Verena Lehmann

Yersinia enterocolitica Type III Secretion Depends on the Proton Motive Force but Not on the Flagellar Motor Components MotA and MotB

ABSTRACT The flagellum is believed to be the common ancestor of all type III secretion systems (TTSSs). In Yersinia enterocolitica, expression of the flagellar TTSS and the Ysc (Yop secretion) TTSS are inversely regulated. We therefore hypothesized that the Ysc TTSS may adopt flagellar motor components in order to use the pathogenicity-related translocon in a drill-like manner. As a prerequisite for this hypothesis, we first tested a requirement for the proton motive force by both systems using the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Motility as well as type III-dependent secretion of Yop proteins was inhibited by CCCP. We deleted motAB, which resulted in an immotile phenotype. This mutant, however, secreted amounts of Yops to the supernatant comparable to those of the wild type. Translocation of Yops into host cells was also not affected by the motAB deletion. Virulence of the mutant was comparable to that of the wild type in the mouse oral infection model. Thus, the hypothesis that the Ysc TTSS might adopt flagellar motor components was not confirmed. The finding that, in addition to consumption of ATP, Ysc TTSS requires the proton motive force is discussed.

Proteomic and functional analysis of the mitotic Drosophila centrosome.

Regulation of centrosome structure, duplication and segregation is integrated into cellular pathways that control cell cycle progression and growth. As part of these pathways, numerous proteins with well‐established non‐centrosomal localization and function associate with the centrosome to fulfill regulatory functions. In turn, classical centrosomal components take up functional and structural roles as part of other cellular organelles and compartments. Thus, although a comprehensive inventory of centrosome components is missing, emerging evidence indicates that its molecular composition reflects the complexity of its functions. We analysed the Drosophila embryonic centrosomal proteome using immunoisolation in combination with mass spectrometry. The 251 identified components were functionally characterized by RNA interference. Among those, a core group of 11 proteins was critical for centrosome structure maintenance. Depletion of any of these proteins in Drosophila SL2 cells resulted in centrosome disintegration, revealing a molecular dependency of centrosome structure on components of the protein translation machinery, actin‐ and RNA‐binding proteins. In total, we assigned novel centrosome‐related functions to 24 proteins and confirmed 13 of these in human cells.

LGALS3BP regulates centriole biogenesis and centrosome hypertrophy in cancer cells

Centrosome morphology and number are frequently deregulated in cancer cells. Here, to identify factors that are functionally relevant for centrosome abnormalities in cancer cells, we established a protein-interaction network around 23 centrosomal and cell-cycle regulatory proteins, selecting the interacting proteins that are deregulated in cancer for further studies. One of these components, LGALS3BP, is a centriole- and basal body-associated protein with a dual role, triggering centrosome hypertrophy when overexpressed and causing accumulation of centriolar substructures when downregulated. The cancer cell line SK-BR-3 that overexpresses LGALS3BP exhibits hypertrophic centrosomes, whereas in seminoma tissues with low expression of LGALS3BP, supernumerary centriole-like structures are present. Centrosome hypertrophy is reversed by depleting LGALS3BP in cells endogenously overexpressing this protein, supporting a direct role in centrosome aberration. We propose that LGALS3BP suppresses assembly of centriolar substructures, and when depleted, causes accumulation of centriolar complexes comprising CPAP, acetylated tubulin and centrin.

Yersinia enterocolitica type III secretion: Evidence for the ability to transport proteins that are folded prior to secretion

BackgroundPathogenic Yersinia species (Y. enterocolitica, Y. pestis, Y. pseudotuberculosis) share a t ype t hree s ecretion s ystem (TTSS) which allows translocation of effector proteins (called Yops) into host cells. It is believed that proteins are delivered through a hollow needle with an inner diameter of 2–3 nm. Thus transport seems to require substrates which are essentially unfolded. Recent work from different groups suggests that the Yersinia TTSS cannot accommodate substrates which are folded prior to secretion. It was suggested that folding is prevented either by co-translational secretion or by the assistance of s pecific Y op c haperones (called Sycs).ResultsIn this study we have fused YopE secretion signals of various length to the mouse dihydrofolate reductase (DHFR) in order to analyse the DHFR folding state prior to secretion. We could demonstrate that secretion-deficient as well as secretion-competent YopE-DHFR fusions complexed to SycE can be efficiently purified from Yersinia cytosol by affinity chromatography using methotrexate-agarose. This implies the folding of the DHFR fusion moiety despite SycE binding and contradicts the previously presented model of folding inhibition by chaperone binding. Secretion-deficient YopE-DHFR fusions caused severe jamming of the TTSS. This observation contradicts the co-translational secretion model.ConclusionsWe present evidence that the Yersinia TTSS is familiar with the processing of transport substrates which are folded prior to secretion. We therefore predict that an unfoldase is involved in type III secretion.

Functional analysis of centrosomal kinase substrates in Drosophila melanogaster reveals a new function of the nuclear envelope component otefin in cell cycle progression

ABSTRACT Phosphorylation is one of the key mechanisms that regulate centrosome biogenesis, spindle assembly, and cell cycle progression. However, little is known about centrosome-specific phosphorylation sites and their functional relevance. Here, we identified phosphoproteins of intact Drosophila melanogaster centrosomes and found previously unknown phosphorylation sites in known and unexpected centrosomal components. We functionally characterized phosphoproteins and integrated them into regulatory signaling networks with the 3 important mitotic kinases, cdc2, polo, and aur, as well as the kinase CkIIβ. Using a combinatorial RNA interference (RNAi) strategy, we demonstrated novel functions for P granule, nuclear envelope (NE), and nuclear proteins in centrosome duplication, maturation, and separation. Peptide microarrays confirmed phosphorylation of identified residues by centrosome-associated kinases. For a subset of phosphoproteins, we identified previously unknown centrosome and/or spindle localization via expression of tagged fusion proteins in Drosophila SL2 cells. Among those was otefin (Ote), an NE protein that we found to localize to centrosomes. Furthermore, we provide evidence that it is phosphorylated in vitro at threonine 63 (T63) through Aurora-A kinase. We propose that phosphorylation of this site plays a dual role in controlling mitotic exit when phosphorylated while dephosphorylation promotes G2/M transition in Drosophila SL2 cells.

Immunoisolation of centrosomes from Drosophila melanogaster.

Classical protocols for the isolation of centrosomes from higher eukaryotic cells are based on enrichment of cell organelles by density gradient centrifugation. Various successful protocols have been described that isolate centrosomes from mammalian tissue culture cells, tissue, clam oocytes, Drosophila, and yeast, to mention only some of the more frequently used sources. The material produced is subsequently used in various assays. These include functional tests such as the microtubule nucleation assay, electron microscopic study of centrosome morphology, and antigen localization; the organelles may also be used for the generation of antibodies. Furthermore, centrosomal preparations have been used for the characterization of their protein composition. The method described here focuses on the isolation of centrosomes from the syncytial stages of the early Drosophila embryo. This is a particularly attractive system because these organelles are not bounded by cellular membranes. Moreover, the abundance of pericentriolar material of these centrosomes produces excellent total protein yields.

Abstract 350: Virtualization of drug testing by predictive systems biology modeling for optimal drug treatment of cancer cells and drug repositioning

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Traditionally, tumors have been classified based on their tissue of origin and their histopathological characteristics and consequently patients are divided into groups where they are treated identically despite substantial differences in genetic profiles. Identification of genes causally implicated in cancer development has led to the development of ‘stratified’ medicine, which adjusts a patient’s therapy based on biomarkers that identify some of the patients expected not respond to a specific therapy. However, many tumor genome sequencing projects have shown that many more gene mutations drive the development of cancer than previously thought, making every tumor unique. These results in low success rates and thereby high cost in drug approval as the identified drugs are only effective for particular patient groups while large patient cohorts have no clear clinical benefit. In the early process of drug development, drugs are therefore screened on large cancer cell line collections to define drug applicability and to determine potential tumor targets. Here, we report the results of a computational modeling platform, ModCell, allowing the prediction of individual drug effects using large-scale genomic and transcriptomic data to virtualize such cell line screening. We validated this approach two-fold: on publicly available data from the cancer cell line encyclopedia, that comprises pharmacological profiles for anti-cancer drugs across a library of cancer cell lines as well as on cancer cell line culture experiments. We provide evidence that ModCell is able to reproducibly predict the effects of individual drugs with a confounding 80% accuracy, to predict combinatory drug action and to identify new applications for existing drugs. Thus, computational modeling using ModCell can improve todays drug development by accelerating and partly replacing work which would have otherwise be conducted in the laboratory and in the clinic. Citation Format: Alexander Kuehn, Felix Dreher, Svetlana Peycheva, Reha Yildiriman, Verena Lehmann, Thomas Kessler, Christoph Wierling, Hans Lehrach, Bodo MH Lange. Virtualization of drug testing by predictive systems biology modeling for optimal drug treatment of cancer cells and drug repositioning. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 350. doi:10.1158/1538-7445.AM2014-350