Mycobacterium tuberculosis encodes a YhhN family membrane protein with lysoplasmalogenase activity that protects against toxic host lysolipids.

The pathogen Mycobacterium tuberculosis (M.tb) resides in human macrophages, wherein it exploits host lipids for survival. However, little is known about the interaction between M.tb and macrophage plasmalogens, a subclass of glycerophospholipids with a vinyl ether bond at the sn-1 position of the glycerol backbone. Lysoplasmalogens, produced from plasmalogens by hydrolysis at the sn-2 carbon by phospholipase A2, are potentially toxic but can be broken down by host lysoplasmalogenase, an integral membrane protein of the YhhN family that hydrolyzes the vinyl ether bond to release a fatty aldehyde and glycerophospho-ethanolamine or glycerophospho-choline. Curiously, M.tb encodes its own YhhN protein (MtbYhhN), despite having no endogenous plasmalogens. To understand the purpose of this protein, the gene for MtbYhhN (Rv1401) was cloned and expressed in Mycobacterium smegmatis (M.smeg). We found the partially purified protein exhibited abundant lysoplasmalogenase activity specific for lysoplasmenylethanolamine or lysoplasmenylcholine (pLPC) (Vmax∼15.5 µmol/min/mg; Km∼83 µM). Based on cell density, we determined that lysoplasmenylethanolamine, pLPC, lysophosphatidylcholine, and lysophosphatidylethanolamine were not toxic to M.smeg cells, but pLPC and LPC were highly toxic to M.smeg spheroplasts, which are cell wall-deficient mycobacterial forms. Importantly, spheroplasts prepared from M.smeg cells overexpressing MtbYhhN were protected from membrane disruption/lysis by pLPC, which was rapidly depleted from the media. Finally, we found that overexpression of full-length MtbYhhN in M.smeg increased its survival within human macrophages by 2.6-fold compared to vector controls. These data support the hypothesis that MtbYhhN protein confers a growth advantage for mycobacteria in macrophages by cleaving toxic host pLPC into potentially energy-producing products.

Local Acceleration of Neurofilament Transport at Nodes of Ranvier.

Myelinated axons are constricted at nodes of Ranvier. These constrictions are important physiologically because they increase the speed of saltatory nerve conduction, but they also represent potential bottlenecks for the movement of axonally transported cargoes. One type of cargo are neurofilaments, which are abundant space-filling cytoskeletal polymers that function to increase axon caliber. Neurofilaments move bidirectionally along axons, alternating between rapid movements and prolonged pauses. Strikingly, axon constriction at nodes is accompanied by a reduction in neurofilament number that can be as much as 10-fold in the largest axons. To investigate how neurofilaments navigate these constrictions, we developed a transgenic mouse strain that expresses a photoactivatable fluorescent neurofilament protein in neurons. We used the pulse-escape fluorescence photoactivation technique to analyze neurofilament transport in mature myelinated axons of tibial nerves from male and female mice of this strain ex vivo Fluorescent neurofilaments departed the activated region more rapidly in nodes than in flanking internodes, indicating that neurofilament transport is faster in nodes. By computational modeling, we showed that this nodal acceleration can be explained largely by a local increase in the duty cycle of neurofilament transport (i.e., the proportion of the time that the neurofilaments spend moving). We propose that this transient acceleration functions to maintain a constant neurofilament flux across nodal constrictions, much as the current increases where a river narrows its banks. In this way, neurofilaments are prevented from piling up in the flanking internodes, ensuring a stable neurofilament distribution and uniform axonal morphology across these physiologically important axonal domains.SIGNIFICANCE STATEMENT Myelinated axons are constricted at nodes of Ranvier, resulting in a marked local decrease in neurofilament number. These constrictions are important physiologically because they increase the efficiency of saltatory nerve conduction, but they also represent potential bottlenecks for the axonal transport of neurofilaments, which move along axons in a rapid intermittent manner. Imaging of neurofilament transport in mature myelinated axons ex vivo reveals that neurofilament polymers navigate these nodal axonal constrictions by accelerating transiently, much as the current increases where a river narrows its banks. This local acceleration is necessary to ensure a stable axonal morphology across nodal constrictions, which may explain the vulnerability of nodes of Ranvier to neurofilament accumulations in animal models of neurotoxic neuropathies and neurodegenerative diseases.

Primary cilia enhance kisspeptin receptor signaling on gonadotropin-releasing hormone neurons.

Most central neurons in the mammalian brain possess an appendage called a primary cilium that projects from the soma into the extracellular space. The importance of these organelles is highlighted by the fact that primary cilia dysfunction is associated with numerous neuropathologies, including hyperphagia-induced obesity, hypogonadism, and learning and memory deficits. Neuronal cilia are enriched for signaling molecules, including certain G protein-coupled receptors (GPCRs), suggesting that neuronal cilia sense and respond to neuromodulators in the extracellular space. However, the impact of cilia on signaling to central neurons has never been demonstrated. Here, we show that the kisspeptin receptor (Kiss1r), a GPCR that is activated by kisspeptin to regulate the onset of puberty and adult reproductive function, is enriched in cilia projecting from mouse gonadotropin-releasing hormone (GnRH) neurons. Interestingly, GnRH neurons in adult animals are multiciliated and the percentage of GnRH neurons possessing multiple Kiss1r-positive cilia increases during postnatal development in a progression that correlates with sexual maturation. Remarkably, disruption of cilia selectively on GnRH neurons leads to a significant reduction in kisspeptin-mediated GnRH neuronal activity. To our knowledge, this result is the first demonstration of cilia disruption affecting central neuronal activity and highlights the importance of cilia for proper GPCR signaling.

Severing and end-to-end annealing of neurofilaments in neurons.

We have shown previously that neurofilaments and vimentin filaments expressed in nonneuronal cell lines can lengthen by joining ends in a process known as "end-to-end annealing." To test if this also occurs for neurofilaments in neurons, we transfected cultured rat cortical neurons with fluorescent neurofilament fusion proteins and then used photoconversion or photoactivation strategies to create distinct populations of red and green fluorescent filaments. Within several hours we observed the appearance of chimeric filaments consisting of alternating red and green segments, which is indicative of end-to-end annealing of red and green filaments. However, the appearance of these chimeric filaments was accompanied by a gradual fragmentation of the red and green filament segments, which is indicative of severing. Over time we observed a progressive increase in the number of red-green junctions along the filaments accompanied by a progressive decrease in the average length of the alternating red and green fluorescent segments that comprised those filaments, suggesting a dynamic cycle of severing and end-to-end-annealing. Time-lapse imaging of the axonal transport of chimeric filaments demonstrated that the red and green segments moved together, confirming that they were indeed part of the same filament. Moreover, in several instances, we also were able to capture annealing and severing events live in time-lapse movies. We propose that the length of intermediate filaments in cells is regulated by the opposing actions of severing and end-to-end annealing, and we speculate that this regulatory mechanism may influence neurofilament transport within axons.

Local regulation of neurofilament transport by myelinating cells.

Axons in the vertebrate nervous system only expand beyond ∼ 1 µm in diameter if they become myelinated. This expansion is due in large part to the accumulation of space-filling cytoskeletal polymers called neurofilaments, which are cargoes of axonal transport. One possible mechanism for this accumulation is a decrease in the rate of neurofilament transport. To test this hypothesis, we used a fluorescence photoactivation pulse-escape technique to compare the kinetics of neurofilament transport in contiguous myelinated and unmyelinated segments of axons in long-term myelinating cocultures established from the dorsal root ganglia of embryonic rats. The myelinated segments contained more neurofilaments and had a larger cross-sectional area than the contiguous unmyelinated segments, and this correlated with a local slowing of neurofilament transport. By computational modeling of the pulse-escape kinetics, we found that this slowing of neurofilament transport could be explained by an increase in the proportion of the time that the neurofilaments spent pausing and that this increase in pausing was sufficient to explain the observed neurofilament accumulation. Thus we propose that myelinating cells can regulate the neurofilament content and morphology of axons locally by modulating the kinetics of neurofilament transport.

Axonal neurofilaments exhibit frequent and complex folding behaviors.

Neurofilaments are flexible cytoskeletal polymers that are capable of folding and unfolding between their bouts of bidirectional movement along axons. Here we present a detailed characterization of this behavior in cultured neurons using kymograph analysis with approximately 30 ms temporal resolution. We analyzed 781 filaments ranging from 0.6-42 µm in length. We observed complex behaviors including pinch folds, hairpin folds, orientation changes (flips), and occasional severing and annealing events. On average, the filaments spent approximately 40% of their time in some sort of folded configuration. A small proportion of filaments (4%) moved while folded, but most (96%) moved in an outstretched configuration. Collectively, our observations suggest that motors may interact with neurofilaments at multiple points along their length, but preferentially at their ends. In addition, the prevalence of neurofilament folding and the tendency of neurofilaments to straighten out when they move, suggest that an important function of the movement of these polymers in axons may be to maintain them in an outstretched and longitudinally co-aligned configuration. Thus, neurofilament movement may function as much to organize these polymers as to move them, and this could explain why they spend so much time engaged in apparently unproductive bidirectional movement.

Live-cell imaging of neurofilament transport in cultured neurons.

Neurofilaments, which are the intermediate filaments of nerve cells, are space-filling cytoskeletal polymers that contribute to the growth of axonal caliber. In addition to their structural role, neurofilaments are cargos of axonal transport that move along microtubule tracks in a rapid, intermittent, and bidirectional manner. Though they measure just 10nm in diameter, which is well below the diffraction limit of optical microscopes, these polymers can reach 100 µm or more in length and are often packed densely, just tens of nanometers apart. These properties of neurofilaments present unique challenges for studies on their movement. In this article, we describe several live-cell fluorescence imaging strategies that we have developed to image neurofilament transport in axons of cultured neurons on short and long timescales. Together, these methods form a powerful set of complementary tools with which to study the axonal transport of these unique intracellular cargos.

Recruitment of ß-Arrestin into Neuronal Cilia Modulates Somatostatin Receptor Subtype 3 Ciliary Localization.

Primary cilia are essential sensory and signaling organelles present on nearly every mammalian cell type. Defects in primary cilia underlie a class of human diseases collectively termed ciliopathies. Primary cilia are restricted subcellular compartments, and specialized mechanisms coordinate the localization of proteins to cilia. Moreover, trafficking of proteins into and out of cilia is required for proper ciliary function, and this process is disrupted in ciliopathies. The somatostatin receptor subtype 3 (Sstr3) is selectively targeted to primary cilia on neurons in the mammalian brain and is implicated in learning and memory. Here, we show that Sstr3 localization to cilia is dynamic and decreases in response to somatostatin treatment. We further show that somatostatin treatment stimulates ß-arrestin recruitment into Sstr3-positive cilia and this recruitment can be blocked by mutations in Sstr3 that impact agonist binding or phosphorylation. Importantly, somatostatin treatment fails to decrease Sstr3 ciliary localization in neurons lacking ß-arrestin 2. Together, our results implicate ß-arrestin in the modulation of Sstr3 ciliary localization and further suggest a role for ß-arrestin in the mediation of Sstr3 ciliary signaling.

FluoroMyelin™ Red is a bright, photostable and non-toxic fluorescent stain for live imaging of myelin.

FluoroMyelin™ Red is a commercially available water-soluble fluorescent dye that has selectivity for myelin. This dye is marketed for the visualization of myelin in brain cryosections, though it is also used widely to stain myelin in chemically fixed tissue. Here we have investigated the suitability of FluoroMyelin™ Red as a vital stain for live imaging of myelin in myelinating co-cultures of Schwann cells and dorsal root ganglion neurons. We show that addition of FluoroMyelin™ Red to the culture medium results in selective staining of myelin sheaths, with an optimal staining time of 2h, and has no apparent adverse effect on the neurons, their axons, or the myelinating cells at the light microscopic level. The fluorescence is bright and photostable, permitting long-term time-lapse imaging. After rinsing the cultures with medium lacking FluoroMyelin™ Red, the dye diffuses out of the myelin with a half life of about 130 min resulting in negligible fluorescence remaining after 18-24h. In addition, the large Stokes shift exhibited by FluoroMyelin™ Red makes it possible to readily distinguish it from popular and widely used green and red fluorescent probes such as GFP and mCherry. Thus FluoroMyelin™ Red is a useful reagent for live fluorescence imaging studies on myelinated axons.