Marie E. Cantino

DYX1C1 is required for axonemal dynein assembly and ciliary motility

DYX1C1 has been associated with dyslexia and neuronal migration in the developing neocortex. Unexpectedly, we found that deleting exons 2–4 of Dyx1c1 in mice caused a phenotype resembling primary ciliary dyskinesia (PCD), a disorder characterized by chronic airway disease, laterality defects and male infertility. This phenotype was confirmed independently in mice with a Dyx1c1 c.T2A start-codon mutation recovered from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Morpholinos targeting dyx1c1 in zebrafish also caused laterality and ciliary motility defects. In humans, we identified recessive loss-of-function DYX1C1 mutations in 12 individuals with PCD. Ultrastructural and immunofluorescence analyses of DYX1C1-mutant motile cilia in mice and humans showed disruptions of outer and inner dynein arms (ODAs and IDAs, respectively). DYX1C1 localizes to the cytoplasm of respiratory epithelial cells, its interactome is enriched for molecular chaperones, and it interacts with the cytoplasmic ODA and IDA assembly factor DNAAF2 (KTU). Thus, we propose that DYX1C1 is a newly identified dynein axonemal assembly factor (DNAAF4).

Synaptic and extrasynaptic GABAA receptor and gephyrin clusters.

Publisher Summary In the brain, most interneurons are GABAergic. Glycinergic neurons are mainly localized in the spinal cord. Apart from the neurotransmittermediated signaling that occurs between interneurons and between interneurons and principal neurons, interneurons can be electrically coupled to each other via gap junctions. Powerful tools are being used to reveal the molecular organization of excitatory and inhibitory synapses and the various signaling pathways involved in these synapses. Recent developments in genomics and proteomics, gene knockout animal models, protein-protein interaction assays (such as yeast two-hybrid), and the production and application of novel specific antibodies in combination with immunofluorescence microscopy (confocal and two-photon) and electron microscopy Immunogold techniques are advancing at a very rapid pace our knowledge of the organization of the molecular machinery of chemical synapses.

A neuroprotective role of the human uncoupling protein 2 (hUCP2) in a Drosophila Parkinson's Disease model

Parkinsons disease (PD), caused by selective loss of dopaminergic (DA) neurons in the substantia nigra pars compacta, is the most common movement disorder. While its etiology remains unknown, mitochondrial dysfunction is recognized as one of the major cellular defects contributing to PD pathogenesis. Mitochondrial uncoupling protein 2 (UCP2) has been implicated in neuroprotection in several neuronal injury models. Here we show that hucp2 expression in Drosophila DA neurons under the control of the tyrosine hydroxylase (TH) promoter protects those flies against the mitochondrial toxin rotenone-induced DA neuron death, head dopamine depletion, impaired locomotor activity and energy deficiency. Under normal conditions, hUCP2 flies maintain an enhanced locomotor activity and have higher steady-state ATP levels suggesting improved energy homeostasis. We show that while no increased mitochondrial DNA content or volume fraction is measured in hUCP2 flies, augmented mitochondrial complex I activity is detected. Those results suggest that it is increased mitochondrial function but not mitochondrial biogenesis that appears responsible for higher ATP levels in hUCP2 flies. Consistent with this notion, an up-regulation of Spargel, the Drosophila peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1) homologue is detected in hUCP2 flies. Furthermore, a Spargel target gene Tfam, the mitochondrial transcription factor A is up-regulated in hUCP2 flies. Taken together, our results demonstrate a neuroprotective effect of hUCP2 in DA neurons in a Drosophila sporadic PD model. Moreover, as the TH promoter activity is present in both DA neurons and epidermis, our results reveal that hucp2 expression in those tissues may act as a stress signal to trigger Spargel activation resulting in enhanced mitochondrial function and increased energy metabolism.

Subsarcomeric distribution of calcium in demembranated fibers of rabbit psoas muscle

Direct measurements were made of the Ca distribution within sarcomeres of glycerinated rabbit psoas muscle fibers in rigor using electron probe x-ray microanalysis. Both analogue raster analysis and digital x-ray imaging were used to quantitate the Ca distribution along thick and thin filaments as a function of the concentration of free Ca2+. Even when corrected for the estimated contribution of Ca bound to thick filaments, the Ca measured in the region of overlap between thick and thin filaments significantly exceeded the Ca in the I-band at subsaturating concentrations of free Ca2+. At saturating levels of free Ca2+, the excess Ca in the overlap region was diminished but still statistically significant. The data thus suggest that the formation of rigor linkages exerts multiple effects on the binding of Ca2+ to thin filaments in the overlap region by increasing the affinity of troponin C for Ca2+ and possibly by unmasking additional Ca2+ binding sites. The data also show that the cooperativity invested in the thin filaments is insufficient to permit the effects of rigor cross-bridge formation on Ca2+ binding to propagate far along the thin filaments into the I-band.

A-band architecture in vertebrate skeletal muscle: polarity of the myosin head array

Despite extensive knowledge of many muscle A-band proteins (myosin molecules, titin, C-protein (MyBP-C)), details of the organization of these molecules to form myosin filaments remain unclear. Recently the myosin head (crossbridge) configuration in a relaxed vertebrate muscle was determined from low-angle X-ray diffraction (Hudson et al. (1997), J Mol Biol273: 440–455). This showed that, even without C-protein, the myosin head array displays a characteristic polar pattern with every third 143 Å-spaced crossbridge level particularly prominent. However, X-ray diffraction cannot determine the polarity of the crossbridge array relative to the neighbouring actin filaments; information crucial to a proper understanding of the contractile event. Here, electron micrographs of negatively-stained goldfish A-segments and of fast-frozen, freeze-fractured plaice A-bands have been used to determine the resting myosin head polarity relative to the M-band. In agreement with the X-ray data, the prominent 429 Å-spaced striations are seen outside the C-zone, where no non-myosin proteins apart from titin are thought to be located. The head orientation is with the concave side of the curved myosin heads (containing the entrance to the ATP-binding site) facing towards the M-band and the convex surface (containing the actin-binding region at one end) facing away from the M-band.

Evidence for a thermally reversible order–order transition between lamellar and perforated lamellar microphases in a triblock copolymer

Abstract The microstructure and microphase behavior of a poly(styrene-b-(ethylene-alt-propylene)-b-styrene) (PS-PEP-PS) triblock copolymer ( M n =50,000 g/mol and 50.8 wt% PS) were characterized by transmission electron microscopy (TEM) and dynamic rheology. The microstructure texture at room temperature and up to about 125°C consisted of alternating PS and PEP lamellae (LAM). Oscillatory shear experiments revealed thermally-reversible post-Tg transitions at 125–130°C and 275°C that are attributed to an order–order transition (OOT) of the mesophase texture and an order–disorder transition (ODT), respectively. Evidence for a perforated lamellar (PL) microstructure of alternating PS and PEP lamellae with hexagonal-packed PS connectors perforating the PEP phase above the ODT and a thermally-reversible LAM ↔ PL OOT was obtained from TEM of quenched and slowly cooled samples. Unlike previous reports of reversible OOT transitions in block copolymers, the reversibility of the OOT for these block copolymers does not require shearing the sample.

Microstructure of Block Copolymers of Polystyrene and Poly(ethylene-alt-propylene)

Abstract The microstructure and microphase behaviour of diblock ( M a ≈ 27 and 50 kg mol −1 ) and triblock ( M a ≈ 50kg mol −1 ) copolymers of polystyrene (PS) and poly(ethylene- alt -propylene) (PEP) were studied by small angle X-ray scattering (SAXS), transmission electron microscopy (TEM) and dynamic mechanical thermal analysis (DMTA). The composition was varied from 10 wt.% to 50 wt.% PS. The morphology of solution-cast and compression-moulded samples was compared. The common packing arrangements of body-centred-cubic (BCC) spheres, hexagonal-close-packed (HCP) cylinders and alternating lamellae were observed for the PS microdomains using SAXS and TEM. A microphase texture of PS spheres with a liquid-like packing (LLP) was obtained at lower molecular weight and PS content. Coexisting textures of LLP spheres and HCP cylinders of PS were observed in a triblock copolymer with low PS content. An order—order transition between conventional lamellar and perforated lamellar textures was detected in a nearly-symmetric triblock copolymer. Conventional order—disorder transitions were identified for most of the other polymers using DMTA and temperature-resolved SAXS.

Distributions of Calcium in A and I Bands of Skinned Vertebrate Muscle Fibers Stretched to Beyond Filament Overlap

Measurements were made of the distributions of total calcium along the length of A and I bands in skinned frog semitendinosus muscles using electron probe x-ray microanalysis. Since calcium in the water space was kept below the detection limit of the technique, the signal was assumed to reflect the distribution of calcium bound to myofilament proteins. Data from sarcomeres with overlap between thick and thin filaments showed enhancement of calcium in this region, as previously demonstrated in rabbit psoas muscle fibers in rigor (Cantino, M. E., T. S. Allen, and A. M. Gordon. 1993. Subsarcomeric distribution of calcium in demembranated fibers of rabbit psoas muscle. Biophys. J. 64:211-222). Such enhancement could arise from intrinsic non-uniformities in calcium binding to either thick or thin filaments or from enhancement of calcium binding to either filament by rigor cross-bridge attachment. To test for intrinsic variations in calcium binding, calcium distributions were determined in fibers stretched to beyond filament overlap. Calcium binding was found to be relatively uniform along both thick and thin filaments, and therefore cannot account for the increased calcium observed in the overlap region. From these results it can be concluded that the observed enhancement of calcium is due to an increase in calcium binding to myofilaments as a result of rigor attachment of cross-bridges to actin. The source of the enhancement is most likely an increase in calcium binding to troponin, although enhancement of calcium binding to myosin light chains cannot be ruled out.

Determination of Myofilament Calcium Distributions from Electron Probe X-ray Microanalysis Images

The quantity and spatial distribution of calcium bound to myofilaments is of considerable interest to physiologists studying the regulation of contraction in striated muscle, but few techniques allow these distributions to be directly measured. Methods are described here by which such data can be derived from electron probe X-ray microanalysis (EPXMA) images. Combination of data from multiple half sarcomeres improves the signal-to-noise level and allows assessment of variation between half sarcomeres. Profiles of calcium counts, calcium counts normalized to local bremstrahlung counts, and calcium counts normalized to the average bremstrahlung counts for the entire half sarcomere provide differing but complementary information. Such data help to identify and characterize spatial variations in Ca binding to regulatory proteins in muscle.

Epitaxial Phase Transformation between Cylindrical and Double Gyroid Mesophases

Complex phase transformation between the hexagonal cylinder (Hex) and double gyroid (G) phases in a polystyrene-block-poly(ethylene oxide) (PS-b-PEO) diblock copolymer was investigated using two-dimensional (2D) synchrotron small-angle X-ray scattering (SAXS), and transmission electron microscope (TEM). The PS-b-PEO sample contained a small population of another bicontinuous cubic phase having an m Im3 symmetry. These two bicontinuous cubic phases (G and m Im3 ) had the same unit cell dimensions. Under a large-amplitude reciprocating shear, the bicontinuous cubic phases transformed into a “single-crystal”-like Hex phase. When annealed at 150 °C for 40 min, the Hex phase partially transformed into well-oriented twinned structures of the G and m Im3 phases without significant loss of orientation in 2D SAXS measurements. Epitaxial phase transformation relationships between the Hex/G and Hex/ m Im3 phases were identified. The mechanism of the Hex → G transformation was examined by TEM.