Motor-dependent anterograde transport, a process that moves cytoplasmic components from sites of biosynthesis to sites of use within cells, is crucial in neurons with long axons. Evidence has emerged that multiple anterograde kinesins can contribute to some transport processes. To test the multi-kinesin possibility for a single vesicle type, we studied the functional relationships of axonal kinesins to dense core vesicles (DCVs) that were filled with a GFP-tagged neuropeptide in the Drosophila nervous system. Past work showed that Unc-104 (a kinesin-3) is a key anterograde DCV motor. Here we show that anterograde DCV transport requires the well-known mitochondrial motor Khc (kinesin-1). Our results indicate that this influence is direct. Khc mutations had specific effects on anterograde run parameters, neuron-specific inhibition of mitochondrial transport by Milton RNA interference had no influence on anterograde DCV runs, and detailed colocalization analysis by superresolution microscopy revealed that Unc-104 and Khc coassociate with individual DCVs. DCV distribution analysis in peptidergic neurons suggest the two kinesins have compartment specific influences. We suggest a mechanism in which Unc-104 is particularly important for moving DCVs from cell bodies into axons, and then Unc-104 and kinesin-1 function together to support fast, highly processive runs toward axon terminals.

The importin-β family members (karyopherins) mediate the majority of nucleocytoplasmic transport. Msn5 and Los1, members of the importin-β family, function in tRNA nuclear export. tRNAs move bidirectionally between the nucleus and the cytoplasm. Nuclear tRNA accumulation occurs upon amino acid (aa) or glucose deprivation. To understand the mechanisms regulating tRNA subcellular trafficking, we investigated whether Msn5 and Los1 are regulated in response to nutrient availability. We provide evidence that tRNA subcellular trafficking is regulated by distinct aa-sensitive and glucose-sensitive mechanisms. Subcellular distributions of Msn5 and Los1 are altered upon glucose deprivation but not aa deprivation. Redistribution of tRNA exportins from the nucleus to the cytoplasm likely provides one mechanism for tRNA nuclear distribution upon glucose deprivation. We extended our studies to other members of the importin-β family and found that all tested karyopherins invert their subcellular distributions upon glucose deprivation but not aa deprivation. Glucose availability regulates the subcellular distributions of karyopherins likely due to alteration of the RanGTP gradient since glucose deprivation causes redistribution of Ran. Thus nuclear-cytoplasmic distribution of macromolecules is likely generally altered upon glucose deprivation due to collapse of the RanGTP gradient and redistribution of karyopherins between the nucleus and the cytoplasm.

Retrograde intraflagellar transport (IFT) is required for assembly of cilia. We identify a Chlamydomonas flagellar protein (flagellar-associated protein 163 [FAP163]) as being closely related to the D1bIC(FAP133) intermediate chain (IC) of the dynein that powers this movement. Biochemical analysis revealed that FAP163 is present in the flagellar matrix and is actively trafficked by IFT. Furthermore, FAP163 copurified with D1bIC(FAP133) and the LC8 dynein light chain, indicating that it is an integral component of the retrograde IFT dynein. To assess the functional role of FAP163, we generated an RNA interference knockdown of the orthologous protein (WD60) in planaria. The Smed-wd60(RNAi) animals had a severe ciliary assembly defect that dramatically compromised whole-organism motility. Most cilia were present as short stubs that had accumulated large quantities of IFT particle-like material between the doublet microtubules and the membrane. The few remaining approximately full-length cilia had a chaotic beat with a frequency reduced from 24 to ∼10 Hz. Thus WD60/FAP163 is a dynein IC that is absolutely required for retrograde IFT and ciliary assembly.

The primary cilium is a nexus for cell signaling and relies on specific protein trafficking for function. The tubby family protein TULP3 transports integral membrane proteins into cilia through interactions with the intraflagellar transport complex-A (IFT-A) and phosphoinositides. It was previously shown that short motifs called ciliary localization sequences (CLSs) are necessary and sufficient for TULP3-dependent ciliary trafficking of transmembrane cargoes. However, the mechanisms by which TULP3 regulates ciliary compartmentalization of nonintegral, membrane-associated proteins and whether such trafficking requires TULP3-dependent CLSs is unknown. Here we show that TULP3 is required for ciliary transport of the Joubert syndrome-linked palmitoylated GTPase ARL13B through a CLS. An N-terminal amphipathic helix, preceding the GTPase domain of ARL13B, couples with the TULP3 tubby domain for ciliary trafficking, irrespective of palmitoylation. ARL13B transport requires TULP3 binding to IFT-A but not to phosphoinositides, indicating strong membrane-proximate interactions, unlike transmembrane cargo transport requiring both properties of TULP3. TULP3-mediated trafficking of ARL13B also regulates ciliary enrichment of farnesylated and myristoylated downstream effectors of ARL13B. The lipidated cargoes show distinctive depletion kinetics from kidney epithelial cilia with relation to Tulp3 deletion-induced renal cystogenesis. Overall, these findings indicate an expanded role of the tubby domain in capturing analogous helical secondary structural motifs from diverse cargoes.

The dependence of neurons on microtubule-based motors for the movement of lysosomes over long distances raises questions about adaptations that allow neurons to meet these demands. Recently, JIP3/MAPK8IP3, a neuronally enriched putative adaptor between lysosomes and motors, was identified as a critical regulator of axonal lysosome abundance. In this study, we establish a human induced pluripotent stem cell (iPSC)-derived neuron model for the investigation of axonal lysosome transport and maturation and show that loss of JIP3 results in the accumulation of axonal lysosomes and the Alzheimer's disease-related amyloid precursor protein (APP)-derived Aβ42 peptide. We furthermore reveal an overlapping role of the homologous JIP4 gene in lysosome axonal transport. These results establish a cellular model for investigating the relationship between lysosome axonal transport and amyloidogenic APP processing and more broadly demonstrate the utility of human iPSC-derived neurons for the investigation of neuronal cell biology and pathology.

Mutations in adenomatous polyposis coli (APC) disrupt regulation of Wnt signaling, mitosis, and the cytoskeleton. We describe a new role for APC in the transport of mitochondria. Silencing of wild-type APC by small interfering RNA caused mitochondria to redistribute from the cell periphery to the perinuclear region. We identified novel APC interactions with the mitochondrial kinesin-motor complex Miro/Milton that were mediated by the APC C-terminus. Truncating mutations in APC abolished its ability to bind Miro/Milton and reduced formation of the Miro/Milton complex, correlating with disrupted mitochondrial distribution in colorectal cancer cells that could be recovered by reconstitution of wild-type APC. Using proximity ligation assays, we identified endogenous APC-Miro/Milton complexes at mitochondria, and live-cell imaging showed that loss of APC slowed the frequency of anterograde mitochondrial transport to the membrane. We propose that APC helps drive mitochondria to the membrane to supply energy for cellular processes such as directed cell migration, a process disrupted by cancer mutations.

The chemokine receptor CXCR4, a G protein-coupled receptor, is targeted for lysosomal degradation via a ubiquitin-dependent mechanism that involves the endosomal sorting complex required for transport (ESCRT) machinery. We have reported recently that arrestin-2 also targets CXCR4 for lysosomal degradation; however, the molecular mechanisms by which this occurs remain poorly understood. Here, we show that arrestin-2 interacts with ESCRT-0, a protein complex that recognizes and sorts ubiquitinated cargo into the degradative pathway. Signal-transducing adaptor molecule (STAM)-1, but not related STAM-2, interacts directly with arrestin-2 and colocalizes with CXCR4 on early endosomal antigen 1-positive early endosomes. Depletion of STAM-1 by RNA interference and disruption of the arrestin-2/STAM-1 interaction accelerates agonist promoted degradation of CXCR4, suggesting that STAM-1 via its interaction with arrestin-2 negatively regulates CXCR4 endosomal sorting. Interestingly, disruption of this interaction blocks agonist promoted ubiquitination of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) but not CXCR4 and STAM-1 ubiquitination. Our data suggest a mechanism whereby arrestin-2 via its interaction with STAM-1 modulates CXCR4 sorting by regulating the ubiquitination status of HRS.

Forkhead transcription factors (FOXOs) alter a diverse array of cellular processes including the cell cycle, oxidative stress resistance, and aging. Insulin/Akt activation directs phosphorylation and cytoplasmic sequestration of FOXO away from its target genes and serves as an endpoint of a complex signaling network. Using a human genome small interfering RNA (siRNA) library in a cell-based assay, we identified an extensive network of proteins involved in nuclear export, focal adhesion, and mitochondrial respiration not previously implicated in FOXO localization. Furthermore, a detailed examination of mitochondrial factors revealed that loss of uncoupling protein 5 (UCP5) modifies the energy balance and increases free radicals through up-regulation of uncoupling protein 3 (UCP3). The increased superoxide content induces c-Jun N-terminal kinase 1 (JNK1) kinase activity, which in turn affects FOXO localization through a compensatory dephosphorylation of Akt. The resulting nuclear FOXO increases expression of target genes, including mitochondrial superoxide dismutase. By connecting free radical defense and mitochondrial uncoupling to Akt/FOXO signaling, these results have implications in obesity and type 2 diabetes development and the potential for therapeutic intervention.

Post-Golgi transport for specific membrane domains, also termed polarized transport, is essential for the construction and maintenance of polarized cells. Highly polarized Drosophila photoreceptors serve as a good model system for studying the mechanisms underlying polarized transport. The Mss4 Drosophila ortholog, Stratum (Strat), controls basal restriction of basement membrane proteins in follicle cells, and Rab8 acts downstream of Strat. We investigated the function of Strat in fly photoreceptors and found that polarized transport in both the basolateral and the rhabdomere membrane domains was inhibited in Strat-deficient photoreceptors. We also observed 79 and 55% reductions in Rab10 and Rab35 levels, respectively, but no reduction in Rab11 levels in whole-eye homozygous clones of Stratnull. Moreover, Rab35 was localized in the rhabdomere, and loss of Rab35 resulted in impaired Rh1 transport to the rhabdomere. These results indicate that Strat is essential for the stable expression of Rab10 and Rab35, which regulate basolateral and rhabdomere transport, respectively, in fly photoreceptors.

Neurons are highly polarized cells with long neurites. Vesicular transport is required for neurite extension. We recently identified protrudin as a key regulator of vesicular transport during neurite extension. Expression of protrudin in nonneuronal cells thus induces formation of neurite-like membrane protrusions. We adopted a proteomics approach to identify proteins that associate with protrudin. Among the protrudin-associated proteins, including many with a function related to intracellular trafficking, we focused on KIF5, a motor protein that mediates anterograde vesicular transport in neurons. A coimmunoprecipitation assay confirmed that endogenous protrudin and KIF5 interact in mouse brain. Overexpression of KIF5 induced the formation of membrane protrusions in HeLa cells, reminiscent of the effect of protrudin overexpression. Forced expression of both protrudin and KIF5 promoted protrusion extension in a synergistic manner, whereas depletion of either protein attenuated protrusion formation. Protrudin facilitated the interaction of KIF5 with Rab11, VAP-A and -B, Surf4, and RTN3, suggesting that protrudin serves as an adaptor protein and that the protrudin-KIF5 complex contributes to the transport of these proteins in neurons. Given that mutation of protrudin or KIF5 is a cause of human hereditary spastic paraplegia, the protrudin-KIF5 axis appears to be integral to neuronal function.

The nuclear pore complex (NPC) is a highly modular assembly of 34 distinct nucleoporins (Nups) to form a versatile transport channel between the nucleus and the cytoplasm. Among them, Nup62 is known as an essential component for nuclear transport, Nup93 for proper nuclear envelope assembly. These Nups constitute various NPC subcomplexes such as the central transport channel (CTC), the cytoplasmic ring (CR), and the inner ring (IR). However, how they play their roles in NPC assembly and transport activity is not clear. Here we delineated the interacting regions and conducted biochemical reconstitution and structural characterization of the mammalian CR complex to reveal its intrinsic dynamic behavior and a distinct "4"-shaped architecture resembling the CTC complex. Our in vitro reconstitution data demonstrate that the Nup62 coiled-coil domain is critical to form both Nup62322-525 •Nup88517-742 and Nup62322-525•Nup88517-742•Nup214693-926 heterotrimers and both can bind to Nup931-150. We therefore propose that Nup93 acts as a "sensor" to bind to Nup62 shared heterotrimers including the Nup62•Nup54 heterotrimer of the CTC, which was not shown previously to be an interacting partner. Altogether, our biochemical study suggests that Nup62 via its coiled-coil domain is central to form compositionally distinct yet structurally similar heterotrimers and Nup93 binds these diverse heterotrimers nonselectively.

In neurons, RNA transport is important for local protein synthesis. mRNAs are transported along dendrites as large RNA granules. The localization and dynamics of Puralpha and Staufen1 (Stau1), major components of RNA transport granules, were investigated in cultured hippocampal neurons. Puralpha-positive granules were localized in both the shafts and spines of dendrites. In contrast, Stau1-positive granules tended to be localized mainly in dendritic shafts. More than 90% of Puralpha-positive granules were positive for Stau1 in immature dendrites, while only half were positive in mature dendrites. Stau1-negative Puralpha granules tended to be stationary with fewer anterograde and retrograde movements than Stau1-positive Puralpha granules. After metabotropic glutamate receptor 5 activation, Stau1-positive granules remained in the dendritic shafts, while Puralpha granules translocated from the shaft to the spine. The translocation of Puralpha granules was dependent on myosin Va, an actin-based molecular motor protein. Collectively our findings suggest the possibility that the loss of Stau1 in Puralpha-positive RNA granules might promote their activity-dependent translocation into dendritic spines, which could underlie the regulation of protein synthesis in synapses.

Many protein factors are required for mRNA biogenesis and nuclear export, which are central to the eukaryotic gene expression program. It is unclear, however, whether all factors have been identified. Here we report on a screen of >1000 essential gene mutants in Saccharomyces cerevisiae for defects in mRNA processing and export, identifying 26 mutants with defects in this process. Single-molecule FISH data showed that the majority of these mutants accumulated mRNA within specific regions of the nucleus, which included 1) mRNAs within the nucleolus when nucleocytoplasmic transport, rRNA biogenesis, or RNA processing and surveillance was disrupted, 2) the buildup of mRNAs near transcription sites in 3'-end processing and chromosome segregation mutants, and 3) transcripts being enriched near nuclear pore complexes when components of the mRNA export machinery were mutated. These data show that alterations to various nuclear processes lead to the retention of mRNAs at discrete locations within the nucleus.

Midkine (MDK) expression is associated with the proliferation of many cancers, including glioma. However, the upstream signaling that leads to MDK accumulation remains elusive. This study investigates the molecular mechanism that induces MDK overexpression in human glioma. The Repository for Molecular Brain Neoplasia Data was analyzed to identify potential MDK regulators. Expression of MDK and specificity protein 1 (SP1) was compared in glioma specimens. Chromatin immunoprecipitation assay was used to confirm the transcriptional regulation. MDK-force-expressed, SP1-silenced glioma cells were used to test rescue effects in vitro and in vivo. MDK and SP1 expression in gliomas was significantly higher than in adjacent tissues and was positively correlated in glioma clinical samples and cell lines. The promoter of the human MDK gene has a putative SP1 binding site. SP1 binds to the promoter of the MDK gene and directly regulates MDK expression. MDK or SP1 gene silencing inhibited the proliferation of glioma cells and reduced the tumor volume in nude mice. Overexpression of MDK in SP1-silenced cells could partially rescue the SP1 inhibition effects in vivo and in vitro. SP1 directly up-regulated the expression of MDK, and the SP1-MDK axis cooperated in glioma tumorigenesis.

Nuclear transporters mediate bidirectional macromolecule traffic through the nuclear pore complex (NPC), thus participating in vital processes of eukaryotic cells. A systematic functional analysis in Aspergillus nidulans permitted the identification of 4 essential nuclear transport pathways of a hypothetical number of 14. The absence of phenotypes for most deletants indicates redundant roles for these nuclear receptors. Subcellular distribution studies of these carriers show three main distributions: nuclear, nucleocytoplasmic, and in association with the nuclear envelope. These locations are not specific to predicted roles as exportins or importins but indicate that bidirectional transport may occur coordinately in all nuclei of a syncytium. Coinciding with mitotic NPC rearrangements, transporters dynamically modified their localizations, suggesting supplementary roles to nucleocytoplasmic transport specifically during mitosis. Loss of transportin-SR and Mex/TAP from the nuclear envelope indicates absence of RNA transport during the partially open mitosis of Aspergillus, whereas nucleolar accumulation of Kap121 and Kap123 homologues suggests a role in nucleolar disassembly. This work provides new insight into the roles of nuclear transporters and opens an avenue for future studies of the molecular mechanisms of transport among nuclei within a common cytoplasm, using A. nidulans as a model organism.

Adipocyte triglyceride lipase (ATGL) is the major enzyme involved in the hydrolysis of triglycerides. The Arf1-coat protein complex I (COPI) machinery is known to be engaged in the recruitment of ATGL to lipid droplets (LDs), but the regulatory mechanism has not been clarified. In the present study, we found that ELMOD2, a putative noncanonical Arf-GTPase activating protein (GAP) localizing in LDs, plays an important role in controlling ATGL transport to LDs. We showed that knockdown of ELMOD2 by RNA interference induced an increase in the amount of ATGL existing in LDs and decreased the total cellular triglycerides. These effects of ELMOD2 knockdown were canceled by transfection of small interfering RNA-resistant cDNA of wild-type ELMOD2 but not by that of mutated ELMOD2 lacking the Arf-GAP activity. ELMOD2 was distributed in the endoplasmic reticulum and mitochondria as well as in LDs, but palmitoylation was required only for distribution to LDs. An ELMOD2 mutant deficient in palmitoylation failed to reconstitute the ATGL transport after the ELMOD2 knockdown, indicating that distribution in LDs is indispensable to the functionality of ELMOD2. These results indicate that ELMOD2 regulates ATGL transport and cellular lipid metabolism by modulating the Arf1-COPI activity in LDs.

Kinesin-driven organelle transport is crucial for neuron development and maintenance, yet the mechanisms by which kinesins specifically bind their organelle cargoes remain undefined. In contrast to other transport kinesins, the neuronal function and specific organelle adaptors of heterodimeric Kinesin-2 family members KIF3AB and KIF3AC remain unknown. We developed a novel microscopy-based assay to define protein-protein interactions in intact neurons. The experiments found that both KIF3AB and KIF3AC bind kinesin-associated protein (KAP). These interactions are mediated by the distal C-terminal tail regions and not the coiled-coil domain. We used live-cell imaging in cultured hippocampal neurons to define the localization and trafficking parameters of KIF3AB and KIF3AC organelle populations. We discovered that KIF3AB/KAP and KIF3AC/KAP bind the same organelle populations and defined their transport parameters in axons and dendrites. The results also show that ∼12% of KIF3 organelles contain the RNA-binding protein adenomatous polyposis coli. These data point toward a model in which KIF3AB and KIF3AC use KAP as their neuronal organelle adaptor and that these kinesins mediate transport of a range of organelles.

The Nxf1 protein is a major nuclear export receptor for the transport of mRNA, and it also is essential for export of retroviral mRNAs with retained introns. In the latter case, it binds to RNA elements known as constitutive transport elements (CTEs) and functions in conjunction with a cofactor known as Nxt1. The NXF1 gene also regulates expression of its own intron-containing RNA through the use of a functional CTE within intron 10. mRNA containing this intron is exported to the cytoplasm, where it can be translated into the 356-amino acid short Nxf1(sNxf1) protein, despite the fact that it is a prime candidate for nonsense-mediated decay (NMD). Here we demonstrate that sNxf1 is highly expressed in nuclei and dendrites of hippocampal and neocortical neurons in rodent brain. Additionally, we show that sNxf1 localizes in RNA granules in neurites of differentiated N2a mouse neuroblastoma cells, where it shows partial colocalization with Staufen2 isoform SS, a protein known to play a role in dendritic mRNA trafficking. We also show that sNxf1 forms heterodimers in conjunction with the full-length Nxf1 and that sNxf1 can replace Nxt1 to enhance the expression of CTE-containing mRNA and promote its association with polyribosomes.

Transport of late endosomes and lysosome-related organelles (LE/LROs) in axons is essential for supplying synaptic cargoes and for removing damaged macromolecules. Defects in this system are implicated in a range of human neurodegenerative and neurodevelopmental disorders. The findings reported here identify a novel mechanism regulating LE/LRO transport based on the coordinated coupling of microtubule motors and vesicle coat proteins to the neuron-enriched, transmembrane protein calcyon (Caly). We found that the cytoplasmic C-terminus of Caly pulled down proteins involved in microtubule-dependent transport (DIC, KIF5A, p150Glued, Lis1) and organelle biogenesis (AP-1 and AP-3) from the brain. In addition, RNA interference-mediated knockdown of Caly increased the percentage of static LE/LROs labeled by LysoTracker in cultured dorsal root ganglion axons. In contrast, overexpression of Caly stimulated movement of organelles positive for LysoTracker or the AP-3 cargo GFP-PI4KIIα. However, a Caly mutant (ATEA) that does not bind AP-3 was unable to pull down motor proteins from brain, and expression of the ATEA mutant failed to increase either LE/LRO flux or levels of associated dynein. Taken together, these data support the hypothesis that Caly is a multifunctional scaffolding protein that regulates axonal transport of LE/LROs by coordinately interacting with motor and vesicle coat proteins.

The nuclear pore complex (NPC) plays a critical role in gene expression by mediating import of transcription regulators into the nucleus and export of RNA transcripts to the cytoplasm. Emerging evidence suggests that in addition to mediating transport, a subset of nucleoporins (Nups) engage in transcriptional activation and elongation at genomic loci that are not associated with NPCs. The underlying mechanism and regulation of Nup mobility on and off nuclear pores remain unclear. Here we show that Nup50 is a mobile Nup with a pronounced presence both at the NPC and in the nucleoplasm that can move between these different localizations. Strikingly, the dynamic behavior of Nup50 in both locations is dependent on active transcription by RNA polymerase II and requires the N-terminal half of the protein, which contains importin α- and Nup153-binding domains. However, Nup50 dynamics are independent of importin α, Nup153, and Nup98, even though the latter two proteins also exhibit transcription-dependent mobility. Of interest, depletion of Nup50 from C2C12 myoblasts does not affect cell proliferation but inhibits differentiation into myotubes. Taken together, our results suggest a transport-independent role for Nup50 in chromatin biology that occurs away from the NPC.