Aging differentially alters the transcriptome and landscape of chromatin accessibility in the male and female mouse hippocampus.

Aging-related memory impairment and pathological memory disorders such as Alzheimer's disease differ between males and females, and yet little is known about how aging-related changes in the transcriptome and chromatin environment differ between sexes in the hippocampus. To investigate this question, we compared the chromatin accessibility landscape and gene expression/alternative splicing pattern of young adult and aged mouse hippocampus in both males and females using ATAC-seq and RNA-seq. We detected significant aging-dependent changes in the expression of genes involved in immune response and synaptic function and aging-dependent changes in the alternative splicing of myelin sheath genes. We found significant sex-bias in the expression and alternative splicing of hundreds of genes, including aging-dependent female-biased expression of myelin sheath genes and aging-dependent male-biased expression of genes involved in synaptic function. Aging was associated with increased chromatin accessibility in both male and female hippocampus, especially in repetitive elements, and with an increase in LINE-1 transcription. We detected significant sex-bias in chromatin accessibility in both autosomes and the X chromosome, with male-biased accessibility enriched at promoters and CpG-rich regions. Sex differences in gene expression and chromatin accessibility were amplified with aging, findings that may shed light on sex differences in aging-related and pathological memory loss.

KymoAnalyzer: a software tool for the quantitative analysis of intracellular transport in neurons.

In axons, proper localization of proteins, vesicles, organelles, and other cargoes is accomplished by the highly regulated coordination of kinesins and dyneins, molecular motors that bind to cargoes and translocate them along microtubule (MT) tracks. Impairment of axonal transport is implicated in the pathogenesis of multiple neurodegenerative disorders including Alzheimer's and Huntington's diseases. To understand how MT-based cargo motility is regulated and to delineate its role in neurodegeneration, it is critical to analyze the detailed dynamics of moving cargoes inside axons. Here, we present KymoAnalyzer, a software tool that facilitates the robust analysis of axonal transport from time-lapse live-imaging sequences. KymoAnalyzer is an open-source software that automatically classifies particle trajectories and systematically calculates velocities, run lengths, pauses, and a wealth of other parameters that are characteristic of motor-based transport. We anticipate that laboratories will easily use this package to unveil previously uncovered intracellular transport details of individually-moving cargoes inside neurons.

A hemi-fission intermediate links two mechanistically distinct stages of membrane fission.

Fusion and fission drive all vesicular transport. Although topologically opposite, these reactions pass through the same hemi-fusion/fission intermediate, characterized by a 'stalk' in which only the outer membrane monolayers of the two compartments have merged to form a localized non-bilayer connection. Formation of the hemi-fission intermediate requires energy input from proteins catalysing membrane remodelling; however, the relationship between protein conformational rearrangements and hemi-fusion/fission remains obscure. Here we analysed how the GTPase cycle of human dynamin 1, the prototypical membrane fission catalyst, is directly coupled to membrane remodelling. We used intramolecular chemical crosslinking to stabilize dynamin in its GDP·AlF4(-)-bound transition state. In the absence of GTP this conformer produced stable hemi-fission, but failed to progress to complete fission, even in the presence of GTP. Further analysis revealed that the pleckstrin homology domain (PHD) locked in its membrane-inserted state facilitated hemi-fission. A second mode of dynamin activity, fuelled by GTP hydrolysis, couples dynamin disassembly with cooperative diminishing of the PHD wedging, thus destabilizing the hemi-fission intermediate to complete fission. Molecular simulations corroborate the bimodal character of dynamin action and indicate radial and axial forces as dominant, although not independent, drivers of hemi-fission and fission transformations, respectively. Mirrored in the fusion reaction, the force bimodality might constitute a general paradigm for leakage-free membrane remodelling.

Characterizing the composition of molecular motors on moving axonal cargo using "cargo mapping" analysis.

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative analysis of motor/cargo associations at the single vesicle level. The goal of this protocol is to use quantitative fluorescence microscopy to correlate ("map") the position and directionality of movement of live cargo to the composition and relative amounts of motors associated with the same cargo. "Cargo mapping" consists of live imaging of fluorescently labeled cargoes moving in axons cultured on microfluidic devices, followed by chemical fixation during recording of live movement, and subsequent immunofluorescence (IF) staining of the exact same axonal regions with antibodies against motors. Colocalization between cargoes and their associated motors is assessed by assigning sub-pixel position coordinates to motor and cargo channels, by fitting Gaussian functions to the diffraction-limited point spread functions representing individual fluorescent point sources. Fixed cargo and motor images are subsequently superimposed to plots of cargo movement, to "map" them to their tracked trajectories. The strength of this protocol is the combination of live and IF data to record both the transport of vesicular cargoes in live cells and to determine the motors associated to these exact same vesicles. This technique overcomes previous challenges that use biochemical methods to determine the average motor composition of purified heterogeneous bulk vesicle populations, as these methods do not reveal compositions on single moving cargoes. Furthermore, this protocol can be adapted for the analysis of other transport and/or trafficking pathways in other cell types to correlate the movement of individual intracellular structures with their protein composition. Limitations of this protocol are the relatively low throughput due to low transfection efficiencies of cultured primary neurons and a limited field of view available for high-resolution imaging. Future applications could include methods to increase the number of neurons expressing fluorescently labeled cargoes.

Dual role of BAR domain-containing proteins in regulating vesicle release catalyzed by the GTPase, dynamin-2.

Dynamin-2 (Dyn2) is ubiquitously expressed and catalyzes membrane fission during clathrin-mediated endocytosis in nonneuronal cells. We have previously shown that Dyn2 inefficiently generates membrane curvature and only mediates fission of highly curved membranes. This led to the hypothesis that other endocytic accessory proteins (EAPs) generate curvature needed to sculpt a sufficiently narrow neck to trigger Dyn2 assembly and fission. Candidates for this activity are EAPs that bind to the dynamin proline/arginine-rich domain (PRD) through their SH3 (src homology-3) domains and also encode curvature-generating BAR (Bin/Amphiphysin/Rvs) domains. We show that at low concentrations, amphiphysin and endophilin, but not SNX9 or the curvature-generating epsin N-terminal homology (ENTH) domain, are able to generate tubules from planar membrane templates and to synergize with Dyn2ΔPRD to catalyze vesicle release. Unexpectedly, SH3-PRD interactions were inhibitory and reciprocally regulate scaffold assembly. Of the three proteins studied, only full-length amphiphysin functions synergistically with full-length Dyn2 to catalyze vesicle release. The differential activity of these proteins correlates with the relative potency of their positive, curvature-generating activity, and the negative regulatory effects mediated by SH3 domain interactions. Our findings reveal opportunities for the spatio-temporal coordination of membrane curvature generation, dynamin assembly, and fission during clathrin-mediated endocytosis.

Analyzing membrane remodeling and fission using supported bilayers with excess membrane reservoir.

A complete understanding of the molecular mechanisms governing vesicle formation requires quantitative assays and vesicle reconstitution using purified components. We describe a simple model membrane template for studying protein-mediated membrane remodeling and vesicle formation or fission that is amenable to both quantitative biochemical analysis and real-time imaging by epifluorescence microscopy. Supported bilayers with excess membrane reservoir (SUPER) templates are compositionally well-defined unilamellar membrane systems prepared on 2-5-µm silica beads under conditions that enable incorporation of excess membrane to form a loosely fitting bilayer that can be used to study membrane remodeling and fission. This protocol describes methods for SUPER template formation and characterization, as well as for the qualitative observation and quantitative measurement of vesicle formation and fission via microscopy and a simple sedimentation assay. SUPER templates can be prepared within 60 min. Results from either sedimentation-based or microscopy-based assays can be obtained within an additional 60 min.

Hyperacidification of trans-Golgi network and endo/lysosomes in melanocytes by glucosylceramide-dependent V-ATPase activity.

Sphingolipids are considered to play a key role in protein sorting and membrane trafficking. In melanocytic cells, sorting of lysosomal and melanosomal proteins requires the sphingolipid glucosylceramide (GlcCer). This sorting information is located in the lumenal domain of melanosomal proteins. We found that two processes dependent on lumenal pH, protein sialylation and lysosomal acid lipase (LAL) activity were aberrant in GM95 melanocyte cells, which do not produce glycosphingolipids. Using fluorescence lifetime imaging microscopy (FLIM), we found that the lumenal pH in the trans-Golgi network and lysosomes of wild-type melanocyte MEB4 cells are >1 pH unit lower than GM95 cells and fibroblasts. In addition to the lower pH found in vivo, the in vitro activity of the proton pump, the vacuolar-type H(+) -translocating ATPase (V-ATPase), was twofold higher in MEB4 compared to GM95 cells. The apparent K(i) for inhibition of the V-ATPase by concanamycin A and archazolid A, which share a common binding site on the c-ring, was lower in glycosphingolipid-deficient GM95 cells. No difference between the MEB4 and GM95 cells was found for the V-ATPase inhibitors apicularen A and salicylihalimide. We conclude that hyperacidification in MEB4 cells requires glycosphingolipids and propose that low pH is necessary for protein sorting and melanosome biogenesis. Furthermore, we suggest that glycosphingolipids are indirectly involved in protein sorting and melanosome biogenesis by stimulating the proton pump, possibly through binding of GlcCer. These experiments establish, for the first time, a link between pH, glycosphingolipids and melanosome biogenesis in melanocytic MEB4 cells, to suggest a role for glycosphingolipids in hyperacidification in melanocytes.

Differential curvature sensing and generating activities of dynamin isoforms provide opportunities for tissue-specific regulation.

Dynamin 1 (Dyn1) and Dyn2 are neuronal and ubiquitously expressed isoforms, respectively, of the multidomain GTPase required for clathrin-mediated endocytosis (CME). Although they are 79% identical, Dyn1 and Dyn2 are not fully functionally redundant. Through direct measurements of basal and assembly-stimulated GTPase activities, membrane binding, self-assembly, and membrane fission on planar and curved templates, we have shown that Dyn1 is an efficient curvature generator, whereas Dyn2 is primarily a curvature sensor. Using Dyn1/Dyn2 chimeras, we identified the lipid-binding pleckstrin homology domain as being responsible for the differential in vitro properties of these two isoforms. Remarkably, their in vitro activities were reversed by a single amino acid change in the membrane-binding variable loop 3. Reconstitution of KO mouse embryo fibroblasts showed that both the pleckstrin homology and the Pro/Arg-rich domains determine the differential abilities of these two isoforms to support CME. These domains are specific to classical dynamins and are involved in regulating their activity. Our findings reveal opportunities for fundamental differences in the regulation of Dyn1, which mediates rapid endocytosis at the synapse, vs. Dyn2, which regulates early and late events in CME in nonneuronal cells.

Mammalian Wnt3a is released on lipoprotein particles.

Little is known about the release and intercellular transport of Wnt proteins from mammalian cells. Lipoproteins may act as carriers for the intercellular movement and gradient formation of the lipid-linked morphogens Wingless and Hedgehog in Drosophila. To investigate whether such a mechanism can occur in mammals, we have studied Wnt release in cultured mammalian cells. Wnt3a associated with lipoproteins in the culture medium and not with extracellular vesicles or exosomes. Although Wnt3a was associated with both high-density lipoproteins (HDL) and low-density lipoproteins, only HDL allowed Wnt3a release from mouse fibroblasts. Remarkably, Wnt3a lacking its palmitate moiety was released in a lipoprotein-independent manner, demonstrating the dual role of palmitoylation in membrane and lipoprotein binding. We additionally found that Wnt3a can be released from enterocyte cell lines on endogenously expressed lipoproteins. We further discuss the physiological implications of our findings.

Sphingolipid management by an orchestra of lipid transfer proteins.

The various membranes in eukaryotic cells have unique lipid compositions. Despite important discoveries in lipid research over recent decades, the basic principles by which cells define their membrane compositions are essentially unknown. Cells must sense the concentration of each lipid, integrate such signals and regulate the activity of their metabolic enzymes and transport routes to dynamically meet their needs in terms of membrane composition. Sphingolipids constitute a lipid category that is essential for eukaryotic life and appears to be key to differences in lipid composition. Here we discuss recent findings that assign an important role to lipid transfer proteins in the regulation of sphingolipid metabolism, organization and function.

Glycolipid-dependent sorting of melanosomal from lysosomal membrane proteins by lumenal determinants.

Melanosomes are lysosome-related organelles that coexist with lysosomes in mammalian pigment cells. Melanosomal and lysosomal membrane proteins share similar sorting signals in their cytoplasmic tail, raising the question how they are segregated. We show that in control melanocytes, the melanosomal enzymes tyrosinase-related protein 1 (Tyrp1) and tyrosinase follow an intracellular Golgi to melanosome pathway, whereas in the absence of glycosphingolipids, they are observed to pass over the cell surface. Unexpectedly, the lysosome-associated membrane protein 1 (LAMP-1) and 2 behaved exactly opposite: they were found to travel through the cell surface in control melanocytes but followed an intracellular pathway in the absence of glycosphingolipids. Chimeric proteins having the cytoplasmic tail of Tyrp1 or tyrosinase were transported like lysosomal proteins, whereas a LAMP-1 construct containing the lumenal domain of Tyrp1 localized to melanosomes. In conclusion, the lumenal domain contains sorting information that guides Tyrp1 and probably tyrosinase to melanosomes by an intracellular route that excludes lysosomal proteins and requires glucosylceramide.

Pre- and post-Golgi translocation of glucosylceramide in glycosphingolipid synthesis.

Glycosphingolipids are controlled by the spatial organization of their metabolism and by transport specificity. Using immunoelectron microscopy, we localize to the Golgi stack the glycosyltransferases that produce glucosylceramide (GlcCer), lactosylceramide (LacCer), and GM3. GlcCer is synthesized on the cytosolic side and must translocate across to the Golgi lumen for LacCer synthesis. However, only very little natural GlcCer translocates across the Golgi in vitro. As GlcCer reaches the cell surface when Golgi vesicular trafficking is inhibited, it must translocate across a post-Golgi membrane. Concanamycin, a vacuolar proton pump inhibitor, blocks translocation independently of multidrug transporters that are known to translocate short-chain GlcCer. Concanamycin did not reduce LacCer and GM3 synthesis. Thus, GlcCer destined for glycolipid synthesis follows a different pathway and transports back into the endoplasmic reticulum (ER) via the late Golgi protein FAPP2. FAPP2 knockdown strongly reduces GM3 synthesis. Overall, we show that newly synthesized GlcCer enters two pathways: one toward the noncytosolic surface of a post-Golgi membrane and one via the ER toward the Golgi lumen LacCer synthase.

Hitch-hiking between cells on lipoprotein particles.

Cell surface proteins containing covalently linked lipids associate with specialized membrane domains. Morphogens like Hedgehog and Wnt use their lipid anchors to bind to lipoprotein particles and employ lipoproteins to travel through tissues. Removal of their lipid anchors or decreasing lipoprotein levels give rise to adverse Hedgehog and Wnt signaling. Some parasites can also transfer their glycosylphosphatidylinositol-anchored surface proteins to host lipoprotein particles. These antigen-loaded lipoproteins spread throughout the circulation, and probably hamper an adequate immune response by killing neutrophils. Together, these findings imply a widespread role for lipoproteins in intercellular transfer of lipid-anchored surface proteins, and may have various physiological consequences. Here, we discuss how lipid-modified proteins may be transferred to and from lipoproteins at the cellular level.