Glia require CerPE for axonal ensheathment.

<p>(A) A genetic dissection study of sphingolipid biosynthetic pathway shows that glial knockdown of <i>Spt-I</i>, <i>schlank</i>, <i>Des1</i> and <i>Pect</i> result in swelling and wrapping defects (<i>repo-GAL4</i>) similar to the <i>lace</i> phenotype. Merged projections of confocal stacks are presented. Scale 20 µm. (B) Sphingolipid biosynthesis pathway. (C) RNAi against these 4 genes were expressed in neurons by using <i>elav-GAL4</i> driver line. HRP (red) is used to visualize the membrane morphology of the neurons. Scale 20 µm. (D) Quantification (mean± SD) showed that the swellings were only observed upon glial specific knockdown but not in neuron-specific knockdown.</p

<i>lace</i> is expressed in glial cells.

<p>(A, B) Quantification of total glial cell number (mean±SD) upon <i>lace</i> knockdown in glia. Counting the number of glial nuclei in A8 peripheral nerve (n = 8) revealed no significant differences between control and <i>lace</i> RNAi larva. (C) Double immunolabeling of anti-β-galactosidase (red) and anti-repo (green) of <i>lace⁵</i> (LacZ enhancer trap line) in L3 larval peripheral nerves. Colocalization shows that lace is expressed in glial cells, present in the peripheral abdominal nerves. (D) RT-PCR analysis showed that lace is expressed in the nervous system of both males and female flies. (E) Hypomorphic combination of <i>lace</i> mutant (<i>lace²/lace⁵</i>) showed axonal defasciculation and increase in the cross-section area of the nerve (arrows). HRP (red) and mCD8-GFP (green) were used to visualize the neuronal and glial morphology, respectively. (F) Quantification showed that the expression of <i>UAS-lace</i> by <i>repo-GAL4</i> could rescue the mutant phenotype. Scale bar 20 µm. All graphs represent mean values ± SD. Unpaired <i>t</i>-test (two groups) and One-way ANOVA followed by Tukey <i>post hoc</i> test (for three groups) were performed for the statistical analysis. Scale 20 µm. ** p<0.01 *** p<0.001. ns not significant.</p

Lipidomics analysis.

<p>Lipidomics analysis using high-resolution shotgun mass spectrometry of dissected brains and peripheral nerves derived from L3 larvae. <i>lace</i>, <i>schlank</i>, <i>Des1</i> and <i>Pect</i> were downregulated in both neuron and glia, L3 larval brain and peripheral nerves were dissected and the amount of sphingolipid (A), sterol (B), neutral lipid (C) and phospholipids (D) were determined. One-way ANOVA with Dunnett <i>post hoc</i> test was used for the statistical analysis. All graphs represent mean values ± SD. * p<0.05 ** p<0.01 *** p<0.001.</p

Glial inhibition of <i>lace</i> delays afferent spike propagation.

<p>(A) Scheme of thoracic and abdominal parts of a dissected larva, electrodes not to scale; ag abdominal ganglion mass, se1, re1 anterior suction electrode and reference electrode, se2, re2 posterior suction electrode and reference electrode. (B) Afferent unit (top) and efferent unit (bottom) recorded simultaneously in 7th right nerve of a <i>repo>mCD8-GFP/+</i> control as sketched in (A). Spike templates were generated for se2 and served as trigger events for averaging both se1 and se2. Δt conduction time; bars 50 µV and 1 ms, respectively. (C) Distribution of conduction speed recorded in <i>repo>mCD8-GFP/+</i> (white bars) and <i>repo>mCD8-GFP/lace</i> RNAi (black bars) animals from afferent (top) and efferent (bottom) units. Horizontal lines indicate medians and their 95% confidence interval of the respective distributions. Difference of medians: * p<0.05 (two-tailed), ns not significant. n number of neurons, recorded from 20 mutant and 9 control larvae.</p

Primary screening and <i>lace</i> phenotype.

<p>(A) Scheme of the screening strategy. (B) Pre-screening result with <i>nejire</i> RNAi with <i>tub-GAL80^ts; repo-GAL4</i>. Survival curves were analyzed with Log-Rank Mantel Cox test. p<0.0001 (C) Results from the primary screening reveal 861 RNAi lines with lethality and 30 RNAi lines with impaired locomotion. (D) Peripheral nerves of L3 larval PNS, glial membrane (green) swelling and axonal (red) wrapping defect were observed upon knockdown of <i>lace</i> with 2 different RNAi lines (Transformant ID 21803, 110181). <i>repo>mCD8-GFP/+</i> served as a control. Merged projection of confocal z-stacks is presented. In insets, orthogonal sections of the nerve region marked by a white line are shown (E) Quantification of the average cross-section of the peripheral nerves after glial specific knockdown of <i>lace</i>. (F) Merged projection of confocal z-stacks did not reveal any visible morphological changes in the neuronal morphology in the PNS after knockdown of <i>lace</i> specifically in the neurons (HRP). (G) Quantification of the average cross-section of the peripheral nerves after neuron-specific knockdown of <i>lace</i>. For the statistical analysis, unpaired <i>t-</i>test was performed with the respective control. Scale bar 20 µm. ns not significant. All graphs represent mean values ± SD. ** p<0.01 *** p<0.001.</p

Sphingosine rescues the glial bulging phenotype.

<p>(A) Addition of sphingosine to diet rescues the <i>lace</i> phenotype. Arrowheads indicate glial swelling region. (B) Orthogonal projections showed that dietary addition of sphingosine rescues the enwrapment defect. (C) Quantification of average cross-section area after knockdown of <i>lace</i> in glia and upon sphingosine treatment is shown. All graphs represent mean values ± SD. One-way ANOVA followed by Tukey <i>post hoc</i> test was performed for statistical analysis. Scale 20 µm (D1–D3) TEM micrographs of L3 larval peripheral nerve cross-sections are shown. Wrapping glia is color-coded. Vacuoles (blue arrowhead) are present in the wrapping glia and there is loss of membrane extension resulting in lack of proper ensheathement in <i>repo-GAL4</i>/<i>lace</i> RNAi flies as compared to control <i>repo-GAL4/+</i> flies. (D3) Addition of 300 µM sphingosine to the diet (Matreya, USA) rescues the ensheathment defect. (E) Quantification of the number of unwrapped axons before and after sphingosine treatment in the in <i>lace</i> knocked down flies. (repo-GAL4/laceRNAi). One-way ANOVA followed by Tukey <i>post hoc</i> test was performed. All graphs represent mean values ±SD. *** p<0.001.</p

CerPE is very essential for axonal ensheathment by wrapping glia.

<p>(A) RNAi against Spt-I, schlank, Des1 and Pect were expressed in wrapping glia (Nrv2-GAL4). Merged projections of the peripheral nerves along with an orthogonal projection are presented. GFP (green) was used to label the wrapping glial membrane, and HRP (red) and repo (blue) were used to label the neuronal membrane and glial nuclei, respectively. (B) TEM micrographs of L3 larval peripheral nerve cross-sections are shown. Wrapping glia is color-coded. Axonal ensheathment is incomplete upon loss of lace function in wrapping glia. Proper ensheathment of axons is observed only in control (+). Scale 1 mm. (C) Quantification of GFP signal intensity of wrapping glial membrane showed a reduction of GFP signal intensity. (D) Quantification of the number of unwrapped axons. One-way ANOVA followed by Dunnett post hoc test was performed. All graphs represent mean values 6 SD. * p,0.05, ** p,0.01, *** p,0.001.</p

Myelin Membrane Assembly Is Driven by a Phase Transition of Myelin Basic Proteins Into a Cohesive Protein Meshwork

<div><p>Rapid conduction of nerve impulses requires coating of axons by myelin. To function as an electrical insulator, myelin is generated as a tightly packed, lipid-rich multilayered membrane sheath. Knowledge about the mechanisms that govern myelin membrane biogenesis is required to understand myelin disassembly as it occurs in diseases such as multiple sclerosis. Here, we show that myelin basic protein drives myelin biogenesis using weak forces arising from its inherent capacity to phase separate. The association of myelin basic protein molecules to the inner leaflet of the membrane bilayer induces a phase transition into a cohesive mesh-like protein network. The formation of this protein network shares features with amyloid fibril formation. The process is driven by phenylalanine-mediated hydrophobic and amyloid-like interactions that provide the molecular basis for protein extrusion and myelin membrane zippering. These findings uncover a physicochemical mechanism of how a cytosolic protein regulates the morphology of a complex membrane architecture. These results provide a key mechanism in myelin membrane biogenesis with implications for disabling demyelinating diseases of the central nervous system.</p> </div

Phase transition of wild-type, but not the F→S mutant of MBP.

<p>(A) In basic solution MBP (5 mg/mL) forms droplets as visualized by phase contrast microscopy. (B) Droplets contain Atto-488-labeled MBP (5 mg/mL) as visualized by wide field (right panel) microscopy. (C) Time-lapse images of two merging droplets. Scale bar, 5 µm.</p

Self-association of MBP molecules via hydrophobic interactions is required for its function.

<p>(A) Quantification of FRET efficiency in PtK2 cells expressing GFP-Tm10 (Donor) and mCherry-GyPTM (Acceptor) both harboring at the C-terminal end either wild-type MBP or MBP F→S. While Tm10 represents the transmembrane domain of Tmem10, GyPTM represents the mutated monomeric transmembrane domain of the glycophorin protein. Bars indicate mean ± SD (<i>n</i> = 20 cells, *<i>p</i><0.05, ANOVA). (B) Comparison of interaction forces between wild-type MBP or F→S mutant molecules pre-adsorbed, both to the mica surface and AFM tip. Inset shows the schematic depiction of shape of the curve as cantilever tip approaches the sample surface (1), as tip touches the surface (2), and as tip is retracted from the sample surface (3). Histogram of peak force measured for MBP (black), MBP F→S (red), and buffer (green). (C) Representative images of a biomimetic assay in which MBP or MBP F→S is sandwiched between SLBs (inner myelin leaflet lipid composition) and GUVs (PC∶PS in 3∶1 mole%). Scale bar, 10 µm. (D) Quantification of percentage GUV bursting. Bars show mean ± SEM (<i>n</i> = 3 experiments, ***<i>p</i><0.001, <i>t</i> test). (E) <i>Shiverer</i> cells at 0 DIV were infected with AAV2 viral particles expressing either wild-type MBP (MBP-HA) or F→S mutant (MBP F→S-HA) containing a C-terminal HA-tag. At 6 DIV, cells were immunostained for CNPase and the HA tags. Expression of MBP-HA induces the depletion of CNPase from regions within the sheets, whereas the F→S mutant fails in extruding CNPase despite entering the sheets of <i>shiverer</i> cells. Enlarged view of the selected regions in merged images is shown on the right side. Scale bar, 10 µm.</p