Characterization of a ligand-gated cation channel based on an inositol receptor in the silkworm,Bombyx mori

Insect herbivores recognize non-volatile compounds in plants to direct their feeding behavior. Gustatory receptors (Gr) appear to be required for nutrient recognition by gustatory organs in the mouthparts of insects. Gr10 is expressed in Bombyx mori (BmGr10) mouthparts such as maxillary galea, maxillary palp, and labrum. BmGr10 is predicted to function in sugar recognition; however, the precise biochemical function remains obscure. Larvae of B. mori are monophagous feeders able to find and feed on mulberry leaves. Soluble mulberry leaf extract contains sucrose, glucose, fructose, and myo-inositol. In this study, we identified BmGr10 as an inositol receptor using electrophysiological analysis with the Xenopus oocyte expression system and Ca²⁺ imaging techniques using mammalian cells. These results demonstrated that Xenopus oocytes or HEK293T cells expressing BmGr10 specifically respond to myo-inositol and epi-inositol but do not respond to any mono-, di-, or tri-saccharides or to some sugar alcohols. These inositols caused Ca²⁺ and Na⁺ influxes into the cytoplasm independently of a G protein-mediated signaling cascade, indicating that BmGr10 is a ligand-gated cation channel. Overall, BmGr10 plays an important role in the myo-inositol recognition required for B. mori larval feeding behavior.     

Expression of arginine vasotocin receptors in the developing zebrafish CNS

Vasotocin/vasopressin is a neuropeptide that regulates social and reproductive behaviors in a variety of animals including fish. Arginine vasotocin (AVT) is expressed by cells in the ventral hypothalamic and preoptic areas in the diencephalon during embryogenesis in zebrafish suggesting that vasotocin might mediate other functions within the CNS prior to the development of social and reproductive behaviors. In order to examine potential early roles for vasotocin we cloned two zebrafish vasotocin receptors homologous to AVPR_1a. The receptors are expressed primarily in the CNS in similar but generally non-overlapping patterns. Both receptors are expressed in the forebrain, midbrain and hindbrain by larval stage. Of note, AVTR_1a-expressing neurons in the hindbrain appear to be contacted by the axons of preoptic neurons in the forebrain that include avt+ neurons and sensory axons in the lateral longitudinal fasciculus (LLF). Furthermore, AVTR_1a-expressing hindbrain neurons extend axons into the medial longitudinal fasciculus (MLF) that contains axons of many neurons thought to be involved in locomotor responses to sensory stimulation. One hypothesis consistent with this anatomy is that AVT signaling mediates or gates sensory input to motor circuits in the hindbrain and spinal cord.     

Expression of arginine vasotocin receptors in the developing zebrafish CNS

Vasotocin/vasopressin is a neuropeptide that regulates social and reproductive behaviors in a variety of animals including fish. Arginine vasotocin (AVT) is expressed by cells in the ventral hypothalamic and preoptic areas in the diencephalon during embryogenesis in zebrafish suggesting that vasotocin might mediate other functions within the CNS prior to the development of social and reproductive behaviors. In order to examine potential early roles for vasotocin we cloned two zebrafish vasotocin receptors homologous to AVPR_1a. The receptors are expressed primarily in the CNS in similar but generally non-overlapping patterns. Both receptors are expressed in the forebrain, midbrain and hindbrain by larval stage. Of note, AVTR_1a-expressing neurons in the hindbrain appear to be contacted by the axons of preoptic neurons in the forebrain that include avt+ neurons and sensory axons in the lateral longitudinal fasciculus (LLF). Furthermore, AVTR_1a-expressing hindbrain neurons extend axons into the medial longitudinal fasciculus (MLF) that contains axons of many neurons thought to be involved in locomotor responses to sensory stimulation. One hypothesis consistent with this anatomy is that AVT signaling mediates or gates sensory input to motor circuits in the hindbrain and spinal cord.     

Slitrk6 expression profile in the mouse embryo and its relationship to that of Nlrr3

Slitrk6 is a member of the Slitrk family of proteins, which are integral membrane proteins possessing two leucine-rich repeat (LRR) domains and a carboxy-terminal domain partially similar to that in the trk neurotrophin receptor proteins. Here, I show that Slitrk6 is uniquely expressed in various organs, different from other Slitrk genes which are predominantly expressed in neural tissues. In the developing mouse embryo, Slitrk6 expression was detected in the otic cyst, lateral trunk epidermis and its underlying mesenchymal tissue, limb bud, maxillary process, pharyngeal arches, cochlea, retina, tongue, tooth primordium, central nervous system (CNS), and the visceral organ primordia including of the lung, gastrointestinal tract (particularly in the enteric neurons) and pancreas. The expression in these organs occurred in a spatially restricted manner. In the CNS, the expression was highly compartmentalized in the dorsal thalamus, cerebellum and medulla. The expression compartment in the thalamus in which Slitrk6 was expressed was closely related to the Gbx2-expressing prosomere 2. Interestingly, the Slitrk6 expression in the CNS, cochlea, tongue, tooth primordial, and other organs was partially complementary to the expression of Nlrr3, which belongs to another family of neuronal LRR-containing transmembrane proteins. The complementary expression of the two proteins in the dorsal thalamus persisted from E13.5 to the adult stage.     

Evolutionary changes in sensory precursor formation in arthropods: embryonic development of leg sensilla in the spider Cupiennius salei

We describe here for the first time the development of mechanosensory organs in a chelicerate, the spider Cupiennius salei. It has been shown previously that the number of external sense organs increases with each moult. While stage 1 larvae do not have any external sensory structures, stage 2 larvae show a stereotyped pattern of touch sensitive ‘tactile hairs’ on their legs. We show that these mechanosensory organs develop during embryogenesis. In contrast to insects, groups of sensory precursors are recruited from the leg epithelium, rather than single sensory organ progenitors. The groups increase by proliferation, and neural cells delaminate from the cluster, which migrate away to occupy a position proximal to the accessory cells of the sense organ. In addition, we describe the development of putative internal sense organs, which do not differentiate until larval stage 2. We show by RNA interference that, similar to Drosophila, proneural genes are responsible for the formation and subtype identity of sensory organs. Furthermore, we demonstrate an additional function for proneural genes in the coordinated invagination and migration of neural cells during sensory organ formation in the spider.     

Morphology of the mouthparts of the spittlebug Philagra albinotata Uhler (Hemiptera: Cercopoidea: Aphrophoridae)

Mouthparts associated with feeding behavior and feeding habits are important sensory and feeding structures in insects. To obtain a better understanding of feeding in Cercopoidea, the morphology of mouthparts of the spittlebug, Philagra albinotata Uhler was examined using scanning electron microscopy. The mouthparts of P. albinotata are of the typical piercing–sucking type found in Hemiptera, comprising a cone-shaped labrum, a tube-like, three-segmented labium with a deep groove on the anterior side, and a stylet fascicle consisting of two mandibular and two maxillary stylets. The mandibles consist of a dorsal smooth region and a ventral serrate region near the apical half of the external convex region, and bear five nodules or teeth on the dorsal external convex region on the distal extremity; these are regarded as unique features that distinguish spittlebugs from other groups of Hemiptera. The externally smooth maxillary stylets, interlocked to form a larger food canal and a smaller salivary canal, are asymmetrical only in the internal position of longitudinal carinae and grooves. One dendritic canal is found in each maxilla and one in each mandible. Two types of sensilla trichodea, three types of sensilla basiconica and groups of multi-peg structures occur in different locations on the labium, specifically the labial tip with two lateral lobes divided into anterior sensory fields with ten small peg sensilla arranged in a 5 + 4 + 1 pattern and one big peg sensillum, and posterior sensory fields with four sensilla trichodea. Compared with those of previously studied Auchenorrhyncha, the mouthparts of P. albinotata may be distinguished by the shape of the mandibles, the multi-peg structures and a tooth between the salivary canal and the food canal on the extreme end of the stylets. The mouthpart morphology is illustrated using scanning electron micrographs, and the taxonomic and putative functional significance of the different structures is briefly discussed.     

Pituitary adenylate cyclase activating polypeptide is expressed in autonomic neurons

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel vasoactive intestinal peptide (VIP)-like peptide, which is present in neuronal elements of several peripheral organs, and thus a putative neurotransmitter/modulator. In the present study, the expression of PACAP in two parasympathetic ganglia (otic, sphenopalatine) and one mixed parasympathetic/sensory ganglion (jugular-nodose) in rat was characterized by use of in situ hybridization and immunocytochemistry and compared to that of VIP and calcitonin gene-related peptide (CGRP). PACAP and VIP were expressed in virtually all nerve cell bodies in the otic and sphenopalatine ganglia; PACAP and VIP were also expressed in subpopulations of nerve cell bodies in the jugular-nodose ganglion. CGRP was expressed in numerous nerve cell bodies in the jugular-nodose ganglion and in a few, scattered, nerve cell bodies in the sphenopalatine ganglion. In the otic and sphenopalatine ganglia, PACAP- and VIP-like immunoreactivities were frequently co-localized; in the jugular-nodose ganglion, PACAP-like immunoreactivity was frequently co-localized with CGRP-like immunoreactivity in presumably sensory neurons and to a lesser extent with VIP in parasympathetic neurons. Thus, PACAP is synthesized and stored in autonomic parasympathetic neurons as well as in vagal sensory neurons, which provides an anatomical basis for the diverse effects of PACAP previously described.     

Genome-wide annotation and functional identification of aphid GLUT-like sugar transporters

Background

Phloem feeding insects, such as aphids, feed almost continuously on plant phloem sap, a liquid diet that contains high concentrations of sucrose (a disaccharide comprising of glucose and fructose). To access the available carbon, aphids hydrolyze sucrose in the gut lumen and transport its constituent monosaccharides, glucose and fructose. Although sugar transport plays a critical role in aphid nutrition, the molecular basis of sugar transport in aphids, and more generally across all insects, remains poorly characterized. Here, using the latest release of the pea aphid, Acyrthosiphon pisum, genome we provide an updated gene annotation and expression profile of putative sugar transporters. Finally, gut expressed sugar transporters are functionally expressed in yeast and screened for glucose and fructose transport activity.

Results

In this study, using a de novo approach, we identified 19 sugar porter (SP) family transporters in the A. pisum genome. Gene expression analysis, based on 214, 834 A. pisum expressed sequence tags, supports 17 sugar porter family transporters being actively expressed in adult female aphids. Further analysis, using quantitative PCR identifies 4 transporters, A. pisum sugar transporter 1, 3, 4 and 9 (ApST1, ApST3, ApST4 and ApST9) as highly expressed and/or enriched in gut tissue. When expressed in a Saccharomyces cerevisiae hexose transporter deletion mutant (strain EBY.VW4000), only ApST3 (previously characterized) and ApST4 (reported here) transport glucose and fructose resulting in functional rescue of the yeast mutant. Here we characterize ApST4, a 491 amino acid protein, with 12 predicted transmembrane regions, as a facilitative glucose/fructose transporter. Finally, phylogenetic reconstruction reveals that ApST4, and related, as yet uncharacterized insect transporters are phylogenetically closely related to human GLUT (SLC2A) class I facilitative glucose/fructose transporters.

Conclusions

The gut enhanced expression of ApST4, and the transport specificity of its product is consistent with ApST4 functioning as a gut glucose/fructose transporter. Here, we hypothesize that both ApST3 (reported previously) and ApST4 (reported here) function at the gut interface to import glucose and fructose from the gut lumen.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-647) contains supplementary material, which is available to authorized users.     

Serotonin- and two putative serotonin receptors-like immunohistochemical reactivities in the ground crickets Dianemobius nigrofasciatus and Allonemobius allardi

Serotonin (5-hydroxytryptamine; 5-HT)- and two putative serotonin receptors, 5-HT1A- and 5-HT1B-like, immunohistochemical reactivities were investigated in the cephalic ganglia of two ground crickets, Dianemobius nigrofasciatus and Allonemobius allardi. 5-HT-ir was strongly expressed in the central body, accessory medulla region of the optic lobe, frontal ganglion, posterior cortex of the protocerebrum, dorsolateral region of the protocerebrum, and the suboesphageal ganglion (SOG) in both crickets. However, 5-HT1A-ir and 5-HT1B-ir showed quite mutually distinct patterns that were also distinct from 5-HT-ir. 5-HT1A-ir was located in the pars intercerebralis, dorsolateral region of the protocerebrum, optic tract, optic lobe, and the midline of the SOG in both crickets. 5-HT1B-ir was located in the pars intercerebralis and dorsolateral region of the protocerebrum, and detected weakly in the optic lobe, tritocerebrum, and the midline of the SOG in both crickets. Interspecific differences were observed with 5-HT1A-ir. 5-HT1A-ir was expressed weakly in two neurons in the mandibular neuromere of the SOG in D. nigrofasciatus, while it was expressed strongly in the tritocerebrum, mandibular neuromere, and maxillary neuromere of the SOG in A. allardi and co-localized with CLOCK-ir (CLK-ir). 5HT-1B-ir was co-localized with CLK-ir in the tritocerebrum, mandibular neuromere, and maxillary neuromere of the SOG when double-labeling was conducted in both crickets. These results indicated that 5-HT and both types of 5-HT receptors may regulate circadian photo-entrainment or photoperiodism in A. allardi, while only 5-HT1B may be involved in circadian photo-entrainment or photoperiodism in D. nigrofasciatus.     

A systematic analysis of <Emphasis Type="Italic">Drosophila</Emphasis> gustatory receptor gene expression in abdominal neurons which project to the central nervous system

In Drosophila, the gustatory receptor (Gr) gene family contains 60 family members that encode 68 proteins through alternative splicing. Some gustatory receptors (Grs) are involved in the sensing of sugars, bitter substrates, CO₂, pheromones, and light. Here, we systematically examined the expression of all 68 Grs in abdominal neurons which project to the abdominal ganglion of the central nervous system using the GAL4/UAS system. Gr gene expression patterns have been successfully analyzed in previous studies by using the GAL4/UAS system to drive reporter gene expression. Interestingly, 21 Gr-GAL4 drivers showed abdominal ganglion projection, and 18 of these 21 Gr-GAL4 drivers labeled multidendritic neurons of the abdominal wall. 4 drivers also labeled neuronal processes innervating the reproductive organs. The peripheral expression of Gr-GAL4 drivers in abdominal multidendritic neurons or neurons innervating the reproductive organs suggests that these Grs have atypical sensory functions in these organs not limited to conventional taste sensing.     

Expression of Rab27B-binding protein Slp1 in pancreatic acinar cells and its involvement in amylase secretion

Slp1 is a putative Rab27 effector protein and implicated in intracellular membrane transport; however, the precise tissue distribution and function of Slp1 protein remain largely unknown. In this study we investigated the tissue distribution of Slp1 in mice and found that Slp1 is abundantly expressed in the pancreas, especially in the apical region of pancreatic acinar cells. Slp1 interacted with Rab27B in vivo and both proteins were co-localized on zymogen granules. Morphological analysis of fasted Slp1 knockout mice showed an increased number of zymogen granules in the pancreatic acinar cells, indicating that Slp1 is part of the machinery of amylase secretion by the exocrine pancreas.     

Hoxa1 lineage tracing indicates a direct role for Hoxa1 in the development of the inner ear, the heart, and the third rhombomere

Loss of Hoxa1 function results in severe defects of the brainstem, inner ear, and cranial ganglia in humans and mice as well as cardiovascular abnormalities in humans. Because Hoxa1 is expressed very transiently during an early embryonic stage, it has been difficult to determine whether Hoxa1 plays a direct role in the precursors of the affected organs or if all defects result from indirect effects due to mispatterning of the hindbrain. In this study we use a Hoxa1-IRES-Cre mouse to genetically label the early Hoxa1-expressing cells and determine their contribution to each of the affected organs, allowing us to conclude in which precursor tissue Hoxa1 is expressed. We found Hoxa1 lineage-labeled cells in all tissues expected to be derived from the Hoxa1 domain, such as the facial and abducens nuclei and nerves as well as r4 neural crest cells. In addition, we detected the lineage in derivatives that were not thought to have expressed Hoxa1 during development. In the brainstem, the anterior border of the lineage was found to be in r3, which is more anterior than previously reported. We also observed an interesting pattern of the lineage in the inner ear, namely a strong contribution to the otic epithelium with the exception of sensory patches. Moreover, lineage-labeled cells were detected in the atria and outflow tract of the developing heart. In conclusion, Hoxa1 lineage tracing uncovered new domains of Hoxa1 expression in rhombomere 3, the otic epithelium, and cardiac precursors, suggesting a more direct role for Hoxa1 in development of these tissues than previously believed.     

Neuronal specificity and its development in the Drosophila wing disc and its derivatives

The imaginal wing disc of flies gives rise to the adult wing blade and dorsal thorax (notum). A great deal has been learned in recent years about the process of neurogenesis in this disc; a number of genes that play crucial roles in the formation of sensory mother cells and in the differentiation of the sensory organs have been identified and their roles defined. Given this extensive background of developmental genetics, it has seemed profitable to summarize what is known about the end-products of neural development, the adult sensory organs. Discussed are their physiological function and role in behavior, the pathways followed by their axons in the CNS, and both genes and epigenetic processes that might play some role in the later stages of neural development and in adult function. The highly individual characteristics of certain of the sensory organs is emphasized, both in the context of their adult roles and as a challenge for future studies in developmental genetics. © 1993 John Wiley & Sons, Inc.     

The Notch ligand JAG1 is required for sensory progenitor development in the mammalian inner ear

In mammals, six separate sensory regions in the inner ear are essential for hearing and balance function. Each sensory region is made up of hair cells, which are the sensory cells, and their associated supporting cells, both arising from a common progenitor. Little is known about the molecular mechanisms that govern the development of these sensory organs. Notch signaling plays a pivotal role in the differentiation of hair cells and supporting cells by mediating lateral inhibition via the ligands Delta-like 1 and Jagged (JAG) 2. However, another Notch ligand, JAG1, is expressed early in the sensory patches prior to cell differentiation, indicating that there may be an earlier role for Notch signaling in sensory development in the ear. Here, using conditional gene targeting, we show that the Jag1 gene is required for the normal development of all six sensory organs within the inner ear. Cristae are completely lacking in Jag1-conditional knockout (cko) mutant inner ears, whereas the cochlea and utricle show partial sensory development. The saccular macula is present but malformed. Using SOX2 and p27^kip1 as molecular markers of the prosensory domain, we show that JAG1 is initially expressed in all the prosensory regions of the ear, but becomes down-regulated in the nascent organ of Corti by embryonic day 14.5, when the cells exit the cell cycle and differentiate. We also show that both SOX2 and p27^kip1 are down-regulated in Jag1-cko inner ears. Taken together, these data demonstrate that JAG1 is expressed early in the prosensory domains of both the cochlear and vestibular regions, and is required to maintain the normal expression levels of both SOX2 and p27^kip1. These data demonstrate that JAG1-mediated Notch signaling is essential during early development for establishing the prosensory regions of the inner ear.     

Sensory organs on the body parts of the bed-bug Cimex hemipterus fabricius (Hemiptera : Cimicidae) and the anatomy of its central nervous system

Anatomy of the sensory organs on the prominent body parts of the adult bed-bug Cimex hemipterus (Hemiptera: Cimicidae) and its central nervous system (CNS) was studied by light, transmission, or scanning electron microscopy. The distal tips of antenna and rostrum were found to have rich complements of sensilla. The antenna has both olfactory and gustatory sensilla. Olfactory sensilla project to the antennal lobe organized in the form of glomeruli, while the 2nd component, presumably from gustatory sensilla, projects to the suboesophageal ganglion. The ultrastructure of the sensory pegs on the rostrum of C. hemipterus does not resemble the chemosensilla of adult insects; rather they resemble the larval sensilla of Drosophila melanogaster in the maxillary organ. Earlier we believed this to be a gustatory organ. A few similar sensilla also occur on the antenna, indicating its multimodal role. Amongst the 3 types of sensory hairs located on legs, there are only a few gustatory hairs (7–10 hairs) on the tibia. The pointed and serrate mechanosensory hair types occur in abundance; the serrate type are prominently present on the lateral surface of the legs. On other parts of the body such as the thorax or abdomen, serrate hairs are most abundant. Both the distal segment of antenna and rostrum are invested by 2 nerves, where the axon counts of the 2 antennal nerves are 380 and 425, while each rostral nerve on average has 205 axons. Abundant clusters of microtubules were found in the brain, thoracio-abdominal ganglia, leg-nerves, and the space between muscles and cuticle. These conspicuous microtubule-clusters occur in interaxonal space, mainly glial cells, in the nervous system. In addition, the glial cells have osmiophilic junctions amongst themselves. A novel “hinge and joint” system, which controls the cross-section of the food canal and the salivary duct in an inversely related manner, was found in the rostrum of the bed-bug.     

Defining the NG2-expressing cell of the adult CNS

The NG2 proteoglycan is believed to be an in vivomarker for oligodendrocyte progenitors found in the developing brain. The prevalence of NG2-expressing cells that remain in the adult CNS following the end of gliogenesis is significant. Current research is focused on how this cell participates in the normal function of the adult CNS and whether it may be activated by injury and/or contribute to repair. Despite substantial evidence for a sub-population of NG2-expressing cells playing a glial progenitor role in the adult CNS, there is much to be learned. Specifically, the heterogeneity of this population has not been adequately addressed for the adult CNS and while NG2 cells continue to divide in the adult CNS it is not clear what function they serve once myelination is complete. Future studies should elucidate the functional importance of NG2 in a variety of cell functions and shed light on the role NG2-expressing cells play in the intact and diseasedCNS.     

Zur Ultrastruktur der seitlichen Sinnesorgane am Augenhügel vonAnoplodactylus pygmaeus (Pycnogonida)

Former light microscopic studies on the lateral sense organs of sea spiders yielded divergent results. Consequently, different authors ascribed different functions to these organs. The present ultrastructural study shows that each lateral sense organ ofA. pygmaeus consists of approximately 15 sensory cells of two different types, approximately 20 sheath cells with numerous long microvilli, and an outer cuticular covering. Essentially the same elements are characteristic features of arthropod sensilla. There are, however, differences between the sense organs described in this paper and the sense organs of other arthropods. The inner dendritic segments of sensory cells S₁ of theA. pygmaeus lateral sense organs are very short, the sensory cilia are invaginated, and the pericarya of the sensory cells contain electron lucent cytoplasmic regions with large granules (glycogen?). In addition, the lateral sense organs ofA. pygmaeus lack a marked receptor lymph cavity and junctions between the cells. The results of the present ultrastructural study clearly indicate that the lateral sense organs ofA. pygmaeus are not glands as was postulated for other sea spider species by earlier authors. Some investigators hypothesized that the lateral sense organs of other sea spider species were auditory organs or rudimentary eyes. The present results do not support such speculations. Some structural details of the sensory cells ofA. pygmaeus resemble those found in chemoreceptive or putative chemoreceptive organs of other arthropods. Accordingly, chemoreceptive or thermoreceptive functions should be taken into consideration for the lateral sense organs ofA. pygmaeus.     

Topological and functional characterization of an insect gustatory receptor

Insect gustatory receptors are predicted to have a seven-transmembrane structure and are distantly related to insect olfactory receptors, which have an inverted topology compared with G-protein coupled receptors, including mammalian olfactory receptors. In contrast, the topology of insect gustatory receptors remains unknown. Except for a few examples from Drosophila, the specificity of individual insect gustatory receptors is also unknown. In this study, the total number of identified gustatory receptors in Bombyx mori was expanded from 65 to 69. BmGr8, a silkmoth gustatory receptor from the sugar receptor subfamily, was expressed in insect cells. Membrane topology studies on BmGr8 indicate that, like insect olfactory receptors, it has an inverted topology relative to G protein-coupled receptors. An orphan GR from the bitter receptor family, BmGr53, yielded similar results. We infer, from the finding that two distantly related BmGrs have an intracellular N-terminus and an odd number of transmembrane spans, that this is likely to be a general topology for all insect gustatory receptors. We also show that BmGr8 functions independently in Sf9 cells and responds in a concentration-dependent manner to the polyalcohols myo-inositol and epi-inositol but not to a range of mono- and di-saccharides. BmGr8 is the first chemoreceptor shown to respond specifically to inositol, an important or essential nutrient for some Lepidoptera. The selectivity of BmGr8 responses is consistent with the known responses of one of the gustatory receptor neurons in the lateral styloconic sensilla of B. mori, which responds to myo-inositol and epi-inositol but not to allo-inositol.     

Human APE2 protein is mostly localized in the nuclei and to some extent in the mitochondria, while nuclear APE2 is partly associated with proliferating cell nuclear antigen

In human cells APE1 is the major AP endonuclease and it has been reported to have no functional mitochondrial targeting sequence (MTS). We found that APE2 protein possesses a putative MTS. When its N-terminal 15 amino acid residues were fused to the N-terminus of green fluorescent protein and transiently expressed in HeLa cells the fusion protein was localized in the mitochondria. By electron microscopic immunocytochemistry we detected authentic APE2 protein in mitochondria from HeLa cells. Western blotting of the subcellular fraction of HeLa cells revealed most of the APE2 protein to be localized in the nuclei. We found a putative proliferating cell nuclear antigen (PCNA)-binding motif in the C-terminal region of APE2 and showed this motif to be functional by immunoprecipitation and in vitro pull-down binding assays. Laser scanning immunofluorescence microscopy of HeLa cells demonstrated both APE2 and PCNA to form foci in the nucleus and also to be co-localized in some of the foci. The incubation of HeLa cells in HAT medium containing deoxyuridine significantly increased the number of foci in which both molecules were co-localized. Our results suggest that APE2 participates in both nuclear and mitochondrial BER and also that nuclear APE2 functions in the PCNA-dependent BER pathway.