Michael Kruhlak - Academia.edu (original) (raw)

Papers by Michael Kruhlak

Research paper thumbnail of Lymphocytes on Chemokine-Activated T Rac1 Mediates Collapse of Microvilli

Research paper thumbnail of Carcinoma AKT Activation Promotes Metastasis in a Mouse Model of Follicular Thyroid

The phosphatidylinositol 3-kinase/AKT pathway is crucial to many cell functions, and its dysregul... more The phosphatidylinositol 3-kinase/AKT pathway is crucial to many cell functions, and its dysregulation in tumors is a common finding. The molecular basis of follicular thyroid cancer metastasis is not well understood but may also be influenced by AKT activation. We previously created a knockin mutant mouse that expresses a mutant thyroid hormone receptor-␤ gene (TR␤PV mouse) that spontaneously develops thyroid cancer and distant metastasis similar to human follicular thyroid cancer. In this study, we investigated whether our mouse model exhibits similar AKT activation as human follicular thyroid cancer. Western blot analysis on thyroids from both wild-type and TR␤ PV/PV mice revealed elevation of activated AKT in TR␤ PV/PV mice. Immunohistochemistry and confocal microscopy reveal activated AKT in both the thyroid and metastatic lesions of TR␤ PV/PV mice. Whereas all three AKT iso-forms were overexpressed in primary tumors from TR␤ PV/PV mice in the cytoplasm of thyroid cancer cells, only AKT1 was also found in the nucleus, matching the localization of activated AKT in a pattern similar to human follicular thyroid cancer. In the metastases, all AKT isoforms correlated with phosphorylated AKT nuclear localization. We created primary thyroid cell lines derived from TR␤ PV/PV mice and found reduction of phosphorylated AKT levels or AKT downstream targets diminishes cell motility. Activated AKT is common to both human and mouse follicular thyroid cancer and is correlated with increased cell motility in vitro and metastasis in vivo. Thus, TR␤ PV/PV mice could be used to further dissect the detailed pathways underlying the progression and metastasis of follicular thyroid carcinoma. (Endocrinology 146: 4456 -4463, 2005)

Research paper thumbnail of Rac1 mediates collapse of microvilli on chemokine-activated T lymphocytes

Proceedings of The National Academy of Sciences, 2004

Lymphocytes circulate in the blood and upon chemokine activation rapidly bind, where needed, to m... more Lymphocytes circulate in the blood and upon chemokine activation rapidly bind, where needed, to microvasculature to mediate immune surveillance. Resorption of microvilli is an early morphological alteration induced by chemokines that facilitates lymphocyte emigration. However, the antecedent molecular mechanisms remain largely undefined. We demonstrate that Rac1 plays a fundamental role in chemokine-induced microvillar breakdown in human T lymphocytes. The supporting evidence includes: first, chemokine induces Rac1 activation within 5 s via a signaling pathway that involves Galphai. Second, constitutively active Rac1 mediates microvilli disintegration. Third, blocking Rac1 function by cell permeant C-terminal "Trojan" peptides corresponding to Rac1 (but not Rac2, Rho, or Cdc42) blocks microvillar loss induced by the chemokine stromal cell-derived factor 1alpha (SDF-1alpha). Furthermore, we demonstrate that the molecular mechanism of Rac1 action involves dephosphorylation-induced inactivation of the ezrin/radixin/moesin (ERM) family of actin regulators; such inactivation is known to detach the membrane from the underlying actin cytoskeleton, thereby facilitating disassembly of actin-based peripheral processes. Specifically, ERM dephosphorylation is induced by constitutively active Rac1 and stromal cell-derived factor 1alpha-induced ERM dephosphorylation is blocked by either the dominant negative Rac1 construct or by Rac1 C-terminal peptides. Importantly, the basic residues at the C terminus of Rac1 are critical to Rac1's participation in ERM dephosphorylation and in microvillar retraction. Together, these data elucidate new roles for Rac1 in early signal transduction and cytoskeletal rearrangement of T lymphocytes responding to chemokine.

Research paper thumbnail of Genetically modified live attenuated L.donovani parasites induce innate immunity through classical activation of macrophages that direct Th1 response in mice

Infection and Immunity, 2015

Visceral Leishmaniasis (VL) causes significant mortality and there is no effective vaccine. Previ... more Visceral Leishmaniasis (VL) causes significant mortality and there is no effective vaccine. Previously, we have shown that, genetically modified Leishmania donovani parasites, hereafter described as live attenuated parasites, induced host protective adaptive immune response in various animal models. In this study, we demonstrate innate immune response upon infection with live attenuated parasites in macrophages from BALB/c mice both in vitro and in vivo. In vitro infection of macrophages with live attenuated parasites induced significantly higher production of pro-inflammatory cytokines (TNF-α, IL-12, IFN-γ, IL-6), chemokines (MCP-1/CCL-2, MIP-1α/CCL-3, IP-10), ROS and nitric oxide concomitantly reducing anti-inflammatory cytokine (IL-10) and arginase-1 activity compared to infection with wild type L. donovani (LdWT) parasites suggesting dominant classically activated/M1 macrophage response which in turn helps in presenting antigen to T cell as observed by robust CD4(+)T cell activation in vitro. Similarly, parasitized splenic macrophages from live attenuated parasite infected mice also demonstrated induction of M1 macrophage phenotype as indicated by up regulation of IL-1β, TNF- α, IL-12, and iNOS2 and down regulation of genes associated with the M2 phenotype i.e. IL-10, YM1, Arg-1 and MRC-1 compared to LdWT infected mice. Furthermore, ex-vivo antigen presentation assay showed macrophages from live attenuated parasite infected mice induced higher IFN-γ and IL-2 but significantly less IL-10 production by OVA specific CD4(+)T cells resulting in proliferation of Th1 cells. These data suggest that infection with live attenuated parasite promotes a state of classical activation (M1 dominant) in macrophages, leading to the generation of protective Th1 responses in BALB/c mice.

Research paper thumbnail of Kaposi's sarcoma-associated herpesvirus ORF57 interacts with cellular RNA export cofactors RBM15 and OTT3 to promote expression of viral ORF59

Journal of virology, 2011

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes ORF57, which promotes the accumulation... more Kaposi's sarcoma-associated herpesvirus (KSHV) encodes ORF57, which promotes the accumulation of specific KSHV mRNA targets, including ORF59 mRNA. We report that the cellular export NXF1 cofactors RBM15 and OTT3 participate in ORF57-enhanced expression of KSHV ORF59. We also found that ectopic expression of RBM15 or OTT3 augments ORF59 production in the absence of ORF57. While RBM15 promotes the accumulation of ORF59 RNA predominantly in the nucleus compared to the levels in the cytoplasm, we found that ORF57 shifted the nucleocytoplasmic balance by increasing ORF59 RNA accumulation in the cytoplasm more than in the nucleus. By promoting the accumulation of cytoplasmic ORF59 RNA, ORF57 offsets the nuclear RNA accumulation mediated by RBM15 by preventing nuclear ORF59 RNA from hyperpolyadenylation. ORF57 interacts directly with the RBM15 C-terminal portion containing the SPOC domain to reduce RBM15 binding to ORF59 RNA. Although ORF57 homologs Epstein-Barr virus (EBV) EB2, herpes...

Research paper thumbnail of Expression of the SNARE Protein SNAP-23 Is Essential for Cell Survival

PloS one, 2015

Members of the SNARE-family of proteins are known to be key regulators of the membrane-membrane f... more Members of the SNARE-family of proteins are known to be key regulators of the membrane-membrane fusion events required for intracellular membrane traffic. The ubiquitously expressed SNARE protein SNAP-23 regulates a wide variety of exocytosis events and is essential for mouse development. Germline deletion of SNAP-23 results in early embryonic lethality in mice, and for this reason we now describe mice and cell lines in which SNAP-23 can be conditionally-deleted using Cre-lox technology. Deletion of SNAP-23 in CD19-Cre expressing mice prevents B lymphocyte development and deletion of SNAP-23 using a variety of T lymphocyte-specific Cre mice prevents T lymphocyte development. Acute depletion of SNAP-23 in mouse fibroblasts leads to rapid apoptotic cell death. These data highlight the importance of SNAP-23 for cell survival and describe a mouse in which specific cell types can be eliminated by expression of tissue-specific Cre-recombinase.

Research paper thumbnail of Spatio-temporal dynamics of chromatin containing DNA breaks

Cell cycle (Georgetown, Tex.), 2006

The cellular response to DNA breaks consists of a complex signaling network that coordinates the ... more The cellular response to DNA breaks consists of a complex signaling network that coordinates the initial recognition of the lesion with the induction of cell cycle checkpoints and DNA repair. With DNA wrapped around histone proteins and packaged into higher order levels of chromatin structure, the detection of a single DNA break (DSB) in the genome is the molecular equivalent of finding a needle in a haystack. A recent study from our laboratory used high-resolution electron microscropy and live cell imaging to demonstrate that chromatin undergoes a marked reorganization in response to a DSB. In an energy dependent manner, chromatin rapidly decondenses to a more open configuration in the regions surrounding the lesion. We propose that this ATP dependent chromatin-remodeling event facilitates the subsequent recognition and processing of damaged DNA. While the chromatin surrounding the lesion remodels to a more open configuration, the DNA break itself remains relatively immobile over t...

Research paper thumbnail of Activation of DNA Damage Response Signaling by Condensed Chromatin

Cell Reports, 2014

Highlights Dynamic compaction of chromatin is an integral step in the DNA damage response Impairm... more Highlights Dynamic compaction of chromatin is an integral step in the DNA damage response Impairment of chromatin condensation attenuates signaling from a DNA break Induced chromatin condensation can stimulate damage-independent upstream signaling Persistent compaction boosts upstream signaling but inhibits repair and recovery

Research paper thumbnail of Compartmentalization of regulatory proteins in the cell nucleus

Journal of Steroid Biochemistry and Molecular Biology, 2001

The cell nucleus is increasingly recognized as a spatially organized structure. In this review, t... more The cell nucleus is increasingly recognized as a spatially organized structure. In this review, the nature and controversies associated with nuclear compartmentalization are discussed. The relationship between nuclear structure and organization of proteins involved in the regulation of RNA polymerase II-transcribed genes is then discussed. Finally, very recent data on the mobility of these proteins within the cell nucleus is

Research paper thumbnail of A Macrohistone Variant Links Dynamic Chromatin Compaction to BRCA1-Dependent Genome Maintenance

Cell Reports, 2014

Appropriate DNA double-strand break (DSB) repair factor choice is essential for ensuring accurate... more Appropriate DNA double-strand break (DSB) repair factor choice is essential for ensuring accurate repair outcome and genomic integrity. The factors that regulate this process remain poorly understood. Here, we identify two repressive chromatin components, the macrohistone variant macroH2A1 and the H3K9 methyltransferase and tumor suppressor PRDM2, which together direct the choice between the antagonistic DSB repair mediators BRCA1 and 53BP1. The macroH2A1/PRDM2 module mediates an unexpected shift from accessible to condensed chromatin that requires the ataxia telangiectasia mutated (ATM)-dependent accumulation of both proteins at DSBs in order to promote DSB-flanking H3K9 dimethylation. Remarkably, loss of macroH2A1 or PRDM2, as well as experimentally induced chromatin decondensation, impairs the retention of BRCA1, but not 53BP1, at DSBs. As a result, mac-roH2A1 and/or PRDM2 depletion causes epistatic defects in DSB end resection, homology-directed repair, and the resistance to poly(ADP-ribose) polymerase (PARP) inhibition-all hallmarks of BRCA1deficient tumors. Together, these findings identify dynamic, DSB-associated chromatin reorganization as a critical modulator of BRCA1-dependent genome maintenance.

Research paper thumbnail of Differential expression of IFN and TRAIL/DR5 in lymphoid tissue of progressor versus nonprogressor HIV1-infected patients

Proceedings of The National Academy of Sciences, 2006

Loss of CD4+ T cells, the hallmark of HIV pathogenesis, was suggested to be partly due to apoptos... more Loss of CD4+ T cells, the hallmark of HIV pathogenesis, was suggested to be partly due to apoptosis. We recently reported that IFN- produced by HIV-1-activated plasmacytoid dendritic cells (pDCs) contributes to CD4+ T cell apoptosis by the TNF-related apoptosis-inducing ligand (TRAIL)/death receptor (DR)5 pathway. Here, we show that HIV-1-induced intracellular expression of IFN- in pDCs is coupled to increased

Research paper thumbnail of Reticulum Prior to Stimulation TLR9 Is Localized in the Endoplasmic

Research paper thumbnail of CtIP-mediated resection is essential for viability and can operate independently of BRCA1

The Journal of Cell Biology, 2014

Research paper thumbnail of Mitotic Transcription Repression In Vivo in the Absence of Nucleosomal Chromatin Condensation

The Journal of Cell Biology, 2000

All nuclear RNA synthesis is repressed during the mitotic phase of the cell cycle. In addition, R... more All nuclear RNA synthesis is repressed during the mitotic phase of the cell cycle. In addition, RNA polymerase II (RNAP II), nascent RNA and many transcription factors disengage from DNA during mitosis. It has been proposed that mitotic transcription repression and disengagement of factors are due to either mitotic chromatin condensation or biochemical modifications to the transcription machinery. In this study, we investigate the requirement for chromatin condensation in establishing mitotic transcription repression and factor loss, by analyzing transcription and RNAP II localization in mitotic cells infected with herpes simplex virus type 1. We find that virus-infected cells enter mitosis and that mitotic viral DNA is maintained in a nu-cleosome-free and noncondensed state. Our data show that RNAP II transcription is repressed on cellular genes that are condensed into mitotic chromosomes and on viral genes that remain nucleosome free and noncondensed. Although RNAP II may interact indirectly with viral DNA during mitosis, it remains transcriptionally unengaged. This study demonstrates that mitotic repression of transcription and loss of transcription factors from mitotic DNA can occur independently of nucleosomal chromatin condensation. 1 Abbreviations used in this paper: 3-D, three-dimensional; DAPI, 4 Ј ,6diamidine-2-phenylindole; ESI, electron spectroscopic imaging; HSV-1, herpes simplex virus type 1; MOI, multiplicity of infection; RNAP, RNA polymerase.

Research paper thumbnail of Reduced Mobility of the Alternate Splicing Factor (ASF) through the Nucleoplasm and Steady State Speckle Compartments

The Journal of Cell Biology, 2000

Compartmentalization of the nucleus is now recognized as an important level of regulation influen... more Compartmentalization of the nucleus is now recognized as an important level of regulation influencing specific nuclear processes. The mechanism of factor organization and the movement of factors in nuclear space have not been fully determined. Splicing factors, for example, have been shown to move in a directed manner as large intact structures from sites of concentration to sites of active transcription, but splicing factors are also thought to exist in a freely diffusible state. In this study, we examined the movement of a splicing factor, ASF, green fluorescent fusion protein (ASF-GFP) using time-lapse microscopy and the technique fluorescence recovery after photobleaching (FRAP). We find that ASF-GFP moves at rates up to 100 times slower than free diffusion when it is associated with speckles and, surprisingly, also when it is dispersed in the nucleoplasm. The mobility of ASF is consistent with frequent but transient interactions with relatively immobile nuclear binding sites. This mobility is slightly increased in the presence of an RNA polymerase II transcription inhibitor and the ASF molecules further enrich in speckles. We propose that the nonrandom organization of splicing factors reflects spatial differences in the concentration of relatively immobile binding sites.

Research paper thumbnail of The Transcription Coactivator Cbp Is a Dynamic Component of the Promyelocytic Leukemia Nuclear Body

The Journal of Cell Biology, 2001

The transcription coactivator and histone acetyltransferase CAMP response element-binding protein... more The transcription coactivator and histone acetyltransferase CAMP response element-binding protein (CBP) has been demonstrated to accumulate in promyelocytic leukemia (PML) bodies. We show that this accumulation is cell type specific. In cells where CBP does not normally accumulate in PML bodies, it can be induced to accumulate in PML bodies through overexpression of either CBP or Pml, but not Sp100.

Research paper thumbnail of Inhibition of the intestinal glucose transporter GLUT2 by flavonoids

The FASEB Journal, 2007

We tested whether the dominant intestinal sugar transporter GLUT2 was inhibited by intestinal lum... more We tested whether the dominant intestinal sugar transporter GLUT2 was inhibited by intestinal luminal compounds that are inefficiently absorbed and naturally present in foods. Because of their abundance in fruits and vegetables, flavonoids were selected as model compounds. Robust inhibition of glucose and fructose transport by GLUT2 expressed in Xenopus laevis oocytes was produced by the flavonols myricetin, fisetin, the widely consumed flavonoid quercetin, and its glucoside precursor isoquercitrin. IC 50 s for quercetin, myricetin, and isoquercitrin were ϳ200to 1000fold less than glucose or fructose concentrations, and noncompetitive inhibition was observed. The two other major intestinal sugar transporters, GLUT5 and SGLT1, were unaffected by flavonoids.

Research paper thumbnail of Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks

Nature Cell Biology, 2003

Histone H2AX is rapidly phosphorylated in the chromatin micro-environment surrounding a DNA doubl... more Histone H2AX is rapidly phosphorylated in the chromatin micro-environment surrounding a DNA double-strand break (DSB). Although H2AX deficiency is not detrimental to life, H2AX is required for the accumulation of numerous essential proteins into irradiation induced foci (IRIF). However, the relationship between IRIF formation, H2AX phosphorylation (gamma-H2AX) and the detection of DNA damage is unclear. Here, we show that the migration of repair and signalling proteins to DSBs is not abrogated in H2AX(-/-) cells, or in H2AX-deficient cells that have been reconstituted with H2AX mutants that eliminate phosphorylation. Despite their initial recruitment to DSBs, numerous factors, including Nbs1, 53BP1 and Brca1, subsequently fail to form IRIF. We propose that gamma-H2AX does not constitute the primary signal required for the redistribution of repair complexes to damaged chromatin, but may function to concentrate proteins in the vicinity of DNA lesions. The differential requirements for factor recruitment to DSBs and sequestration into IRIF may explain why essential regulatory pathways controlling the ability of cells to respond to DNA damage are not abolished in the absence of H2AX.

Research paper thumbnail of 53BP1 facilitates long-range DNA end-joining during V(D)J recombination

Nature, 2008

V(D)J recombination and class switch recombination employ overlapping but distinct nonhomologous ... more V(D)J recombination and class switch recombination employ overlapping but distinct nonhomologous end-joining (NHEJ) pathways to repair DNA double strand break (DSB) intermediates. 53BP1 is a DNA damage response protein that is rapidly recruited to sites of chromosomal DSBs, where it appears to function in a subset of ataxia-telangiectasia mutated (ATM) kinase, H2AX-and MDC1-dependent events 1,2 . A 53BP1 dependent end joining pathway has been described that is dispensable for V(D)J recombination but essential for class-switch recombination CSR 3, 4 . Here, we report a previously unrecognized defect in the joining phase of V(D)J recombination in 53BP1 deficient lymphocytes distinct from that found in classical NHEJ-, H2AX-, MDC1and Atm-deficient mice. Absence of 53BP1 leads to impairment of distal V-DJ joining with extensive degradation of un-repaired coding ends and episomal signal joint reintegration at V(D)J junctions. This results in apoptosis, loss of T-cell receptor alpha locus integrity and lymphopenia. Further impairment of the apoptotic checkpoint causes propagation of lymphocytes bearing antigen receptor breaks. These data suggest a more general role for 53BP1 in maintaining genomic stability during long range joining of DNA breaks. RAG1 and RAG2 (RAG1/2) proteins perform the pair wise cleavage step in V(D)J recombination, whereas activation-induced cytidine deaminase (AID) triggers the formation of DSBs in the switch regions during CSR 5 . RAG1/2-and AID-induced lesions in antigen receptor loci initiate nuclear focus formation of the DNA damage response proteins γ-H2AX, NBS1 and 53BP1 over a large chromosome domain 5-8 . Based on the analysis of CSR in H2AX-9 , Atm-7 , and 53BP1-deficient 3, 4, 10, 11 lymphocytes, it was proposed that focus forming factors might promote and/or maintain synapsis of distal switch regions 9 . In the case of 53BP1-deficiency, there is an almost complete loss of long range CSR 3, 4, 12 and a concomitant increase in the frequency of short-range intra switch recombination 12 . Paradoxically, although V(D)J recombination and CSR employ similar DSB repair

Research paper thumbnail of The ATM repair pathway inhibits RNA polymerase I transcription in response to chromosome breaks

Nature, 2009

DNA lesions interfere with DNA and RNA polymerase activity. Cyclobutane pyrimidine dimers and pho... more DNA lesions interfere with DNA and RNA polymerase activity. Cyclobutane pyrimidine dimers and photoproducts generated by ultraviolet irradiation cause stalling of RNA polymerase II, activation of transcription-coupled repair enzymes, and inhibition of RNA synthesis 1,2 . During the S phase of the cell cycle, collision of replication forks with damaged DNA blocks ongoing DNA replication while also triggering a biochemical signal that suppresses the firing of distant origins of replication 3,4 . Whether the transcription machinery is affected by the presence of DNA doublestrand breaks remains a long-standing question. Here we monitor RNA polymerase I (Pol I) activity in mouse cells exposed to genotoxic stress and show that induction of DNA breaks leads to a transient repression in Pol I transcription. Surprisingly, we find Pol I inhibition is not itself the direct result of DNA damage but is mediated by ATM kinase activity and the repair factor proteins NBS1 (also known as NLRP2) and MDC1. Using live-cell imaging, laser micro-irradiation, and photobleaching technology we demonstrate that DNA lesions interfere with Pol I initiation complex assembly and lead to a premature displacement of elongating holoenzymes from ribosomal DNA. Our data reveal a novel ATM/NBS1/MDC1-dependent pathway that shuts down ribosomal gene transcription in response to chromosome breaks.

Research paper thumbnail of Lymphocytes on Chemokine-Activated T Rac1 Mediates Collapse of Microvilli

Research paper thumbnail of Carcinoma AKT Activation Promotes Metastasis in a Mouse Model of Follicular Thyroid

The phosphatidylinositol 3-kinase/AKT pathway is crucial to many cell functions, and its dysregul... more The phosphatidylinositol 3-kinase/AKT pathway is crucial to many cell functions, and its dysregulation in tumors is a common finding. The molecular basis of follicular thyroid cancer metastasis is not well understood but may also be influenced by AKT activation. We previously created a knockin mutant mouse that expresses a mutant thyroid hormone receptor-␤ gene (TR␤PV mouse) that spontaneously develops thyroid cancer and distant metastasis similar to human follicular thyroid cancer. In this study, we investigated whether our mouse model exhibits similar AKT activation as human follicular thyroid cancer. Western blot analysis on thyroids from both wild-type and TR␤ PV/PV mice revealed elevation of activated AKT in TR␤ PV/PV mice. Immunohistochemistry and confocal microscopy reveal activated AKT in both the thyroid and metastatic lesions of TR␤ PV/PV mice. Whereas all three AKT iso-forms were overexpressed in primary tumors from TR␤ PV/PV mice in the cytoplasm of thyroid cancer cells, only AKT1 was also found in the nucleus, matching the localization of activated AKT in a pattern similar to human follicular thyroid cancer. In the metastases, all AKT isoforms correlated with phosphorylated AKT nuclear localization. We created primary thyroid cell lines derived from TR␤ PV/PV mice and found reduction of phosphorylated AKT levels or AKT downstream targets diminishes cell motility. Activated AKT is common to both human and mouse follicular thyroid cancer and is correlated with increased cell motility in vitro and metastasis in vivo. Thus, TR␤ PV/PV mice could be used to further dissect the detailed pathways underlying the progression and metastasis of follicular thyroid carcinoma. (Endocrinology 146: 4456 -4463, 2005)

Research paper thumbnail of Rac1 mediates collapse of microvilli on chemokine-activated T lymphocytes

Proceedings of The National Academy of Sciences, 2004

Lymphocytes circulate in the blood and upon chemokine activation rapidly bind, where needed, to m... more Lymphocytes circulate in the blood and upon chemokine activation rapidly bind, where needed, to microvasculature to mediate immune surveillance. Resorption of microvilli is an early morphological alteration induced by chemokines that facilitates lymphocyte emigration. However, the antecedent molecular mechanisms remain largely undefined. We demonstrate that Rac1 plays a fundamental role in chemokine-induced microvillar breakdown in human T lymphocytes. The supporting evidence includes: first, chemokine induces Rac1 activation within 5 s via a signaling pathway that involves Galphai. Second, constitutively active Rac1 mediates microvilli disintegration. Third, blocking Rac1 function by cell permeant C-terminal "Trojan" peptides corresponding to Rac1 (but not Rac2, Rho, or Cdc42) blocks microvillar loss induced by the chemokine stromal cell-derived factor 1alpha (SDF-1alpha). Furthermore, we demonstrate that the molecular mechanism of Rac1 action involves dephosphorylation-induced inactivation of the ezrin/radixin/moesin (ERM) family of actin regulators; such inactivation is known to detach the membrane from the underlying actin cytoskeleton, thereby facilitating disassembly of actin-based peripheral processes. Specifically, ERM dephosphorylation is induced by constitutively active Rac1 and stromal cell-derived factor 1alpha-induced ERM dephosphorylation is blocked by either the dominant negative Rac1 construct or by Rac1 C-terminal peptides. Importantly, the basic residues at the C terminus of Rac1 are critical to Rac1's participation in ERM dephosphorylation and in microvillar retraction. Together, these data elucidate new roles for Rac1 in early signal transduction and cytoskeletal rearrangement of T lymphocytes responding to chemokine.

Research paper thumbnail of Genetically modified live attenuated L.donovani parasites induce innate immunity through classical activation of macrophages that direct Th1 response in mice

Infection and Immunity, 2015

Visceral Leishmaniasis (VL) causes significant mortality and there is no effective vaccine. Previ... more Visceral Leishmaniasis (VL) causes significant mortality and there is no effective vaccine. Previously, we have shown that, genetically modified Leishmania donovani parasites, hereafter described as live attenuated parasites, induced host protective adaptive immune response in various animal models. In this study, we demonstrate innate immune response upon infection with live attenuated parasites in macrophages from BALB/c mice both in vitro and in vivo. In vitro infection of macrophages with live attenuated parasites induced significantly higher production of pro-inflammatory cytokines (TNF-α, IL-12, IFN-γ, IL-6), chemokines (MCP-1/CCL-2, MIP-1α/CCL-3, IP-10), ROS and nitric oxide concomitantly reducing anti-inflammatory cytokine (IL-10) and arginase-1 activity compared to infection with wild type L. donovani (LdWT) parasites suggesting dominant classically activated/M1 macrophage response which in turn helps in presenting antigen to T cell as observed by robust CD4(+)T cell activation in vitro. Similarly, parasitized splenic macrophages from live attenuated parasite infected mice also demonstrated induction of M1 macrophage phenotype as indicated by up regulation of IL-1β, TNF- α, IL-12, and iNOS2 and down regulation of genes associated with the M2 phenotype i.e. IL-10, YM1, Arg-1 and MRC-1 compared to LdWT infected mice. Furthermore, ex-vivo antigen presentation assay showed macrophages from live attenuated parasite infected mice induced higher IFN-γ and IL-2 but significantly less IL-10 production by OVA specific CD4(+)T cells resulting in proliferation of Th1 cells. These data suggest that infection with live attenuated parasite promotes a state of classical activation (M1 dominant) in macrophages, leading to the generation of protective Th1 responses in BALB/c mice.

Research paper thumbnail of Kaposi's sarcoma-associated herpesvirus ORF57 interacts with cellular RNA export cofactors RBM15 and OTT3 to promote expression of viral ORF59

Journal of virology, 2011

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes ORF57, which promotes the accumulation... more Kaposi's sarcoma-associated herpesvirus (KSHV) encodes ORF57, which promotes the accumulation of specific KSHV mRNA targets, including ORF59 mRNA. We report that the cellular export NXF1 cofactors RBM15 and OTT3 participate in ORF57-enhanced expression of KSHV ORF59. We also found that ectopic expression of RBM15 or OTT3 augments ORF59 production in the absence of ORF57. While RBM15 promotes the accumulation of ORF59 RNA predominantly in the nucleus compared to the levels in the cytoplasm, we found that ORF57 shifted the nucleocytoplasmic balance by increasing ORF59 RNA accumulation in the cytoplasm more than in the nucleus. By promoting the accumulation of cytoplasmic ORF59 RNA, ORF57 offsets the nuclear RNA accumulation mediated by RBM15 by preventing nuclear ORF59 RNA from hyperpolyadenylation. ORF57 interacts directly with the RBM15 C-terminal portion containing the SPOC domain to reduce RBM15 binding to ORF59 RNA. Although ORF57 homologs Epstein-Barr virus (EBV) EB2, herpes...

Research paper thumbnail of Expression of the SNARE Protein SNAP-23 Is Essential for Cell Survival

PloS one, 2015

Members of the SNARE-family of proteins are known to be key regulators of the membrane-membrane f... more Members of the SNARE-family of proteins are known to be key regulators of the membrane-membrane fusion events required for intracellular membrane traffic. The ubiquitously expressed SNARE protein SNAP-23 regulates a wide variety of exocytosis events and is essential for mouse development. Germline deletion of SNAP-23 results in early embryonic lethality in mice, and for this reason we now describe mice and cell lines in which SNAP-23 can be conditionally-deleted using Cre-lox technology. Deletion of SNAP-23 in CD19-Cre expressing mice prevents B lymphocyte development and deletion of SNAP-23 using a variety of T lymphocyte-specific Cre mice prevents T lymphocyte development. Acute depletion of SNAP-23 in mouse fibroblasts leads to rapid apoptotic cell death. These data highlight the importance of SNAP-23 for cell survival and describe a mouse in which specific cell types can be eliminated by expression of tissue-specific Cre-recombinase.

Research paper thumbnail of Spatio-temporal dynamics of chromatin containing DNA breaks

Cell cycle (Georgetown, Tex.), 2006

The cellular response to DNA breaks consists of a complex signaling network that coordinates the ... more The cellular response to DNA breaks consists of a complex signaling network that coordinates the initial recognition of the lesion with the induction of cell cycle checkpoints and DNA repair. With DNA wrapped around histone proteins and packaged into higher order levels of chromatin structure, the detection of a single DNA break (DSB) in the genome is the molecular equivalent of finding a needle in a haystack. A recent study from our laboratory used high-resolution electron microscropy and live cell imaging to demonstrate that chromatin undergoes a marked reorganization in response to a DSB. In an energy dependent manner, chromatin rapidly decondenses to a more open configuration in the regions surrounding the lesion. We propose that this ATP dependent chromatin-remodeling event facilitates the subsequent recognition and processing of damaged DNA. While the chromatin surrounding the lesion remodels to a more open configuration, the DNA break itself remains relatively immobile over t...

Research paper thumbnail of Activation of DNA Damage Response Signaling by Condensed Chromatin

Cell Reports, 2014

Highlights Dynamic compaction of chromatin is an integral step in the DNA damage response Impairm... more Highlights Dynamic compaction of chromatin is an integral step in the DNA damage response Impairment of chromatin condensation attenuates signaling from a DNA break Induced chromatin condensation can stimulate damage-independent upstream signaling Persistent compaction boosts upstream signaling but inhibits repair and recovery

Research paper thumbnail of Compartmentalization of regulatory proteins in the cell nucleus

Journal of Steroid Biochemistry and Molecular Biology, 2001

The cell nucleus is increasingly recognized as a spatially organized structure. In this review, t... more The cell nucleus is increasingly recognized as a spatially organized structure. In this review, the nature and controversies associated with nuclear compartmentalization are discussed. The relationship between nuclear structure and organization of proteins involved in the regulation of RNA polymerase II-transcribed genes is then discussed. Finally, very recent data on the mobility of these proteins within the cell nucleus is

Research paper thumbnail of A Macrohistone Variant Links Dynamic Chromatin Compaction to BRCA1-Dependent Genome Maintenance

Cell Reports, 2014

Appropriate DNA double-strand break (DSB) repair factor choice is essential for ensuring accurate... more Appropriate DNA double-strand break (DSB) repair factor choice is essential for ensuring accurate repair outcome and genomic integrity. The factors that regulate this process remain poorly understood. Here, we identify two repressive chromatin components, the macrohistone variant macroH2A1 and the H3K9 methyltransferase and tumor suppressor PRDM2, which together direct the choice between the antagonistic DSB repair mediators BRCA1 and 53BP1. The macroH2A1/PRDM2 module mediates an unexpected shift from accessible to condensed chromatin that requires the ataxia telangiectasia mutated (ATM)-dependent accumulation of both proteins at DSBs in order to promote DSB-flanking H3K9 dimethylation. Remarkably, loss of macroH2A1 or PRDM2, as well as experimentally induced chromatin decondensation, impairs the retention of BRCA1, but not 53BP1, at DSBs. As a result, mac-roH2A1 and/or PRDM2 depletion causes epistatic defects in DSB end resection, homology-directed repair, and the resistance to poly(ADP-ribose) polymerase (PARP) inhibition-all hallmarks of BRCA1deficient tumors. Together, these findings identify dynamic, DSB-associated chromatin reorganization as a critical modulator of BRCA1-dependent genome maintenance.

Research paper thumbnail of Differential expression of IFN and TRAIL/DR5 in lymphoid tissue of progressor versus nonprogressor HIV1-infected patients

Proceedings of The National Academy of Sciences, 2006

Loss of CD4+ T cells, the hallmark of HIV pathogenesis, was suggested to be partly due to apoptos... more Loss of CD4+ T cells, the hallmark of HIV pathogenesis, was suggested to be partly due to apoptosis. We recently reported that IFN- produced by HIV-1-activated plasmacytoid dendritic cells (pDCs) contributes to CD4+ T cell apoptosis by the TNF-related apoptosis-inducing ligand (TRAIL)/death receptor (DR)5 pathway. Here, we show that HIV-1-induced intracellular expression of IFN- in pDCs is coupled to increased

Research paper thumbnail of Reticulum Prior to Stimulation TLR9 Is Localized in the Endoplasmic

Research paper thumbnail of CtIP-mediated resection is essential for viability and can operate independently of BRCA1

The Journal of Cell Biology, 2014

Research paper thumbnail of Mitotic Transcription Repression In Vivo in the Absence of Nucleosomal Chromatin Condensation

The Journal of Cell Biology, 2000

All nuclear RNA synthesis is repressed during the mitotic phase of the cell cycle. In addition, R... more All nuclear RNA synthesis is repressed during the mitotic phase of the cell cycle. In addition, RNA polymerase II (RNAP II), nascent RNA and many transcription factors disengage from DNA during mitosis. It has been proposed that mitotic transcription repression and disengagement of factors are due to either mitotic chromatin condensation or biochemical modifications to the transcription machinery. In this study, we investigate the requirement for chromatin condensation in establishing mitotic transcription repression and factor loss, by analyzing transcription and RNAP II localization in mitotic cells infected with herpes simplex virus type 1. We find that virus-infected cells enter mitosis and that mitotic viral DNA is maintained in a nu-cleosome-free and noncondensed state. Our data show that RNAP II transcription is repressed on cellular genes that are condensed into mitotic chromosomes and on viral genes that remain nucleosome free and noncondensed. Although RNAP II may interact indirectly with viral DNA during mitosis, it remains transcriptionally unengaged. This study demonstrates that mitotic repression of transcription and loss of transcription factors from mitotic DNA can occur independently of nucleosomal chromatin condensation. 1 Abbreviations used in this paper: 3-D, three-dimensional; DAPI, 4 Ј ,6diamidine-2-phenylindole; ESI, electron spectroscopic imaging; HSV-1, herpes simplex virus type 1; MOI, multiplicity of infection; RNAP, RNA polymerase.

Research paper thumbnail of Reduced Mobility of the Alternate Splicing Factor (ASF) through the Nucleoplasm and Steady State Speckle Compartments

The Journal of Cell Biology, 2000

Compartmentalization of the nucleus is now recognized as an important level of regulation influen... more Compartmentalization of the nucleus is now recognized as an important level of regulation influencing specific nuclear processes. The mechanism of factor organization and the movement of factors in nuclear space have not been fully determined. Splicing factors, for example, have been shown to move in a directed manner as large intact structures from sites of concentration to sites of active transcription, but splicing factors are also thought to exist in a freely diffusible state. In this study, we examined the movement of a splicing factor, ASF, green fluorescent fusion protein (ASF-GFP) using time-lapse microscopy and the technique fluorescence recovery after photobleaching (FRAP). We find that ASF-GFP moves at rates up to 100 times slower than free diffusion when it is associated with speckles and, surprisingly, also when it is dispersed in the nucleoplasm. The mobility of ASF is consistent with frequent but transient interactions with relatively immobile nuclear binding sites. This mobility is slightly increased in the presence of an RNA polymerase II transcription inhibitor and the ASF molecules further enrich in speckles. We propose that the nonrandom organization of splicing factors reflects spatial differences in the concentration of relatively immobile binding sites.

Research paper thumbnail of The Transcription Coactivator Cbp Is a Dynamic Component of the Promyelocytic Leukemia Nuclear Body

The Journal of Cell Biology, 2001

The transcription coactivator and histone acetyltransferase CAMP response element-binding protein... more The transcription coactivator and histone acetyltransferase CAMP response element-binding protein (CBP) has been demonstrated to accumulate in promyelocytic leukemia (PML) bodies. We show that this accumulation is cell type specific. In cells where CBP does not normally accumulate in PML bodies, it can be induced to accumulate in PML bodies through overexpression of either CBP or Pml, but not Sp100.

Research paper thumbnail of Inhibition of the intestinal glucose transporter GLUT2 by flavonoids

The FASEB Journal, 2007

We tested whether the dominant intestinal sugar transporter GLUT2 was inhibited by intestinal lum... more We tested whether the dominant intestinal sugar transporter GLUT2 was inhibited by intestinal luminal compounds that are inefficiently absorbed and naturally present in foods. Because of their abundance in fruits and vegetables, flavonoids were selected as model compounds. Robust inhibition of glucose and fructose transport by GLUT2 expressed in Xenopus laevis oocytes was produced by the flavonols myricetin, fisetin, the widely consumed flavonoid quercetin, and its glucoside precursor isoquercitrin. IC 50 s for quercetin, myricetin, and isoquercitrin were ϳ200to 1000fold less than glucose or fructose concentrations, and noncompetitive inhibition was observed. The two other major intestinal sugar transporters, GLUT5 and SGLT1, were unaffected by flavonoids.

Research paper thumbnail of Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks

Nature Cell Biology, 2003

Histone H2AX is rapidly phosphorylated in the chromatin micro-environment surrounding a DNA doubl... more Histone H2AX is rapidly phosphorylated in the chromatin micro-environment surrounding a DNA double-strand break (DSB). Although H2AX deficiency is not detrimental to life, H2AX is required for the accumulation of numerous essential proteins into irradiation induced foci (IRIF). However, the relationship between IRIF formation, H2AX phosphorylation (gamma-H2AX) and the detection of DNA damage is unclear. Here, we show that the migration of repair and signalling proteins to DSBs is not abrogated in H2AX(-/-) cells, or in H2AX-deficient cells that have been reconstituted with H2AX mutants that eliminate phosphorylation. Despite their initial recruitment to DSBs, numerous factors, including Nbs1, 53BP1 and Brca1, subsequently fail to form IRIF. We propose that gamma-H2AX does not constitute the primary signal required for the redistribution of repair complexes to damaged chromatin, but may function to concentrate proteins in the vicinity of DNA lesions. The differential requirements for factor recruitment to DSBs and sequestration into IRIF may explain why essential regulatory pathways controlling the ability of cells to respond to DNA damage are not abolished in the absence of H2AX.

Research paper thumbnail of 53BP1 facilitates long-range DNA end-joining during V(D)J recombination

Nature, 2008

V(D)J recombination and class switch recombination employ overlapping but distinct nonhomologous ... more V(D)J recombination and class switch recombination employ overlapping but distinct nonhomologous end-joining (NHEJ) pathways to repair DNA double strand break (DSB) intermediates. 53BP1 is a DNA damage response protein that is rapidly recruited to sites of chromosomal DSBs, where it appears to function in a subset of ataxia-telangiectasia mutated (ATM) kinase, H2AX-and MDC1-dependent events 1,2 . A 53BP1 dependent end joining pathway has been described that is dispensable for V(D)J recombination but essential for class-switch recombination CSR 3, 4 . Here, we report a previously unrecognized defect in the joining phase of V(D)J recombination in 53BP1 deficient lymphocytes distinct from that found in classical NHEJ-, H2AX-, MDC1and Atm-deficient mice. Absence of 53BP1 leads to impairment of distal V-DJ joining with extensive degradation of un-repaired coding ends and episomal signal joint reintegration at V(D)J junctions. This results in apoptosis, loss of T-cell receptor alpha locus integrity and lymphopenia. Further impairment of the apoptotic checkpoint causes propagation of lymphocytes bearing antigen receptor breaks. These data suggest a more general role for 53BP1 in maintaining genomic stability during long range joining of DNA breaks. RAG1 and RAG2 (RAG1/2) proteins perform the pair wise cleavage step in V(D)J recombination, whereas activation-induced cytidine deaminase (AID) triggers the formation of DSBs in the switch regions during CSR 5 . RAG1/2-and AID-induced lesions in antigen receptor loci initiate nuclear focus formation of the DNA damage response proteins γ-H2AX, NBS1 and 53BP1 over a large chromosome domain 5-8 . Based on the analysis of CSR in H2AX-9 , Atm-7 , and 53BP1-deficient 3, 4, 10, 11 lymphocytes, it was proposed that focus forming factors might promote and/or maintain synapsis of distal switch regions 9 . In the case of 53BP1-deficiency, there is an almost complete loss of long range CSR 3, 4, 12 and a concomitant increase in the frequency of short-range intra switch recombination 12 . Paradoxically, although V(D)J recombination and CSR employ similar DSB repair

Research paper thumbnail of The ATM repair pathway inhibits RNA polymerase I transcription in response to chromosome breaks

Nature, 2009

DNA lesions interfere with DNA and RNA polymerase activity. Cyclobutane pyrimidine dimers and pho... more DNA lesions interfere with DNA and RNA polymerase activity. Cyclobutane pyrimidine dimers and photoproducts generated by ultraviolet irradiation cause stalling of RNA polymerase II, activation of transcription-coupled repair enzymes, and inhibition of RNA synthesis 1,2 . During the S phase of the cell cycle, collision of replication forks with damaged DNA blocks ongoing DNA replication while also triggering a biochemical signal that suppresses the firing of distant origins of replication 3,4 . Whether the transcription machinery is affected by the presence of DNA doublestrand breaks remains a long-standing question. Here we monitor RNA polymerase I (Pol I) activity in mouse cells exposed to genotoxic stress and show that induction of DNA breaks leads to a transient repression in Pol I transcription. Surprisingly, we find Pol I inhibition is not itself the direct result of DNA damage but is mediated by ATM kinase activity and the repair factor proteins NBS1 (also known as NLRP2) and MDC1. Using live-cell imaging, laser micro-irradiation, and photobleaching technology we demonstrate that DNA lesions interfere with Pol I initiation complex assembly and lead to a premature displacement of elongating holoenzymes from ribosomal DNA. Our data reveal a novel ATM/NBS1/MDC1-dependent pathway that shuts down ribosomal gene transcription in response to chromosome breaks.