BK channels in human glioma cells

Gliomzellen menschlicher Herkunft sind mit einer Reihe verschiedener spannungsabhängiger Ionenkanäle ausgestattet. Besonders BK Kanäle wurden in menschlichen Gliomzellinien regelmäßig beobachtet. Dies legt nahe, daß BK Kanäle auch in vivo ein fester Bestandteil solcher Zellen sind. Wir konnten das Vorkommen von BK Kanälen für 1321N1 Zellen bestätigen. Die Kanäle, die wir in unseren Experimenten charakterisierten, wiesen typische Merkmale von BK Kanälen auf : Sie öffneten erst bei Spannungen von mehr als +60 mV, waren ausgesprochen abhängig von [Ca2+]i, und liessen sich mit 100 nM IBTX sowie 1 mM TEA spezifisch blocken. Weil jedoch diese Kanäle eine so ausgeprägte Spannungsabhängigkeit zeigten, lag die Annahme nahe, daß diese Kanäle bei physiologischen Membranpotentialen geschlossen sind und deshalb auch keine physiologische Funktion haben können. Excised patch Experimente belegten jedoch, daß diese Annahme falsch ist. Anhand dieser Experimente, die eine gleichzeitige Variation von [Ca2+]i und des Membranpotentials erlauben, konnten wir zeigen, daß unter physiologischen Bedingungen diese Kanäle offen sein können. Dies ist die Bedingung für eine Funktion von BK Kanälen in menschlichen Gliomzellinien, welche bisher nicht untersucht worden war. Wir konnten beobachten, daß durch eine Erhöhung von [K+]e im Medium von 5 auf 20 mM die Zellzahl nach 6 Tagen um 20 % zunahm. Diese Zunahme der Zellzahl konnte durch spezifische BK Kanal Blocker verhindert werden. Dieser Befund deutet auf eine Rolle der BK Kanäle bei der durch bestimmte Wachstumsfaktoren ausgelösten Proliferation hin. Der genaue Mechanismus, der der Beteiligung von BK Kanälen bei der Proliferation zugrundeliegt ist noch nicht aufgeklärt. Die Untersuchungen zur Migration von 1321N1 Zellen ergaben, daß hierbei BK Kanäle keine Rolle zu spielen scheinen

The new citizens : a study of the architectural identity of public philanthropic institutions built by two Isma'ili communities in contemporary Bombay

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Architecture, 2009.Includes bibliographical references (p. 143-146).Just across the railway lines at Charni road, forming a backdrop to the Marine Drive, stands the Saifee Hospital in all its splendor. Across the city in the neighborhood of Mazgaon, nestled behind the St. Mary's school along the central railway line, and in an equal splendor, stands the Center of Excellence, Diamond Jubilee High School. Both buildings were built in the last five years. Both use a similar quantity of glass and cement plaster and establish their contemporaneity so. Both institutions were built by Shi'i Isma'ili communities, the hospital by the Bohras and the school by the Khojas. Both buildings represent a significant phase in the history of these mercantile communities in Bombay where their emergence as public philanthropists echoes the rapid increase in wealth and the creation of global diasporic networks in a liberal Indian economy. But while the Saifee hospital is cloaked in its massive pastel colored facade punctured by numerous arched windows and capped by ornamental domes, the Diamond Jubilee High School displays a playful juxtaposition of geometrical forms and volumes with dashes of color composed so as to expose structure and skin. The thesis explains how these two buildings, which have such different appearances, are comparable strategies for expressing Isma'ili communal identity. In Chapter two, I construct a detailed comparison of the two buildings with respect to their location in the city, aspects of siting, faqade, interior, spatial organization, program, client and architect teams.(cont.) In Chapter three, I investigate and conclude that the two institutions mediate Isma'ili faith, citizenship and mercantilism in architecturally different but functionally comparable ways that respond to the complex social 'condition' in contemporary Bombay. This thesis thus studies the expression of communal identity through its patronage of public architecture. It claims that architecture is instrumental in the creation, sustenance and subversion of communal identity and is an effective social construction used to communicate within the public sphere. I argue that for post-partition Indian Muslims, to contend with their identity in a rising tide of Hindu nationalism in the country, requires mediation of faith, citizenship and in the case of the Isma'ilis, mercantilism. Isma'ili public philanthropy, I propose, is a mode for expressing this communal identity. I explain Isma' ili architectural expression as a product of a condition distinctive of contemporary Bombay, where the simultaneous marginalization of the two Isma'ili communities by the Hindus and the other Muslims, creates a space for them to perform within the public sphere.by Zameer Basrai.S.M

Skp, Cullin, F-box (SCF)-Met30 and SCF-Cdc4-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A for Chromosomal Stability in Budding Yeast

Restricting the localization of the histone H3 variant CENP-A (Cse4 in yeast, CID in flies) to centromeres is essential for faithful chromosome segregation. Mislocalization of CENP-A leads to chromosomal instability (CIN) in yeast, fly and human cells. Overexpression and mislocalization of CENP-A has been observed in many cancers and this correlates with increased invasiveness and poor prognosis. Yet genes that regulate CENP-A levels and localization under physiological conditions have not been defined. In this study we used a genome-wide genetic screen to identify essential genes required for Cse4 homeostasis to prevent its mislocalization for chromosomal stability. We show that two Skp, Cullin, F-box (SCF) ubiquitin ligases with the evolutionarily conserved F-box proteins Met30 and Cdc4 interact and cooperatively regulate proteolysis of endogenous Cse4 and prevent its mislocalization for faithful chromosome segregation under physiological conditions. The interaction of Met30 with Cdc4 is independent of the D domain, which is essential for their homodimerization and ubiquitination of other substrates. The requirement for both Cdc4 and Met30 for ubiquitination is specifc for Cse4; and a common substrate for Cdc4 and Met30 has not previously been described. Met30 is necessary for the interaction between Cdc4 and Cse4, and defects in this interaction lead to stabilization and mislocalization of Cse4, which in turn contributes to CIN. We provide the first direct link between Cse4 mislocalization to defects in kinetochore structure and show that SCF-mediated proteolysis of Cse4 is a major mechanism that prevents stable maintenance of Cse4 at non-centromeric regions, thus ensuring faithful chromosome segregation. In summary, we have identified essential pathways that regulate cellular levels of endogenous Cse4 and shown that proteolysis of Cse4 by SCF-Met30/Cdc4 prevents mislocalization and CIN in unperturbed cells

The Yeast Nuclear Pore Complex Functionally Interacts with Components of the Spindle Assembly Checkpoint

Aphysical and functional link between the nuclear pore complex (NPC) and the spindle checkpoint machinery has been established in the yeast Saccharomyces cerevisiae. We show that two proteins required for the execution of the spindle checkpoint, Mad1p and Mad2p, reside predominantly at the NPC throughout the cell cycle. There they are associated with a subcomplex of nucleoporins containing Nup53p, Nup170p, and Nup157p. The association of the Mad1p–Mad2p complex with the NPC requires Mad1p and is mediated in part by Nup53p. On activation of the spindle checkpoint, we detect changes in the interactions between these proteins, including the release of Mad2p (but not Mad1p) from the NPC and the accumulation of Mad2p at kinetochores. Accompanying these events is the Nup53p-dependent hyperphosphorylation of Mad1p. On the basis of these results and genetic analysis of double mutants, we propose a model in which Mad1p bound to a Nup53p-containing complex sequesters Mad2p at the NPC until its release by activation of the spindle checkpoint. Furthermore, we show that the association of Mad1p with the NPC is not passive and that it plays a role in nuclear transport

Yeast hEST1A/B (SMG5/6)- Like proteins contribute to environment-sensing adaptive gene expression responses

During its natural life cycle, budding yeast (Saccharomyces cerevisiae) has to adapt to drastically changing environments, but how environmental-sensing pathways are linked to adaptive gene expression changes remains incompletely understood. Here, we des

Spd2 assists Spd1 in modulation of RNR architecture but does not regulate deoxynucleotide pools

In yeasts, small intrinsically disordered proteins (IDPs) modulate ribonucleotide reductase (RNR) activity to ensure an optimal supply of dNTPs for DNA synthesis. The S. pombe Spd1 protein can directly inhibit the large RNR subunit (R1), import the small subunit (R2) into the nucleus and induce an architectural change in the R1-R2 holocomplex. Here, we report the characterization of Spd2, a protein with homology to Spd1. We show that Spd2 is a CRL4Cdt2 controlled IDP that functions together with Spd1 in the DNA damage response and in modulation of RNR architecture. However, Spd2 does not regulate dNTP pools and R2 nuclear import. Furthermore, deletion of spd2 only weakly suppresses the Rad3ATR checkpoint dependency of CRL4Cdt2 mutants. However, when we raised intracellular dNTP pools by inactivation of RNR feedback inhibition, deletion of spd2 could suppress the checkpoint dependency of CRL4Cdt2 mutant cells to the same extent as spd1. Collectively, these observations suggest that Spd1 on its own regulates dNTP pools, while it together with Spd2 modulates RNR architecture and sensitizes cells to DNA damage

N-terminal Sumoylation of Centromeric Histone H3 Variant Cse4 Regulates Its Proteolysis To Prevent Mislocalization to Non-centromeric Chromatin

Stringent regulation of cellular levels of evolutionarily conserved centromeric histone H3 variant (CENP-A in humans, CID in flies, Cse4 in yeast) prevents its mislocalization to non-centromeric chromatin. Overexpression and mislocalization of CENP-A has been observed in cancers and leads to aneuploidy in yeast, flies, and human cells. Ubiquitin-mediated proteolysis of Cse4 by E3 ligases such as Psh1 and Sumo-Targeted Ubiquitin Ligase (STUbL) Slx5 prevent mislocalization of Cse4. Previously, we identified Siz1 and Siz2 as the major E3 ligases for sumoylation of Cse4. In this study, we have identified lysine 65 (K65) in Cse4 as a site that regulates sumoylation and ubiquitin-mediated proteolysis of Cse4 by Slx5. Strains expressing cse4 K65R exhibit reduced levels of sumoylated and ubiquitinated Cse4 in vivo. Furthermore, co-immunoprecipitation experiments reveal reduced interaction of cse4 K65R with Slx5, leading to increased stability and mislocalization of cse4 K65R under normal physiological conditions. Based on the increased stability of cse4 K65R in psh1 strains but not in slx5 strains, we conclude that Slx5 targets sumoylated Cse4 K65 for ubiquitination-mediated proteolysis independent of Psh1. In summary, we have identified and characterized the physiological role of Cse4 K65 in sumoylation, ubiquitin-mediated proteolysis, and localization of Cse4 for genome stability

The Journey to smORFland

The genome sequences completed so far contain more than 20 000 genes with unknown function and no similarity to genes in other genomes. The origin and evolution of the orphan genes is an enigma. Here, we discuss the suggestion that some orphan genes may represent pseudogenes or short fragments of genes that were functional in the genome of a common ancestor. These may be the remains of unsuccessful duplication or horizontal gene transfer events, in which the acquired sequences have entered the fragmentation process and thereby lost their similarity to genes in other species. This scenario is supported by a recent case study of orphan genes in several closely related species of Rickettsia, where full-length ancestral genes were reconstructed from sets of short, overlapping orphan genes. One of these was found to display similarity to genes encoding proteins with ankyrin-repeat domains

SUMO-Targeted Ubiquitin Ligases (STUbLs) Reduce the Toxicity and Abnormal Transcriptional Activity Associated With a Mutant, Aggregation-Prone Fragment of Huntingtin

Cell viability and gene expression profiles are altered in cellular models of neurodegenerative disorders such as Huntington\u27s Disease (HD). Using the yeast model system, we show that the SUMO-targeted ubiquitin ligase (STUbL) Slx5 reduces the toxicity and abnormal transcriptional activity associated with a mutant, aggregation-prone fragment of huntingtin (Htt), the causative agent of HD. We demonstrate that expression of an aggregation-prone Htt construct with 103 glutamine residues (103Q), but not the non-expanded form (25Q), results in severe growth defects in slx5Delta and slx8Delta cells. Since Slx5 is a nuclear protein and because Htt expression affects gene transcription, we assessed the effect of STUbLs on the transcriptional properties of aggregation-prone Htt. Expression of Htt 25Q and 55Q fused to the Gal4 activation domain (AD) resulted in reporter gene auto-activation. Remarkably, the auto-activation of Htt constructs was abolished by expression of Slx5 fused to the Gal4 DNA-binding domain (BD-Slx5). In support of these observations, RNF4, the human ortholog of Slx5, curbs the aberrant transcriptional activity of aggregation-prone Htt in yeast and a variety of cultured human cell lines. Functionally, we find that an extra copy of SLX5 specifically reduces Htt aggregates in the cytosol as well as chromatin-associated Htt aggregates in the nucleus. Finally, using RNA sequencing, we identified and confirmed specific targets of Htt\u27s transcriptional activity that are modulated by Slx5. In summary, this study of STUbLs uncovers a conserved pathway that counteracts the accumulation of aggregating, transcriptionally active Htt (and possibly other poly-glutamine expanded proteins) on chromatin in both yeast and in mammalian cells