Siddiqui, Shahid (2021) International Worm Meeting "Caenorhabditis elegans Fluorescent Mutants: Tryptophan Kynurenine Pathway"

Siddiqui, Shahid

[International Worm Meeting, 2021]

Caenorhabditis elegans fluorescent mutants (flu-1- flu-4) show altered fluorescence of the lysosome like intestinal granules, when observed under UV light [Babu, 1974, Siddiqui, 1978; Siddiqui and von Ehrenstein, 1980; Siddiqui and Babu, 1980 a, b; and Babu and Bhat, 1980]. Using biochemical techniques, it was shown that the flu-1 mutant alleles characterized by a bluish purple gut fluorescence possess reduced kynurenine hydroxylase enzyme activity [Siddiqui and Babu, 1980], and the flu-2 mutant alleles characterized by a pale dull green fluorescence had a block in kyunreniase enzyme activity [Babu and Bhat, 1980]. The C. elegans genomic sequence encodes these two enzymes in the proximity of flu-1 and flu-2 loci. R07B7.5 encodes kynurenine hydroxylase, and C15H9.7 encodes kynureninase, respectively [Altschul et al., 1990; Kanehisa, 2012]. Kynurenine pathway is the primary route for tryptophan catabolism in and the starting point for the synthesis of nicotinamide adenine dinucleotide (NAD) in mammals. Dysregulation or overactivation of this pathway can result in immune system activation and accumulation of potentially neurotoxic compounds, resulting in inflammation, depression, cancer, neurodegenerative and behavioral alterations, SARS-COV-2 resistance, and epigenetic modifications. Kynurenine pathway genes are also conserved in C. elegans [van der Goot and Nolan, 2013]. We are investigating genetic and epigenetic consequences of manipulating kyunrenine pathway in C. elegans to reveal key components of the catabolic pathway in C. elegans, to reveal molecules and mechanisms that are shared in human disease. For instance, we may use sequence optimized human ortholg cDNA in a flu-1 deletion knockout mutant that shows altered bluish purple intestinal fluorescence and reduced kynurenine hydroxylase activity. Human gene variants cDNA can be used to rescue and be examined for the restoration of intestinal fluorescence and the kynurenine hydroxylase enzyme activity, if the cDNA carrying human variant can restore the normal function in the nematode. Such approach may allow examining human lethal and sublethal variants using the C. elegans model, and could elucidate critical molecular mechanisms underlying human disease.

Higashi KI et al. (1995) International C. elegans Meeting "PVR A SINGLE SENSORY LUMBAR NEURON PROVIDES A 'SCAFFOLD'IN FORMATION OF THE TOUCH CIRCUITRY IN C. ELEGANS"

Miwa J, Siddiqui SS, Higashi KI

[International C. elegans Meeting, 1995]

Both cell-extrinsic and cell intrinsic mechanisms mediate the formation of precise neural networks. In C. elegans cell-cell interactions for a single lumbar neuron PVR have been studied in the touch circuitry, consisting of a set of six mechanosensory microtubule cells (ALML, ALMR, AVM, PLML, PLMR, & PVM), that are brightly stained with the McAb 6-11B-1 soecific to the acetylated alpha tubulin (Siddiqui et al., 1989). Since, the six touch receptor cells have synaptic interactions with the PVR, which also stains with the 6-11B-1 McAb, we have laser killed the PVR, or its precursor cells during embryogenesis, resulting in operated animals that lack normal touch response in the tail region. However, post-embryonic ablation of PVR has no effect on touch sensitivity in the operated animals. Therefore we propose that PVR may have little to do with the touch neural network directly, but provides the essential 'scaffold' in formation of the touch circuitry. This is similar to the role of BDU neurons in providing the guidance cues to the AVM neuron in the anterior touch ciruitry.

Shahid S Siddiqui et al. (2004) West Coast Worm Meeting "Expression of kinesin motor proteins during embryogenesis and neural function in C. elegans"

Shahid S Siddiqui, Fumio Hori, Robert Jhonsen, Asrar B Malik, M Yusuf Ali, Zhongying Zhao, Zeba K Siddiqui, Shams T Khan, David Baillie, M L Khan, Douglas Yau

[West Coast Worm Meeting, 2004]

Kinesin is an ATP hydrolyzing enzyme that transduces chemical energy of ATP hydrolysis into mechanical force to carry intracellular cargo on microtubule tracks. Why do multiple kinesin motors exist in a given cell type or a tissue, and whether these motors perform distinct or overlapping functions, remains to be understood. In C. elegans a superfamily of 21 kinesin motor proteins has been described that participate in a variety of cellular function such as chromosomal movement, cell and growth cone migration, process outgrowth and axonal guidance (Siddiqui, 2002). Members of the nematode kinesin family represent all major groups of kinesin motors found in the vertebrates. Accordingly, we have determined the temporal and spatial pattern of expression of the nematode kinesin family members by in situ hybridization, or by studying kinesin promoter fusion with GFP or dsRed protein markers in transgenic animals. Our results suggest that members of the kinesin family (klp-1-klp-20) have distinct role in mitosis, chromosomal dynamics and are essential for neural development and function.

Gogonea CB et al. (1997) International C. elegans Meeting "EXPRESSION OF THE REPORTER GENE MEC-12::LACZ IN LOCOMOTORY AND CHEMOSENSORY MUTANTS OF C. ELEGANS"

Siddiqui SS, Siddiqui ZK, Gogonea CB

[International C. elegans Meeting, 1997]

Mutations in close to 100 genes affect locomotion. These mutants show uncoordinated locomotion, ranging from slow movement, poor backing, colier, kinkier, and paralyzed phenes (J. Hodgkin, 1997: Appendix in C. elegans II). Previously it was shown that mutants in at least 25 genes harbor defects in the axonal growth and process placement of the six touch receptor neurons (Siddiqui, 1990). A reporter gene mec-12::lacZ has been shown to express in the set of six touch receptor neurons (Fukushige et al., 1997). So far, a variety of cellular defects in the process placement, axonal guidance and axonal growth of the touch cells and the PVR in 15 genes (unc-4, unc-5,unc-30, unc-33, unc-44, unc-51, unc-53, unc-55, unc-61, unc-71, unc-76, unc-83, unc-86, and unc-119) have been observed. Defects include ectopic branching of axons, fore- shortened processes, misplaced cell body positions, extra commissures, and a lack of staining. In addition, we have examined the reporter gene expression in the background of various mechano- sensory mutations (Chalfie and Au, 1989). These results will be summarized. We thank the Caenorhabdtis Genetics Center, and Martin Chalfie for various mutant strains.

Ayesha N Shajahan et al. (2001) International C. elegans Meeting "Regulation of endocytosis of albumin in the intestinal epithelial cells of C.elegans by TGFbeta-like signaling"

Shahid S Siddiqui, Ayesha N Shajahan

[International C. elegans Meeting, 2001]

In C. elegans , TGF b -like signaling pathways regulate a range of developmental processes such as dauer formation (Estevez et al., 1993), body size growth and male tail formation (Morita et al., 1999; Suzuki et al., 1999), and axon guidance (Colavita et al., 1998). Recently, Siddiqui et al. have shown that daf-4 (TGF b -like-RII) and daf-1 (TGF b -like-RI) (at 25 o C) mutants show poor uptake of FITC-BSA (fluorescein-5-isothiocyanate isomer- bovine serum albumin) in the intestinal cells when compared to wild-type animals. Our aim is to identify the genes in the TGF b -like pathways that affect endocytosis of albumin in the intestinal epithelial cells. We will examine the uptake of FITC-BSA in the mutants of the TGF b -like pathway in comparison to its uptake in wild type. daf-4::gfp (Patterson et al., 1997) and daf-1::gfp (Gunther et al., 2000) are expressed in the intestinal cells. Expression and regulation of other TGF b -like receptors, Smad proteins and the downstream trancription factors in the TGF b -like signaling pathway will be examined in the intestinal cells of wild type and various daf mutants, in the presence and absence of albumin, by immunofluorescence and immunoprecipitation assays. This approach will determine the role of TGF b -like signaling in endocytosis of albumin, and also uncover any possible cross talk between such pathways in the process.

Fukushige T et al. (1991) International C. elegans Meeting "STRUCTURE AND EXPRESSION OE ALPHA TUBULIN GENES IN C. ELEGANS."

Fukushige T, Siddiqui SS, Yasuda H

[International C. elegans Meeting, 1991]

Microtubules are cylindrical filamentous structures composed mainly of alpha and beta tubulin proteins, and serve a variety of functions in multicellular organisms. In C. elegans, like other higher organisms, alpha and beta tubulins are coded by a family of genes, and individual isotypic variants of tubulins can be separated on isoelectric focusing gels (Siddiqui et al. 1989). Here we report characterization of two members of the alpha tubulin gene family in C. elegans. Tubulin alpha -1 was cloned by direct screening of a C. eleFans cDNA expression library with a anti-alpha tubulin McAb 3A5 (M. Fuller), and Tubulin alpha -2 was originally identified by L. Gremke and J. Culotti, by screening C. eleFans genomic library with chicken alpha - tubulin as a probe. We have determined the physical map and the nucleotide sequence of the two alpha tubulin genes. The amino acid sequence deduced from the nucleotide sequence data has been compared with tubulin sequences from other organisms, and shows strong similarity with mammalian tubulins. A novel feature (pointed out to us by Dr. T. Blumenthal) in the 5' flanking sequence of both tubulin genes was the presence of typical eight nucleotide C. elegans acceptor sequence in the trans-splicing reaction of mRNA. We have now found that indeed a 22 nucleotide leader sequence is trans-spliced to 5'-flanking region of both alpha tubulin mRNA in C. elegans. Finally, Northern hybridization using alpha tubulin probes, has shown that these genes are expressed increasingly from embryonic to Ll-L3 stages. Experiments to determine the in situ expression of these genes are in progress. We thank the support to our work provided by the laboratories of Drs. J. Miwa and G. Eguchi.

Shahid S Siddiqui et al. (2005) International Worm Meeting "KLP-4 a Kinesin-3 family member is required for embryogenesis and neural development in C. elegans"

Asrar B Malik, Zhongying Zhao, Shahid S Siddiqui, Douglas Yau, Robert Johnsen, David Baillie, Hae K Lee, Athar Chishti, Toshio Hanada

[International Worm Meeting, 2005]

Members of the UNC-104/KIF-1 family play important role in neural growth, axonal transport and synaptic function. In C. elegans in addition to UNC-104, two similar motors KLP-4 and KLP-6 comprise the Kinesin-3 family subgroup (Siddiqui 2002) characterized by an amino-terminal Motor head domain (ATP and microtubule binding sites), followed by a coiled coil stalk hinge region for multimer formation and a carboxyl terminal tail domain of variable length and composition and is similar to the GAKIN 13A family of mammalian motors (Hanada et al., 2002). We have shown that inhibition of klp-4 function by RNAi an rrf-3 mutant background results in embryonic arrest and reduction in brood size. A klp-4::gfp promoter fusion transgene expresses in the sensory neurons in the head, a set of ventral cord motor neurons, and the sensory ray neurons in the male tail ray neurons and the intestine from larval stages L1-L4 and in adults. Polyclonal antibodies raised against the carboxyl terminal of the KLP-4 detect a 180-kDa band. Immunostaining with an affinity purified anti-KLP-4 antibody shows that the KLP-4 motor is present in the sensory neurons in the head, developing embryos, germ cells and intestine, consistent with the klp-4::gfp expression results. The KLP-4 also contains the fork-head association domain (residues 534-594) suggesting a motif for protein-protein interaction. We are examining the stalk and tail region of the KLP-4 motor using klp-4::gfp constructs to determine the cell specificity of klp-4 expression. Expression of klp-4 closely resembles that of klp-6 another member of the UNC-104 family of kinesins enriched in sensory neurons and motor neurons. A mutation in klp-6 aka lov-2(sy511) has been described (Paden and Barr, 2004). Expression of klp-4::gfp in lov-2 background may reveal epistasis. Similar interactions between klp-6 and unc-104 are under investigation.

Siddiqui SS (1991) International C. elegans Meeting "MULTIPLE REQUIREMENT OF GENES AFFECTING AXONAL GUIDANCE AND GROWTH OF MOTOR AND SENSORY NEURONS."

Siddiqui SS

[International C. elegans Meeting, 1991]

I have been studying the pattern of axonal guidance and process outgrowth in the set of 'unc' mutants in C. elegans, by screening these immunocytochemically, using a variety of antibodies that are specific for different sets of motor and sensory neurons. Remarkably, it appears that neural circuitry formation requires the concerted activity of a number of genes, including those affecting axonal growth and guidance. Secondly, most of these genes are required in more than one neuronal type. Thirdly, although mutatlons in these genes may affect the locomotion of animals with a high degree of expressivity and penetrance, the effect on axonal guidance and growth is highly variable. It is likely that the defects observed in neuronal phenotypes may also be caused by secondary defects in non-neural tissue, including the hypodermis and muscle. Mutations affecting neurons are listed: (a) Touch receptor cells(ALM, PLM, AVM): unc-l, unc-3, unc-5, unc-6, unc-13, unc-18, unc-27, unc- 30, unc-33, unc-34, unc-40, unc-44, unc- 49, unc-51, unc-55, unc-59, unc-61, unc-62, unc-69, unc-71, unc-73, unc-76, unc-85, unc-98, and unc-104 (Stained with anti- tubulin McAb 6-11B-1). (b) Motor neurons*(DD & VD) : unc-5, unc-6, unc-13, unc-25, unc-30, unc-34, unc-40, unc-51, unc-62 unc-69, unc-71, unc-73, and unc-76 ( Stained with anti- tubulin McAb 2-28-33). (c) Lumbar neurons** (PHA, PHB, PHC, & PVN): unc-6, unc-13, unc-33, unc-44, unc-51 , unc-59, unc-61, unc-71, unc-73, unc-76, and unc-98 ( Stained with anti-HRP Abs. ; normalnormalnormalnormalnormalnormalnormalnormalnormal Siddiqui & Culotti, 1991). * Some of these genes were previously identified by S. McIntire, J. White, and R. Horvitz using antiGABA antiserum. ** see also Hedgecock et al.(1985) I thank J. Culotti, E. Hedgecock, R. Horvitz, and J White for many useful discussions and ideas.

Siddiqui, Shahid S. et al. (2013) International Worm Meeting "Antimicrobial compound screens using C. elegans model system for periodontal pathogens -."

Al-Beyari, Mohammad, Siddiqui, Shahid S., Faskhani, Fathy A.

[International Worm Meeting, 2013]

Chronic periodontal disease (PD) is an inflammatory condition that is highly widespread. Periodontal disease is a result of sub-gingival plaque pathogens, leading to alveolar bone loss. PD is also associated with diabetes, obesity, cardiovascular disease, respiratory disease, rheumatic arthritis and pre-term birth. The mechanism of periodontal infection is poorly understood due to the complexity of microbial factors, and highly complex inflammatory and anti-inflammatory signaling between pathogens and the host. New microbial pathogens are emerging that are resistant to conventional antibiotics; thus there is a need for drugs that are new class of antibiotics, and novel compounds that reduce pathogenicity of invading microbes. Pioneering studies in Frederick Ausubel's Laboratory (Harvard Medical School) have shown the effective use of C. elegans as a simple and powerful genetic model to screen for antimicrobial compounds, such as the discovery of RPW-24 that protects C. elegans from bacterial infections by modulating the p38 MAPK, and ATF7 transcription regulatory pathways [Pukkila-Worley et al. 2012]. We have previously used C. elegans to screen chemicals by using the nematode egg laying assay, and have identified novel compounds that affect egg laying behavior in a high throughput screen of commercially available chemical library [Siddiqui et al., C. elegans meeting UCLA, 2011]. We are screening chemical libraries in C. elegans for novel compounds that may inhibit the growth of oral pathogens such as Pseudomonas aeruginosa, found in the periodontal infections; and look for modulators of NSY-1, SEK-1, PMK-1 Mitogen Activated Protein (MAP) Kinase pathway that is comparable to the mammalian ASK-1(MAP kinase kinase kinase)/MKK3/6 (MAP kinase kinase) p38 MAP kinase signaling pathway. To validate the discovery of novel compounds in C. elegans high throughput screens for periodontal pathogens, we will use in vitro PLC (periodontal ligament cell) cell culture screens. Results will be presented on the efficacy of screened compounds on oral pathogens specific to chronic periodontal disease.

MY Ali et al. (1999) International C. elegans Meeting "Multiple kinesins play critical role in chromosomal movement during embryonic cell divisions in C.elegans."

AS Mohammed, ST Khan, MY Ali, SS Siddiqui

[International C. elegans Meeting, 1999]

A superfamily of genes klp-1 - klp-20 encoding kinesin proteins has been identified in C. elegans (see abstract by Siddiqui et al.), that has representative members belonging to all nine groups of kinesins characterized in mammals. A major question in kinesin biology is how many kinesins are expressed in a single cell type, and if they are unique, or have overlapping functions? Early embryogenesis in C. elegans is well characterized and offers a good opportunity to elucidate kinesin motor function at genetic, cellular and molecular levels. For example, the unc-116 KHC has been previously shown to affect first mitotic cell division (Hall and Hedgecock, 1991; Patel et al., 1993, Ali et al. 1999). Here we have charcterized several members of the kinesin family, such as klp-3 (Khan et al., 1997) klp-7, klp-12 , and klp-14 , which function in chromosome movement during early embryogenesis. Cloning of a C.elegans cDNA of the klp-7 gene reveals that it encodes a CeKLP7 of 689 amino acids. which is a member of MCAK/KIF2 subfamily. Members of this group have a distinct primary and secondary structure, as their motor domain is located in the middle of the protein sequence. MCAK kinesin that lacks the ATP binding motor domain, binds centromeres but not the microtubules and can disrupt chromosome segregation during anaphase (Hunter et al. 1998). Our insitu hybridization result suggests that CeKLP-7 functions in early embryogenesis. In a parallel experiment, klp-7 gene inactivation with the double stranded RNAi in adult animals, produced dead eggs, arrested in different developmental stages, suggesting a requirement of the klp-7 gene in embryogenesis. Similar to the klp-7 , we have analysed the role of CeKLP12, an ortholog of the chicken chromoskinesin, and the CeKLP-14, an ortholog of the BimC family of kinesins. Results on in situ hybridization, RNAi, immunocytochemistry, and fusion gene expression suggest that CeKLP-12, and CeKLP-14 are directly involved in early cell divsions in C. elegans during embryogenesis. We thank J. Sulston, Y. Kohara, A. Fire and Y. Ohshima for support. Funds from Monbusho, Japan were provided for grant in aid for basic research to SSS.