Chairman’s Travel Awards

2015 Meritorious Research Travel Award Recipients

Name: Zanetta Chang
Lab: Heitman
Conference: FASEB conference on Molucular Pathogenesis: Mechanisms of Infections Disease
Date: July 12-17, 2015
Location: Keystone, Colorado
Purpose: To present a poster

Abstract:

Zanetta Chang_Heitman LabDrug resistance via RNAi-dependent epimutations in pathogenic fungi: The opportunistic fungal infection mucormycosis is notable for its high morbidity and mortality as well as its increasing incidence. Treatment is complicated by the fact that Mucor circinelloides, one of the major causes of mucormycosis, demonstrates intrinsic resistance to a number of common antifungal drugs. Previous work demonstrated that Mucor is capable of developing transient resistance to the antifungal FK506 through RNA interference-dependent epimutations. These resistant strains, which silence the drug target gene encoding FKBP12, can be selected by exposure to FK506; however, the FKBP12 gene in these strains is re-expressed once FK506 is not supplemented in the growth media. The silencing process requires the generation of small RNAs (sRNA) against the FKBP12 gene via core RNAi pathway proteins including RNA-dependent RNA polymerases, Dicer, and Argonaute. It is unknown whether this novel mechanism of resistance is involved in Mucor resistance to other antifungal drugs, or if it can be found in other species of fungi. We have isolated mutant strains of Mucor that exhibit transient resistance to the antifungal 5-fluoroorotic acid but do not carry mutations in the target genes, pyrG (URA3) and pyrF (URA5). We are currently evaluating the mechanism of resistance to determine if these mutants silence the pyr genes at a transcriptional level via generation of sRNAs mapped on either the pyrG or the pyrF locus. We are also investigating the potential for epimutations in a second opportunistic fungal pathogen with established RNAi pathways, Cryptococcus neoformans, where we have isolated strains transiently resistant to FK506. Discovery of epimutations as a cause of resistance against multiple drugs or in multiple species of fungi will provide a novel forum for understanding drug resistance mechanisms in pathogenic fungi and will have implications in the development of unique classes of antifungals.

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Name: Sarah Jaslow
Lab: Ko
Conference: FASEB conference on Molucular Pathogenesis: Mechanisms of Infections Disease
Date: July 12-17, 2015
Location: Keystone, Colorado
Purpose: To present talk and poster

Abstract:

Sarah Jaslow_Ko LabContribution to virulence of a novel anti-inflammatory effector in Salmonella enterica
Background: S. enterica manipulate the inflammatory response through the injection of effector proteins that alter host signaling pathways. Characterizing the functions of these effectors and their role in virulence can reveal novel mechanisms important in infection and autoimmunity.

Methods: Luminex screening of cytokine levels was used to identify cytokines regulated by S. enterica in immortalized B cell lines. To identify and characterize S. enterica genetic regions important in regulating the production of these cytokines, we used comparative genomics of S. enterica serovars, gene knockout mutants, and functional testing in cultured cells. Furthermore, C57BL/6 mice were used as a model for infection to test the effects of the putative factors in vivo.

Results: Salmonella enterica serovar Typhimurium induced the anti-inflammatory cytokines interleukin-10 (IL-10) and interleukin-1 receptor antagonist (IL-1RA) in immortalized cultured B cells. A previously uncharacterized gene, designated sara (Salmonella anti-inflammatory response activator) was determined to only be present in IL-10 inducing serovars of S. enterica. When this gene was deleted, S. Typhimurium was unable to induce IL-10, demonstrating that the protein is required for the IL-10 inducing phenotype. In addition, sara mutants showed altered intracellular survival and replication. These intracellular phenotypes were not restored upon addition of IL-10 to the media of Δsara-infected cells, indicating additional unidentified effects of SARA beyond cytokine regulation. To see the effect of SARA during a full host infection, I compared the amount of colony forming units (CFUs) in the spleen after infection with either wildtype Salmonella or Δsara. The Δsara S. Typhimurium showed consistently higher spleen CFU counts. Complementary to these studies we are also examining host variation in the IL-10 production pathway and the consequences of this for autoimmune disease.

Conclusion: Together, these data suggest that SARA contributes to Salmonella-driven manipulation of the delicate balance of pro- and anti-inflammatory host immune responses. These findings are likely relevant to the ability of people to respond and clear Salmonella infections, and may have longer term consequences for the host immune response.

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Name: Arun Haldar
Lab: Coers
Conference: FASEB conference on Molucular Pathogenesis: Mechanisms of Infections Disease
Date: July 12-17, 2015
Location: Keystone, Colorado
Purpose: To present talk and poster

Abstract:

Arun Haldar_Charimans Travel Award 2015The E3 ubiquitin ligase TRAF6 and the ubiquitin-binding protein p62 regulate the recruitment of Guanylate Binding Proteins to pathogen-containing vacuoles: Many microbes create and maintain pathogen-containing vacuoles (PVs) as an intracellular niche permissive for microbial growth and survival. The destruction of PVs by IFNg-inducible GBP and IRG host proteins is central to a successful immune response directed against numerous PV-resident pathogens. However, the mechanism by which IRGs and GBPs cooperatively detect and destroy PVs is unclear. We find that host cell priming with IFNg results in IRG-dependent ubiquitination of Toxoplasma- and Chlamydia-containing vacuoles through regulated translocation of the E3 ubiquitin ligase TRAF6. This initial ubiquitin labelling elicits p62-mediated escort and deposition of GBPs to PVs thereby conferring cell-autonomous immunity. Hypervirulent strains of Toxoplasma gondii evade this process via specific rhoptry protein kinases that inhibit IRG function resulting in blockage of downstream PV ubiquitination and GBP delivery. Our results define an ubiquitin-centered mechanism by which host cells deliver GBPs to PVs and explain how hypervirulent parasites evade GBP-mediated immunity.

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Name: Xiou Cao
Lab: Aballay
Conference: 20th international C. elegans Meeting
Date: June 24-28, 2015
Location: Los Angeles, California
Purpose: To present talk and poster

Abstract:

Xiou Cao_CTA2015Investigation of the dopamine signaling in immune regulation of Caenorhabditis elegans: Increasing evidence suggests that active interactions exist between the nervous and immune systems. Previous data from our lab indicate that neuronal G protein-coupled receptors (GPCRs) in C. elegans play an important role in regulating immune response to pathogen Pseudomonas aeruginosa PA14. Upon pathogen infection, the immune system of C. elegans responds by activating conserved pathways that control the expression of immune effectors. It was found that many of these pathways are fine-tuned by the nervous system through GPCRs that function in a subset of neurons. Recently, a chemical screen was performed in our lab to identify potential immune activators in C. elegans. In this screen, we found that dopamine antagonists have the ability to increase the expression of a gene (F35E12.5) that is a marker of the p38/PMK-1 MAPK pathway, implying a role of dopamine signaling in the regulation of immune response. Consistent with this notion, the worms treated with dopamine antagonists during survival assays were protected from P. aeruginosa PA14 infection in a dose-dependent manner. Strain FG58, which has a deletion mutation in a conserved D1-like dopamine receptor DOP-4 was found to be significantly resistant to P. aeruginosa infection. In addition, the mutation in DOP-4 also suppressed the enhanced resistance conferred by dopamine antagonists.

Furthermore, we showed that the level of phosphorylation of p38/PMK-1 in C. elegans was significantly up-regulated upon either dop-4 mutation or the treatment of dopamine antagonists. Knocking down of pmk-1 using RNAi fully suppressed the enhanced resistance of dop-4 mutant worms. These results provide the evidence that dopamine signaling may functions to control conserved immune response upon pathogen infection. We are in the process of studying the tissue and cell specificity of DOP-4 in mediating immune response. Ongoing experiments also include the qRT-PCR confirmation of immune gene expression and genomic survey of novel pathways that are involved in the immune modulation of dopamine signaling.

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Name: Keisha McSweeney
Lab: Goldstein
Conference: European Society for Human Genetics Annual Meeting
Date: June 4-9, 2015
Location: Glasgow, Scotland
Purpose: To present a poster

Abstract:

Keisha Melodi McSweeney_CTA2015Microelectrode arrays (MEAs) are powerful tools for detecting spontaneous neuronal activity. MEAs have been used to detect the response of primary dissociated neurons to neurotoxins and are emerging as reliable drug screening platforms. Recent studies indicating the involvement of microRNAs in epilepsy phenotypes encouraged us to investigate the effect of modulating microRNA-128, on neuronal activity. Mice deficient in microRNA-128 are prone to fatal seizures. Modulating microRNA-128 in the MEA system allowed us to directly test whether MEAs are useful to screen other microRNAs that influence risk of seizures. In particular, we sought to evaluate whether down-regulation of microRNA-128 results in clear excitability phenotypes by MEA analysis. We used primary cortical neurons from wild-type post-natal day zero C57BL6 mice and a lentivirus delivered microRNA sponge to target the mature microRNA-128. Sponges are competitive microRNA inhibitors which express tandem repeats that are partially complementary to the microRNA sequence. We recorded neuronal activity on the MEA for 15 minutes each day to collect spike, burst, and network synchronicity data. Our data show that down regulation of microRNA-128 by inhibition with a sponge results in significantly increased neuronal activity. These features are consistent with the previous observations that microRNA-128 deficiency promotes neuronal excitability 1. These experiments illustrate the utility of the MEA platform in evaluation of microRNAs in regulation of network phenotypes.

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Name: Katherine Rempe
Lab: St. Geme
Conference: American Society for Microbiology
Date: May 30 – June 2, 2015
Location: New Orleans, LA
Purpose: To present a poster

Abstract:

Katherine Rempe_CTA2015Cytoplasmic N-linked Glycosylation is Necessary for Proper Function of TrimericAutotransporters in Kingella kingae and Aggregatibacter aphrophilus: Glycosylation is a widespread mechanism employed by both eukaryotes and bacteria to increase the functional diversity of their proteomes.  The non-typeable Haemophilus influenzae HMW1C glycosyltransferase mediates unconventional N-linked glycosylation of the two-partner secretion system protein HMW1 and is the prototype for glycosyltransferases involved in modification of two-partner secretion proteins. HMW1C-mediated glycosylation takes place in the cytoplasm and results in the sequential transfer of hexose molecules from UDP-hexose donors to asparagine residues within the HMW1 protein.  Homology analysis demonstrates that many gram-negative species contain a gene that encodes an HMW1C homologue and is not located near genes for a two-partner secretion system.  To identify the target acceptor proteins for these HMW1C homologues, we focused on the proteins in Kingella kingae and Aggregatibacter aphrophilus.  We found both homologues to be functional glycosyltransferases and identified their substrates as the trimeric autotransporter proteins Knh in K. kingae and EmaA in A. aphrophilus. LC-MS/MS analysis revealed multiple sites of N-linked hexose modification on Knh and EmaA. Without glycosylation Knh and EmaA failed to facilitate autoaggregation or adherence to Chang cells, establishing that glycosylation is essential for proper protein function.  This work describes the first examples where an HMW1C-like enzyme glycosylates a protein that is not encoded in the same gene cluster as the enzyme and is not a member of a two-partner secretion system. Furthermore, Knh and EmaA are the first examples of trimeric autotransporters that are N-glycosylated.

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Name: Yue Jiang
Lab: Matsunami
Conference: AChemS (Association for Chemoreception Sciences) 37th Annual meeting
Date: April 22-26, 2015
Location: Bonita Springs, Florida
Purpose: To present a poster

Abstract:

Yue Jiang_CTA2015

Molecular profiling of activated olfactory neurons using phosphorylated ribosome immunoprecipitation and RNA-Seq identifies odorant receptors responding to odors in vivo: The mammalian olfactory system uses a large family of odorant receptors to detect and discriminate amongst a myriad of volatile odor molecules. Understanding odor coding requires comprehensive mapping between odorant receptors and corresponding odors. Here we present high–throughput in vivo identification of odorant receptor repertoires responding to odorants, using phosphorylated ribosome immunoprecipitation of mRNA from olfactory epithelium of odor–stimulated mice followed by RNA–Seq. This approach screens the endogenously expressed odorant receptors against an odor in one set of experiments, using awake and freely behaving mice. In combination with validations in a heterologous system, we identify sets of odorant receptors for two odorants, acetophenone and 2,5–dihydro–2,4,5–trimethylthiazoline (TMT), encompassing 69 odorant receptor–odorant pairs. We also identified shared amino acid residues specific to the acetophenone or TMT receptors, and developed models to predict receptor activation by acetophenone. This study provides a means to understand the combinatorial coding of odors in vivo.

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2014 Meritorious Research Travel Award Recipients

Name: Kyla Selvig
Lab: Alspaugh
Conference: 9th International Conference on Cryptococcus and Cryptococcosis
Date: May 15-19, 2014
Location: Amsterdam, Netherlands
Purpose: To present talk and poster

Abstract:

The basics of the Cryptococcus neoformans alkaline response pathway: Adapting to a bitter host Kyla Selvig_Alspaugh LabBackground: Cryptococcus neoformans must adapt to dramatic changes in environmental conditions to establish infection. One of these host-associated stresses includes a significant increase in ambient pH, which can disrupt membrane potential, decrease iron availability, and alter ion homeostasis. To combat this stress, C. neoformans and other fungal pathogens utilize the Rim101 alkaline response pathway. In neutral/alkaline conditions, this signaling pathway activates the Rim101 transcription factor, which regulates the expression of genes necessary for adaptation to alkaline pH stress. In addition to this conserved Rim101 pathway role, C. neoformans has repurposed this pathway to regulate cell wall changes necessary for polysaccharide capsule formation and immune system evasion. It is unknown how C. neoformans senses and activates the Rim101 pathway, despite its being a vital pathway for survival and proliferation during infection. Furthermore, the majority of the research on this conserved fungal signaling pathway was performed in ascomycetes, such as S. cerevisiae and C. albicans, and much less is known about pH response in basidiomycetes such as C. neoformans.

Aim: Elucidate the Rim101 signaling pathway responsible for Rim101 activation in C. neoformans.

Methods: We used targeted mutagenesis to disrupt genes with predicted homology to canonical Rim101 pathway proteins. We screened these mutants for phenotypes that require Rim101 activation (growth on alkaline pH, salt tolerance, capsule formation). We utilized a GFP-Rim101 construct to assess Rim101 cleavage and subcellular localization in these mutants. Finally, to identify members of the Rim101 pathway that could not be identified using homology, we used arandom mutagenesis screen to identify mutants with phenotypes similar to the rim101 mutant.

Results:  Of the six canonical members of the Rim101 pathway, only Rim13, Rim20 and Rim23/PalC were conserved and required for C. neoformans Rim101 activation. Deletion of the genes encoding each of these proteins phenocopied therim101 mutant. Each mutation also disrupted Rim101 proteolytic activation and nuclear localization. Furthermore, therim20, rim23, and rim13 mutant phenotypes were rescued by expressing the active form of the Rim101 transcription factor, strongly supporting their role as Rim101 activators. These highly conserved proteins compose the most downstream portion of the pathway immediately prior to Rim101 activation. However, the membrane-associated proteins required for sensing extracellular pH were not identified by homology. Using an A. tumefaciens random mutagenesis screen, we identified a novel component of the C. neoformans Rim101 pathway that, like the previously described pathway components, is completely required for Rim101 activation. This protein, named Rra1, is predicted to be structurally similar to the ascomycete pH sensor, despite having distinct amino acid sequence. Our results strongly support a model in which Rra1 serves as the C. neoformans pH sensor responsible for activating the Rim101 pathway. In addition, the RRA1 gene appears to be highly conserved throughout the Basidiomycota phylum, indicating that it is likely serving a similar role in these fungi.

Conclusion: Previously, our lab found that C. neoformans specifically uses the Rim101 pathway to regulate the expression of target genes favoring its survival within its host. Here, we have found that only some of the canonical activators of Rim101 are present in C. neoformans. We identified a novel component of this pathway, Rra1, which is likely acting as the alkaline pH sensor in C. neoformans and other basidiomycetes. These findings have significantly advanced our knowledge of how this fungal pathogen senses and responds to the alkaline environment within its infected host.

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Name: Alexander Price
Lab: Luftig
Conference: Epstein Barr Virus 50th Anniversary
Date: March 23-25, 2014
Location: Keble College, Oxford
Purpose: To present talk and poster

Abstract:

Characterizing survival in the absence of LMP1-mediated NFkB signaling early after B-cell infection
Alexander Price_Luftig LabIt has long been known that early after B-cell infection EBV expresses a set of latency-associated genes that mimic normal B-cell maturation and aid in immortalization. In particular, Latent Membrane Protein 1 (LMP1) is a constitutively active version of the host CD40 receptor that signals chronically through the downstream NFkB pathway. This LMP1-induced NFkB signaling is absolutely critical for the generation and survival of LCLs. Indeed, inhibition of NFkB in LCLs induces apoptosis. As such, it was quite surprising when recent work from our laboratory identified a period early after infection where EBV-infected B-cells proliferated extensively despite low levels of LMP1 and were immune to apoptosis induced by NFkB inhibition.

To ascertain how these cells survive in the absence of LMP1-induced NFkB activation, we performed BH3 profiling to query the state of mitochondrial priming of apoptosis at different times post infection and validated these profiles with specific small molecule antagonists. Our data support a model where uninfected B cells are characterized by active BCL-2, suggesting dependence on this anti-apoptotic molecule. As proliferation commences early after EBV infection, the cells are characterized by combined BCL-2 and MCL1 dependence. Finally, the resultant LCLs are characterized by BFL1/A1 dependence. Thus, EBV appears to promote survival at the mitochondria by different mechanisms throughout B-cell immortalization. Overall, these data provide insight into how EBV prevents apoptosis and supports a new model for EBV-driven outgrowth where restricted viral gene expression early after infection precedes the full latency III growth program found in LCLs.

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2013 Meritorious Research Travel Award Recipients

Name: Yi-Shan Chen
Lab: Valdivia
Conference: American Society for Cell Biology (ASCB)
Date: December 14 – 18, 2013
Location: New Orleans, LA
Purpose: To present talk and poster

Abstract:

A bacterial effector protein co-opts a host scaffolding protein and innate immune signaling during Chlamydia infection
Yi-Shan Chen_Valdiva LabChlamydia trachomatis, the causative agent of trachoma and sexually transmitted infections, employs a type III secretion (T3S) system to deliver effector proteins into host epithelial cells to establish a replicative vacuole (“inclusion”). Aside from the phosphoprotein TARP, a Chlamydia effector that promotes actin re-arrangements, very few factors mediating bacterial entry and early inclusion establishment have been identified or characterized. Like many T3S effectors, TARP requires a chaperone (Slc1) for efficient translocation into host cells. In this study, we defined proteins that associate with Slc1 in invasive Chlamydia elementary bodies (EB) by immunoprecipitation coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this manner, we identified a previously uncharacterized protein, which we renamed TepP (Translocated early phosphoprotein), as a new T3S effector. We show that TepP is translocated early during bacterial entry into epithelial cells and is phosphorylated at two distinct tyrosine residues. Phosphorylated TepP co-immunoprecipitated with CrkI and II, isoforms of a SH2 domain and SH3 domain-containing host scaffolding protein, during infection. CrkI and II were recruited to bacteria entry sites and remained associated till the nascent inclusions had trafficked to microtubule organizing center. Importantly, a Chlamydia mutant lacking TepP failed to recruit CrkI-II to inclusions, providing genetic confirmation of a direct role for this effector in the recruitment of this signaling scaffolding protein. Finally, endocervical epithelial cells infected with a tepP mutant showed altered expression of a subset of genes associated with innate immune responses. We propose a model wherein TepP acts downstream of TARP to recruit scaffolding proteins at entry sites to initiate and amplify signaling cascades important for the regulation of innate immune responses to Chlamydia.​

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Name: Marcela Kokes
Lab: Valdivia
Conference: American Society for Cell Biology (ASCB)
Date: December 14 – 18, 2013
Location: New Orleans, LA
Purpose: To present talk and poster

Abstract:

Host and bacterial factors mediate cytoskeletal rearrangements at the surface of the Chlamydia trachomatis pathogenic vacuole
Marcela_ Kokes_Valdivia LabThe obligate intracellular bacterial pathogen Chlamydia trachomatis resides within a membrane-bound vacuole (“inclusion”). A cytoskeletal cage of actin and intermediate filaments surrounds the inclusion, and disruption of this cage leads to increased exposure of bacterial products to the host cell’s innate immune receptors and increased expression of pro-inflammatory cytokines.

To identify Chlamydia factors involved in cytoskeletal cage assembly at inclusions, we screened an arrayed library of chemically-mutagenized C. trachomatis strains for changes in F-actin assembly at inclusions and identified two mutants with altered F-actin cages. Using new genetic tools developed for Chlamydia, we determined that loss of F-actin cage assembly at the inclusion is caused by a mutation in an integral inclusion membrane protein, renamed INAC (Inclusion membrane protein for actin cages). Re-introduction via transformation of wild-type INAC under its endogenous promoter on a chlamydial plasmid rescued F-actin cage formation.

In parallel, we sought to define the host factors contributing to F-actin cage formation around the inclusion. Previous studies determined that RhoA is necessary for the formation of F-actin cages at inclusions and that EGFP-RhoA localized to the inclusion periphery. We identified proteins that co-immunoprecipitated with an EGFP-tagged RhoA variant specifically during Chlamydia infection. Most of these cytoskeletal proteins localized around inclusions and co-localized with F-actin cages when tagged and expressed ectopically including non-muscle myosin II, and we have confirmed the same endogenous localization of a few, including Alpha-actinin, and Filamin. Many identified proteins are structural and interact directly with actin filaments, often in the context of adhesion. Taken together, these data are consistent with a model in which INAC recruits large adhesion-like complexes to play structural or signaling roles fundamental in F-actin cage formation.

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Name: Emily Snavely
Lab: Valdivia
Conference: 2013 Chlamydia Basic Research Society Meeting
Date: March 19 – 22, 2013
Location: San Antonio, TX
Purpose: To present talk and poster

Abstract:

Reassessing the role of CPAF in Chlamydia infections through genetic approaches
Emily Snavely_Valdivia LabThe Chlamydia protease CPAF has been proposed to modulate multiple mammalian cellular functions based on the identification of a broad range of host substrates. Recent findings suggest that CPAF activity can persist after cell lysis and thus confound the extent to which CPAF cleavage and degradation of its putative targets occurs in living cells. Furthermore, small molecule and peptide inhibitors of CPAF have yielded divergent results and their off-target effects are difficult to predict and control. Here we sought to clarify the role played by CPAF in the manipulation of host cell functions by analyzing Chlamydia mutants lacking CPAF activity. From a broad screen of small plaque forming, EMS-derived Chlamydia mutants, we identified a strain with a loss-of-function truncation mutation at the amino-terminal end of CPAF. CPAF cannot be detected in this strain by western blot or immunofluorescence microscopy, and infected cell lysates do not possess any detectable CPAF activity. Cells infected with these CPAF-deficient strains were still resistant to staurosporine-induced apoptosis, exhibited Golgi fragmentation and were still resistant to reinfection, suggesting that these chlamydial activities are not mediated by CPAF. In contrast, we observed that late in infection remodeling of the intermediate filament protein vimentin was dependent on CPAF. This cleavage correlated with loss of inclusion membrane integrity, suggesting that CPAF may play a role during the late stages of infection.

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Name: Chris Frank
Lab: Crawford
Conference: Keystone Symposia: Epigenetic Marks and Cancer Drugs
Date: March 20 – 25, 2013
Location: Santa Fe, NM
Purpose: To present talk and poster

Abstract:

Histone deacetylase inhibitor-induced chromatin remodeling
Chris Frank_Crawford LabHistone deacetylase inhibitors (HDACi) are among the most promising anticancer drugs in development – capable of inducing apoptosis, cell cycle arrest, and differentiation pathways preferentially in cancer cells. These compounds block the action of endogenous histone deacetylases, resulting in both histone and nonhistone protein hyperacetylation that is thought to impact chromatin structure. Histone hyperacetylation has previously been associated with local chromatin structure relaxation and increased access of transcription factors to the DNA template. Despite observation of extensive histone hyperacetylation genome-wide following HDACi treatment, transcription level changes only occur at a small fraction of expressed genes. To investigate this discrepancy and better understand the mechanism of action for HDACi, we identified and characterized genome-wide chromatin accessibility changes that result from HDACi treatments.

DNase I hypersensitive site sequencing (DNase-seq) and RNA-seq were performed on human myelogenous leukemia K562 cells following 72 hour treatments with the HDACi butyrate and suberoylanilide hydroxamic acid (SAHA). This identified overlapping sets of ~16,000 DNase I hypersensitive (DHS) sites that either “open” or “close” in response to treatment. Changes in DHS sites were statistically enriched for nearby genes that change in transcription level. Intriguingly, the opened DHS exhibit enrichment for transcription factor PU.1 binding site motifs, while the closed DHS are enriched for GATA1/2 motifs. These factors are known to be key for hematopoietic differentiation, regulated by acetylation, and often have aberrant function in hematological cancers. DNase footprinting and ChIP-seq data provide evidence these factors indeed bind to remodeled chromatin sites. Luciferase reporter assays confirm individual PU.1 motif-containing genomic regions are sufficient to increase gene expression in response to HDACi treatment. Together these data implicate PU.1 and GATA1/2 in mediating the epigenetic reprogramming following HDACi treatment that confers anticancer effects.

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Name: Yuvon Mobley
Lab: Abraham
Conference: American Society for Microbiology
Date: May 18 – 21, 2013
Location: Denver, CO
Purpose: To present talk and poster

Abstract:

Antibacterial activity of wasp venom peptide
Yuvon Mobley_Abraham LabAntimicrobial peptides (AMPs) are part of the innate immune system that is widely distributed in nature, acting as a defense mechanism against invading microorganisms. AMPs have potent antimicrobial activity against a range of microorganisms including fungi, bacteria and viruses. In view of growing multidrug resistance, AMPs are increasingly being viewed as potential therapeutic agents with a novel mechanism of action. Mastoparan is a natural, highly positively charged AMP derived from the venom of wasps. It was originally of interest based on its inherent mast cell degranulation activity. Previously, mastoparan has been shown to exhibit antimicrobial activity in vitro however these studies have been limited in scope. Here we hypothesize that mastoparan possess the capacity to be a potent broad spectrum antibacterial agent including activity against multidrug resistant bacteria.

We examined the scope of antibacterial activity exhibited by mastoparan using a variety of antimicrobial susceptibility tests and have utilized a bacterial skin infection (S. aureus) model to determine the potential of mastoparan to serve as a therapeutic agent. We tested mastoparan against 4 Gram-positive clinical isolates (e.g., S. aureus, and E. faecium), 9 Gram-negative clinical isolates (e.g., E. coli, P. aeruginosa, and B. cepacia), and 4 multidrug resistant clinical isolates (e.g., MRSA, ESBL E.coli, and ESBL K. pneumonia). These studies reveal that mastoparan exhibits broad spectrum activity against both Gram-negative (MIC: 1.9 – 125 μg/ml) and Gram-positive (MIC: 15.6 – 125 μg/ml) bacteria and against multidrug resistant bacteria (MIC: 7.8 – 125 μg/ml). We also demonstrated that mastoparan disrupts the bacterial membrane, exhibits fast acting antibacterial activity and is highly effective against both multiplying and non-multiplying bacteria. Furthermore, we have shown that mastoparan demonstrates efficacy as a topical antimicrobial agent reducing lesion size by up to 79% and the amount of bacteria recovered from skin lesions by up to a 98% reduction.

Based on these results, we conclude that mastoparan is a highly effective antibacterial agent and is therefore a potential alternative to currently antibiotics. Mastoparan offers a promising new therapeutic option for treating bacterial infections.

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2012 Meritorious Research Travel Award Recipients

Name: Bartlomiej (Bart) Bartkowiak
Lab: Greenleaf
Conference: 10th EMBL Conference – Transcription & Chromatin
Date: August 25 – 28, 2012
Location: Heidelberg, Germany
Purpose: To present talk and poster

Abstract:

CDK12: The transcriptional role of an elongation phase CTD kinase
Bartlomiej Bart Bartkowiak_Greenleaf LabEukaryotic RNA polymerase II (RNAPII) not only synthesizes mRNA but also coordinates transcription-related processes via its unique C-terminal repeat domain (CTD). The CTD (a series of heptad repeats with the consensus sequence Y1S2P3T4S5P6S7) is extensively post-translationally modified during transcription resulting in the binding and recruitment of transcription associated factors. The CTD has been implicated in a wide variety of transcriptional processes including mRNA and snRNA processing, termination, and chromatin modification.

The major elongation-phase CTD kinase in S. cerevisiae is the Ser2 specific CTDK-I complex whose catalytic subunit is the CDK-like Ctk1. Drosophila contains one (CDK12), and humans contain two (CDK12 & CDK13) proteins that are the closest evolutionary relatives of yeast Ctk1, however, neither CDK12 nor CDK13 had been shown to be a bona fide CTD kinase.  Employing Drosophila we have recently demonstrated that CDK12 is a transcription-associated CTD kinase, the ortholog of yeast Ctk1. We have also identified the cyclin partner of CDK12 to be the Ctk2-like cyclinK. The discovery of these novel CTD kinase activities presents an opportunity for the study of the interplay between CTD phosphorylation and transcription associated events in higher eukaryotes.

We are currently working to further characterize CDK12 in the context of transcription. We find that RNAi-mediated knockdown of CDK12 in Drosophila cell culture results in the preferential down-regulation of long and highly expressed genes.  Correspondingly, knockdown of CyclinK affects the same gene set in the same manner. Efforts are underway to map the global distribution of RNAPII and Ser2 CTD phosphorylation post CDK12 depletion via ChIP-chip and to investigate potential splicing defects via RNA-seq. We anticipate that these experiments will clarify the connections between elongation-phase CTD phosphorylation, CTD kinases, and a wide range of transcription-associated events.

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Name: Yi Yin
Lab: Petes
Conference: Yeast Chromosome Structure, Replication & Segregation, FASEB
Date: July 15 – 20, 2012
Location: Steamboat Springs, CO
Purpose: To present talk and poster

Abstract:

A genome-wide high-resolution mapping of UV-induced mitotic recombination in Saccharomyces cerevisiae
Yi Yin_Petes LabGenetic recombination occurs in both meiotic and mitotic cells. In diploid yeast cells, mitotic recombination is very important for repairing double-strand breaks (DSB). When repair of a DSB results in reciprocal crossovers (RCO), it may cause loss of heterozygosity (LOH) of markers centromere-distal to the DSB in both daughter cells. Gene conversion also causes LOH for a small section of a chromosome. Alternatively, DSBs can initiate break-induced replication (BIR), causing LOH in only one of the daughter cells. Mapping mitotic LOH enables understanding of mechanisms for repairing DSBs and distribution of these recombinogenic lesions. Methods for selecting mitotic crossovers and mapping the positions of crossovers have recently been developed in our lab. Our current approach uses a hybrid diploid yeast heterozygous for 55,000 SNPs, and SNP-Microarrays to map LOH events throughout the genome. These methods allow us to examine selected crossovers on chromosome V and unselected mitotic recombination events (RCO, conversions and BIR) at about 1 kb resolution across the genome.

Mitotic recombination can be greatly induced by UV radiation. However, the nature of the recombinogenic lesions with respect to different stages of the cell cycle and the resulting distribution of recombination events remain to be characterized. Using SNP-Microarrays, I constructed maps of UV-induced LOH events in G1- and G2-synchronized wild type cells. The initial mapping shows that a low dosage of UV stimulates crossovers at least 1000-fold in G1 cells in the first cell cycle after UV treatment but has much less effect in later cell cycles. However, in G2 cells, the rate of generating RCOs by UV lesions is significantly lower than that in G1 cells during the first cell cycle, although they can still stimulate both gene conversion and BIR; interestingly, the G2-induced lesions can persist and continue to be recombinogenic during the second cell division. In addition, G1 and G2 events also differ in that a high fraction of G1 events affect both sister chromatids while most of the G2 events affect only one of the two sister chromatids.

The primary pathway to remove pyrimidine dimers introduced by UV is the nucleotide excision repair (NER) pathway. In NER, the dimer is excised to generate a 30 nt gap that can be replicated to form DSBs if not filled in before DNA replication. The NER gaps are generally considered as the recombinogenic lesions by current models. To investigate whether the substantial recombinogenic effect in UV-treated G1 cells requires NER, I mapped UV-induced LOH events in an NER-deficient mutant. Surprisingly, mitotic recombination between homologs is greatly stimulated to a similar extent as in NER-proficient cells, which suggests that dimers themselves can also cause recombination without going through an NER intermediate.

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Name: Omür Kayıkçı
Lab: Magwene
Conference: 13th International Congress on Yeasts
Date: August 26 – 30, 2012
Location: Madison, WI
Purpose: To present talk and poster

Abstract:

Evolution of cAMP signaling
Omur Kayikci_Magwene LabPseudohyphal growth is a developmental response to limiting nutrient conditions in yeast. In Saccharomyces cerevisiae, cAMP signaling is a critical regulator of pseudohyphal responses. We conducted a comparative investigation on the function of cAMP-PKA signaling in pseudohyphal development using three yeast species belonging to the Saccharomyces sensu stricto (S. cerevisiae, S. paradoxus, and S. bayanus). We find that these Saccharomyces species exhibit different propensities for pseudohyphal growth and our analysis suggest that these differences are related to interspecific variation in cAMP dynamics and interactions at the genetic, transcriptional and signaling levels. Moreover, our data suggest that rewiring of the cAMP-PKA signaling system may have led to a markedly distinct role for the pathway in pseudohyphal development in S. bayanus. This study provides a powerful platform to investigate the evolution and interactions of an important nutrient signaling system, and underpins the critical role of the cAMP-PKA pathway in nutrient-induced responses.

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Name: Mayya Shveygert
Lab: Gromeier
Conference: Keystone Symposia: Protein-RNA Interactions in Biology and Disease
Date: March 4 – 9, 2012
Location: Santa Fe, NM
Purpose: To present talk and poster

Abstract:

Cdk1/cyclin B1 may regulate translation in mitosis through phosphorylation of eukaryotic initiation factor 4G1
Mayya Shveygert_Gromeier LabDuring mitosis global protein synthesis is suppressed, however, translation of certain mRNAs, such as p58 PITSLRE/Cdk11 and ODC, is increased. We hypothesize that in mitosis, unknown kinases affect translation through phosphorylation of the translation initiation machinery. Specifically, we have focused on eukaryotic initiation factor 4G1 (eIF4G1), which brings together multiple initiation factors and delivers the small ribosomal subunit to the 5′ end of mRNAs. Thus, eIF4G1 serves as a central scaffolding protein, however, signal transduction to eIF4G1 in mitosis has not been investigated.

We utilized phosphoproteomics to study the phosphorylation of eIF4G1 in either asynchronous or nocodazole-treated mitotic HEK-293 cells. Our approach has detected 10 phosphosites; interestingly, phosphorylation of one of the major phosphosites, Ser1232, was greatly increased in mitosis. Further experiments with a specific antibody to phosphorylated Ser1232 have validated mitotic phosphorylation in both synchronized cells and untreated mitotic cells prepared by shake-off. Amino acid sequence for this phosphosite closely resembles Cdk1/cyclin B1 consensus and Cdk1-specific inhibitors abolished Ser1232 phosphorylation in vivo and in vitro. Moreover, in vitroimmunodepletion and reconstitution experiments have confirmed that Cdk1 phosphorylates eIF4G1 on Ser1232 in mitosis. To analyze the functional role of eIF4G1 phosphorylation on Ser1232 we created a “knock-out/knock-in” HEK-293 cell line in which endogenous eIF4G1 was substituted with an eIF4G1 Ser1232A mutant. We are currently conducting functional studies to identify a role for Ser1232 phosphorylation in mitotic translation.

This work was supported by PHS grants CA124756 and CA140510.

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Name: Jonathan Kotula
Lab: Sullenger
Conference: American Society of Gene and Cell Therapy 15th Annual Meeting
Date: May 15 – 19, 2012
Location: Philadelphia, PA
Purpose: To present talk and poster

Abstract:

Aptamer-Mediated Delivery of Splice-Switching Oligonucleotides to the Nuclei of Cancer Cells
Jonathan Kotula_Sullenger LabTo reduce the adverse effects of cancer therapies and increase their efficacy, new delivery agents that specifically target cancer cells are needed. We and others have shown that aptamers can selectively deliver therapeutic oligonucleotides to the endosome and cytoplasm of cancer cells that express a particular cell surface receptor. Identifying a single aptamer that can internalize into many different cancer cell-types would increase the utility of aptamer-mediated delivery of therapeutic agents. We investigated the ability of the nucleolin aptamer (AS1411) to internalize into multiple cancer cell-types and observed that it internalizes into a wide variety of cancer cells and migrates to the nucleus. To determine if the aptamer could be utilized to deliver therapeutic oligonucleotides to modulate events in the nucleus, we evaluated the ability of the aptamer to deliver splice-switching oligonucleotides. We observed that aptamer-splice-switching oligonucleotide chimeras can alter splicing in the nuclei of treated cells and are effective at lower doses than the splice switching oligonucleotides alone. Our results suggest that aptamers can be utilized to deliver oligonucleotides to the nucleus of a wide variety of cancer cells to modulate nuclear events such as RNA splicing.

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2011 Meritorious Research Travel Award Recipients

Name: Maria M. Bednar
Lab: McCafferty
Conference: 242nd American Chemical Society National Meeting
Date: August 27 – September 1, 2011
Location: Denver, CO
Purpose: To present talk

Abstract:

Chlamydial protease-like activity factor (CPAF): enzymatic activity, inhibitor design, and specificity
Maria Bednar_McCafferty LabChlamydia trachomatis is a leading sexually transmitted pathogen. Here we report that the effector Chlamydia protease-like activity factor (CPAF), a protease that cleaves multiple host and chlamydial proteins, is an essential virulence factor and an attractive candidate for the development of protease inhibitors. These results indicated a need for further investigation into CPAF substrate specificity and an understanding of the enzymatic mechanism.
Through the design of a cell permeable CPAF-specific inhibitory peptide we observed that the peptide blocked the cleavage of bacterial and host proteins. The downstream effects of this inhibition caused severely compromised inclusion integrity and ultimately led to a Caspase1-dependent death of infected epithelial cells. Further investigation of CPAF substrate specificity and mechanism will facilitate our understanding of not only the development of inhibitors, but also toward the identification of additional host and chlamydial targets.

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2010 Meritorious Research Travel Award Recipients

Name: Pavel A. Nikitin
Lab: Luftig
Conference: 14th Biennial Conference of the International Association for Research on Epstein-Barr Virus and Associated Diseases
Date: September 4 – 7, 2010
Location: Birmingham, United Kingdom
Purpose: To present talk and poster

Abstract:

An ATM/Chk2-mediated DNA damage responsive signaling pathway suppresses Epstein-Barr virus transformation of primary human B cells
Pavel Nikitin_Luftig LabEpstein-Barr virus infection of primary B cells leads to the outgrowth of indefinitely proliferating lymphoblastoid cell lines (LCLs). However, the efficiency of immortalization is less than 10% of infected cells. We hypothesize that a robust innate tumor suppressor response prevents long-term outgrowth of the majority of infected cells. In this study we identify the DNA damage response (DDR) as a major component of this response. EBV infection of primary B cells activated hallmarks of the DDR including phosphorylated ATM, Chk2, g-H2AX, and 53BP1 foci. DDR activation was not due to lytic viral DNA replication nor did its marks co-localize with latent viral episomes. Rather, EBV induced a period of hyper-proliferation early after infection responsible for DDR activation. Microarray data supported the transient activation and subsequent attenuation of proliferation and DDR-associated mRNAs during LCL outgrowth. Importantly, activation of this pathway suppressed transformation as small molecule antagonism of the DNA damage responsive kinases ATM and Chk2 increased EBV transformation efficiency. Thus, we propose a model whereby EBV infection initially drives aberrant cellular DNA replication activating an anti-proliferative DNA damage response. Long-term outgrowth depends on attenuation of this hyper-proliferative signal through full latency III gene expression.

Name: Edmond Byrnes
Lab: Heitman
Conference: Infectious Diseases Society of America (IDSA) 48th Annual Meeting
Date: October 21 – 24, 2010
Location: Vancouver, Canada
Purpose: To present talk and poster

Abstract:

Examination of Cryptococcus gattii isolates from HIV/AIDS patients uncovers a diverse population of VGIII molecular type isolates endemic in Southern California
Edmond Byrnes_Heitman LabC. gattii infections in Southern California and Mexico have been reported to occur in immunocompromised patients, specifically patients with HIV/AIDS. In this study, we examined the molecular epidemiology, population structure, and virulence attributes of C. gattii isolates collected from a cohort of HIV/AIDS patients in Los Angeles County, California. We show that these isolates consist almost exclusively of VGIII molecular type, in contrast to the vast majority of VGII molecular type isolates found in the outbreak region of the North American Pacific Northwest. Upon molecular phylogenetic analysis, the global VGIII population structure can be divided into two groups, VGIIIa and VGIIIb. We show that isolates from the CA patients are virulent in murine and macrophage models of infection, with VGIIIa significantly more virulent than VGIIIb. Several VGIII isolates are highly fertile and able to produce large numbers of spores that may serve as infectious propagules. Based on molecular analysis, the a and a VGIII MAT loci are largely syntenic with the known VGI and VGII MAT loci. Our studies indicate that C. gattii VGIII is endemic in Southern California, with other isolates originating from the neighboring regions of Mexico, and in some rarer cases from Oregon and Washington state. Given that > 1,000,000 cases of cryptococcal infection occur annually in the context of the global AIDS pandemic, our findings suggest a significant burden of C. gattii infection in AIDS patients may be unrecognized, with potential prognostic and therapeutic implications. These results signify the need to classify pathogenic Cryptococcus cases in the region and elsewhere and highlight possible host differences among the C. gattii molecular types, influencing infection of immunocompetent (VGI/VGII) vs. immunocompromised (VGIII/VGIV) hosts.

Name: Teresa O’Meara
Lab: Alspaugh
Conference: Woods Hole Molecular Mycology Course
Date: August 4 – 21, 2010
Location: Marine Biological Laboratory, Woods Hole, MA
Purpose: Training course

Abstract:

Importance of chromatin remodeling by the conserved histone acetyltransferase Gcn5 in regulating the expression of specific genes that allow C. neoformans to respond appropriately to the human host
Teresa O'Meara_Alspaugh LabCryptococcus neoformans is an environmental fungus and an opportunistic human pathogen. Previous studies have demonstrated major alterations in its transcriptional profile as this microorganism enters the hostile environment of the human host. To assess the role of chromatin remodeling on host-induced transcriptional responses, we identified the C. neoformans Gcn5 histone acetyltransferase and demonstrated its function by complementation studies in S. cerevisiae. The C. neoformans gcn5mutant strain has defects in high temperature growth and capsule attachment to the cell surface, in addition to increased sensitivity to FK506 and oxidative stress. Treatment of wild type cells with the histone acetyltransferase inhibitor garcinol mimics cellular effects of the gcn5 mutation. Gcn5 regulates the expression of many genes that are important in responding to the specific environmental conditions encountered by C. neoformans inside the host. Accordingly, the gcn5 mutant is avirulent in animal models of cryptococcosis. Our study demonstrates the importance of chromatin remodeling by the conserved histone acetyltransferase Gcn5 in regulating the expression of specific genes that allow C. neoformans to respond appropriately to the human host.

Name: Irina Evsyukova
Lab: Garcia-Blanco
Conference: 7th International Congress on Autoimmunity
Date: May 5 – 9, 2010
Location: Ljubljana, Slovenia
Purpose: To present talk and poster

Abstract:

Alternative splicing of interleukin 7 receptor alpha chain (IL7Rα) and multiple sclerosis
Irina Evsyukova_Garcia-Blanco Lab_CTA 2010Multiple sclerosis (MS) is a prototypic neurodegenerative disease with a strong genetic component. Various genetic studies have failed to identify consistently linked regions outside of the major histocompatibility complex on chromosome 6p. We have uncovered allelic association of a polymorphism in the gene encoding the interleukin 7 receptor alpha chain (IL7Rα) with MS in four independent family-based or case-control data sets. The likely causal SNP is located within the alternatively spliced exon 6 of IL7Rα and has a functional effect on gene expression. Alternative splicing of exon 6 leads to the mutually exclusive production of the membrane-bound (exon 6 included) or soluble (exon 6 skipped) protein isoforms. The SNP disrupts a putative exonic splicing silencer within exon 6 and thus alters the amount of soluble and membrane-bound isoforms.

We have performed extensive mutagenesis studies to characterize cis-acting regulatory elements that control alternative splicing of exon 6. Three cis-acting elements have been uncovered in the vicinity of the SNP: two exonic splicing enhancers and one exonic splicing silencer. This splicing regulation can be recapitulated both in vivo and in vitro. Proteomics studies are now underway to identify trans-acting protein factors that bind to these cis-acting regulatory elements and regulate alternative splicing of exon 6. These protein factors will then be tested as candidates for genetic association with MS. Since many disease-associated missense mutations exert their deleterious effects by altering the splicing pattern of the gene, these studies will shed light on the importance of alternative splicing in the development of autoimmunity.

Name: Neel Mukherjee
Lab: Keene
Conference: 2010 Cold Spring Harbor Laboratory/Wellcome Trust Conference Systems, Biology: Networks
Date: August 11 – 15, 2010
Location: Cold Spring Harbor, NY
Purpose: To present talk and poster

Abstract:

High-resolution transcriptome-wide identification of HuR regulatory elements
Neel Mukherjee_Keene LabRNA-binding proteins (RBPs) and noncoding RNAs are posttranscriptional regulatory factors that control the fate of each mRNA species. RBPs coordinate all aspects of RNA processing including splicing, export, localization, stability, and translation. HuR, a member of the ELAV/Hu family of RBPs, has been shown to positively regulate the stability and translation of target mRNAs through AU-rich elements (AREs) typically found in the 3’ untranslated region (UTR) of mRNAs encoding many immediate early genes, inflammatory cytokines, and growth factors. While many studies have identified mRNAs associated with HuR, the precise binding sites have not been identified on a genome-wide scale. Furthermore, the extent to which HuR regulates mRNA processing events, such as splicing and poly-adenylation, has not been examined globally.

We identified precise HuR-RNA interaction sites transcriptome-wide at a high resolution utilizing a recently developed technology, PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation). As expected there were many binding sites in the 3’ UTR of mRNAs. Surprisingly, there were a substantial number of intronic binding sites, suggesting HuR is involved in pre-mRNA processing.

Utilizing Affymetrix exon arrays, we identified mRNAs that require HuR for maintaining their expression levels and RNA processing. Integration of binding data with knockdown expression data reveals that HuR positively regulates the expression of target mRNAs. Furthermore, the degree of binding was proportional to extent of HuR-dependent regulation of gene expression. Unexpectedly, the positive regulation remained true for mRNAs that only contained intronic HuR binding sites.

Integration of the data revealed known functions of HuR, for example HuR positively regulates the expression of functionally related proteins involved in the regulation of cell cycle. We also discovered novel potential cellular functions of HuR. Our data suggest that HuR directly regulates the expression of mRNAs encoding amino-acyl tRNA-synthetases (ARSs). The majority of the twenty ARSs are positively regulated by HuR and contain HuR binding sites. Nine ARSs form a macromolecular complex which include 3 other non-ARS proteins. The mRNAs encoding these three proteins are also directly bound and regulated by HuR. The tRNA multi-synthetase macromolecular complex, a highly multifunctional complex important in many diseases, may represent a post-transcriptional RNA operon coordinated by HuR.

Name: Jayme Johnson
Lab: Lew
Conference: Yeast Genetics and Molecular Biology Meeting
Date: July 27 – August 1, 2010
Location: Vancouver, Canada
Purpose: To present talk and poster

Abstract:

Chemotropic reorientation utilizes a mobile polarity patch
Jayme Johnson_Lew LabDuring chemotropism, yeast direct polarized growth up a gradient of pheromone towards a mating partner. Cells exposed to low doses of pheromone sometimes misdirect growth but can re-orient it over time, implying that cells continuously monitor and adapt to the pheromone gradient.

The pheromone receptor influences the direction of polarized growth. Ligand-bound receptor releases free Gβγ, which recruits the guanine-nucleotide-exchange factor for the polarity regulator Cdc42p. Polarized GTP-Cdc42p and other polarity proteins (the “polarity patch”) orient the actin cytoskeleton and vesicle delivery, determining the direction of polarized growth. Thus, to re-orient growth cells must alter the cortical location of the polarity patch.

We show that cells exposed to low doses of pheromone have a mobile polarity patch. Mobility of the patch decreases as pheromone concentration is increased, and this stabilization depends on the connection between the receptor and the polarity patch via Gβγ. Mutants that disrupt the receptor-polarity-patch connection display very mobile patches, whose movement is compromised in Latrunculin A or when lacking the actin cable stabilizer TPM1. Thus, actin cables promote patch mobility.

What causes polarity patch mobility? We speculated that vesicle delivery to the polarity patch might perturb its precise geometry. Amplification of these perturbations by positive feedback loops might shift the patch centroid, causing apparent “movement.” To eliminate the disruptive effects of vesicle delivery, we fused the v-SNARE Snc2p to the scaffold polarity protein Bem1p. We reasoned that newly delivered Bem1p-Snc2p would positively reinforce the patch’s position on the cortex rather than disrupt it. Indeed, we found that Bem1p-Snc2p cells had very stable polarity patches even in low pheromone doses and in mutant cells.

We propose a model whereby vesicle delivery causes constitutive patch mobility in response to pheromone. Patch stabilization and subsequent growth occurs towards the highest concentration of pheromone due to the receptor-polarity-patch connection.