Kao G et al. (2007) Cell "ASNA-1 positively regulates insulin secretion in C. elegans and mammalian cells."

Nordenson C, Still M, Ronnlund A, Kao G, Tuck S, Naredi P

[Cell, 2007]

C. elegans worms hatching in the absence of food show growth arrest during the first larval stage (L1). While much has been learned about the later diapause, dauer, which worms enter under adverse conditions, much less is known about the mechanisms governing L1 arrest. Here we show that worms lacking activity of the asna-1 gene arrest growth reversibly at the L1 stage even when food is abundant. asna-1 encodes an ATPase that functions nonautonomously to regulate growth. asna-1 is expressed in a restricted set of sensory neurons and in insulin-producing intestinal cells. asna-1 mutants are reduced in insulin secretion while overexpression of asna-1 mimics the effects of insulin overexpression. Human ASNA1 is highly expressed in pancreatic beta cells, but not in other pancreatic endocrine cell types, and regulates insulin secretion in cultured cells. We propose that ASNA1 is an evolutionarily conserved modulator of insulin signaling.

Kao CY et al. (2011) PLoS Pathog "Global functional analyses of cellular responses to pore-forming toxins."

Wachi S, Hsieh M, Schwartz JL, Hsu CS, Los FC, Husmann M, Ha C, Huffman DL, Kloft N, Bellier A, Aroian RV, Fan H, Sagong Y, Karabrahimi V, Kao CY, Ghosh P, Chen L

[PLoS Pathog, 2011]

Here we present the first global functional analysis of cellular responses to pore-forming toxins (PFTs). PFTs are uniquely important bacterial virulence factors, comprising the single largest class of bacterial protein toxins and being important for the pathogenesis in humans of many Gram positive and Gram negative bacteria. Their mode of action is deceptively simple, poking holes in the plasma membrane of cells. The scattered studies to date of PFT-host cell interactions indicate a handful of genes are involved in cellular defenses to PFTs. How many genes are involved in cellular defenses against PFTs and how cellular defenses are coordinated are unknown. To address these questions, we performed the first genome-wide RNA interference (RNAi) screen for genes that, when knocked down, result in hypersensitivity to a PFT. This screen identifies 106 genes (0.5% of genome) in seven functional groups that protect Caenorhabditis elegans from PFT attack. Interactome analyses of these 106 genes suggest that two previously identified mitogen-activated protein kinase (MAPK) pathways, one (p38) studied in detail and the other (JNK) not, form a core PFT defense network. Additional microarray, real-time PCR, and functional studies reveal that the JNK MAPK pathway, but not the p38 MAPK pathway, is a key central regulator of PFT-induced transcriptional and functional responses. We find C. elegans activator protein 1 (AP-1; c-jun, c-fos) is a downstream target of the JNK-mediated PFT protection pathway, protects C. elegans against both small-pore and large-pore PFTs and protects human cells against a large-pore PFT. This in vivo RNAi genomic study of PFT responses proves that cellular commitment to PFT defenses is enormous, demonstrates the JNK MAPK pathway as a key regulator of transcriptionally-induced PFT defenses, and identifies AP-1 as the first cellular component broadly important for defense against large- and small-pore PFTs.

Kao AW et al. (2011) Proc Natl Acad Sci U S A "A neurodegenerative disease mutation that accelerates the clearance of apoptotic cells."

Cabello J, Kao AW, Neukomm LJ, Farese RV, Eisenhut RJ, Huang A, Bagley JA, Martens LH, Nakamura A, Zhou P, de Luis A, Kenyon C

[Proc Natl Acad Sci U S A, 2011]

Frontotemporal lobar degeneration is a progressive neurodegenerative syndrome that is the second most common cause of early-onset dementia. Mutations in the progranulin gene are a major cause of familial frontotemporal lobar degeneration [Baker M, et al. (2006) Nature 442:916-919 and Cruts M, et al. (2006) Nature 442:920-924]. Although progranulin is involved in wound healing, inflammation, and tumor growth, its role in the nervous system and the mechanism by which insufficient levels result in neurodegeneration are poorly understood [Eriksen and Mackenzie (2008) J Neurochem 104:287-297]. We have characterized the normal function of progranulin in the nematode Caenorhabditis elegans. We found that mutants lacking pgrn-1 appear grossly normal, but exhibit fewer apoptotic cell corpses during development. This reduction in corpse number is not caused by reduced apoptosis, but instead by more rapid clearance of dying cells. Likewise, we found that macrophages cultured from progranulin KO mice displayed enhanced rates of apoptotic-cell phagocytosis. Although most neurodegenerative diseases are thought to be caused by the toxic effects of aggregated proteins, our findings suggest that susceptibility to neurodegeneration may be increased by a change in the kinetics of programmed cell death. We propose that cells that might otherwise recover from damage or injury are destroyed in progranulin mutants, which in turn facilitates disease progression.

Kao BT et al. (2006) Lipids "Mice raised on milk transgenically enriched with n-3 PUFA have increased brain docosahexaenoic acid."

DePeters EJ, Kao BT, Van Eenennaam AL

[Lipids, 2006]

The brain contains high levels of the long-chain n-3 FA DHA (22:6n-3), mainly in the gray matter and synaptosomes. Adequate intake of DHA is crucial for optimal nervous system function, particularly in infants. Supplementation of infant formulas with DHA at levels similar to human breast milk is recommended for biochemical and functional benefits to neonates. We generated transgenic mice that produce elevated levels of n-3 PUFA in their milk by expressing the Caenorhabditis elegans n-3 FA desaturase under the control of a lactation-induced goat beta-casein promoter. To examine the postnatal effects of consuming the n-3-enriched milk, we compared the growth and brain and plasma FA composition of mouse pups raised on milk from transgenic dams with those observed for pups raised on milk from nontransgenic dams. A significant decrease in arachidonic acid (ARA, 20:4n-6) and concomitant increases in n-3 PUFA were observed in the phospholipid fraction of transgenic mouse milk. The n-6:n-3 FA ratios were 4.7 and 34.5 for the transgenic and control milk phospholipid fractions, respectively. DHA and DPA (22:5n-6) comprised 15.1% and 2.8% of brain FA from weanling mice nursed on transgenic dams, as compared with 6.9% and 9.2% for weanling mice nursed on control dams, respectively. This transgenic mouse model offers a unique approach to disassociate the effects and fetal programming resulting from a high n-6:n-3 FA ratio gestational environment from the postnatal nutritional effects of providing milk with differing n-6:n-3 FA ratios.

Kao HT et al. (1999) J Exp Zool "Molecular evolution of the synapsin gene family."

Kao HT, Stefani G, DeSalle R, Greengard P, Porton B, Pieribone VA, Hilfiker S

[J Exp Zool, 1999]

Synapsins, a family of synaptic vesicle proteins, play a crucial role in the regulation of neurotransmission and synaptogenesis. They have been identified in a variety of invertebrate and vertebrate species, including human, rat (Rattus norvegicus), cow (Bos taurus), longfin squid (Loligo pealei), and fruit fly (Drosophila melanogaster). Here, synapsins were cloned from three additional species: frog (Xenopus laevis), lamprey (Lampetra fluviatilis), and nematode (Caenorhabditis elegans). Synapsin protein sequences from all these species were then used to explore the molecular phylogeny of these important neuronal phosphoproteins. The ancestral condition of a single synapsin gene probably gave rise to the vertebrate synapsin gene family comprised of at least three synapsin genes (I, II, and III) in higher vertebrates. Synapsins possess multiple domains, which have evolved at different rates throughout evolution. In invertebrate synapsins, the most conserved domains are C and E. During the evolution of vertebrates, at least two gene duplication events are hypothesized to have given rise to the synapsin gene family. This was accompanied by the emergence of an additional conserved domain, termed A. J. Exp. Zool. ( Mol. Dev. Evol. ) 285:360-377, 1999.

Kao G et al. (2006) Dev Biol "The role of the laminin beta subunit in laminin heterotrimer assembly and basement membrane function and development in C. elegans."

Huang CC, Hall DH, Kao G, Wadsworth WG, Hedgecock EM

[Dev Biol, 2006]

Laminins are components of basement membranes that are required for morphogenesis, organizing cell adhesions and cell signaling. Studies have suggested that laminins function as alpha(x) beta(y) gamma(z) heterotrimers in vivo. In C. elegans, there is only one laminin beta gene, suggesting that it is required for all laminin functions. Our analysis is consistent with the role of the laminin beta as a subunit of laminin heterotrimers; the same cells express the laminin alpha, beta, and gamma subunits, the laminin beta subunit localizes to all basement membranes throughout development, and secretion of the beta subunit requires an alpha subunit. RNAi inhibition of the beta subunit gene or of the other subunit genes causes an embryonic lethality phenotype. Furthermore, a distinctive set of phenotypes is caused by both viable laminin alpha and beta partial loss-of-function mutations. These results show developmental roles for the laminin beta subunit, and they provide further genetic evidence for the importance of heterotrimer assembly in vivo.

Kao G et al. (2004) Development "C. elegans SUR-6/PR55 cooperates with LET-92/protein phosphatase 2A and promotes Raf activity independently of inhibitory Akt phosphorylation sites."

Tuck S, Sundaram MV, Baillie D, Kao G

[Development, 2004]

Protein phosphatase 2A (PP2A) can both positively and negatively influence the Ras/Raf/MEK/ERK signaling pathway, but its relevant substrates are largely unknown. In C elegans, the PR55/B regulatory subunit of PP2A, which is encoded by sur-6, positively regulates Ras-mediated vulval induction and acts at a step between Ras and Raf. We show that the catalytic subunit (C) of PP2A, which is encoded by let-92, also positively regulates vulval induction. Therefore SUR-6/PR55 and LET-92/PP2A-C probably act together to dephosphorylate a Ras pathway substrate. PP2A has been proposed to activate the Raf kinase by removing inhibitory phosphates from Ser259 from Raf-1 or from equivalent Akt phosphorylation sites in other Raf family members. However, we find that mutant forms of C. elegans LIN-45 RAF that lack these sites still require sur-6. Therefore, SUR-6 must influence Raf activity via a different mechanism. SUR-6 and KSR (kinase suppressor of Ras) function at a similar step in Raf activation but our genetic analysis suggests that KSR activity is intact in sur-6 mutants. We identify the kinase PAR-1 as a negative regulator of vulval induction and show that it acts in opposition to SUR-6 and KSR-1. In addition to their roles in Ras signaling, SUR-6/PR55 and LET-92/PP2A-C cooperate to control mitotic progression during early embryogenesis.

Hsu TY et al. (2018) Methods Mol Biol "The Use of Caenorhabditis elegans to Study Progranulin in the Regulation of Programmed Cell Death and Stress Response."

Kao AW, Hsu TY, Butler VJ

[Methods Mol Biol, 2018]

The nematode Caenorhabditis elegans (C. elegans) has proven to be a powerful model organism for the study of many biological processes, with major implications for human health and disease. As progranulin is a pleiotropic, secreted protein with both cell autonomous and non-autonomous roles, a multicellular organism such as C. elegans is ideal for the investigation of its normal function and pathological effects. The C. elegans genome contains a progranulin-like gene known as pgrn-1. The nematode pgrn-1 encodes a protein with three cysteine-rich granulin domains, compared to the seven and a half granulins in the human protein. We have shown that C. elegans mutants lacking pgrn-1 appear grossly normal, but exhibit accelerated apoptotic cell engulfment as well as a stress resistance phenotype (Kao et al., Proc Natl Acad Sci U S A 108:4441-4446, 2011; Judy et al., PLoS Genet 9:e1003714, 2013). In addition, the roles of individual granulins can also be dissected in C. elegans (Salazar et al., J Neurosci 35:9315-9328, 2015). Here, we describe methods for studying apoptosis and stress response in C. elegans.

Osman MA et al. (1998) J Cell Biol "Iqg1p, a yeast homologue of the mammalian IQGAPs, mediates cdc42p effects on the actin cytoskeleton."

Cerione RA, Osman MA

[J Cell Biol, 1998]

The Rho-type GTPase Cdc42p has been implicated in diverse cellular functions including cell shape, cell motility, and cytokinesis, all of which involve the reorganization of the actin cytoskeleton. Targets of Cdc42p that interface the actin cytoskeleton are likely candidates for mediating cellular activities. In this report, we identify and characterize a yeast homologue for the mammalian IQGAP, a cytoskeletal target for Cdc42p. The yeast IQGAP homologue, designated Iqg1p, displays a two-hybrid interaction with activated Cdc42p and coimmunoprecipitates with actin filaments. Deletion of IQG1 results in a temperature-sensitive lethality and causes aberrant morphologies including elongated and round multinucleated cells. This together with its localization at the mother-bud neck, suggest that Iqg1p promotes budding and cytokinesis. At restrictive temperatures, the vacuoles of the mutant cells enlarge and vesicles accumulate in the bud. Interestingly, Iqg1p shows two-hybrid interactions with the ankyrin repeat-containing protein, Akr1p (Kao, L.-R., J. Peterson, J. Ruiru, L. Bender, and A. Bender. 1996. Mol. Cell. Biol. 16:168-178), which inhibits pheromone signaling and appears to promote cytokinesis and/or trafficking. We also show two-hybrid interactions between Iqg1p and Afr1p, a septin-binding protein involved in projection formation (Konopka, J.B., C. DeMattei, and C. Davis. 1995. Mol. Cell. Biol. 15:723-730). We propose that Iqg1p acts as a scaffold to recruit and localize a protein complex involved in actin-based cellular functions and thus mediates the regulatory effects of Cdc42p on the actin cytoskeleton.

Gabel CV et al. (2007) J Neurosci "Neural circuits mediate electrosensory behavior in Caenorhabditis elegans."

Kao A, Pavlichin D, Gabel CV, Gabel H, Clark DA, Samuel AD

[J Neurosci, 2007]

The nematode Caenorhabditis elegans deliberately crawls toward the negative pole in an electric field. By quantifying the movements of individual worms navigating electric fields, we show that C. elegans prefers to crawl at specific angles to the direction of the electric field in persistent periods of forward movement and that the preferred angle is proportional to field strength. C. elegans reorients itself in response to time-varying electric fields by using sudden turns and reversals, standard reorientation maneuvers that C. elegans uses during other modes of motile behavior. Mutation or laser ablation that disrupts the structure and function of amphid sensory neurons also disrupts electrosensory behavior. By imaging intracellular calcium dynamics among the amphid sensory neurons of immobilized worms, we show that specific amphid sensory neurons are sensitive to the direction and strength of electric fields. We extend our analysis to the motor level by showing that specific interneurons affect the utilization of sudden turns and reversals during electrosensory steering. Thus, electrosensory behavior may be used as a model system for understanding how sensory inputs are transformed into motor outputs by the C. elegans nervous system.