Bidirectional Trafficking Research Articles

Environmental responsiveness of tubulin glutamylation in sensory cilia is regulated by the p38 MAPK pathway

Glutamylation is a post-translational modification found on tubulin that can alter the interaction between microtubules (MTs) and associated proteins. The molecular mechanisms regulating tubulin glutamylation in response to the environment are not well understood. Here, we show that in the sensory cilia of Caenorhabditis elegans, tubulin glutamylation is upregulated in response to various signals such as temperature, osmolality, and dietary conditions. Similarly, tubulin glutamylation is modified in mammalian photoreceptor cells following light adaptation. A tubulin glutamate ligase gene ttll-4, which is essential for tubulin glutamylation of axonemal MTs in sensory cilia, is activated by p38 MAPK. Amino acid substitution of TTLL-4 has revealed that a Thr residue (a putative MAPK-phosphorylation site) is required for enhancement of tubulin glutamylation. Intraflagellar transport (IFT), a bidirectional trafficking system specifically observed along axonemal MTs, is required for the formation, maintenance, and function of sensory cilia. Measurement of the velocity of IFT particles revealed that starvation accelerates IFT, which was also dependent on the Thr residue of TTLL-4. Similarly, starvation-induced attenuation of avoidance behaviour from high osmolality conditions was also dependent on ttll-4. Our data suggest that a novel evolutionarily conserved regulatory system exists for tubulin glutamylation in sensory cilia in response to the environment.

Ca2+ dependent surface trafficking of norepinephrine transporters depends on threonine 30 and Ca2+ calmodulin kinases

The antidepressant-sensitive norepinephrine (NE) transporter (NET) inactivates NE released during central and peripheral neuronal activity by transport into presynaptic cells. Altered NE clearance due to dysfunction of NET has been associated with the development of mental illness and cardiovascular diseases. NET activity in vivo is influenced by stress, neuronal activity, hormones and drugs. We investigated the mechanisms of Ca2+ regulation of NET and found that Ca2+ influenced both Vmax and Km for NE transport into cortical synaptosomes. Changes in extracellular Ca2+ triggered rapid and bidirectional surface trafficking of NET expressed in cultured cells. Deletion of residues 28–47 in the NET NH2-terminus abolished the Ca2+ effect on surface trafficking. Mutagenesis studies identified Thr30 in this region as the essential residue for both Ca2+- dependent phosphorylation and trafficking of NET. Depolarization of excitable cells increased surface NET in a Thr30 dependent manner. A proteomic analysis, RNA interference, and pharmacological inhibition supported roles of CaMKI and CaMKII in Ca2+-modulated NE transport and NET trafficking. Depolarization of primary noradrenergic neurons in culture with elevated K+ increased NET surface expression in a process that required external Ca2+ and depended on CaMK activity. Hippocampal NE clearance in vivo was also stimulated by depolarization, and inhibitors of CaMK signaling prevented this stimulation. In summary, Ca2+ signaling influenced surface trafficking of NET through a CaMK-dependent mechanism requiring Thr30.

Quantitative metabolic flux analysis reveals an unconventional pathway of fatty acid synthesis in cancer cells deficient for the mitochondrial citrate transport protein

The mitochondrial citrate transport protein (CTP), encoded by SLC25A1, accommodates bidirectional trafficking of citrate between the mitochondria and cytosol, supporting lipid biosynthesis and redox homeostasis. Genetic CTP deficiency causes a fatal neurodevelopmental syndrome associated with the accumulation of L- and D-2-hydroxyglutaric acid, and elevated CTP expression is associated with poor prognosis in several types of cancer, emphasizing the importance of this transporter in multiple human pathologies. Here we describe the metabolic consequences of CTP deficiency in cancer cells. As expected from the phenotype of CTP-deficient humans, somatic CTP loss in cancer cells induces broad dysregulation of mitochondrial metabolism, resulting in accumulation of lactate and of the L- and D- enantiomers of 2-hydroxyglutarate (2HG) and depletion of TCA cycle intermediates. It also eliminates mitochondrial import of citrate from the cytosol. To quantify the impact of CTP deficiency on metabolic flux, cells were cultured with a set of 13C-glucose and 13C-glutamine tracers with resulting data integrated by metabolic flux analysis (MFA). CTP-deficient cells displayed a major restructuring of central carbon metabolism, including suppression of pyruvate dehydrogenase (PDH) and induction of glucose-dependent anaplerosis through pyruvate carboxylase (PC). We also observed an unusual lipogenic pathway in which carbon from glucose supplies mitochondrial production of alpha-ketoglutarate (AKG), which is then trafficked to the cytosol and used to supply reductive carboxylation by isocitrate dehydrogenase 1 (IDH1). The resulting citrate is cleaved to produce lipogenic acetyl-CoA, thereby completing a novel pathway of glucose-dependent reductive carboxylation. In CTP deficient cells, IDH1 inhibition suppresses lipogenesis from either glucose or glutamine, implicating IDH1 as a required component of fatty acid synthesis in states of CTP deficiency.

O-GlcNAc-ylation in the Nuclear Pore Complex.

O-GlcNAc-ylation is the post-translational addition of an O-linked β-N-acetylglucosamine to the serine and threonine residues of thousands of proteins in eukaryotic cells. Specifically, half of the thirty different types of protein components in the nuclear pore complex (NPC) are modified by O-GlcNAc, of which the majority are intrinsically disordered nucleoporins (Nups) containing multiple phenylalanine-glycine (FG) repeats. Moreover, these FG-Nups form a strict selectivity barrier with a high density of O-GlcNAc in the NPC to mediate bidirectional trafficking between the cytoplasm and nucleus. However, the roles that O-GlcNAc plays in the structure and function of the NPC remain obscure. In this review paper, we will discuss the current knowledge of O-GlcNAc-ylated Nups, highlight some new techniques used to probe O-GlcNAc's roles in the nuclear pore, and finally propose a new model for the effect of O-GlcNAc on the NPC's permeability.

SPEED Microscopy and Its Application in Nucleocytoplasmic Transport.

In eukaryotic cells, the nuclear pore complexes (NPCs) selectively mediate the bidirectional trafficking of macromolecules between the cytoplasm and the nucleus. The selective barrier is formed by intrinsically disordered phenylalanine-glycine (FG) nucleoporins anchored on the wall of the submicrometer NPC, which allows for passive diffusion and facilitated translocation through the nuclear pore. Dysfunction of nucleocytoplasmic transport has been associated with many human diseases. However, due to the technical challenge of imaging the native tomography of the FG-nucleoporin barrier and its interactions with transiting molecules in the native NPC, the precise nucleocytoplasmic transport mechanism remains unresolved. To refine the transport mechanism, single-molecule fluorescence microscopy methods have been employed to obtain the transport kinetics and the spatial transport route of individual fluorescent molecules through the NPC. In this method paper, we particularly highlight a newly developed high-speed super-resolution three-dimensional microscopy approach, termed as SPEED (single-point edge-excitation subdiffraction) microscopy, and its application in characterizing nucleocytoplasmic transport.

Two classes of short intraflagellar transport train with different 3D structures are present in Chlamydomonas flagella.

Intraflagellar transport (IFT) is responsible for the bidirectional trafficking of molecular components required for the elongation and maintenance of eukaryotic cilia and flagella. Cargo is transported by IFT 'trains', linear rows of multiprotein particles moved by molecular motors along the axonemal doublets. We have previously described two structurally distinct categories of 'long' and 'short' trains. Here, we analyse the relative number of these trains throughout flagellar regeneration and show that long trains are most abundant at the beginning of flagellar growth whereas short trains gradually increase in number as flagella elongate. These observations are incompatible with the previous hypothesis that short trains are derived solely from the reorganization of long trains at the flagellar tip. We demonstrate with electron tomography the existence of two distinct ultrastructural organizations for the short trains, we name these 'narrow' and 'wide', and provide the first 3D model of the narrow short trains. These trains are characterized by tri-lobed units, which repeat longitudinally every 16 nm and contact protofilament 7 of the B-tubule. Functional implications of the new structural evidence are discussed.

Distinct Bone Marrow Blood Vessels Differentially Regulate Normal and Malignant Hematopoietic Stem and Progenitor Cells

Bone marrow (BM) endothelial cells (BMECs) form a network of blood vessels (BVs) that regulate both leukocyte trafficking and hematopoietic stem and progenitor cell (HSPC) maintenance. However, it is not clear how BMECs balance between these dual regulatory roles and if these events occur at the same vascular site. We define the BM architecture of functionally distinct BVs, their spatial localization and association with specific stromal precursors, which cooperatively regulate HSPC fate. BM stem and progenitor cell maintenance in a metabolically non-active state and leukocyte trafficking occur at separate sites and are differentially regulated by specific BVs with distinct permeability properties. BM arteries were found to be mostly encircled by aSMA+ pericytes whereas the ensuing small-diameter endosteal and trabecular arterioles were predominantly surrounded by stem cell-niche supporting stromal precursor cells. Live imaging and flow analysis revealed that endosteal arteriole BVs exhibited high flow rate, low permeability to external plasma from the peripheral blood, and high levels of adhesion- and tight-junction molecules. Primitive HSPCs located in peri-arteriole regions were found in a non-activated, low reactive oxygen species (ROS) state. Exposure of BM HSPCs to peripheral blood plasma, enhanced their metabolic activity, exhibited by enhanced intracellular ROS levels, and glucose uptake. The same was also evident for circulating HSPCs in the blood. Plasma-exposed HSPCs displayed enhanced motility alongside with reduced long-term repopulation potential. Live imaging showed that all immature and mature leukocyte bi-directional trafficking occurred exclusively at the more permeable sinusoids, located downstream to the endosteal arterioles. Of note, BM sinusoids contained a higher prevalence of ROShigh cells in their microenvironment, including HSPCs. Rapid AMD3100-induced HSPC mobilization preferentially affected sinusoidal but not arterial BVs permeability and CXCL12 chemokine release. Endothelial specific in vivo interference with CXCL12-CXCR4 interactions, via conditional CXCR4 genetic deletion, hampered BM barrier integrity resulting in enhanced HSPC egress. In line with these results we found that during conditions favoring BM stem and progenitor cells expansion, endothelial integrity was enhanced along with reduced HSPC bi-directional trafficking. Conversely, conditional endothelial specific induced genetic or pharmacologic disruption of barrier integrity augmented ROS levels in HSPCs, enhancing their bi-directional trafficking and differentiation while reducing their BM pool size and maintenance in a metabolically non-active state. Of note, humanized mice engrafted with pre-B ALL cells exhibited reduced BM barrier permeability most probably due to BM endothelium modification via FGF-2 secretion by the leukemic clone. Interestingly, human pre-B ALL cells displayed hypersensitivity to plasmatic exposure. We hypothesize that malignant cells modify BM endothelium to provide themselves with a supportive and protective microenvironment composed of undifferentiated BM stromal progenitors and tightly sealed endothelial barrier. In conclusion, our study identifies anatomically distinct BM BVs with different barrier functions serving as systemic leukocyte trafficking or HSPC BM maintenance sites with clinical therapeutic relevance. DisclosuresRafii:Angiocrine Bioscience: Consultancy, Equity Ownership.

Platelet-derived CXCL12 regulates monocyte function, survival, differentiation into macrophages and foam cells through differential involvement of CXCR4-CXCR7.

Platelets store and release CXCL12 (SDF-1), which governs differentiation of hematopoietic progenitors into either endothelial or macrophage-foam cells. CXCL12 ligates CXCR4 and CXCR7 and regulates monocyte/macrophage functions. This study deciphers the relative contribution of CXCR4–CXCR7 in mediating the effects of platelet-derived CXCL12 on monocyte function, survival, and differentiation. CXCL12 and macrophage migration inhibitory factor (MIF) that ligate CXCR4–CXCR7 induced a dynamic bidirectional trafficking of the receptors, causing CXCR4 internalization and CXCR7 externalization during chemotaxis, thereby influencing relative receptor availability, unlike MCP-1. In vivo we found enhanced accumulation of platelets and platelet-macrophage co-aggregates in peritoneal fluid following induction of peritonitis in mice. The relative surface expression of CXCL12, CXCR4, and CXCR7 among infiltrated monocytes was also enhanced as compared with peripheral blood. Platelet-derived CXCL12 from collagen-adherent platelets and recombinant CXCL12 induced monocyte chemotaxis specifically through CXCR4 engagement. Adhesion of monocytes to immobilized CXCL12 and CXCL12-enriched activated platelet surface under static and dynamic arterial flow conditions were mediated primarily through CXCR7 and were counter-regulated by neutralizing platelet-derived CXCL12. Monocytes and culture-derived-M1–M2 macrophages phagocytosed platelets, with the phagocytic potential of culture-derived-M1 macrophages higher than M2 involving CXCR4–CXCR7 participation. CXCR7 was the primary receptor in promoting monocyte survival as exerted by platelet-derived CXCL12 against BH3-mimetic induced apoptosis (phosphatidylserine exposure, caspase-3 activation, loss of mitochondrial transmembrane potential). In co-culture experiments with platelets, monocytes predominantly differentiated into CD163+ macrophages, which was attenuated upon CXCL12 neutralization and CXCR4/CXCR7 blocking antibodies. Moreover, OxLDL uptake by platelets induced platelet apoptosis, like other platelet agonists TRAP and collagen-related peptide (CRP). CXCL12 facilitated phagocytosis of apoptotic platelets by monocytes and M1–M2 macrophages, also promoted their differentiation into foam cells via CXCR4 and CXCR7. Thus, platelet-derived CXCL12 could regulate monocyte-macrophage functions through differential engagement of CXCR4 and CXCR7, indicating an important role in inflammation at site of platelet accumulation.

Ultrastructural studies of Intraflagellar Transport trains in Chlamydomonas reinhardtii suggest a revision of the current model for IFT trafficking in the flagellar compartment

Intraflagellar Transport (IFT) is the molecular process responsible for the active bidirectional trafficking of structural and functional components that occurs in the flagellar compartment of eukaryotic cells. Flagellar components undergo a constant turnover at flagellar tip and multiple evidences indicate that flagellar elongation, maintenance and reabsorption depend on the correct balance between anterograde and retrograde trafficking. IFT particles are formed by >22 polypeptides assembled into two subcomplexes, A and B, and are moved bidirectionally along the outer surface of axonemal doublets as linear rows of IFT particles, for which we proposed the term train. Anterograde IFT trains are moved by kinesin II and carry to flagellar tip the retrograde motor cytoplasmic dynein 1b, responsible for retrograde IFT. In a previous study carried out on Chlamydomonas flagella we identified two types of IFT trains we named long and short trains, each characterized by a specific ultrastructure and a definite internal repeat, and proposed that long, less compact trains could represent anterograde IFT while the short, more compact trains could be retrograde. To challenge such model, we monitored by transmission electron microscopy the IFT trains expressed both in wt regenerating flagella and during flagellar reabsorption induced in the ts mutant pf1-fla10. We also progressed in our electron tomographic 3D modeling of short IFT trains. Our data suggest that long IFT trains are not the only anterograde IFT component. Rather, anterograde IFT is contributed also by a subclass of short trains that is expressed in a flagellar length-dependent fashion.

Microchimerism and thyroid disorders: current knowledge and future prospects

This review will provide an overview of a novel phenomenon in medicine and endocrinology known as microchimerism (MC). MC is defined as the long-term presence of genetically distinct populations of cells in peripheral blood and tissues of individual. This condition may be caused by the transplacental bidirectional cell trafficking between mother and fetus during pregnancy. Other sources are the passage from a twin or a vanished twin and iatrogenic interventions such as organ transplantation and blood transfusion. Long-term consequences of MC and its influence on woman's health are under active investigation. Recent studies have shown a broad spectrum of its biological effects either beneficial or adverse. MC has been suggested to play a key role in the pathogenesis of autoimmune diseases. It is assumed that microchi-meric cells could initiate a versus or host versus graft reactions. MC has been extensively studied in autoimmune thyroid diseases (AITD). MC could explain the higher prevalence of AITD in females and frequent manifestation during the postpartum period. It is proposed that microchimeric cells could reduce the risk of cancer development and be a part of tissue repair. MC has also been investigated in papillary thyroid cancer. Further studies of this phenomenon are mandatory to get more insights about its role in thyroid cancer and AITD development. This could provide novel therapeutic, preventive or prognostic perspectives regarding these diseases.

Mutations in IFT172 cause isolated retinal degeneration and Bardet-Biedl syndrome.

Primary cilia are sensory organelles present on most mammalian cells. The assembly and maintenance of primary cilia are facilitated by intraflagellar transport (IFT), a bidirectional protein trafficking along the cilium. Mutations in genes coding for IFT components have been associated with a group of diseases called ciliopathies. These genetic disorders can affect a variety of organs including the retina. Using whole exome sequencing in three families, we identified mutations in Intraflagellar Transport 172 Homolog [IFT172 (Chlamydomonas)] that underlie an isolated retinal degeneration and Bardet-Biedl syndrome. Extensive functional analyses of the identified mutations in cell culture, rat retina and in zebrafish demonstrated their hypomorphic or null nature. It has recently been reported that mutations in IFT172 cause a severe ciliopathy syndrome involving skeletal, renal, hepatic and retinal abnormalities (Jeune and Mainzer-Saldino syndromes). Here, we report for the first time that mutations in this gene can also lead to an isolated form of retinal degeneration. The functional data for the mutations can partially explain milder phenotypes; however, the involvement of modifying alleles in the IFT172-associated phenotypes cannot be excluded. These findings expand the spectrum of disease associated with mutations in IFT172 and suggest that mutations in genes originally reported to be associated with syndromic ciliopathies should also be considered in subjects with non-syndromic retinal dystrophy.

MiRNA nano-packages for maternal-placental-fetal communication

Positioned at the maternal-fetal interface, the placenta generates regulatory signals and serves as a communication junction that governs pregnancy health. In addition to hormones, growth factors and diverse proteins, we and others have recently shown that small non-coding RNA molecules, including microRNAs (miRNAs), traffic among the maternalplacental-fetal compartments, and may have a central role in maternalfetal adaptation to homeostatic perturbations. Although common and unique types of miRNAs are expressed by the placenta during pregnancy, the function of placental miRNA species remains largely unknown. We recently showed that primary human trophoblasts are resistant to infection by a heterogeneous panel of viruses, and that this resistance can be partly conferred to non-trophoblast cells by exogenous expression of trophoblast-specific miRNAs, expressed from the chromosome 19 miRNA cluster (C19MC). This effect was not observed during cell infection by nonviral placental pathogens, including Listeria monocytogenes, and Toxoplasma gondii. Trophoblastic miRNAs can be released to the extracellular space within several types of vesicles, including cell fragments, apoptotic bodies, microvesicles, and nanovesicles (exosomes), or can be packaged with non-vesicular protein complexes, such as argonaute-2. To examine the trafficking of trophoblastic miRNAs to maternal-fetal tissues we engineered a transgenic mouse that expresses a 160 kb segment of human genomic DNA harboring the human C19MC cluster. We coupled this technology with RNAseq analysis of miRNA expression in triads of placenta, maternal plasma and fetal plasma from pregnant women at term. Together, these analyses shed new light on transplacental bidirectional miRNA trafficking patterns (Supported by NIH HD06589, HD075665, HD071707, AI081759, and the Pennsylvania Department of Health Research Formula Funds)

Microtubule initiation from the nuclear surface controls cortical microtubule growth polarity and orientation in Arabidopsis thaliana.

The nuclear envelope in plant cells has long been known to be a microtubule organizing center (MTOC), but its influence on microtubule organization in the cell cortex has been unclear. Here we show that nuclear MTOC activity favors the formation of longitudinal cortical microtubule (CMT) arrays. We used green fluorescent protein (GFP)-tagged gamma tubulin-complex protein 2 (GCP2) to identify nuclear MTOC activity and GFP-tagged End-Binding Protein 1b (EB1b) to track microtubule growth directions. We found that microtubules initiate from nuclei and enter the cortex in two directions along the long axis of the cell, creating bipolar longitudinal CMT arrays. Such arrays were observed in all cell types showing nuclear MTOC activity, including root hairs, recently divided cells in root tips, and the leaf epidermis. In order to confirm the causal nature of nuclei in bipolar array formation, we displaced nuclei by centrifugation, which generated a corresponding shift in the bipolarity split point. We also found that bipolar CMT arrays were associated with bidirectional trafficking of vesicular components to cell ends. Together, these findings reveal a conserved function of plant nuclear MTOCs and centrosomes/spindle pole bodies in animals and fungi, wherein all structures serve to establish polarities in microtubule growth.

Hook is an adapter that coordinates kinesin-3 and dynein cargo attachment on early endosomes

Bidirectional membrane trafficking along microtubules is mediated by kinesin-1, kinesin-3, and dynein. Several organelle-bound adapters for kinesin-1 and dynein have been reported that orchestrate their opposing activity. However, the coordination of kinesin-3/dynein-mediated transport is not understood. In this paper, we report that a Hook protein, Hok1, is essential for kinesin-3- and dynein-dependent early endosome (EE) motility in the fungus Ustilago maydis. Hok1 binds to EEs via its C-terminal region, where it forms a complex with homologues of human fused toes (FTS) and its interactor FTS- and Hook-interacting protein. A highly conserved N-terminal region is required to bind dynein and kinesin-3 to EEs. To change the direction of EE transport, kinesin-3 is released from organelles, and dynein binds subsequently. A chimaera of human Hook3 and Hok1 rescues the hok1 mutant phenotype, suggesting functional conservation between humans and fungi. We conclude that Hok1 is part of an evolutionarily conserved protein complex that regulates bidirectional EE trafficking by controlling attachment of both kinesin-3 and dynein.

Selective Protection of an ARF1-GTP Signaling Axis by a Bacterial Scaffold Induces Bidirectional Trafficking Arrest

SUMMARYBidirectional vesicular transport between the endoplasmic reticulum (ER) and Golgi is mediated largely by ARF and Rab GTPases, which orchestrate vesicle fission and fusion, respectively. How their activities are coordinated in order to define the successive steps of the secretory pathway and preserve traffic directionality is not well understood in part due to the scarcity of molecular tools that simultaneously target ARF and Rab signaling. Here, we take advantage of the unique scaffolding properties of E. coli secreted protein G (EspG) to describe the critical role of ARF1/Rab1 spatiotemporal coordination in vesicular transport at the ER-Golgi intermediate compartment. Structural modeling and cellular studies show that EspG induces bidirectional traffic arrest by tethering vesicles through select ARF1-GTP/effector complexes and local inactivation of Rab1. The mechanistic insights presented here establish the effectiveness of a small bacterial catalytic scaffold for studying complex processes and reveal an alternative mechanism of immune regulation by an important human pathogen.

Characterization of the Selective Barrier in Nuclear Pore Complex

The nuclear pore complexes (NPCs) selectively mediate the bidirectional trafficking of macromolecules between the cytoplasm and nucleus in eukaryotic cells. The selective barrier formed by natively unfolded phenylalanine-glycine (FG) nucleoporins (Nups) inside the NPC allows for passive and facilitated transport through the NPC. However, the mechanism of formation and spatial distribution of FG-barrier in the NPC remains unresolved. By a newly developed single-molecule microscopy, single-point edge-excitation sub-diffraction (SPEED) microscopy, we have used various fluorescent transport receptors and FG segments, as probes to determine the structure of FG-Nup barrier in the native NPC with a spatiotemporal resolution of 9 nm and 400 μs. The interactions among FG-filaments enhance a donut-like structure formed at the central of NPC, acting as the primary selective gate for nuclear transport. On either side of the NPC, additionally FG Nups may function as the secondary selectivity for incoming molecules. Finally, the conformations of both selective barriers can be further regulated by major transport receptors.

A Research Study of the Association between Maternal Microchimerism and Systemic Lupus Erythematosus in Adults: A Comparison between Patients and Healthy Controls Based on Single-Nucleotide Polymorphism Using Quantitative Real-Time PCR

BackgroundNaturally acquired microchimerism may arise in the mother and her child during pregnancy when bidirectional trafficking of cells occurs through the placental barrier. The occurrence of maternal microchimerism (maternal cells in the offspring) has been associated with several autoimmune diseases, especially in children. Systemic Lupus erythematosus (SLE) is an autoimmune disorder with a resemblance to graft-versus-host disease. The aim of this study was to investigate the association between maternal microchimerism in the blood and SLE.Methodology/Principal FindingsThirty-two patients with SLE, 17 healthy brothers of the patients, and an additional 12 unrelated healthy men were the subjects in this study. A single-nucleotide polymorphism unique to each mother was identified, and maternal microchimerism in the study group and in the control group was detected using a quantitative real-time polymerase chain reaction technique. No differences in the frequency or the concentration of maternal cells were apparent in the blood of patients with SLE or in that of the controls. Two patients and one control tested positive for maternal microchimerism, but the positive subjects were all negative at a follow-up 16 years later. The sensitivity of the method was estimated to 1/10.000.Conclusions/SignificanceThese results show no association between SLE and maternal microchimerism. The frequency of maternal microchimerism in the blood of adults overall may be lower than earlier reported.

Inherited human sex reversal due to impaired nucleocytoplasmic trafficking of SRY defines a male transcriptional threshold

Human testis determination is initiated by SRY (sex determining region on Y chromosome). Mutations in SRY cause gonadal dysgenesis with female somatic phenotype. Two subtle variants (V60L and I90M in the high-mobility group box) define inherited alleles shared by an XY sterile daughter and fertile father. Whereas specific DNA binding and bending are unaffected in a rat embryonic pre-Sertoli cell line, the variants exhibited selective defects in nucleocytoplasmic shuttling due to impaired nuclear import (V60L; mediated by Exportin-4) or export (I90M; mediated by chromosome region maintenance 1). Decreased shuttling limits nuclear accumulation of phosphorylated (activated) SRY, in turn reducing occupancy of DNA sites regulating Sertoli-cell differentiation [the testis-specific SRY-box 9 (Sox9) enhancer]. Despite distinct patterns of biochemical and cell-biological perturbations, V60L and I90M each attenuated Sox9 expression in transient transfection assays by twofold. Such attenuation was also observed in studies of V60A, a clinical variant associated with ovotestes and hence ambiguity between divergent cell fates. This shared twofold threshold is reminiscent of autosomal syndromes of transcription-factor haploinsufficiency, including XY sex reversal associated with mutations in SOX9. Our results demonstrate that nucleocytoplasmic shuttling of SRY is necessary for robust initiation of testicular development. Although also characteristic of ungulate orthologs, such shuttling is not conserved among rodents wherein impaired nuclear export of the high-mobility group box and import-dependent phosphorylation are compensated by a microsatellite-associated transcriptional activation domain. Human sex reversal due to subtle defects in the nucleocytoplasmic shuttling of SRY suggests that its transcriptional activity lies near the edge of developmental ambiguity.

Axonal trafficking of NMNAT2 and its roles in axon growth and survival in vivo

The NAD-synthesizing enzyme NMNAT2 is critical for axon survival in primary culture and its depletion may contribute to axon degeneration in a variety of neurodegenerative disorders. Here we discuss several recent reports from our laboratory that establish a critical role for NMNAT2 in axon growth in vivo in mice and shed light on the delivery and turnover of this survival factor in axons. In the absence of NMNAT2, axons fail to extend more than a short distance beyond the cell body during embryonic development, implying a requirement for NMNAT2 in axon maintenance even during development. Furthermore, we highlight findings regarding the bidirectional trafficking of NMNAT2 in axons on a vesicle population that undergoes fast axonal transport in primary culture neurites and in mouse sciatic nerve axons in vivo. Surprisingly, loss of vesicle association boosts the axon protective capacity of NMNAT2, an effect that is at least partially mediated by a longer protein half-life of cytosolic NMNAT2 variants. Analysis of wild-type and variant NMNAT2 in mouse sciatic nerves and Drosophila olfactory receptor neuron axons supports the existence of a similar mechanism in vivo, highlighting the potential for regulation of NMNAT2 stability and turnover as a mechanism to modulate axon degeneration in vivo.

Slide to the Left and Slide to the Right: Motor Coordination in Neurons

Molecular motors employ specific adaptor proteins to dock on transport cargos. Reporting in The Journal of Cell Biology, Fu and Holzbaur (2013) show that the adaptor JNK interacting protein 1 (JIP1) binds kinesin-1 and dynactin and controls bidirectional axonal amyloid precursor protein trafficking, suggesting a regulatory role for adaptors during cargo transport.