Immune related endonucleases and GTPases are not associated with tumor response in patients with advanced non-small cell lung cancer treated with checkpoint inhibitors.

Immune related endonucleases have recently been described as potential therapeutic targets and predictors of response to treatment with immune checkpoint inhibitors (ICI). The aim is to evaluate the association between the expression of 5 biomarkers involved in the immune response (CD73, CD39, VISTA, Arl4d and Cytohesin-3) in parallel with the more common ICI-predictive markers, PD-L1 expression and Tumor Mutation Burden (TMB) with response to ICI therapy in an advanced non-small cell lung cancer (NSCLC) cohort. METHODS: Patients with advanced NSCLC treated with ICI single agent were divided into responders and non-responders according to RECIST v1.1 and duration of response (DOR) criteria. Immunohistochemistry was performed on pretreatment tumor tissue samples for PD-L1, CD73, CD39, VISTA, Arl4d, and Cytohesin-3 expression. TMB was estimated with NEOplus v2 RUO (NEO New Oncology GmbH) hybrid capture next generation sequencing assay. Resistance mutations in STK11/KEAP1 and positive predictive mutations in ARID1A/POLE were also evaluated. RESULTS: Included were 56 patients who were treated with ICI single agent. The median progression-free and overall survival for the whole cohort was 3.0 (95% CI, 2.4-3.6) and 15 (95% CI, 9.7-20.2) months, respectively. The distribution of CD73 in tumor cells and CD39, VISTA, Arl4d and Cytohesin-3 expression in immune cells were not different between responders and non-responders. Also, PD-L1 and TMB were not predictive for response. The frequency of STK11, KEAP1 and ARID1A mutations was low and only observed in the non-responder group. CONCLUSION: Separate and combined expression of 5 biomarkers involved in the immune response (CD73, CD39, VISTA, Arl4d, and Cytohesin-3) was not associated with response in our cohort of advanced NSCLC patients receiving single agent ICI. To confirm our findings the analysis of independent larger cohorts is warranted.

Quantification of Vibrio cholerae cholix exotoxin by sandwich bead-ELISA.

Introduction. Cholix toxin (ChxA) is an ADP-ribosylating exotoxin produced by Vibrio cholerae. However, to date, there is no quantitative assay available for ChxA, which makes it difficult to detect and estimate the level of ChxA produced by V. cholerae.Hypothesis/Gap Statement. It is important to develop a reliable and specific quantitative assay to measure the production level of ChxA, which will help us to understand the role of ChxA in V. cholerae pathogenesis.Aim. The aim of this study was to develop a bead-based sandwich ELISA (bead-ELISA) for the quantification of ChxA and to evaluate the importance of ChxA in the pathogenesis of V. cholerae infection.Methodology. Anti-rChxA was raised in New Zealand white rabbits, and Fab-horse radish peroxidase conjugate was prepared by the maleimide method to use in the bead-ELISA. This anti-ChxA bead-ELISA was applied to quantify the ChxA produced by various V. cholerae strains. The production of ChxA was examined in different growth media such as alkaline peptone water (APW), Luria-Bertani broth and AKI. Finally, the assay was evaluated using a mouse lethality assay with representative V. cholerae strains categorized as low to high ChxA-producers based on anti-ChxA bead-ELISA.Results. A sensitive bead-ELISA assay, which can quantify from 0.6 to 60 ng ml-1 of ChxA, was developed. ChxA was mostly detected in the extracellular cell-free supernatant and its production level varied from 1.2 ng ml-1 to 1.6 µg ml-1. The highest ChxA production was observed when V. cholerae strains were cultured in LB broth, but not in APW or AKI medium. The ChxA-producer V. cholerae strains showed 20-80 % lethality and only the high ChxA II-producer was statistically more lethal than a non-ChxA-producer, in the mice model assay. ChxA I and II production levels were not well correlated with mice lethality, and this could be due to the heterogeneity of the strains tested.Conclusion. ChxA I to III was produced mostly extracellularly at various levels depending on strains and culture conditions. The bead-ELISA developed in this study is useful for the detection and quantification of ChxA in V. cholerae strains.

Neutrophil gelatinase-associated lipocalin (NGAL) is localised to the primary cilium in renal tubular epithelial cells - A novel source of urinary biomarkers of renal injury.

BACKGROUND: Primary cilia have been shown to play a central role in regulating epithelial cell differentiation during injury and repair. Growing evidence implicates structural and functional abnormalities of primary cilia in kidney epithelial cells in the onset and development of various kidney diseases including polycystic kidney disease (PKD). Neutrophil-gelatinase associated lipocalin (NGAL) has been identified as a reliable urinary biomarker of kidney injury. However, the mechanism by which this protein accumulates in patient urine samples has not been fully elucidated. METHODS: Human renal tubular epithelial cells (RPTECs) were exposed to previously characterized deciliating agents to assess mechanisms of primary cilium loss. Confocal immunofluorescent imaging was employed to visualise the effects on cilia. Western blot analysis was utilised to quantify the ciliary protein Arl13b in both RPTEC whole cell lysates and supernatants. Co-immunoprecipitation was used to demonstrate co-localisation of Arl13b and NGAL in urinary samples from a clinical Chronic Allograft Nephropathy (CAN) cohort. RESULTS: Immunofluorescent analysis revealed that NGAL was localised to the primary cilium in RPTECs, co-localizing with a ciliary specific protein, Arl13b. Deciliation experiments showed that loss of the cilia coincided with loss of NGAL from the cells. CONCLUSION: The accumulation of NGAL in supernatants in vitro and in the urine of CAN patients was concurrent with loss of Arl13b, a specific ciliary protein. The findings of this study propose that increased NGAL urinary concentrations are directly linked to deciliation of the renal epithelial cells as a result of injury.

TULP3 is required for localization of membrane-associated proteins ARL13B and INPP5E to primary cilia.

The primary cilia are known as biosensors that transduce signals through the ciliary membrane proteins in vertebrate cells. The ciliary membrane contains transmembrane proteins and membrane-associated proteins. Tubby-like protein 3 (TULP3), a member of the tubby family, has been shown to interact with the intraflagellar transport-A complex (IFT-A) and to be involved in the ciliary localization of transmembrane proteins, although its role in the ciliary entry of membrane-associated proteins has remained unclear. Here, to determine whether TULP3 is required for the localization of ciliary membrane-associated proteins, we generated and analyzed TULP3-knockout (KO) hTERT RPE-1 (RPE1) cells. Immunofluorescence analysis demonstrated that ciliary formation was downregulated in TULP3-KO cells and that membrane-associated proteins, ADP-ribosylation factor-like 13B (ARL13B) and inositol polyphosphate-5-phosphatase E (INPP5E), failed to localize to primary cilia in TULP3-KO cells. These defects in the localization of ARL13B and INPP5E in TULP3-KO cells were rescued by the exogenous expression of wild-type TULP3, but not that of mutant TULP3 lacking the ability to bind IFT-A. In addition, the expression of TUB protein, another member of the tubby family whose endogenous expression is absent in RPE1 cells, also rescued the defective ciliary localization of ARL13B and INPP5E in TULP3-KO cells, suggesting that there is functional redundancy between TULP3 and TUB. Our findings indicate that TULP3 participates in ciliogenesis, and targets membrane-associated proteins to primary cilia via binding to IFT-A.

Cellular and subcellular localization of ADP-ribosylation factor 6 in mouse peripheral tissues.

ADP-ribosylation factor 6 (Arf6) is a small GTPase that regulates endosomal trafficking and actin cytoskeleton remodeling. In the present study, we comprehensively examined the cellular and subcellular localization of Arf6 in adult mouse peripheral tissues by immunofluorescence and immunoelectron microscopy using the heat-induced antigen retrieval method with Tris-EDTA buffer (pH 9.0). Marked immunolabeling of Arf6 was observed particularly in epithelial cells of several tissues including the esophagus, stomach, small and large intestines, trachea, kidney, epididymis, oviduct, and uterus. In most epithelial cells of simple or pseudostratified epithelia, Arf6 exhibited predominant localization to the basolateral membrane and a subpopulation of endosomes. At an electron microscopic level, Arf6 was localized along the basolateral membrane, with dense accumulation at interdigitating processes and infoldings. Arf6 was present in a ring-like appearance at intercellular bridges in spermatogonia and spermatocytes in the testis and at the Flemming body of cytokinetic somatic cells in the ovarian follicle, thymus, and spleen. The present study provides anatomical clues to help understand the physiological roles of Arf6 at the whole animal level.

Depletion of the human N-terminal acetyltransferase hNaa30 disrupts Golgi integrity and ARFRP1 localization.

The organization of the Golgi apparatus (GA) is tightly regulated. Golgi stack scattering is observed in cellular processes such as apoptosis and mitosis, and has also been associated with disruption of cellular lipid metabolism and neurodegenerative diseases. Our studies show that depletion of the human N-α-acetyltransferase 30 (hNaa30) induces fragmentation of the Golgi stack in HeLa and CAL-62 cell lines. The GA associated GTPase ADP ribosylation factor related protein 1 (ARFRP1) was previously shown to require N-terminal acetylation for membrane association and based on its N-terminal sequence, it is likely to be a substrate of hNaa30. ARFRP1 is involved in endosome-to-trans-Golgi network (TGN) traffic. We observed that ARFRP1 shifted from a predominantly cis-Golgi and TGN localization to localizing both Golgi and non-Golgi vesicular structures in hNaa30-depleted cells. However, we did not observe loss of membrane association of ARFRP1. We conclude that hNaa30 depletion induces Golgi scattering and induces aberrant ARFRP1 Golgi localization.

High expression of EPB41L5, an integral component of the Arf6-driven mesenchymal program, correlates with poor prognosis of squamous cell carcinoma of the tongue.

BACKGROUND: Squamous cell carcinoma of the tongue (tongue SCC) is a major subtype of head and neck squamous cell carcinoma (HNSCC), which is an intractable cancer under current therapeutics. ARF6 and its effector AMAP1 are often overexpressed in different types of cancers, such as breast cancer and renal cancer, and in these cancers, AMAP1 binds to EPB41L5 to promote invasion, metastasis, and drug resistance. EPB41L5 is a mesenchymal-specific protein, normally induced during epithelial-mesenchymal transition (EMT) to promote focal adhesion dynamics. Similarly to breast cancer and renal cancer, the acquisition of mesenchymal phenotypes is the key process that drives the malignancy of HNSCC. We previously showed that the overexpression of AMAP1 in tongue SCC is statistically correlated with the poor outcome of patients. In this study, we examined whether tongue SCC also expresses EPB41L5 at high levels. RESULTS: Immunohistochemical staining of clinical specimens of tongue SCC demonstrated that high expression levels of EPB41L5 statistically correlate with poor disease-free survival and poor overall survival rates of patients. The tongue SCC cell line SCC-9, which overexpress Arf6 and AMAP1, also expressed EPB41L5 at high levels to promote invasiveness, whereas the weakly invasive SCC-25 cells did not express EPB41L5 at notable levels. Among the different EMT-associated transcriptional factors, ZEB1 was previously found to be most crucial in inducing EPB41L5 in breast cancer and renal cancer. In contrast, expression levels of ZEB1 did not correlate with the expression levels of EPB41L5 in tongue SCC, whereas KLF8 and FOXO3 levels showed positive correlations with EPB41L5 levels. Moreover, silencing of EPB41L5 only marginally improved the drug resistance of SCC-9 cells, even when coupled with ionizing radiation. CONCLUSION: Our results indicate that activation of the cancer mesenchymal program in tongue SCC, which leads to EPB41L5 expression, closely correlates with the poor prognosis of patients. However, ZEB1 was not the major inducer of EPB41L5 in tongue SCC, unlike in breast cancer and renal cancer. Thus, processes that trigger the mesenchymal program of tongue SCC, which drives their malignancies, seem to be substantially different from those of other cancers.

Distinct subcellular localization of alternative splicing variants of EFA6D, a guanine nucleotide exchange factor for Arf6, in the mouse brain.

EFA6D (guanine nucleotide exchange factor for ADP-ribosylation factor 6 [Arf6]D) is also known as EFA6R, Psd3, and HCA67. It is the fourth member of the EFA6 family with guanine nucleotide exchange activity for Arf6, a small guanosine triphosphatase (GTPase) that regulates endosomal trafficking and actin cytoskeleton remodeling. We propose a classification and nomenclature of 10 EFA6D variants deposited in the GenBank database as EFA6D1a, 1b, 1c, 1d, 1s, 2a, 2b, 2c, 2d, and 2s based on the combination of N-terminal and C-terminal insertions. Polymerase chain reaction analysis showed the expression of all EFA6D variants except for variants a and d in the adult mouse brain. Immunoblotting analysis with novel variant-specific antibodies showed the endogenous expression of EFA6D1b, EFA6D1c, and EFA6D1s at the protein level, with the highest expression being EFA6D1s, in the brain. Immunoblotting analysis of forebrain subcellular fractions showed the distinct subcellular distribution of EFA6D1b/c and EFA6D1s. The immunohistochemical analysis revealed distinct but overlapping immunoreactive patterns between EFA6D1b/c and EFA6D1s in the mouse brain. In immunoelectron microscopic analyses of the hippocampal CA3 region, EFA6D1b/c was present predominantly in the mossy fiber axons of dentate granule cells, whereas EFA6D1s was present abundantly in the cell bodies, dendritic shafts, and spines of hippocampal pyramidal cells. These results provide the first anatomical evidence suggesting the functional diversity of EFA6D variants, particularly EFA6D1b/c and EFA6D1s, in neurons. J. Comp. Neurol. 524:2531-2552, 2016. © 2016 Wiley Periodicals, Inc.

Approaches to studying Arf GAPs in cells: in vitro assay with isolated focal adhesions.

The Arf GAPs are a family of proteins with a common catalytic function of hydrolyzing GTP bound to ADP-ribosylation factors (Arf) with proposed cellular functions that are diverse (Inoue and Randazzo, 2007; Kahn et al., 2008). Understanding the biochemistry of the Arf GAPs is valuable for designing and interpreting experiments using standard cell biology techniques described elsewhere. The following briefly reviews some common approaches for in vivo studies of Arf GAPs and discusses the use of isolated cellular organelles to complement in vivo experiments. Detailed protocols for examining the activity of Arf GAPs in whole cell lysates and in association with isolated focal adhesions are provided.

Grp1-associated scaffold protein (GRASP) is a regulator of the ADP ribosylation factor 6 (Arf6)-dependent membrane trafficking pathway.

GRASP interacts with Grp1 (general receptor for phosphoinositides 1; cytohesin 3), which catalyses nucleotide exchange on and activation of Arf6 (ADP-ribosylation factor-6). Arf6 is a low-molecular-mass GTPase that regulates key aspects of endocytic recycling pathways. Overexpressed GRASP accumulated in the juxtanuclear ERC (endocytic recycling compartment). GRASP co-localized with a constitutively inactive mutant of Arf6 in the ERC such that it was reversed by expression of wild-type Grp1. Co-expression of GRASP and Grp1 promoted membrane ruffling, a cellular hallmark of Arf6 activation. GRASP accumulation in ERC was found to block recycling of the MHC-I (major histocompatibility complex-I), which is trafficked by the Arf6-dependent pathway. In contrast, overexpression of GRASP had no effect on the recycling of transferrin receptors, which are trafficked by a clathrin-dependent pathway. The findings suggest that GRASP regulates the non-clathrin/Arf6-dependent, plasma membrane recycling and signalling pathways.

Carcinogens induce loss of the primary cilium in human renal proximal tubular epithelial cells independently of effects on the cell cycle.

The primary cilium is an immotile sensory and signaling organelle found on the majority of mammalian cell types. Of the multitude of roles that the primary cilium performs, perhaps some of the most important include maintenance of differentiation, quiescence, and cellular polarity. Given that the progression of cancer requires disruption of all of these processes, we have investigated the effects of several carcinogens on the primary cilium of the RPTEC/TERT1 human proximal tubular epithelial cell line. Using both scanning electron microscopy and immunofluorescent labeling of the ciliary markers acetylated tubulin and Arl13b, we confirmed that RPTEC/TERT1 cells express primary cilium upon reaching confluence. Treatment with the carcinogens ochratoxin A (OTA) and potassium bromate (KBrO(3)) caused a significant reduction in the number of ciliated cells, while exposure to nifedipine, a noncarcinogenic renal toxin, had no effect on primary cilium expression. Flow cytometric analysis of the effects of all three compounds on the cell cycle revealed that only KBrO(3) resulted in an increase in the proportion of cells entering the cell cycle. Microarray analysis revealed dysregulation of multiple pathways affecting ciliogenesis and ciliary maintenance following OTA and KBrO(3) exposure, which were unaffected by nifedipine exposure. The primary cilium represents a unique physical checkpoint with relevance to carcinogenesis. We have shown that the renal carcinogens OTA and KBrO(3) cause significant deciliation in a model of the proximal tubule. With KBrO(3), this was followed by reentry into the cell cycle; however, deciliation was not found to be associated with reentry into the cell cycle following OTA exposure. Transcriptomic analysis identified dysregulation of Wnt signaling and ciliary trafficking in response to OTA and KBrO(3) exposure.

An in vitro assay system as a potential replacement for the histamine sensitisation test for acellular pertussis based combination vaccines.

The histamine sensitisation test (HIST) for pertussis toxin is currently an official batch release test for acellular pertussis containing combination vaccines in Europe and North America. However, HIST, being a lethal endpoint assay, often leads to repeated tests due to large variations in test performance. Although a more precise HIST test based on measurement of temperature reduction after the histamine challenge is used in Asian countries, this test still uses animals. An in vitro test system based on a combination of enzyme coupled-HPLC and carbohydrate-binding assays with results analysed by a mathematical formula showed a good agreement with the in vivo HIST results based on measurement of temperature reduction after histamine challenge. The new in vitro test system was shown to be a potential alternative to the current in vivo HIST.

Molecular cloning, sequence and expression analysis of ZmArf2, a maize ADP-ribosylation factor.

A full-length cDNA encoding a maize GTP-binding protein of the ADP-ribosylation factor family was cloned by suppression subtractive hybridization and an in silico cloning approach. The cDNA was 938 bp in length and contained a complete ORF of 612 bp, which encodes a protein of 203 amino acid residues. Its deduced amino acids sequence had an 83% identity with that of a GTP-binding protein in rice. The gene was designated ZmArf2. The ZmArf2 gene consists of G1, G2, G3, G4 and G5 boxes, and Switch I and Switch II regions. Eight nucleotides differed and five amino acids changed between the popcorn inbred N04 and the dent corn inbred Dan232. One changed amino acid was in the G1 box. RT-PCR analysis showed that ZmArf2 expression increased in the early stages of endosperm development and was not tissue-specific.

NAD+ -dependent post-translational modification of Escherichia coli glyceraldehyde-3-phosphate dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional housekeeping protein reported to be a target of several covalent modifications in many organisms. In a previous study, enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli strains were shown to secrete GAPDH and the protein to bind human plasminogen and fibrinogen. Here we report that GAPDH of these pathogens is ADP-ribosylated either in the cytoplasm or in the extracellular medium. GAPDH catalyzes its own modification, which involves Cys-149 at the active site. ADP-ribosylation of extracellular GAPDH may play an important role in the host-pathogen interaction, as also proposed in other pathogens (AU)

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The localization of the Golgin GCC185 is independent of Rab6A/A' and Arl1.

Mammalian golgins of the trans-Golgi network (TGN) are small G protein effectors that are required for membrane transport and contain a Golgi targeting C-terminal GRIP domain. The localization of two TGN golgins, p230/golgin-245 and golgin-97, is mediated by the small GTPase Arl1, whereas recruitment of the TGN golgin GCC185 is controversial. Recently, GCC185 was proposed to localize to the Golgi by the co-operation of two small GTPases, Rab6A/A' and Arl1 (Burguete et al., 2008), a model based predominantly on in vitro interactions. Here we demonstrate that Golgi recruitment of endogenous GCC185 does not involve Rab6A/A' and Arl1. We find minimal colocalization between Rab6A/A' and endogenous GCC185 on Golgi membranes and failed to detect an interaction between Rab6A/A' and C-terminal domains of GCC185 by yeast two-hybrid analyses. Moreover, depletion of both Rab6A/A' and Arl1 also had no effect on the localization of endogenous GCC185 or the isolated GRIP domain of GCC185.

Yeast and human Ysl2p/hMon2 interact with Gga adaptors and mediate their subcellular distribution.

The Gga proteins represent a family of ubiquitously expressed clathrin adaptors engaged in vesicle budding at the tubular endosomal network/trans Golgi network. Their membrane recruitment is commonly thought to involve interactions with Arf and signals in cargo through the so-called VHS domain. For yeast Gga proteins, however, partners binding to its VHS domain have remained elusive and Gga localization does not absolutely depend on Arf. Here, we demonstrate that yeast Gga recruitment relies on a network of interactions between the scaffold Ysl2p/Mon2p, the small GTPase Arl1p, and the flippase Neo1p. Deletion of either YSL2 or ARL1 causes mislocalization of Gga2p, whereas a neo1-69 mutant accumulates Gga2p on aberrant structures. Remarkably, Ysl2p directly interacts with human and yeast Ggas through the VHS domain, and binding to Gga proteins is also found for the human Ysl2p orthologue hMon2. Thus, Ysl2p represents an essential, evolutionarily conserved member of a network controlling direct binding and membrane docking of Ggas. Because activated Arl1p is part of the network that binds Gga2p, Arf and Arf-like GTPases may interact in a regulatory cascade.

Localization and function of ADP ribosylation factor A in Aspergillus nidulans.

Filamentous fungi undergo polarized hyphal growth throughout the majority of their life cycle. The Spitzenkörper is a structure unique to filamentous fungi that participates in hyphal growth and is composed largely of vesicles. An important class of proteins involved in vesicle assembly and trafficking are the ADP-ribosylation factors (Arfs). In Saccharomyces cerevisiae, Arf1p and Arf2p are involved in secretion. Aspergillus nidulans ArfA is a homolog of ScArf1p and ScArf2p with 75% of amino acid sequence similarity to each. ArfA::GFP localizes to cellular compartments consistent with Golgi equivalents. An N-terminal myristoylation motif is critical for localization of ArfA. Treatment with Brefeldin A, an inhibitor of Golgi transport, leads to ArfA::GFP diffusing through the cytosol and accumulating into a subcellular compartment further suggesting the ArfA localizes to and functions in the Golgi network. Costaining with FM4-64 revealed that ArfA::GFP likely localized to subcellular compartments participating in exocytosis. We were unable to recover arfA gene disruption strains indicating that the gene is essential in A. nidulans. The overexpression of ArfA protein partially suppresses the polarity defect phenotype of an N-myristoyltransferase mutant. Taken together, these results suggest that ArfA participates in hyphal growth through the secretory system.

Increased expression of the glioma-associated antigen ARF4L after loss of the tumor suppressor PTEN. Laboratory investigation.

OBJECT: Despite recent advances in cancer immunotherapy, cellular mechanisms controlling expression of tumor-associated antigens are poorly understood. Mutations in cancer cells, such as loss of PTEN, may increase expression of tumor-associated antigens. The authors investigated the relationship between PTEN status and the expression of a glioma-associated antigen, adenosine diphosphate-ribosylation factor 4-like (ARF4L) protein. METHODS: Human glioma cell lines with confirmed PTEN status were examined by Northern blot analysis and quantitative polymerase chain reaction. Western blot analysis was used to measure ARF4L protein levels across multiple cell lines. RESULTS: The loss of PTEN was shown to lead to increased levels of ARF4L protein but no change in transcript levels. Cell lines with serial mutations, including activation of Ras and Akt pathways, also demonstrated increased levels of ARF4L protein, which decreased after treatment with rapamycin. The ARF4L transcript preferentially localized to the polysomal compartment after PTEN loss in glioma or activation of Akt in human astrocytes. CONCLUSIONS: Expression of ARF4L is controlled by the activated Akt/mTOR pathway, which is a downstream effect of the loss of PTEN function. Mutations leading to oncogenesis may impact the regulation and expression of tumor specific antigens. Screening of mutation status in glioma may be helpful in selecting patients for immunotherapy trials in the future.

A fluorescence resonance energy transfer activation sensor for Arf6.

The involvement of the small GTPase Arf6 in Rac activation, cell migration, and cancer invasiveness suggests that it is activated in a spatially and temporally regulated manner. Small GTPase activation has been imaged in cells using probes in which the GTPase and a fragment of a downstream effector protein are fused to fluorescent reporter proteins that constitute a fluorescence resonance energy transfer (FRET) donor/acceptor pair. Unlike other Ras family GTPases, the N terminus of Arf6 is critical for membrane targeting and, thus, cannot be modified by fusion to a fluorescent protein. We found that the previously described C-terminal green fluorescent protein (GFP) derivative also shows diminished membrane targeting. Therefore, we inserted a fluorescent protein into an inert loop within the Arf6 sequence. This fusion showed normal membrane targeting, nucleotide-dependent interaction with the downstream effector GGA3, and normal regulation by a GTPase-activating protein (GAP) and a guanine nucleotide exchange factor (GEF). Using the recently developed CyPET/YPET fluorescent proteins as a FRET pair, we found that Arf6-CyPET underwent efficient energy transfer when bound to YPET-GGA3 effector domain in intact cells. The addition of platelet-derived growth factor (PDGF) to fibroblasts triggered a rapid and transient increase in FRET, indicative of Arf6 activation. These reagents should be useful for investigations of Arf6 activation and function.

The involvement of arl-5b in the repair of hair cells in sea anemones.

The subcellular processes involved in repair of hair cells are not well understood. Sea anemones repair hair bundle mechanoreceptors on their tentacles after severe trauma caused by 1-h exposure to calcium-depleted seawater. Repair is dependent on the synthesis and secretion of large protein complexes named "repair proteins." A cDNA library on traumatized anemone tissue was probed using polyclonal antibodies raised to a specific chromatographic fraction of the repair protein mixture. An ADP-ribosylation factor-like protein, Arl-5b, was identified. The amino acid sequence of the Arl-5b protein in sea anemones is similar to that among several model vertebrates and humans. A polyclonal antibody raised to a peptide of the anemone Arl-5b labels some but not all hair bundles in healthy control animals. The abundance of labeled hair bundles significantly increases above healthy controls after trauma and continuing through the first hour of recovery. Dilute anti-Arl-5b blocks the spontaneous repair of hair bundle mechanoreceptors, suggesting that Arl-5b acts on the extracellular face of the plasma membrane. Immunoelectron microscopy indicates that Arl-5b is located along the length of stereocilia including sites in the vicinity of tip links. We propose that Arl-5b is involved in installing replacement linkages into damaged hair bundle mechanoreceptors.