Raich WB et al. (1995) International C. elegans Meeting "CHARACTERIZATION OF ALPHA-CATENIN IN C.ELEGANS"

Raich WB, Hardin JD

[International C. elegans Meeting, 1995]

Cadherin mediated cell-cell adhesion plays a central role in coordinating the reorganization of cells during the development of multicellular animals. In vertebrates, three proteins have been identified which bind to the cytoplasmic tail of the cadherins and which are obligatory for cadherin mediated adhesion. The largest of these proteins, a-catenin, is required for cadherin association with the actin cytoskeleton, suggesting that it acts as a linker for the transmission of mechanical force from the exterior of the cell to its interior. We have sequenced a C. elegans a-catenin homologue identified by the Kohara cDNA project, and are currently cloning the upstream sequence in preparation for a Tc1 knockout. Our current sequence consists of 1172 base pairs, approximately 40% of the expected length. At the amino acid level, this region shows 52% identity with Drosophila a-catenin. In addition, we have made GST and His-tagged fusion proteins with an 801 base pair C-terminal fragment. These fusion proteins will be used to generate antibodies in an effort to characterize the subcellular location and the proteins associated with a-catenin in C. elegans.

Raich WB et al. (1997) International C. elegans Meeting "CHARACTERIZATION OF zen-4, A MUTATION THAT SPECIFICALLY DISRUPTS VENTRAL ENCLOSURE"

Raich WB, Hardin JD, Rothman JH

[International C. elegans Meeting, 1997]

The hypodermis of C. elegans forms as six rows of cells on the dorsolateral surface of the embryo. In a process known as ventral enclosure, the hypodermal sheet spreads bilaterally from the dorsal surface, ultimately meeting and establishing adherens junctions at the ventral midline. Laser ablation studies and treatment with a variety of cytoskeletal inhibitors demonstrate that ventral enclosure is a necessary prerequisite for the subsequent elongation of C. elegans into the vermiform shape achieved at hatching. In an effort to identify the molecular players involved in hypodermal enclosure, we are in the process of characterizing a zygotic lethal mutation called zen-4 required for this process. Immunostaining with the monoclonal antibody MH27, which recognizes a component of the adherens junction, indicates that the correct number of hypodermal cells are generated in homozygous zen-4 embryos. Time-lapse Nomarski movies demonstrate that the hypodermal cells begin to migrate ventrally; however, they cease their migration less than halfway towards the ventral midline. After a variable interval, the hypodermal cells retract towards the dorsal surface, where they remain as a hypodermal patch. Elongation of the zen-4 embryos is completely blocked, presumably as a consequence of the enclosure failure. The mutation has been mapped the right arm of chromosome IV between unc-44 and bli-6, and the mutation fails to complement the deficiency nDf41. Attempts to rescue the mutation by germline transformation are underway, and we are actively screening for additional zen-4 alleles.

Raich WB et al. (1996) Midwest Worm Meeting "CHARACTERIZATION OF hmp-1, A C. ELEGANS a-CATENIN HOMOLOGUE, REQUIRED FOR ELONGATION OF THE EMBRYO"

Costa M, Raich WB, Hardin JD, Priess JR

[Midwest Worm Meeting, 1996]

Numerous circumferential bundles of actin filaments arise during the course of embryogenesis in C. elegans. These filaments are required for elongation from an approximately spherical ball of cells into the vermiform, or worm-like shape, achieved at hatching. In vertebrates, a-catenin is thought to link transmembrane cadherins to the actin cytoskeleton. Given that actin filaments arise in circumferential arrays, we wondered if a-catenin might be required for elongation of the embryo. The Kohara cDNA project identified a clone showing homology with a-catenin. Subsequent library screening has resulted in a 1.7 kb cDNA fragment (Northern blot predicts a 3 kb message) that shows 42% identity with Drosophila a-catenin. Affinity purified antibodies raised against a GST / a-catenin fusion protein recognize a 105 kD protein on a Western blot, consistent with the expected size based on homology with other organisms. Immunostaining indicates that hmp-1 appears to be turned on weakly in the 2-28 cell stage, localizing to cell junctions. Staining during embryonic proliferation drops off markedly, but returns strongly at the onset of elongation. a-catenin is present in the hypodermis, intestine, and pharynx, all of which contain adherens junctions. Staining is strongest in the seam cells, perhaps because junctions are turned over at a rapid rate in the rearranging dorsal and ventral hypodermis. a-catenin maps to cosmid R13H4 on chromosome V. We have obtained rescue of a zygotic lethal mutation called hmp-1 by germline injection of R13H4. Offspring of a-catenin antisense injected animals mimic the hmp-1 phenotype, confirming that hmp-1 encodes a-catenin. hmp-1 mutants form the normal pattern of hypodermal cells, but appear to arrest at 1.25-fold and develop abnormal humps in the dorsal hypodermis. Analysis of the deficiency ctDf1 indicates that this phenotype results from a complete loss of zygotic gene function. Circumferential actin filament bundles form properly in the hypodermal cells, but filaments in the dorsal hypodermis appear to detach from the hypodermis prior to elongation, suggesting that the failure to elongate results from the failure to channel the actin contraction into elongation. In addition, time-lapse Nomarski movies of the offspring of a germline mosaic animal indicate that maternal a-catenin may be required to enclose the head, suggesting that elongation is required for normal head enclosure. We are currently examining protein expression in the mutant embryos, conducting a library screen to identify the 5' end of the cDNA, and sequencing a 7.5 kb fragment of cosmid R13H4.

Raich WB et al. (2003) Genetics "Characterization of Caenorhabditis elegans homologs of the Down syndrome candidate gene DYRK1A."

Moorman C, Plasterk RH, Lehrer J, Hobert O, Bartsch D, Kandel ER, Raich WB, Lacefield CO

[Genetics, 2003]

The pathology of trisomy 21/Down syndrome includes cognitive and memory deficits. Increased expression of the dual-specificity protein kinase DYRK1A kinase (DYRK1A) appears to play a significant role in the neuropathology of Down syndrome. To shed light on the cellular role of DYRK1A and related genes we identified three DYPLK/minibrain-like genes in the genome sequence of Caenorhabditis elegans, termed mbk-1, mbk-2, and hpk-1. We found these genes to be widely expressed and to localize to distinct subcellular compartments. We isolated deletion alleles in all three genes and show that loss of mbk-1, the gene most closely related to DYRK1A, causes no obvious defects, while another gene, mbk-2, is essential for viability. The overexpression of DYRK1A in Down syndrome led us to examine the effects of overexpression of its C. elegans ortholog mbk-1. We found that animals containing additional copies of the mbk-1 gene display behavioral defects in chemotaxis toward volatile chemoattractants and that the extent of these defects correlates,with mbk-1 gene dosage. Using tissue-specific and inducible promoters, we show that additional copies of mbk-1 can impair olfaction cell-autonomously in mature, fully differentiated neurons and that this impairment is reversible. Our results suggest that increased gene dosage of human DYRK1A in trisomy 21 may disrupt the function of fully differentiated neurons and that this disruption is reversible.

Raich WB et al. (1998) Midwest Worm Meeting "zen-4 encodes a kinesin-like protein with functions in dividing and non-dividing cells"

Moran AN, Hardin JD, Raich WB, Rothman JH

[Midwest Worm Meeting, 1998]

MKLP1 kinesin proteins bundle antiparallel microtubules in vitro and slide them past one another in an ATP-dependent fashion. They have been implicated in cell division as well as in the formation of vertebrate dendrites. Here, we report the identification of a null allele in zen-4 (zygotic enclosure defective), a C. elegans MKLP1 subfamily member. Homozygous mutant zen-4(w35) embryos produce the normal number of hypodermal cells, but fail to undergo ventral enclosure. This suggests that ZEN-4 is required for the cytoskeletal rearrangements needed to complete hypodermal migration. Antibodies raised against the ZEN-4 peptide SRDQVRRKKLSIEET demonstrate that ZEN-4 localizes to all hypodermal cells during ventral enclosure, and a zen-4::gfp translational fusion confirms this observation. Depletion of maternal ZEN-4 by RNA interference or mosaic analysis unmasks the requirement for ZEN-4 in cell division. Complete absence of ZEN-4 results in the production of a single, multinucleate cell, as embryos continue to cycle through mitosis but fail to complete cytokinesis. Time-lapse Nomarski microscopy reveals that the cleavage furrow during the first cell division initiates its ingression at the normal time, but fails to progress into the region of the spindle midzone. In addition, polar bodies fail to be segregated from the embryo proper. Tubulin staining reveals that microtubule bundles in the midzone of the mitotic spindle appear diminished in number and disorganized, suggesting that ZEN-4 is required to stabilize these microtubules. ZEN-4 co-localizes with the midzone microtubules, and is expressed at high levels in the midbody remnant following cell division. It is intriguing that the same motor is utilized during cell division and in non-dividing hypodermal cells, and future efforts will be directed towards elucidating the role of zen-4 during hypodermal enclosure.

Raich WB et al. (1998) Mol Biol Cell "Cytokinesis and midzone microtubule organization in Caenorhabditis elegans require the kinesin-like protein ZEN-4."

Hardin JD, Raich WB, Moran AN, Rothman JH

[Mol Biol Cell, 1998]

Members of the MKLP1 subfamily of kinesin motor proteins localize to the equatorial region of the spindle midzone and are capable of bundling antiparallel microtubules in vitro. Despite these intriguing characteristics, it is unclear what role these kinesins play in dividing cells, particularly within the context of a developing embryo. Here, we report the identification of a null allele of zen-4, an MKLP1 homologue in the nematode Caenorhabditis elegans, and demonstrate that ZEN-4 is essential for cytokinesis. Embryos deprived of ZEN-4 form multinucleate single-celled embryos as they continue to cycle through mitosis but fail to complete cell division. Initiation of the cytokinetic furrow occurs at the normal time and place, but furrow propagation halts prematurely. Time-lapse recordings and microtubule staining reveal that the cytokinesis defect is preceded by the dissociation of the midzone microtubules. We show that ZEN-4 protein localizes to the spindle midzone during anaphase and persists at the midbody region throughout cytokinesis. We propose that ZEN-4 directly cross-links the midzone microtubules and suggest that these microtubules are required for the completion of cytokinesis.

Raich WB et al. (1999) Curr Biol "Rapid epithelial-sheet sealing in the Caenorhabditis elegans embryo requires cadherin-dependent filopodial priming."

Hardin JD, Agbunag C, Raich WB

[Curr Biol, 1999]

BACKGROUND: During embryonic development, epithelia with free edges must join together to create continuous tissues that seal the interior of the organism from the outside environment; failure of epithelial sealing underlies several common human birth defects. Sealing of epithelial sheets in embryos can be extremely rapid, dramatically exceeding the rate of adherens junction formation by epithelial cells in culture or during healing of epithelial wounds. Little is known about the dynamic redistribution of cellular junctional components during such events in living embryos. RESULTS: We have used time-lapse, multiphoton laser-scanning microscopy and green fluorescent protein fusion proteins to analyze the sealing of the Caenorhabditis elegans epidermis in living embryos. Rapid recruitment of alpha-catenin to sites of filopodial contact between contralateral migrating epithelial cells, concomitant with clearing of cytoplasmic alpha-catenin, resulted in formation of nascent junctions; this preceded the formation of mature junctions. Surprisingly, upon inactivation of the entire cadherin-catenin complex, only adhesive strengthening between filopodia was reproducibly affected. Other ventral epidermal cells, which did not extend filopodia and appeared to seal along the ventral midline by coordinated changes in cell shape, successfully adhered in the absence of these proteins. CONCLUSIONS: We propose that 'filopodial priming' - prealignment of bundled actin in filopodia combined with the rapid recruitment of alpha-catenin from cytoplasmic reserves at sites of filopodial contact - accounts for the rapid rate of sealing of the embryonic epidermis of C. elegans. Filopodial priming may provide a general mechanism for rapid creation of adherens junctions during epithelial-sheet sealing in embryos.

WB Raich et al. (1999) International C. elegans Meeting "The cadherin-catenin complex stabilizes filopodial adhesive contacts during hypodermal morphogenesis"

WB Raich, C Agbunag, J Hardin, JR Priess

[International C. elegans Meeting, 1999]

The sealing of epithelial sheets via the formation of adherent cell-cell junctions is an important morphogenetic event during animal development, yet little is known about how migratory epithelia modulate their junctional connections. The three dimensional shape of embryonic epithelia makes their analysis difficult, and perturbation of junctional molecules via mutation or RNA interference typically disrupts epithelia prior to sheet sealing. Mutations in the C. elegans cadherin hmr-1 , a -catenin hmp-1 , and b -catenin/armadillo hmp-2 are a valuable exception, as these molecules are not essential for general cell adhesion (M. Costa et al., 1998. J. Cell Biol. 141 , 297-308). We have used time-lapse multiphoton laser scanning microscopy to analyze the mechanisms of migration and sealing of the C. elegans hypodermis during ventral enclosure at the subcellular level. Cell-cell junctions were visualized using two reporters: HMP-1-GFP and JAM-1-GFP (for "junction associated molecule"; JAM-1 is recognized by the MH27 monoclonal antibody). HMP-1-GFP is distributed uniformly throughout filopodia extended by the first ventral hypodermal cells to reach the midline ("leading cells"). By 5 min after their contact at the ventral midline, HMP-1 a -catenin rapidly accumulates at future sites of junction formation between contralateral cells. JAM-1 is inserted at ventral midline junctions 15 min following a -catenin accumulation. Cadherin-mediated adhesive strengthening is specifically required to stabilize initial adhesion events between filopodia; in embryos lacking hmr-1 or hmp-1 mRNA, leading cells approach the midline, establish filopodial contact, but do not form stable junctional connections. In addition, in the absence of HMR-1, ectopic and precocious cell-cell fusions occur between hypodermal cells that reach the ventral midline. In contrast, posterior ventral hypodermal cells, which do not display long filopodia, can make correct attachments at the ventral midline in the absence of HMR-1 or HMP-1. Our results indicate that embryonic epithelial cells that use filopodia for migration require recruitment of a -catenin at sites of nascent junction formation to correctly regulate mature junction assembly.

Rao KV et al. (2000) Mol Biochem Parasitol "The Wuchereria bancrofti orthologue of Brugia malayi SXP1 and the diagnosis of bancroftian filariasis."

Gnanasekhar B, Kaliraj P, Ravi V, Scott AL, Marson A, Rao KV, Jayaraman K, Raghavan N, Eswaran M, Narayanan RB

[Mol Biochem Parasitol, 2000]

The gene encoding the Wuchereria bancrofti orthologue of the Brugia malayi-derived diagnostic antigen SXP1 was identified from a W. bancrofti L3 cDNA library and characterized. The Wb-sxp-1 cDNA encoded a basic protein with a calculated molecular mass of 20.8 kDa. Wb-SXP-1 was 85% identical to the SXP1 protein described from B. malayi (Bm-SXP-1). The Wb-SXP-1 sequence also showed significant identity with proteins described from B. pahangi, Onchocerca volvulus, Acanthochilonema vitea, Ascaris suum, Loa loa, Litomosoides sigmodontis and Caenorhabditis elegans. The presence of a number of invariant and conserved residues in all of these nematode-derived molecules suggests that Wb-SXP-1 is a member of a new protein family. A recombinant form of Wb-SXP-1 was produced and it was determined that the anti-Wb-SXP-1 antibody response in patients with W. bancrofti infections was restricted to the IgG4 subclass. An anti-Wb-SXP-1 IgG4 ELISA was developed and this assay was found to be 100% sensitive for patients with patent W. bancrofti infection. Sera from individuals experiencing chronic pathology, endemic normals or patients with non-filarial nematode infections had no detectable IgG4 against Wb-SXP-1. While patients with patent Onchocerca volvulus infections were uniformly negative in the Wb-SXP-1 assay, 40% of sera from patent Loa loa infections were positive. When Bm-SXP-1 was used as the antigen under identical conditions, the assay was 88% specific for patent W. bancrofti infections and the antigen was recognized by antibodies from both O. volvulus and L. loa infections. The results strongly suggested that, for certain diagnostic filarial antigens, the use of same-species molecules can enhance the specificity of diagnostic tests.

Bhargavi R et al. (2005) Bioinformation "Modeling analysis of GST (glutathione-S-transferases) from Wuchereria bancrofti and Brugia malayi."

Vishwakarma S, Murty US, Bhargavi R

[Bioinformation, 2005]

GST (glutathione S-transferases) are a family of detoxification enzymes that catalyze the conjugation of reduced GSH (glutathione) to xenobiotic (endogenous electrophilic) compounds. GST from Wb (Wuchereria bancrofti) and Bm (Brugia malayi) are significantly different from human GST in sequence and structure. Thus, Wb-GST and Bm-GST are potential chemotherapeutic targets for anti-filarial treatment. Comparison of modeled Wb and Bm GST with human GST show structural difference between them. Analysis of the active site residues for the binding of electrophilic co-substrates provides insight towards the design of parasite specific GST inhibitors.