Hrs- and CD63-dependent competing mechanisms make different sized endosomal intraluminal vesicles

Multivesicular endosomes/bodies (MVBs) contain intraluminal vesicles (ILVs) that bud away from the cytoplasm. Multiple mechanisms of ILV formation have been identified, but the relationship between different populations of ILVs and MVBs remains unclear. Here we show in HeLa cells that different ILV subpopulations can be distinguished by size. EGF stimulation promotes the formation of large ESCRT-dependent ILVs, while depletion of the ESCRT-0 component, Hrs, promotes the formation of a uniformly sized population of small ILVs, the formation of which requires CD63. CD63 has previously been implicated in ESCRT-independent sorting of PMEL in MVBs and transfected PMEL is present on the small ILVs that form on Hrs depletion. Upregulation of CD63-dependent ILV formation by Hrs depletion indicates that Hrs and CD63 regulate competing machineries required for the generation of distinct ILV subpopulations. Taken together our results indicate that ILV size is influenced by their cargo and mechanism of formation and suggest a competitive relationship between ESCRT-dependent and -independent mechanisms of ILV formation within single MVBs

Comparative physiology of Australian quolls (Dasyurus; Marsupialia)

Quolls (Dasyurus) are medium-sized carnivorous dasyurid marsupials. Tiger (3,840 g) and eastern quolls (780 g) are mesic zone species, northern quolls (516 g) are tropical zone, and chuditch (1,385 g) were once widespread through the Australian arid zone. We found that standard physiological variables of these quolls are consistent with allometric expectations for marsupials. Nevertheless, inter-specific patterns amongst the quolls are consistent with their different environments. The lower T ^sub b^ of northern quolls (34°C) may provide scope for adaptive hyperthermia in the tropics, and they use torpor for energy/water conservation, whereas the larger mesic species (eastern and tiger quolls) do not appear to. Thermolability varied from little in eastern (0.035°C °C^sup -1^) and tiger quolls (0.051°C ºC^sup -1^) to substantial in northern quolls (0.100°C ºC^sup -1^) and chuditch (0.146°C ºC^sup -1^), reflecting body mass and environment. Basal metabolic rate was higher for eastern quolls (0.662 ± 0.033 ml O^sub 2^ g^sup -1^ h^sup -1^), presumably reflecting their naturally cool environment. Respiratory ventilation closely matched metabolic demand, except at high ambient temperatures where quolls hyperventilated to facilitate evaporative heat loss; tiger and eastern quolls also salivated. A higher evaporative water loss for eastern quolls (1.43 ± 0.212 mg H^sub 2^O g^sup -1^ h^sup -1^) presumably reflects their more mesic distribution. The point of relative water economy was low for tiger (-1.3°C), eastern (-12.5°C) and northern (+3.3) quolls, and highest for the chuditch (+22.6°C). We suggest that these differences in water economy reflect lower expired air temperatures and hence lower respiratory evaporative water loss for the arid-zone chuditch relative to tropical and mesic quolls

Interactions among mitochondrial proteins altered in glioblastoma

Mitochondrial dysfunction is putatively central to glioblastoma (GBM) pathophysiology but there has been no systematic analysis in GBM of the proteins which are integral to mitochondrial function. Alterations in proteins in mitochondrial enriched fractions from patients with GBM were defined with label-free liquid chromatography mass spectrometry. 256 mitochondrially-associated proteins were identified in mitochondrial enriched fractions and 117 of these mitochondrial proteins were markedly (fold-change ≥2) and significantly altered in GBM (p ≤ 0.05). Proteins associated with oxidative damage (including catalase, superoxide dismutase 2, peroxiredoxin 1 and peroxiredoxin 4) were increased in GBM. Protein–protein interaction analysis highlighted a reduction in multiple proteins coupled to energy metabolism (in particular respiratory chain proteins, including 23 complex-I proteins). Qualitative ultrastructural analysis in GBM with electron microscopy showed a notably higher prevalence of mitochondria with cristolysis in GBM. This study highlights the complex mitochondrial proteomic adjustments which occur in GBM pathophysiology

Time-Lapse Imaging of the Dynamics of CNS Glial-Axonal Interactions In Vitro and Ex Vivo

Myelination is an exquisite and dynamic example of heterologous cell-cell interaction, which consists of the concentric wrapping of multiple layers of oligodendrocyte membrane around neuronal axons. Understanding the mechanism by which oligodendrocytes ensheath axons may bring us closer to designing strategies to promote remyelination in demyelinating diseases. The main aim of this study was to follow glial-axonal interactions over time both in vitro and ex vivo to visualize the various stages of myelination.We took two approaches to follow myelination over time: i) time-lapse imaging of mixed CNS myelinating cultures generated from mouse spinal cord to which exogenous GFP-labelled murine cells were added, and ii) ex vivo imaging of the spinal cord of shiverer (Mbp mutant) mice, transplanted with GFP-labelled murine neurospheres. We demonstrate that oligodendrocyte-axonal interactions are dynamic events with continuous retraction and extension of oligodendroglial processes. Using cytoplasmic and membrane-GFP labelled cells to examine different components of the myelin-like sheath, we provide evidence from time-lapse fluorescence microscopy and confocal microscopy that the oligodendrocytes' cytoplasm-filled processes initially spiral around the axon in a corkscrew-like manner. This is followed subsequently by focal expansion of the corkscrew process to form short cuffs, which then extend longitudinally along the axons. We predict from this model that these spiral cuffs must extend over each other first before extending to form internodes of myelin.These experiments show the feasibility of visualizing the dynamics of glial-axonal interaction during myelination over time. Moreover, these approaches complement each other with the in vitro approach allowing visualization of an entire internodal length of myelin and the ex vivo approach validating the in vitro data

Functional divergence in the role of N-linked glycosylation in smoothened signaling

The G protein-coupled receptor (GPCR) Smoothened (Smo) is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh) pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Gαi. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. Herein, we present a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, we found that all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Gαi, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, we postulate a profound shift in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice