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Identification of cell cycle dependent interaction partners of the septins by quantitative mass spectrometry

ABSTRACT: The septins are a conserved family of GTP-binding proteins that, in the baker's yeast, assemble into a highly ordered array of filaments at the mother bud neck. These filaments undergo dramatic structural rearrangements during the cell cycle which are mechanistically not yet understood. We aimed at identifying key components that are involved in the transitions of the septins. Combining cell synchronization and quantitative affinity-purification mass-spectrometry (AP-MS), we identified interaction partners of the septins at three distinct stages of the cell cycle. A total of 83 interaction partners of the septins were assigned. We detected DNA-interacting/nuclear proteins and proteins involved in ribosome biogenesis and protein synthesis predominantly present in G1 arrested cells without an assembled septin structure. In addition, two distinct sets of regulatory proteins that are specific for cells at S-phase with a stable septin collar or at mitosis with split septin rings were identified. Complementary methods such as the SPLIFF method allowed us to more exactly define the spatial and temporal characteristics of selected candidate proteins of the AP-MS screen.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Friedel Drepper

LAB HEAD: Thomas Gronemeyer

PROVIDER: PXD002561 | Pride | 2016-01-28

REPOSITORIES: Pride

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Identification of Cell Cycle Dependent Interaction Partners of the Septins by Quantitative Mass Spectrometry.

Renz Christian C Oeljeklaus Silke S Grinhagens Sören S Warscheid Bettina B Johnsson Nils N Gronemeyer Thomas T

PloS one 20160212 2

The septins are a conserved family of GTP-binding proteins that, in the baker's yeast, assemble into a highly ordered array of filaments at the mother bud neck. These filaments undergo significant structural rearrangements during the cell cycle. We aimed at identifying key components that are involved in or regulate the transitions of the septins. By combining cell synchronization and quantitative affinity-purification mass-spectrometry, we performed a screen for specific interaction partners of ...[more]

PMID: 26871441

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Project description:Tertiary lymphoid organs (TLOs) emerge in response to nonresolving inflammation but their roles in adaptive immunity remain unknown. Here, we explored artery TLOs (ATLOs) to delineate atherosclerosis T cell responses in apoe-/- mice during aging. Though the T cell repertoire showed systemic age-associated contractions in size and modifications in subtype composition and activation, wt and apoe-/- mice were equally affected. In contrast, ATLOs - but not wt aortae, apoe-/- aorta segments without ATLOs or atherosclerotic plaques - promoted T cell recruitment, altered characteristics of T cell motility, primed and imprinted T cells in situ, generated CD4+/FoxP3-, CD4+/FoxP3+, CD8+/FoxP3- effector and central memory cells, and converted naïve CD4+/FoxP3- T cells into induced Treg cells. ATLOs also showed substantially increased antigen presentation capability by conventional dendritic cells (DCs) and monocyte-derived DCs but not by plasmacytoid DCs. Thus, the senescent immune system specifically employs ATLOs to control dichotomic atherosclerosis T cell immune responses. We assembled transcriptome maps of wt and apoe-/- aortae and aorta-draining RLNs and identified ATLOs as major sites of atherosclerosis-specific T cell responses during aging: Transcriptome atlases of wt and apoe-/- abdominal aortae and associated draining RLNs were constructed from laser capture microdissection (LCM)-based whole genome mRNA expression microarrays yielding 6 maps: wt adventitia (tissue-1); wt RLN (tissue-2); apoe-/- ATLOs (tissue-3); apoe-/- RLN (tissue-4); apoe-/- adventitia without adjacent plaques (tissue-5), and plaques (tissue-6). Several two-tissue comparisons within the transcriptome atlases are noteworthy: Unexpectedly, transcriptomes of wt and apoe-/- RLNs were virtually identical; additonal data revealed that transcriptomes of RLNs were strikingly similar to those of inguinal LNs which do not drain the aorta adventitia (as shown of India ink injection experiments of surgically exposed aortae); in sharp contrast, wt adventitia versus ATLOs revealed 1405 differentially expressed transcripts many of which encoded members of GO terms immune response and inflammatory response; the ATLO-plaque comparison also showed > 1000 differentially expressed transcripts; however, wt adventitia versus apoe-/- adventitia without plaque showed few genes (< 5 % of differentially expressed transcripts of the wt adventitia-ATLO comparison). Thus, the aorta transcriptome atlases support the conclusion that neither aorta-draining apoe-/- RLNs nor ILNs participate in atherosclerosis-specific T cell responses. In addition, they demonstrate that T cell responses in the diseased aorta are highly territorialized. Finally, these data show that the immune responses carried out in ATLOs differ significantly from those carried out in plaques. We next identified three major clusters within the transcriptome atlases through ANOVA analyses and application of strict filters: An adventitia cluster, a plaque/ATLO cluster, and a LN/plaque cluster. The total number of differentially expressed genes in each cluster were examined for GO terms immune response, inflammatory response, T cell activation, positive regulation of T cell response, and T cell proliferation. Within the adventitia cluster, similarities of transcriptomes of wt adventitia and apoe-/- adventitia without associated plaque versus ATLOs indicate that a robust number of immune response-regulating genes are selectively expressed in ATLOs which are located within a distance of few µm of the adventitia without associated plaques indicating a very high degree of territoriality of the atherosclerosis T cell response. Furthermore, unlike the total number of differentially regulated transcripts, the majority of transcripts among GO terms immune response and inflammatory response, was up-regulated. Inspection of the plaque/ATLO cluster provided further information: The majority of immune response regulating genes where expressed at a higher level in ATLOs when compared to plaques though plaques also contained a significant number of immune response regulating genes; the reverse is true for genes regulating inflammation. Finally, the lymph node cluster revealed that though the majority of immune response regulating genes resides in both wt and apoe-/- RLNs (with little differences between them) ATLOs express a selected set of immune response regulating genes at a higher level when compared to LNs. In addition, the inflammatory component of ATLOs when compared to LNs is documented by the finding that many more genes regulating inflammation reside in ATLOs even when compared to those of plaques. Key words: T cell response in atherosclerosis; Laser capture microdissection; transcriptome atlas of atherosclerosis. Citations: Gräbner R. et al. Lymphotoxin beta receptor signaling promotes tertiary lymphoid organogenesis in the aorta adventitia of aged ApoE-/- mice. J Exp Med 2009 Jan 16;206(1):233-48. PMID: 19139167; Moos M. et al. The lamina adventitia is the major site of immune cell accumulation in standard chow-fed apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2005 Nov;25(11):2386-91. Epub 2005 Sep 22. PMID: 16179593; Beer M. et al. Laser-capture microdissection of hyperlipidemic/ApoE-/- mouse aorta atherosclerosis. Methods Mol Biol. 2011;755:417-28. PMID: 21761324; Weih F. et al. Control of Dichotomic Innate and Adaptive Immune Responses by Artery Tertiary Lymphoid Organs in Atherosclerosis. Front Physiol. 2012;3:226. Epub 2012 Jul 6. PMID: 22783198. Wild-type and apoE-deficient mice on the C57BL/6J genetic background were maintained on a standard mouse chow. Total aortae were removed at the age of 6 (n=3), 32 (n=3), or 78 (n=3) w and microarrays were prepared from total RNA extracts or extracts of atherosclerotic lesions and adventitiae obtained by the use of a laser dissection microscope. In addition, aorta associated LNs or distant LNs were prepared from both mouse genotypes.

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