Geetanjali Sundaram

Abstract: TRAPP is a multi-subunit complex that acts as a Ypt/Rab activator at the Golgi apparatus. TRAPP exists in two forms: TRAPP I is comprised of five essential and conserved subunits and TRAPP II contains two additional essential and conserved subunits, Trs120 and Trs130. Previously, we have shown that Trs65, a nonessential fungi-specific TRAPP subunit, plays a role in TRAPP II assembly. TRS33 encodes another nonessential but conserved TRAPP subunit whose function is not known. Here, we show that one of these two subunits, nonessential individually, is required for TRAPP II assembly. Trs33 and Trs65 share sequence, intracellular localization and interaction similarities. Specifically, Trs33 interacts genetically with both Trs120 and Trs130 and physically with Trs120. In addition, trs33 mutant cells contain lower levels of TRAPP II and exhibit aberrant localization of the Golgi Ypts. Together, our results indicate that in yeast, TRAPP II assembly is an essential process that can be accomplished by either of two related TRAPP subunits. Moreover, because humans express two Trs33 homologues, we propose that the requirement of Trs33 for TRAPP II assembly is conserved from yeast to humans.

Abstract: The objective of this study was to investigate genotoxicity, especially DNA damage, in drinking water samples collected from tap by using fission yeast Schizosaccharomyces pombe as a model organism. Generally raw water potabolization is done by treatment with polymeric coagulant, alum, chlorine, etc. In the comet test, highly significant (P<0.001) effects of DNA damage were detected in treated water (tap water) when compared to negative control (raw water) as well as laboratory control (distilled water) samples for both 1 h and 2 h exposure. In the water treatment plant, raw water treatment is done by the process of prechlorination, alum and polymeric coagulant (CatflocT) dosing, postchlorination, filtration and final discharge for consumption. In conclusion it can be stated from the results that chlorinated disinfectant, alum and polymeric coagulant (CatflocT) mixture used in drinking water has a potent cumulative genotoxic effect in the eukaryotic cells and may pose potential genotoxic risk for human health following long-term consumption.

Abstract: Progression into mitosis is a major point of regulation in the Schizosaccharomyces pombe cell cycle, and its proper control is essential for maintenance of genomic stability. Investigation of the G(2)/M progression event in S. pombe has revealed the existence of a complex regulatory process that is responsible for making the decision to enter mitosis. Newer aspects of this regulation are still being revealed. In this paper, we report the discovery of a novel mode of regulation of G(2)/M progression in S. pombe. We show that the mitogen-activated protein kinase (MAPK)-regulated transcription factor Atf1 is a regulator of Cdc13 (mitotic cyclin) transcription and is therefore a prominent player in the regulation of mitosis in S. pombe. We have used genetic approaches to study the effect of overexpression or deletion of Atf1 on the cell length and G(2)/M progression of S. pombe cells. Our results clearly show that Atf1 overexpression accelerates mitosis, leading to an accumulation of cells with shorter lengths. The previously known major regulators of entry into mitosis are the Cdc25 phosphatase and the Wee1 kinase, which modulate cyclin-dependent kinase (CDK) activity. The significantly striking aspect of our discovery is that Atf1-mediated G(2)/M progression is independent of both Cdc25 and Wee1. We have shown that Atf1 binds to the Cdc13 promoter, leading to activation of Cdc13 expression. This leads to enhanced nuclear localization of CDK Cdc2, thereby promoting the G(2)/M transition.

Abstract: Cigarette smoke has long been recognized as a major environmental pollutant that can cause significant damage to the cellular macromolecules Although much is known about the types of damage, little is known about the cellular responses to the stress caused by cigarette smoke We have used the fission yeast Schizosaccharomyces pombe to elucidate the overall cellular responses towards cigarette smoke Here, we demonstrate that fission yeast cells exposed to aqueous extract of cigarette smoke exhibit cell cycle arrest and cell death in a dose-dependent manner Cigarette smoke treatment also results in accumulation of reactive oxygen species, unusual nuclear morphology and altered cellular structure Our data further establish activation of the S phase checkpoint in cigarette smoke-exposed Sz pombe cells The checkpoint proteins Rad3, Rad26, Rad17, Rad1, Hus1 and Cds1 play key roles in this process, as evidenced by cell survival and biochemical analysis, although another checkpoint protein, Rad9, seems to be less required Our results also suggest involvement of the stress-activated protein kinase Spc1/Sty1 and the bZIP transcription factors Atf1 and Pap1 in the cellular response towards cigarette smoke extract These findings indicate activation of the critical S phase checkpoint and cell cycle arrest in Sz pombe following CSE assault

Abstract: Genotoxic stress caused by carcinogens like cigarette smoke activate both the MAPK pathway and the S phase checkpoint in Schizosacchaomyces pombe. But the cross talk between these two pathways has not been investigated in great detail in fission yeast. This study deals with the molecular mechanism of co-ordination between the two regulatory pathways. We show that both the pathways have a common effector molecule, namely Cdc25, the cell cycle regulatory phosphatase. We demonstrate that the MAPK Sty1 interacts with Cdc25 and prevents mitotic entry in S.pombe cells exposed to CSE. To our knowledge, this is the first demonstration of interaction between Sty1 and Cdc25 in S. pombe. The functional significance of this interaction lies in effecting Cdc25 turnover after CSE exposure in S.pombe. We show that Cdc25 turnover after CSE treatment is dependent on the presence of Rad3 activity and Sty1-Cdc25 interaction. Our study suggests that the cigarette smoke extract (CSE) induced stress is counteracted by the simultaneous activation of a mitotic checkpoint in addition to the previously described S phase checkpoint. We also show that Sty1 activity is not essential for activation of the S phase checkpoint.

Abstract: This review considers the potential of the Comet assay (or Single Cell Gel Electrophoresis, SCGE) to evaluate the environmental impact of genotoxins in aquatic environments. It focuses on in vivo and in situ studies that have been carried out in various marine and freshwater sentinel species, published in the last 5 years. A large number of the studies reviewed report that the Comet assay is more sensitive when compared with other biomarkers commonly used in genetic ecotoxicology, such as sister chromatid exchanges or micronucleus test. Due to its high sensitivity, the Comet assay is widely influenced by laboratory procedures suggesting that standard protocols are required for both fish and mussel cells. However, there are still a wide variety of personalised Comet procedures evident in the literature reviewed, making comparison between published results often very difficult. Standardization and inter-laboratory calibration of the Comet assay as applied to aquatic species will be required if the Comet assay is to be used routinely by national bodies charged with monitoring water quality.

Abstract: New chemicals are being added each year to the existing burden of toxic substances in the environment. This has led to increased pollution of ecosystems as well as deterioration of the air, water, and soil quality. Excessive agricultural and industrial activities adversely affect biodiversity, threatening the survival of species in a particular habitat as well as posing disease risks to humans. Some of the chemicals, e.g., pesticides and heavy metals, may be genotoxic to the sentinel species and/or to non-target species, causing deleterious effects in somatic or germ cells. Test systems which help in hazard prediction and risk assessment are important to assess the genotoxic potential of chemicals before their release into the environment or commercial use as well as DNA damage in flora and fauna affected by contaminated/polluted habitats. The Comet assay has been widely accepted as a simple, sensitive, and rapid tool for assessing DNA damage and repair in individual eukaryotic as well as some prokaryotic cells, and has increasingly found application in diverse fields ranging from genetic toxicology to human epidemiology. This review is an attempt to comprehensively encase the use of Comet assay in different models from bacteria to man, employing diverse cell types to assess the DNA-damaging potential of chemicals and/or environmental conditions. Sentinel species are the first to be affected by adverse changes in their environment. Determination of DNA damage using the Comet assay in these indicator organisms would thus provide information about the genotoxic potential of their habitat at an early stage. This would allow for intervention strategies to be implemented for prevention or reduction of deleterious health effects in the sentinel species as well as in humans.

Abstract: TRAPP is a highly conserved modular multi-subunit protein complex. Originally identified as a “transport protein particle” with a role in endoplasmic reticulum-to-Golgi transport, its multiple subunits and their conservation from yeast to humans were characterized in the late 1990s. TRAPP attracted attention when it was shown to act as a Ypt/Rab GTPase nucleotide exchanger, GEF, in the 2000s. Currently, three TRAPP complexes are known in yeast, I, II, and III, and they regulate two different intracellular trafficking pathways: secretion and autophagy. Core TRAPP contains four small subunits that self assemble to a stable complex, which has a GEF activity on Ypt1. Another small subunit, Trs20/Sedlin, is an adaptor required for the association of core TRAPP with larger subunits to form TRAPP II and TRAPP III. Whereas the molecular structure of the core TRAPP complex is resolved, the architecture of the larger TRAPP complexes, including their existence as dimers and multimers, is less clear. In addition to its Ypt/Rab GEF activity, and thereby an indirect role in vesicle tethering through Ypt/Rabs, a direct role for TRAPP as a vesicle tether has been suggested. This idea is based on TRAPP interactions with vesicle coat components. While much of the basic information about TRAPP complexes comes from yeast, mutations in TRAPP subunits were connected to human disease. In this review we will summarize new information about TRAPP complexes, highlight new insights about their function and discuss current controversies and future perspectives.

Abstract: The transport protein particle (TRAPP) was initially identified as a vesicle tethering factor in yeast and as a guanine nucleotide exchange factor (GEF) for Ypt1/Rab1. In mammals, structures and functions of various TRAPP complexes are beginning to be understood. We found that mammalian TRAPPII was a GEF for both Rab18 and Rab1. Inactivation of TRAPPII‐specific subunits by various methods including siRNA depletion and CRISPR–Cas9‐mediated deletion reduced lipolysis and resulted in aberrantly large lipid droplets. Recruitment of Rab18 onto lipid droplet (LD) surface was defective in TRAPPII‐deleted cells, but the localization of Rab1 on Golgi was not affected. COPI regulates LD homeostasis. We found that the previously documented interaction between TRAPPII and COPI was also required for the recruitment of Rab18 to the LD. We hypothesize that the interaction between COPI and TRAPPII helps bring TRAPPII onto LD surface, and TRAPPII, in turn, activates Rab18 and recruits it on the LD surface to facilitate its functions in LD homeostasis. ![][1] Mammalian TRAPPII serves as a GEF for Rab18 and, together with COPI, regulates its recruitment onto the lipid droplet surface, thereby controlling lipid homeostasis. [1]: /embed/graphic-1.gif

Abstract: This work describes the efficiency of photoelectrocatalysis based on Ti/TiO2 nanotubes in the degradation of the azo dyes Disperse Red 1, Disperse Red 13 and Disperse Orange 1 and to remove their toxic properties, as an alternative method for the treatment of effluents and water. For this purpose, the discoloration rate, total organic carbon (TOC) removal, and genotoxic, cytotoxic and mutagenic responses were determined, using the comet, micronucleus and cytotoxicity assays in HepG2 cells and the Salmonella mutagenicity assay. In a previous study it was found that the surfactant Emulsogen could contribute to the low mineralization of the dyes (60% after 4 h of treatment), which, in turn, seems to account for the mutagenicity of the products generated. Thus this surfactant was not added to the chloride medium in order to avoid this interference. The photoelectrocatalytic method presented rapid discoloration and the TOC reduction was ≥87% after 240 min of treatment, showing that photoelectrocatalysis is able to mineralize the dyes tested. The method was also efficient in removing the mutagenic activity and cytotoxic effects of these three dyes. Thus it was concluded that photoelectrocatalysis was a promising method for the treatment of aqueous samples.