Endophilin 1 is a presynaptically enriched protein which binds the GTPase dynamin and the polyphosphoinositide phosphatase synptojanin. Perturbation of endophilin function in cell-free systems and in a living synapse has implicated endophilin in endocytic vesicle budding (Ringstad, N., H. Gad, P. Low, G. Di Paolo, L. Brodin, O. Shupliakov, and P. De Camilli. 1999. Neuron. 24:143–154; Schmidt, A., M. Wolde, C. Thiele, W. Fest, H. Kratzin, A.V. Podtelejnikov, W. Witke, W.B. Huttner, and H.D. Soling. 1999. Nature. 401:133–141; Gad, H., N. Ringstad, P. Low, O. Kjaerulff, J. Gustafsson, M. Wenk, G. Di Paolo, Y. Nemoto, J. Crun, M.H. Ellisman, et al. 2000. Neuron. 27:301–312). Here, we show that purified endophilin can directly bind and evaginate lipid bilayers into narrow tubules similar in diameter to the neck of a clathrin-coated bud, providing new insight into the mechanisms through which endophilin may participate in membrane deformation and vesicle budding. This property of endophilin is independent of its putative lysophosphatydic acid acyl transferase activity, is mediated by its NH2-terminal region, and requires an amino acid stretch homologous to a corresponding region in amphiphysin, a protein previously shown to have similar effects on lipid bilayers (Takei, K., V.I. Slepnev, V. Haucke, and P. De Camilli. 1999. Nat. Cell Biol. 1:33–39). Endophilin cooligomerizes with dynamin rings on lipid tubules and inhibits dynamin's GTP-dependent vesiculating activity. Endophilin B, a protein with homology to endophilin 1, partially localizes to the Golgi complex and also deforms lipid bilayers into tubules, underscoring a potential role of endophilin family members in diverse tubulovesicular membrane-trafficking events in the cell.

Octopamine biosynthesis requires tyrosine decarboxylase to convert tyrosine into tyramine and tyramine beta-hydroxylase to convert tyramine into octopamine. We identified and characterized a Caenorhabditis elegans tyrosine decarboxylase gene, tdc-1, and a tyramine beta-hydroxylase gene, tbh-1. The TBH-1 protein is expressed in a subset of TDC-1-expressing cells, indicating that C. elegans has tyraminergic cells that are distinct from its octopaminergic cells. tdc-1 mutants have behavioral defects not shared by tbh-1 mutants. We show that tyramine plays a specific role in the inhibition of egg laying, the modulation of reversal behavior, and the suppression of head oscillations in response to anterior touch. We propose a model for the neural circuit that coordinates locomotion and head oscillations in response to anterior touch. Our findings establish tyramine as a neurotransmitter in C. elegans, and we suggest that tyramine is a genuine neurotransmitter in other invertebrates and possibly in vertebrates as well.

The GTPase dynamin I and the inositol 5-phosphatase synaptojanin are nerve terminal proteins implicated in synaptic vesicle recycling. Both proteins contain COOHterminal proline-rich domains that can interact with a variety of Src homology 3 (SH3) domains. A major physiological binding partner for dynamin I and synaptojanin in the nervous system is amphiphysin I, an SH3 domain-containing protein also concentrated in nerve terminals. We have used the proline-rich tail of synaptojanin to screen a rat brain library by the two-hybrid method to identify additional interacting partners of synaptojanin. Three related proteins containing SH3 domains that are closely related to the SH3 domains of Grb2 were isolated: SH3p4, SH3p8, and SH3p13. Further biochemical studies demonstrated that the SH3p4͞8͞13 proteins bind to both synaptojanin and dynamin I. The SH3p4͞8͞13 transcripts are differentially expressed in tissues: SH3p4 mRNA was detected only in brain, SH3p13 mRNA was present in brain and testis, and the SH3p8 transcript was detected at similar levels in multiple tissues. Members of the SH3p4͞8͞13 protein family were found to be concentrated in nerve terminals, and pools of synaptojanin and dynamin I were coprecipitated from brain extracts with antibodies recognizing SH3p4͞8͞13. These findings underscore the important role of SH3-mediated interactions in synaptic vesicle recycling.

Cell transformation by Rous sarcoma virus results in a dramatic change of adhesion structures with the substratum. Adhesion plaques are replaced by dot-like attachment sites called podosomes. Podosomes are also found constitutively in motile nontransformed cells such as leukocytes, macrophages, and osteoclasts. They are represented by columnar arrays of actin which are perpendicular to the substratum and contain tubular invaginations of the plasma membrane. Given the similarity of these tubules to those generated by dynamin around a variety of membrane templates, we investigated whether dynamin is present at podosomes. Immunoreactivities for dynamin 2 and for the dynamin 2–binding protein endophilin 2 (SH3P8) were detected at podosomes of transformed cells and osteoclasts. Furthermore, GFP wild-type dynamin 2aa was targeted to podosomes. As shown by fluorescence recovery after photobleaching, GFP-dynamin 2aa and GFP-actin had a very rapid and similar turnover at podosomes. Expression of the GFP-dynamin 2aaG273D abolished podosomes while GFP-dynaminK44A was targeted to podosomes but delayed actin turnover. These data demonstrate a functional link between a member of the dynamin family and actin at attachment sites between cells and the substratum.

Coordination between sequential steps in synaptic vesicle endocytosis, including clathrin coat formation, fission, and uncoating, appears to involve proteinprotein interactions. Here, we show that compounds that disrupt interactions of the SH3 domain of endophilin with dynamin and synaptojanin impair synaptic vesicle endocytosis in a living synapse. Two distinct endocytic intermediates accumulated. Free clathrin-coated vesicles were induced by a peptide-blocking endophilin's SH3 domain and by antibodies to the proline-rich domain (PRD) of synaptojanin. Invaginated clathrin-coated pits were induced by the same peptide and by the SH3 domain of endophilin. We suggest that the SH3 domain of endophilin participates in both fission and uncoating and that it may be a key component of a molecular switch that couples the fission reaction to uncoating.

Endophilin/SH3p4 is a protein highly enriched in nerve terminals that binds the GTPase dynamin and the polyphosphoinositide phosphatase synaptojanin, two proteins implicated in synaptic vesicle endocytosis. We show here that antibody-mediated disruption of endophilin function in a tonically stimulated synapse leads to a block in the invagination of clathrin-coated pits adjacent to the active zone and therefore to a block of synaptic vesicle recycling. We also show that in a cell-free system, endophilin is not associated with clathrin coats and is a functional partner of dynamin. Our findings suggest that endophilin is part of a biochemical machinery that acts in trans to the clathrin coat from early stages to vesicle fission.

Multiple antibiotic resistance in Escherichia coli can be mediated by induction of the SoxS or MarA protein, triggered by oxygen radicals (in the soxRS regulon) or certain antibiotics (in the marRAB regulon), respectively. These small proteins (SoxS, 107 residues; MarA, 127 residues) are homologous to the C terminus of the XylS-AraC family of proteins and are more closely related to a ϳ100-residue segment in the N terminus of Rob protein, which binds the right arm of the replication origin, oriC. We investigated whether the SoxS-MarA homology in Rob might extend to the regulation of some of the same inducible genes. Overexpression of Rob indeed conferred multiple antibiotic resistance similar to that known for SoxS and MarA (against chloramphenicol, tetracycline, nalidixic acid, and puromycin), as well as resistance to the superoxide-generating compound phenazine methosulfate. The Rob-induced antibiotic resistance depended only partially on the micF antisense RNA that down-regulates the OmpF outer membrane porin to limit antibiotic uptake. Similar antibiotic resistance was conferred by expression of a Rob fragment containing only the N-terminal 123 residues that constitute the SoxS-MarA homology. Both intact Rob and the N-terminal fragment activated expression of stress genes (inaA, fumC, sodA) but with a pattern distinct from that found for SoxS and MarA. Purified Rob protein bound a DNA fragment containing the micF promoter (50% bound at ϳ10 ؊9 M Rob) as strongly as it did oriC, and it bound more weakly to DNA containing the sodA, nfo, or zwf promoter (50% bound at 10 ؊8 to 10 ؊7 M). Rob formed multiple DNA-protein complexes with these fragments, as seen previously for SoxS. These data point to a DNA-binding gene activator module used in different protein contexts.

CO 2 is both a critical regulator of animal physiology and an important sensory cue for many animals for host detection, food location, and mate finding. The free-living soil nematode Caenorhabditis elegans shows CO 2 avoidance behavior, which requires a pair of ciliated sensory neurons, the BAG neurons. Using in vivo calcium imaging, we show that CO 2 specifically activates the BAG neurons and that the CO 2 -sensing function of BAG neurons requires TAX-2/ TAX-4 cyclic nucleotide-gated ion channels and the receptor-type guanylate cyclase GCY-9. Our results delineate a molecular pathway for CO 2 sensing and suggest that activation of a receptor-type guanylate cyclase is an evolutionarily conserved mechanism by which animals detect environmental CO 2 .guanylyl cyclase | olfaction | transcriptional profiling | regulator of G protein signaling | chemosensation T he ability to detect and respond to changing concentrations of environmental CO 2 is widespread among animals and plays a critical role in locating food, finding hosts and mates, and avoiding danger (1-4). CO 2 exposure can also have profound physiological effects, including altered respiration, motility, fecundity, and emotional state (5-7). CO 2 is sensed as an aversive cue by many free-living animals, including humans (3,6,8,9). By contrast, many parasites and disease vectors are attracted to CO 2 , which serves as a sensory cue for host location (1, 10).Nematodes constitute a large and highly diverse phylum that includes both free-living and parasitic species. Many parasitic nematodes, including some of the most devastating human-and plant-parasitic nematodes, are attracted to CO 2 . By contrast, adults of the free-living species Caenorhabditis elegans are repelled by CO 2 (11-14). CO 2 avoidance by C. elegans requires a pair of head neurons called the BAG neurons (13), which also mediate responses to decreases in ambient oxygen levels (15). Whether the BAG neurons directly sense CO 2 is not known, and the signaling pathways that mediate CO 2 detection are poorly understood.We show here that environmental CO 2 specifically activates the BAG neurons and not other neurons that drive avoidance behavior, suggesting that the BAG neurons are primary sensory neurons that detect CO 2 . Prolonged CO 2 exposure causes desensitization of avoidance behavior and the BAG neurons themselves, indicating that behavioral adaptation to CO 2 occurs at the level of the BAG neurons. In addition, we show that the CO 2 -evoked activity of the BAG neurons requires a cGMP signaling pathway consisting of the receptor guanylate cyclase GCY-9 and the cGMP-gated cation channel TAX-2/TAX-4. Insects detect CO 2 using a pair of gustatory receptors (16, 17), whereas some mammals detect CO 2 using the receptor-type guanylate cyclase, guanylate cyclase D (GC-D), and soluble adenylate cyclase (18)(19)(20). Our results show that the mechanism of CO 2 detection in C. elegans more closely resembles that of mammals than insects and suggest an evolutionarily ancient role for receptor-type guanyl...