dihydropyridine receptors (DHPRs) in transverse tubule membrane and Ca2+ release channel ryanodine receptor 1 (RyR1) in the sarcoplasmic reticulum (SR). Stac3 protein (SH3 and cysteine-rich domain 3) is an essential component of the EC coupling apparatus and a mutation in human STAC3 causes the debilitating Native American myopathy (NAM), but the nature of how Stac3 acts on the DHPR and/or RyR1 is unknown. Using electron microscopy, electrophysiology, and dynamic imaging of zebrafish muscle fibers, we find significantly reduced DHPR levels, functionality, and stability in stac3 mutants. Furthermore, stac3NAM myofibers exhibited increased caffeine-induced Ca2+ release across a wide range of concentrations in the absence of altered caffeine sensitivity as well as increased Ca2+ in internal stores, which is consistent with increased SR luminal Ca2+ These findings define critical roles for Stac3 in EC coupling and human disease.

Skeletal Muscle Microalterations in Patients Carrying Malignant Hyperthermia-Related Mutations of the e-c Coupling Machinery.

We have compared the ultrastructure of skeletal muscle biopsies from patients that have survived a [Malignant Hyperthermia, MH] episode and siblings that test positive for MH susceptibility with those from siblings that tested negatives. The aim is to establish whether life long exposure to the MH-related mutation effects may result in subtle abnormalities even in the absence of active episodes and/or clinically detectable deficiencies. Although a specific ultrastructural signature for MH mutants cannot be demonstrated, an MH related pattern of minor alterations does exist. These include the tendency for micro damage to the contractile apparatus and a higher than normal level of mitochondrial abnormalities.

Nanoscale patterning of STIM1 and Orai1 during store-operated Ca2+ entry.

Stromal interacting molecule (STIM) and Orai proteins constitute the core machinery of store-operated calcium entry. We used transmission and freeze-fracture electron microscopy to visualize STIM1 and Orai1 at endoplasmic reticulum (ER)-plasma membrane (PM) junctions in HEK 293 cells. Compared with control cells, thin sections of STIM1-transfected cells possessed far more ER elements, which took the form of complex stackable cisternae and labyrinthine structures adjoining the PM at junctional couplings (JCs). JC formation required STIM1 expression but not store depletion, induced here by thapsigargin (TG). Extended molecules, indicative of STIM1, decorated the cytoplasmic surface of ER, bridged a 12-nm ER-PM gap, and showed clear rearrangement into small clusters following TG treatment. Freeze-fracture replicas of the PM of Orai1-transfected cells showed extensive domains packed with characteristic "particles"; TG treatment led to aggregation of these particles into sharply delimited "puncta" positioned upon raised membrane subdomains. The size and spacing of Orai1 channels were consistent with the Orai crystal structure, and stoichiometry was unchanged by store depletion, coexpression with STIM1, or an Orai1 mutation (L273D) affecting STIM1 association. Although the arrangement of Orai1 channels in puncta was substantially unstructured, a portion of channels were spaced at ∼15 nm. Monte Carlo analysis supported a nonrandom distribution for a portion of channels spaced at ∼15 nm. These images offer dramatic, direct views of STIM1 aggregation and Orai1 clustering in store-depleted cells and provide evidence for the interaction of a single Orai1 channel with small clusters of STIM1 molecules.

Schwann cells and deleted in colorectal carcinoma direct regenerating motor axons towards their original path.

After complete nerve transection, a major challenge for regenerating peripheral axons is to traverse the injury site and navigate toward their original trajectory. Denervated Schwann cells distal to the lesion site secrete factors promoting axonal growth and serve as an axonal substrate, yet whether Schwann cells also actively direct axons toward their original trajectory is unclear. Using live-cell imaging in zebrafish, we visualize for the first time how in response to nerve transection distal Schwann cells change morphology as axons fragment, and how Schwann cell morphology reverses once regenerating growth cones have crossed the injury site and have grown along distal Schwann cells outlining the original nerve path. In mutants lacking Schwann cells, regenerating growth cones extend at rates comparable with wild type yet frequently fail to cross the injury site and instead stray along aberrant trajectories. Providing growth-permissive yet Schwann cell-less scaffolds across the injury site was insufficient to direct regenerating growth cones toward the original path, providing compelling evidence that denervated Schwann cells actively direct regenerating axons across the injury site toward their original trajectory. To identify signals that guide regenerating axons in vivo, we examined mutants lacking the deleted in colorectal carcinoma (DCC) guidance receptor. In these dcc mutants, a significant fraction of regenerating motor axons extended along aberrant trajectories, similar to what we observe in mutants lacking Schwann cells. Thus, Schwann cell and dcc-mediated guidance are critical early during regeneration to direct growth cones across the transection gap and onto their original axonal trajectory.

A method for the ultrastructural preservation of tiny percutaneous needle biopsy material from skeletal muscle.

Skeletal muscle biopsies require transecting the muscle fibers resulting, in structural damage near the cut ends. Classically, the optimal ultrastructural preservation has been obtained by the use of relatively large biopsies in which the tissue fibers are restrained by ligating to a suitable retaining support prior to excision, and by examining regions at some distance from the cut ends. However, these methods require invasive surgical procedures. In the present study, we present and substantiate an alternative approach that allows for the excellent ultrastructural preservation of needle biopsy samples, even the very small samples obtained through tiny percutaneous needle biopsy (TPNB). TPNB represents an advantage, relative to standard muscle biopsy techniques and to other needle biopsies currently in use, as in addition to not requiring a skin incision, it leaves no scars in the muscle and requires an extremely brief recovery period. It is most appropriate for obtaining repeated samples in horizontal studies, e.g., in order to follow changes with athletic training and/or aging in a single individual and for studies of sarcopenic muscles in elderly patients. Due to the small size of the sample, TPNB may present limited usefulness for classical pathology diagnostics. However, it offers the major advantage of allowing multiple samples within a single session and this may be useful under specific circumstances.

Stac3 is a component of the excitation-contraction coupling machinery and mutated in Native American myopathy.

Excitation-contraction coupling, the process that regulates contractions by skeletal muscles, transduces changes in membrane voltage by activating release of Ca(2+) from internal stores to initiate muscle contraction. Defects in excitation-contraction coupling are associated with muscle diseases. Here we identify Stac3 as a novel component of the excitation-contraction coupling machinery. Using a zebrafish genetic screen, we generate a locomotor mutation that is mapped to stac3. We provide electrophysiological, Ca(2+) imaging, immunocytochemical and biochemical evidence that Stac3 participates in excitation-contraction coupling in muscles. Furthermore, we reveal that a mutation in human STAC3 is the genetic basis of the debilitating Native American myopathy (NAM). Analysis of NAM stac3 in zebrafish shows that the NAM mutation decreases excitation-contraction coupling. These findings enhance our understanding of both excitation-contraction coupling and the pathology of myopathies.

Domain cooperativity in the ß1a subunit is essential for dihydropyridine receptor voltage sensing in skeletal muscle.

The dihydropyridine receptor (DHPR) ß1a subunit is crucial for enhancement of DHPR triad expression, assembly of DHPRs in tetrads, and elicitation of DHPRα1S charge movement--the three prerequisites of skeletal muscle excitation-contraction coupling. Despite the ability to fully target α1S into triadic junctions and tetradic arrays, the neuronal isoform ß3 was unable to restore considerable charge movement (measure of α1S voltage sensing) upon expression in ß1-null zebrafish relaxed myotubes, unlike the other three vertebrate ß-isoforms (ß1a, ß2a, and ß4). Thus, we used ß3 for chimerization with ß1a to investigate whether any of the five distinct molecular regions of ß1a is dominantly involved in inducing the voltage-sensing function of α1S. Surprisingly, systematic domain swapping between ß1a and ß3 revealed a pivotal role of the src homology 3 (SH3) domain and C terminus of ß1a in charge movement restoration. More interestingly, ß1a SH3 domain and C terminus, when simultaneously engineered into ß3 sequence background, were able to fully restore charge movement together with proper intracellular Ca(2+) release, suggesting cooperativity of these two domains in induction of the α1S voltage-sensing function in skeletal muscle excitation-contraction coupling. Furthermore, substitution of a proline by alanine in the putative SH3-binding polyproline motif in the proximal C terminus of ß1a (also of ß2a and ß4) fully obstructed α1S charge movement. Consequently, we postulate a model according to which ß subunits, probably via the SH3-C-terminal polyproline interaction, adapt a discrete conformation required to modify the α1S conformation apt for voltage sensing in skeletal muscle.

Mutation of sec63 in zebrafish causes defects in myelinated axons and liver pathology.

Mutations in SEC63 cause polycystic liver disease in humans. Sec63 is a member of the endoplasmic reticulum (ER) translocon machinery, although it is unclear how mutations in SEC63 lead to liver cyst formation in humans. Here, we report the identification and characterization of a zebrafish sec63 mutant, which was discovered in a screen for mutations that affect the development of myelinated axons. Accordingly, we show that disruption of sec63 in zebrafish leads to abnormalities in myelinating glia in both the central and peripheral nervous systems. In the vertebrate nervous system, segments of myelin are separated by the nodes of Ranvier, which are unmyelinated regions of axonal membrane containing a high density of voltage-gated sodium channels. We show that sec63 mutants have morphologically abnormal and reduced numbers of clusters of voltage-gated sodium channels in the spinal cord and along peripheral nerves. Additionally, we observed reduced myelination in both the central and peripheral nervous systems, as well as swollen ER in myelinating glia. Markers of ER stress are upregulated in sec63 mutants. Finally, we show that sec63 mutants develop liver pathology. As in glia, the primary defect, detectable at 5 dpf, is fragmentation and swelling of the ER, indicative of accumulation of proteins in the lumen. At 8 dpf, ER swelling is severe; other pathological features include disrupted bile canaliculi, altered cytoplasmic matrix and accumulation of large lysosomes. Together, our analyses of sec63 mutant zebrafish highlight the possible role of ER stress in polycystic liver disease and suggest that these mutants will serve as a model for understanding the pathophysiology of this disease and other abnormalities involving ER stress.

In vivo nerve-macrophage interactions following peripheral nerve injury.

In vertebrates, the peripheral nervous system has retained its regenerative capacity, enabling severed axons to reconnect with their original synaptic targets. While it is well documented that a favorable environment is critical for nerve regeneration, the complex cellular interactions between injured nerves with cells in their environment, as well as the functional significance of these interactions, have not been determined in vivo and in real time. Here we provide the first minute-by-minute account of cellular interactions between laser transected motor nerves and macrophages in live intact zebrafish. We show that macrophages arrive at the lesion site long before axon fragmentation, much earlier than previously thought. Moreover, we find that axon fragmentation triggers macrophage invasion into the nerve to engulf axonal debris, and that delaying nerve fragmentation in a Wld(s) model does not alter macrophage recruitment but induces a previously unknown 'nerve scanning' behavior, suggesting that macrophage recruitment and subsequent nerve invasion are controlled by separate mechanisms. Finally, we demonstrate that macrophage recruitment, thought to be dependent on Schwann cell-derived signals, occurs independently of Schwann cells. Thus, live cell imaging defines novel cellular and functional interactions between injured nerves and immune cells.

Mice expressing T4826I-RYR1 are viable but exhibit sex- and genotype-dependent susceptibility to malignant hyperthermia and muscle damage.

Mutation T4825I in the type 1 ryanodine receptor (RYR1(T4825I/+)) confers human malignant hyperthermia susceptibility (MHS). We report a knock-in mouse line that expresses the isogenetic mutation T4826I. Heterozygous RYR1(T4826I/+) (Het) or homozygous RYR1(T4826I/T4826I) (Hom) mice are fully viable under typical rearing conditions but exhibit genotype- and sex-dependent susceptibility to environmental conditions that trigger MH. Hom mice maintain higher core temperatures than WT in the home cage, have chronically elevated myoplasmic[Ca(2+)](rest), and present muscle damage in soleus with a strong sex bias. Mice subjected to heat stress in an enclosed 37°C chamber fail to trigger MH regardless of genotype, whereas heat stress at 41°C invariably triggers fulminant MH in Hom, but not Het, mice within 20 min. WT and Het female mice fail to maintain euthermic body temperature when placed atop a bed whose surface is 37°C during halothane anesthesia (1.75%) and have no hyperthermic response, whereas 100% Hom mice of either sex and 17% of the Het males develop fulminant MH. WT mice placed on a 41°C bed maintain body temperature while being administered halothane, and 40% of the Het females and 100% of the Het males develop fulminant MH within 40 min. Myopathic alterations in soleus were apparent by 12 mo, including abnormally distributed and enlarged mitochondria, deeply infolded sarcolemma, and frequent Z-line streaming regions, which were more severe in males. These data demonstrate that an MHS mutation within the S4-S5 cytoplasmic linker of RYR1 confers genotype- and sex-dependent susceptibility to pharmacological and environmental stressors that trigger fulminant MH and promote myopathy.

Electron tomography of cryofixed, isometrically contracting insect flight muscle reveals novel actin-myosin interactions.

BACKGROUND: Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ. METHODOLOGY: We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the "target zone", situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening. CONCLUSION: We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very different from strong binding attachments.

Characterization and temporal development of cores in a mouse model of malignant hyperthermia.

Malignant hyperthermia (MH) and central core disease are related skeletal muscle diseases often linked to mutations in the type 1 ryanodine receptor (RYR1) gene, encoding for the Ca(2+) release channel of the sarcoplasmic reticulum (SR). In humans, the Y522S RYR1 mutation is associated with malignant hyperthermia susceptibility (MHS) and the presence in skeletal muscle fibers of core regions that lack mitochondria. In heterozygous Y522S knock-in mice (RYR1(Y522S/WT)), the mutation causes SR Ca(2+) leak and MHS. Here, we identified mitochondrial-deficient core regions in skeletal muscle fibers from RYR1(Y522S/WT) knock-in mice and characterized the structural and temporal aspects involved in their formation. Mitochondrial swelling/disruption, the initial detectable structural change observed in young-adult RYR1(Y522S/WT) mice (2 months), does not occur randomly but rather is confined to discrete areas termed presumptive cores. This localized mitochondrial damage is followed by local disruption/loss of nearby SR and transverse tubules, resulting in early cores (2-4 months) and small contracture cores characterized by extreme sarcomere shortening and lack of mitochondria. At later stages (1 year), contracture cores are extended, frequent, and accompanied by areas in which contractile elements are also severely compromised (unstructured cores). Based on these observations, we propose a possible series of events leading to core formation in skeletal muscle fibers of RYR1(Y522S/WT) mice: Initial mitochondrial/SR disruption in confined areas causes significant loss of local Ca(2+) sequestration that eventually results in the formation of contractures and progressive degradation of the contractile elements.

Full-length myosin VI dimerizes and moves processively along actin filaments upon monomer clustering.

Myosin VI is a reverse direction actin-based motor capable of taking large steps (30-36 nm) when dimerized. However, all dimeric myosin VI molecules so far examined have included non-native coiled-coil sequences, and reports on full-length myosin VI have failed to demonstrate the existence of dimers. Herein, we demonstrate that full-length myosin VI is capable of forming stable, processive dimers when monomers are clustered, which move up to 1-2 mum in approximately 30 nm, hand-over-hand steps. Furthermore, we present data consistent with the monomers being prevented from dimerizing unless they are held in close proximity and that dimerization is somewhat inhibited by the cargo binding tail. A model thus emerges that cargo binding likely clusters and initiates dimerization of full-length myosin VI molecules. Although this mechanism has not been previously described for members of the myosin superfamily, it is somewhat analogous to the proposed mechanism of dimerization for the kinesin Unc104.

Electron tomography of fast frozen, stretched rigor fibers reveals elastic distortions in the myosin crossbridges.

As a first step toward freeze-trapping and 3-D modeling of the very rapid load-induced structural responses of active myosin heads, we explored the conformational range of longer lasting force-dependent changes in rigor crossbridges of insect flight muscle (IFM). Rigor IFM fibers were slam-frozen after ramp stretch (1000 ms) of 1-2% and freeze-substituted. Tomograms were calculated from tilt series of 30 nm longitudinal sections of Araldite-embedded fibers. Modified procedures of alignment and correspondence analysis grouped self-similar crossbridge forms into 16 class averages with 4.5 nm resolution, revealing actin protomers and myosin S2 segments of some crossbridges for the first time in muscle thin sections. Acto-S1 atomic models manually fitted to crossbridge density required a range of lever arm adjustments to match variably distorted rigor crossbridges. Some lever arms were unchanged compared with low tension rigor, while others were bent and displaced M-ward by up to 4.5 nm. The average displacement was 1.6 +/- 1.0 nm. "Map back" images that replaced each unaveraged 39 nm crossbridge motif by its class average showed an ordered mix of distorted and unaltered crossbridges distributed along the 116 nm repeat that reflects differences in rigor myosin head loading even before stretch.

Junctate is a key element in calcium entry induced by activation of InsP3 receptors and/or calcium store depletion.

In many cell types agonist-receptor activation leads to a rapid and transient release of Ca(2+) from intracellular stores via activation of inositol 1,4,5 trisphosphate (InsP(3)) receptors (InsP(3)Rs). Stimulated cells activate store- or receptor-operated calcium channels localized in the plasma membrane, allowing entry of extracellular calcium into the cytoplasm, and thus replenishment of intracellular calcium stores. Calcium entry must be finely regulated in order to prevent an excessive intracellular calcium increase. Junctate, an integral calcium binding protein of endo(sarco)plasmic reticulum membrane, (a) induces and/or stabilizes peripheral couplings between the ER and the plasma membrane, and (b) forms a supramolecular complex with the InsP(3)R and the canonical transient receptor potential protein (TRPC) 3 calcium entry channel. The full-length protein modulates both agonist-induced and store depletion-induced calcium entry, whereas its NH(2) terminus affects receptor-activated calcium entry. RNA interference to deplete cells of endogenous junctate, knocked down both agonist-activated calcium release from intracellular stores and calcium entry via TRPC3. These results demonstrate that junctate is a new protein involved in calcium homeostasis in eukaryotic cells.

Cross-bridge number, position, and angle in target zones of cryofixed isometrically active insect flight muscle.

Electron micrographic tomograms of isometrically active insect flight muscle, freeze substituted after rapid freezing, show binding of single myosin heads at varying angles that is largely restricted to actin target zones every 38.7 nm. To quantify the parameters that govern this pattern, we measured the number and position of attached myosin heads by tracing cross-bridges through the three-dimensional tomogram from their origins on 14.5-nm-spaced shelves along the thick filament to their thin filament attachments in the target zones. The relationship between the probability of cross-bridge formation and axial offset between the shelf and target zone center was well fitted by a Gaussian distribution. One head of each myosin whose origin is close to an actin target zone forms a cross-bridge most of the time. The probability of cross-bridge formation remains high for myosin heads originating within 8 nm axially of the target zone center and is low outside 12 nm. We infer that most target zone cross-bridges are nearly perpendicular to the filaments (60% within 11 degrees ). The results suggest that in isometric contraction, most cross-bridges maintain tension near the beginning of their working stroke at angles near perpendicular to the filament axis. Moreover, in the absence of filament sliding, cross-bridges cannot change tilt angle while attached nor reach other target zones while detached, so may cycle repeatedly on and off the same actin target monomer.

Functional implications of RyR-DHPR relationships in skeletal and cardiac muscles

Dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs) interact during EC coupling within calcium release units, CRUs. The location of the two channels and their positioning are related to their role in EC coupling. als DHPR and RyR1 of skeletal muscle form interlocked arrays. Groups of four DHPRs (forming a tetrad) are located on alternate RyR1s. This association provides the structural framework for reciprocal signaling between the two channels. RyR3 are present in some skeletal muscles in association with RyR1 and in ratios up to 1:1. RyR3 neither induce formation of tetrads by DHPRs nor sustain EC coupling. RyR3 are located in a parajunctional position, in proximity of the RyR1-DHPR complexes, and they may be indirectly activated by calcium liberated via the RyR1 channels. RyR2 have two locations in cardiac muscle. One is at CRUs that contain DHPRs and RyRs. In these cardiac CRUs, RyR2 and a1c DHPR are in proximity of each other, but not closely linked, so that they may not have a direct molecular interaction. A second location of RyR2 is on SR cisternae that are not attached to surface membrane/T tubules. The RyR2 in these cisternae, which are often several microns away from any DHPRs, must necessarily be activated indirectly.