Podcasts about u2os

Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Tareg Omer Mohammed, You-Rong Lin, and Clemens M. Franz at the Nano Life Science Institute (WPI-NanoLSI), at Kanazawa University.The research described in this podcast was published in the Journal of Cell Science in January 2024.Kanazawa University NanoLSI websitehttps://nanolsi.kanazawa-u.ac.jp/en/A novel role for S100A11 in focal adhesion regulationResearchers at Kanazawa University report in the Journal of Cell Science on a novel role of the small Ca2+ion-binding protein S100A11 [S one hundred A eleven] in focal adhesion disassembly.S100A11 is a small Ca2+ion-activatable protein with an established role in different cellular processes involving actin cytoskeleton remodeling, such as cell migration, membrane protrusion formation, and plasma membrane repair. It also displays F-actin binding activity and localizes to actin stress fibers, but its precise role in regulating these structures remained unclear.In their study, Tareg Omer Mohammed, You-Rong Lin, and Clemens M. Franz together with colleagues from the Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, in Japan, and Karlsruhe Institute of Technology, in Germany, report a novel localization of S100A11 to disassembling focal adhesions at the end of contractile stress fibers in HeLa and U2OS cells. Specifically, S100A11 transiently appears at the onset of focal adhesion disassembly, reliably marking the targeted adhesion sites for subsequent disassembly. Interestingly, S100A11 leaves focal adhesion sites before the completion of disassembly, indicating that S100A11 plays a specific role in the initiation of adhesion site disassembly, rather than the disassembly process itself.So what are focal adhesions anyway and what can we learn from them?Focal adhesions are integrin-containing cell/matrix adhesion sites enabling cells to adhere to the cellular environment and to apply cellular contraction forces during extracellular matrix remodeling. Directed cell migration requires the coordinated assembly of new adhesion sites at the front, and disassembly at the rear of the cell, and better understanding mechanisms regulating focal adhesion turnover is, therefore, an important goal in cell migration and invasion research. The newly discovered role of S100A11 in focal adhesion disassembly extends insights into the molecular mechanisms underlying focal adhesion site disassembly.The authors furthermore delineate a force-dependent recruitment mechanism for S100A11 to adhesion sites involving non-muscle myosin II-driven stress fiber contraction, activation of mechanosensitive, Ca2+ ion-permeable Piezo1 channels, and intracellular Ca2+ ion influx at mechanically stressed focal adhesions. In turn, locally elevated Ca2+ ion levels activates and recruits S100A11 to the adhesion sites targeted for disassembly. So how did they work this out?The force-dependent recruitment of S100A11 to stressed focal adhesions was confirmed using a micropipette pulling assay able to apply pulling forces onto individual focal adhesion sites. Even when myosin II-dependent intracellular contractility was inhibited, external pulling forces still recruited S100A11 to stretched focal adhesion sites, corroborating the mechanosensitive recruitment mechanism of S100A11. However, extracellular Ca2+ ion and Piezo1 function was still indispensable, indicating that myosin II-dependent contraction forces act upstream of Piezo1-mediated Ca2+ ion influx, in turn leading to S100A11 activation and focal adhesion recruitment.Lastly, the authors show impaired focal adhesion translocation and disassembly ratNanoLSI Podcast website

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.17.549432v1?rss=1 Authors: Mohammed, T. O., Lin, Y.-R., Weissenbruch, K., Ngo, K. X., Zhang, Y., Kodera, N., Bastmeyer, M., Miyanari, Y., Taoka, A., Franz, C. M. Abstract: S100A11 is a small Ca2+-activatable protein with an established role in different cellular processes involving actin cytoskeleton remodeling, such as cell migration, membrane protrusion formation, and plasma membrane repair. It also displays Ca2+-dependent F-actin binding activity and localizes to actin stress fibers (SFs), but its precise role in regulating these structures remains unclear. Analyzing endogenous S100A11 localization in HeLa and U2OS osteosarcoma cells confirmed SF association but in addition revealed steady localization to stable focal adhesions (FAs), typically at the end of dorsal stress fibers. In contrast, S100A11 levels at FAs increased sharply, but transiently, at the onset of peripheral FA disassembly. Elevating intracellular Ca2+ levels using the Ca2+ ionophore ionomycin reliably stimulated both S100A11 recruitment and subsequent FA disassembly. However, pre-incubation with the non-muscle myosin II (NM II) inhibitor blebbistatin, or with an inhibitor to the stretch-activatable Ca2+ channel Piezo1 effectively suppressed S100A11 recruitment, implicating S100A11 in an actomyosin contractility-driven FA disassembly mechanism involving Piezo1-dependent Ca2+ influx. Applying external mechanical forces on peripheral FAs via a micropipette likewise recruited S100A11 to FAs, even when NM II was inhibited by blebbistatin or in NM IIA knockout cells, corroborating the mechanosensitive recruitment mechanism of S100A11. However, extracellular Ca2+ and Piezo1 function was still indispensable, indicating that NM II-dependent contraction forces act upstream of Piezo1-mediated Ca2+ influx, in turn leading to S100A11 activation and FA recruitment. Moreover, S100A11 knockout cells feature enlarged FAs and display delayed FA disassembly during cell membrane retraction, consistent with impaired FA turnover in these cells. Our results thus demonstrate a novel mechano-sensitive function for S100A11 in promoting actomyosin contractility-driven FA disassembly. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.26.546598v1?rss=1 Authors: Shangguan, J., Rock, R. S. Abstract: Myosin 10 (Myo10) is a vertebrate-specific motor protein well known for its role in filopodia formation. Although Myo10-driven filopodial dynamics have been characterized, there is no information about the numbers of Myo10 in filopodia. To better understand molecular stoichiometries and packing restraints in filopodia, we measured Myo10 abundance in these structures. Here we combined SDS-PAGE analysis with epifluorescence microscopy to quantitate HaloTag-labeled Myo10 in U2OS cells. About 6% of total intracellular Myo10 localizes to filopodia, where it tends to be enriched at opposite ends of the cell. Hundreds of Myo10 are found in a typical filopodium, and their distribution across filopodia is log-normal. Some filopodial tips even contain more Myo10 than accessible binding sites on the actin filament bundle. Our estimates of Myo10 molecules in filopodia provide insight into the physics of packing Myo10, its cargo, and other filopodia-associated proteins in narrow membrane deformations in addition to the numbers of Myo10 required for filopodia initiation. Our protocol provides a framework for future work analyzing Myo10 abundance and distribution upon perturbation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.21.545954v1?rss=1 Authors: Thompson, J., Boisvert, F.-M., Salsman, J., Dellaire, G., Ridgway, N. D. Abstract: When exposed to excess fatty acids, specific cell types produce nuclear lipid droplets (nLDs) that associate with promyelocytic leukemia (PML) protein to form Lipid Associated PML Structures (LAPS) that are enriched in lipid biosynthetic enzymes but deficient in canonical proteins associated with PML nuclear bodies (PML NBs). To identify the PML interactome during lipid stress, we employed proximity-dependent biotin identification (BioID) in U2OS cells expressing PMLI and PMLII fused to the ascorbate peroxidase APEX2 and cultured in the absence or presence of oleate to enhance lipid droplet formation. The resulting interactome included proteins enriched under oleate-treated conditions, such mitogen activated protein kinase-activated protein kinase 2 (MK2), ESCRT proteins and the COPII vesicle transport proteins Sec23B, Sec24A and USO1. COPII proteins co-localized with both PML-NBs and LAPS but were selectively enriched in PML-NBs following oleate treatment. The nuclear localization of USO1 was uniquely dependent on PML expression. Thus, the APEX2-PML proximity interactome implicates PML domains in the nuclear function of a non-canonical network of COPII vesicle trafficking proteins. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.15.532715v1?rss=1 Authors: Yamashita, A., Ignatenko, O., Nguyen, M., Lambert, R., Watt, K., Daneault, C., Robillard-Frayne, I., Topisirovic, I., Des Rosiers, C., McBride, H. M. Abstract: Peroxisomes play a central role in tuning metabolic and signaling programs in a tissue- and cell type-specific manner. However, the mechanisms by which the status of peroxisomes is communicated and integrated into cellular signaling pathways is not yet understood. Herein, we report the cellular responses to acute peroxisomal proteotoxic stress upon silencing the peroxisomal protease/chaperone LONP2. Depletion of LONP2 triggered accumulation of its substrates, alterations in peroxisome size and numbers, and luminal protein import failure. Gene expression changes and lipidomic analysis revealed striking cell specific differences in the response to siLONP2. Specific to COS-7 cells was a strong activation of the integrated stress response (ISR) and upregulation of ribosomal biogenesis gene expression levels. Common changes between COS-7 and U2OS cell lines included repression of the retinoic acid signaling pathway, and upregulation of sphingolipids. Cholesterol accumulated in the endomembrane compartments in both cell lines, consistent with evidence that peroxisomes are required for cholesterol flux out of late endosomes. These unexpected consequences of peroxisomal stress provide an important insight for our understanding of the tissue-specific responses seen in peroxisomal disorders. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.22.525054v1?rss=1 Authors: Kliszczak, M., Moralli, D., Jankowska, J., Bryjka, P., Subha, L., Goncalves, T., Hester, S., Fischer, R., Clynes, D., Green, C. M. Abstract: Hereditary fibrosing poikiloderma (HFP) is a rare human dominant negative disorder caused by mutations in the FAM111B gene that encodes a nuclear trypsin-like serine protease. HFP patients present with symptoms including skin abnormalities, tendon contractures, myopathy and lung fibrosis. We characterised the cellular roles of human FAM111B using U2OS and MCF7 cell lines and report here that the protease interacts with components of the nuclear pore complex. Loss of FAM111B expression resulted in abnormal nuclear shape and reduced telomeric DNA content suggesting that FAM111B protease is required for normal telomere length; we show that this function is independent of telomerase or recombination driven telomere extension. Even though FAM111B-deficient cells were proficient in DNA repair, they showed hallmarks of genomic instability such as increased levels of micronuclei and ultra-fine DNA bridges. Interestingly, FAM111B variants, including mutations that cause HFP, showed more frequent localisation to the nuclear lamina suggesting that accumulation of mutant FAM111B at the nuclear periphery may drive the disease pathology. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.27.522042v1?rss=1 Authors: Labitigan, R. L. D., Sanborn, A. L., Hao, C. V., Chan, C. K., Belliveau, N. M., Brown, E. M., Mehrotra, M., Theriot, J. A. Abstract: The contributions of individual genes to cell-scale morphology and cytoskeletal organization are challenging to define due to the wide intercellular variation of these complex phenotypes. We leveraged the controlled nature of image-based pooled screening to assess the impact of CRISPRi knockdown of 366 genes on cell and nuclear morphology in human U2OS osteosarcoma cells. Screen scale-up was facilitated by a new, efficient barcode readout method that successfully genotyped 85% of cells. Phenotype analysis using a deep learning algorithm, the {beta}-variational autoencoder, produced a feature embedding space distinct from one derived from conventional morphological profiling, but detected similar gene hits while requiring minimal design decisions. We found 45 gene hits and visualized their effect by rationally constrained sampling of cells along the direction of phenotypic shift. By relating these phenotypic shifts to each other, we construct a quantitative and interpretable space of morphological variation in human cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.09.333203v1?rss=1 Authors: Weissenbruch, K., Grewe, J., Stricker, K., Baulesch, L., Schwarz, U. S., Bastmeyer, M. Abstract: Nonmuscle myosin II (NM II) is an integral part of essential cellular processes, including adhesion and migration. Mammalian cells express up to three isoforms termed NM IIA, B, and C. We used U2OS cells to create CRISPR/Cas9-based knockouts of all three isoforms and analyzed the phenotypes on homogeneous and micropatterned substrates. We find that NM IIA is essential to build up cellular tension during initial stages of force generation, while NM IIB is necessary to elastically stabilize NM IIA-generated tension. The knockout of NM IIC has no detectable effects. A scale-bridging mathematical model explains our observations by relating actin fiber stability to the molecular rates of the myosin crossbridge cycle. We also find that NM IIA initiates and guides co-assembly of NM IIB into heterotypic minifilaments. We finally use mathematical modeling to explain the different exchange dynamics of NM IIA and B in minifilaments, as measured in FRAP experiments. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.25.311803v1?rss=1 Authors: Moroz-Omori, E. V., Huang, D., Bedi, R. K., Cheriyamkunnel, S. J., Bochenkova, E., Dolbois, A., Rzeczkowski, M. D., Wiedmer, L., Sledz, P., Caflisch, A. Abstract: The methylase METTL3 is the writer enzyme of the N6-methyladenosine (m6A) modification of RNA. Using a structure-based drug discovery approach, we identified a METTL3 inhibitor (UZH1a) with potency in a biochemical assay of 280 nM, while its enantiomer UZH1b is 100 times less active. The crystal structure of the complex of METTL3 with UZH1a illustrates the interactions that make it selective against protein methyltransferases. We observed a dose-dependent reduction in m6A methylation level of mRNA in several cell lines treated with UZH1a already after 16 h of exposure, as determined by triple-quadrupole LC mass spectrometry, while its enantiomer UZH1b was essentially inactive at concentrations up to 100 M. Interestingly, the kinetics of m6A level reduction in mRNAs followed a first-order reaction model, with a half-decay time {tau} of 1.8 h and a maximum m6A inhibition level of 70%, which is in line with the previously observed shorter half-life of m6A-modified mRNAs. Notably, treatment with the compounds did not alter cellular METTL3 levels, ruling out indirect effects on m6A levels. The effect of the m6A level depletion by UZH1a directly translated into growth inhibition of MOLM-13 leukemia cells, under short-term and long-term culture. Incubation of the MOLM-13 cells with UZH1a, but not with UZH1b, resulted in increased cell apoptosis and cell cycle arrest already after 16 h of incubation. Interestingly, other cell lines sensitive to METTL3 level (U2Os, HEK293T) did not reveal statistically significant differences between UZH1a and UZH1b in a cell viability assay, confirming that the degree of reliance on m6A signalling for survival can vary between cancers/cell types. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.14.289330v1?rss=1 Authors: Vinje, J., Guadagno, N. A., Progida, C., Sikorski, P. Abstract: Cells in their natural environment are embedded in a complex surrounding consisting of biochemical and biomechanical cues directing cell properties and cell behaviour. Nonetheless, in vitro cell studies are typically performed on flat surfaces, with clear differences from the more complex situation cells experience in vivo. To increase the physiological relevance of these studies, a number of advanced cellular substrates for in vitro studies have been applied. One of these approaches include flat surfaces decorated with vertically aligned nanostructures. In this work, we explore how U2OS cells are affected by arrays of polymer nanopillars fabricated on flat glass surfaces. We focus on describing changes to the organisation of the actin cytoskeleton and in the location, number and shape of focal adhesions. From our findings we identify that the cells can be categorised into different regimes based on their spreading and adhesion behaviour on nanopillars. A quantitative analysis suggests that cells seeded on dense nanopillar arrays are suspended on top of the pillars with focal adhesions forming closer to the cell periphery compared to flat surfaces or sparse pillar arrays. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.26.268086v1?rss=1 Authors: Hummert, J., Yserentant, K., Fink, T., Euchner, J., Herten, D.-P. Abstract: The composition of cellular structures on the nanoscale is a key determinant of macroscopic functions in cell biology and beyond. Different fluorescence single-molecule techniques have proven ideally suited for measuring protein copy numbers of cellular structures in intact biological samples. Of these, photobleaching step analysis poses minimal demands on the microscope and its counting range has significantly improved with more sophisticated algorithms for step detection, albeit at an increasing computational cost. Here, we present a comprehensive framework for photobleaching step analysis, optimizing both data acquisition and analysis. To make full use of the potential of photobleaching step analysis, we evaluate various labelling strategies with respect to their molecular brightness and photostability. The developed analysis algorithm focuses on automation and computational efficiency. Moreover, we benchmark the framework with experimental data acquired on DNA origami labelled with defined fluorophore numbers to demonstrate counting of up to 35 fluorophores. Finally, we show the power of the combination of optimized trace acquisition and automated data analysis for robust protein counting by counting labelled nucleoporin 107 in nuclear pore complexes of intact U2OS cells. The successful in situ application promotes this framework as a new resource enabling cell biologists to robustly determine the stoichiometries of molecular assemblies at the single-molecule level in an automated fashion. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.11.246504v1?rss=1 Authors: Campbell, A., Ferraz Franco, C., Su, L.-I., Perkins, S., Jones, A. R., Brownridge, P. J., Perkins, N. D., Eyers, C. E. Abstract: Different types of DNA damage can initiate phosphorylation-mediated signalling cascades that result in stimulus specific pro- or anti-apoptotic cellular responses. Amongst its many roles, the NF-{kappa}B transcription factor RelA is central to these DNA damage response pathways. However, we still lack understanding of the co-ordinated signalling mechanisms that permit different DNA damaging agents to induce distinct cellular outcomes through RelA. Here, we use label-free quantitative phosphoproteomics to examine the temporal effects of exposure of U2OS cells to either etoposide (ETO) or hydroxyurea (HU) by monitoring the phosphorylation status of RelA and its protein binding partners. Although few stimulus-specific differences were identified in the constituents of phosphorylated RelA interactome after exposure to these DNA damaging agents, we observed subtle, but significant, changes in their phosphorylation states, as a function of both type and duration of treatment. The DNA double strand break (DSB)-inducing ETO invoked more rapid, sustained responses than HU, with regulated targets primarily involved in transcription, cell division and canonical DSB repair. Kinase substrate prediction of ETO-regulated phosphosites suggest abrogation of CDK1 and ERK1 signalling, in addition to the known induction of ATM/ATR. In contrast, HU-induced replicative stress mediated temporally dynamic regulation, with phosphorylated RelA binding partners having roles in rRNA/mRNA processing and translational initiation, many of which contained a 14-3-3{varepsilon} binding motif, and were putative substrates of the dual specificity kinase CLK1. Our data thus point to differential regulation of key cellular processes and the involvement of distinct signalling pathways in modulating DNA damage-specific functions of RelA. Copy rights belong to original authors. Visit the link for more info

The cover for issue 31 of Oncotarget features Figure 4, "Concentration dose-response curves of sirolimus effect [55 nM–1 nM] on the number of cells per surviving colony in U2OS cell line after 2 weeks exposure," by Vasuri, et al. which reported that the authors evaluated the long-term effects of sirolimus on three different cell in vitro models, cultured in physiological conditions mimicking sirolimus-eluted stent, in order to clarify the effectiveness of sirolimus in blocking cell proliferation and survival. Three cell lines were selected and growth in 10 ml of Minimum Essential Medium for 5 weeks with serial dilutions of sirolimus. The number of colonies and the number of cells per colony were counted. As a result, the number of WPMY-1 surviving colonies increased in a dose-dependent manner when treated with sirolimus, while the number of U2OS colonies progressively decreased. In conclusion sirolimus showed the well-known cytostatic effect, but with an effect on clonogenic potential different among the different cell types. Dr. Gianandrea Pasquinelli from The Bologna University said, "Rapamycin (sirolimus) is a widely used cytostatic drug blocking the cell cycle in the phase G1/S through the inhibition of the mammalian target of Rapamycin (mTOR) pathway, that has found several clinical applications, from immunosuppression in diabetes and organ transplantation to cancer therapy and drug-eluting stents (DES)" Beside to its cytostatic activity, sirolimus was also discovered to protect normal human oral keratinocytes from apoptosis by activating autophagy, and to act as a basal stem cell keratinocyte-protecting drug in irradiated mice. The effect of sirolimus on mesenchymal cells is unknown, but it is an important issue, since mesenchymal cells such as myofibroblasts and cells promoting vascular calcification play an important role in atherogenesis and vascular restenosis. Sirolimus seems to block the proliferation and the migration of vascular smooth muscle cells, but we lack information concerning the effects on other cells composing atherosclerotic plaques. The aim of the present paper is to evaluate the long-term effects of sirolimus, rather than short-term cell survival, on three different cell in vitro models, cultured in Minimum Essential Medium, which simulates physiological conditions (w/o CO2 and glucose, in order to clarify the effectiveness of sirolimus in blocking cell proliferation and survival. The Pasquinelli Research Team concluded in their Oncotarget Research Paper that the plaque typology and the different cell composition of the plaque, e. g., the presence of inflammatory cells, angiogenesis, prevalence of fibrosis, presence of osteogenic progenitors, may influence the response to sirolimus. Moreover, it is known that the clonal capacity varies between cells and we should consider this matter when evaluating the effectiveness of eluted stent. Finally, additional mechanisms can have a role, such as amitotic cell division. These mechanisms were also observed in human atherogenesis and could be fundamental to evaluate the in vivo effect of sirolimus too. Full text - https://www.oncotarget.com/article/27554/text/ Correspondence to - Gianandrea Pasquinelli - gianandr.pasquinelli@unibo.it Keywords - atherosclerosis, cell proliferation, sirolimus, stents About Oncotarget Oncotarget is a weekly, peer-reviewed, open access biomedical journal covering research on all aspects of oncology. To learn more about Oncotarget, please visit https://www.oncotarget.com or connect with: SoundCloud - https://soundcloud.com/oncotarget Facebook - https://www.facebook.com/Oncotarget/ Twitter - https://twitter.com/oncotarget LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Oncotarget is published by Impact Journals, LLC please visit http://www.ImpactJournals.com or connect with @ImpactJrnls

Adulte Weichgewebesarkome (engl. soft tissue sarcoma; STS) werden zu einer Gruppe seltener maligner und teilweise aggressiver Tumoren klassifiziert, die eine Tendenz zur Bildung von hämatogenen Fernmetastasen aufweisen. Die Kombination der Regionalen Hyperthermie mit einer Chemotherapie erwies sich in vorangegangenen Studien als eine vielversprechende Behandlungsoption beim lokalisierten Hochrisiko STS. Es wurde gezeigt, dass eine neoadjuvante Chemotherapie mit Regionaler Hyperthermie bei diesen Sarkomen das Tumoransprechen, das lokale progressionsfreie und das krankheitsfreie Überleben im Vergleich zu einer alleinigen Chemotherapie signifikant verbessert. Auf zellulärer Ebene induziert ein Hitzeschock (HS) bei klinisch relevanten Temperaturen (41,8°C/43°C) unter anderem eine temporäre Defizienz der Homologen Rekombinationsreparatur (HR), einem essentiellen Mechanismus für die fehlerfreie Reparatur von DNA-Doppelstrangbrüchen (DSB). Dies steht im Zusammenhang mit einer hitzeinduzierten proteosomalen Degradierung von BRCA2, einer unerlässlichen Komponente der HR. Trabectedin (Tr) ist eine antiproliferativ wirksame Substanz, die ursprünglich aus dem marinen Tunikat Ecteinascidia turbinata isoliert wurde. Die vielfältigen zytotoxischen Aktivitäten von Tr umfassen neben dem Interferieren mit der aktivierten Transkription und der Modulation der Tumor-Mikroumgebung hauptsächlich die Induktion von DSBs. Seit 2007 wird Tr in der Zweitlinientherapie zur Behandlung refraktärer STS, sowie bei Patienten eingesetzt, bei denen die Erstlinientherapie (Ifosfamid und/oder Doxorubicin) nicht angewendet werden kann. In Anbetracht der hitzeinduzierten Inaktivierung von BRCA2 und den DNA schädigenden Eigenschaften von Tr wurde in dieser Arbeit untersucht, ob und wie die Hyperthermie zu einer Wirkungsverstärkung der zytotoxischen Effekte von Tr beitragen kann. Tr bewirkt in vitro bei Zelllinien unterschiedlicher Sarkomentitäten (U2Os, SW872, SW982) eine dosisabhängige Reduktion des klonogenen Überlebens, das durch einen HS zusätzlich verstärkt wird. Die erhöhte antiproliferative Aktivität von Tr nach einem HS wird als thermale Chemosenitivierung definiert. Zudem konnte durch die Analyse der DNA-Verteilung bei U2Os und SW872 Zellen eine Intensivierung und Verlängerung der Tr-induzierten G2/M-Blockade nachgewiesen werden. Darüber hinaus wurden Zelllinien-spezifische Unterschiede bezüglich einer behandlungsinduzierten Apoptoseinduktion oder Senseszenzantwort identifiziert. SW872 Zellen weisen einen dosis- und temperaturabhängigen Anstieg des Anteiles apoptotischer Zellen auf, der mit einer starken Aktivierung der Effektorcaspasen 3 und 7 einhergeht. Dem entgegen gehen U2Os Zellen in eine ausgeprägte behandlungsinduzierte zelluläre Seneszenz über. Anhand der quantitativen Analyse Tr-induzierter H2AX Foci hat sich ein relevanter Anstieg an DSBs durch eine zusätzliche Hitzeexposition herausgestellt, der eine Beeinträchtigung der BRCA2-vermittelten vollständigen Assemblierung der DNA-Reparaturfoci vermuten lässt. Die Hypothese einer thermalen Chemosensitivierung gegenüber Tr durch eine hitzeinduzierte HR-Defizienz – insbesondere im Rahmen der hitzeinduzierten BRCA2 Degradierung – wurde zudem durch das Ausbleiben der hitzebedingten Verstärkung der Tr-induzierten Zytotoxizität bei BRCA2-defizienten Zellen bekräftigt. Darüber hinaus wurde durch Hochdurchsatzanalysen bestätigt, dass eine hitzevermittelte, erhöhte antiproliferative Aktivität von Tr nach einem Knockdown zahlreicher HR-spezifischer Komponenten ausbleibt. Durch Hochdurchsatzanalysen sowie durch anschließende Validierungsexperimente wurden Proteine identifiziert, die sich als relevant für weitere präklinische und klinische Untersuchungen herausgestellt haben. Die Proteine BRCA1, PARP1 und CHEK1 stellen dabei potentielle molekulare Marker für ein Tumoransprechen auf die Kombinationstherapie von Tr und Hyperthermie dar. Deren Inhibition erwies sich zudem als eine weitere Strategie, um die Effektivität der ursprünglichen Behandlung zusätzlich zu erhöhen. Darüber hinaus wurde die Funktion von FANCD2 als prädiktiver Marker und von ERCC1 als Resistenzmarker für das Therapieansprechen einer alleinigen Tr-Behandlung in vitro bestätigt. Die herausgearbeitete thermale Chemosensitivierung gegenüber Tr mit Hyperthermie durch die induzierte HR-Defizienz mittels passagerer BRCA2 Degradierung (induzierte synthetische Letalität) sowie die Identifizierung weiterer Proteine, deren medikamentöse Inhibition die Effektivität der Kombinationsbehandlung zusätzlich erhöhen könnte, eröffnen neue Möglichkeiten in der Therapie solider Tumoren.

Super-resolution imaging, the ability to resolve structures well below the diffraction limit, has changed fluorescence microscopy as we know it. Diffraction-unlimited microscopy, termed nanoscopy, is able to image deep inside the cell with a resolution approaching that of electron microscopes. A variety of different methods currently exist, the first proposed and demonstrated technique being stimulated emission depletion (STED) microscopy. In STED microscopy, the resolution limit of a laser scanning microscope is overcome by transiently silencing fluorescence in parts of the focus. Practical implementations of this method are limited by optical aberrations and the characteristics of the fluorophores used. In this thesis, I present a fully working, custom built, 3D dual-color STED microscope. A super-continuum source is used to provide all spectral bands necessary for excitation and efficient depletion to achieve a lateral and axial resolution of ~ 35 nm and ~ 90 nm, respectively. I characterize the system’s performance by imaging colloidal particles and single fluorescent molecules. Its biological applicability is demonstrated by imaging of nuclear pore complexes in U2OS and yeast cells, replication complexes in C2C12 cells, C2 toxin component C2I in HeLa cells and scaffold proteins in chemical synapses of hippocampal neurons. Advice on how to build such a microscope is given in the appendix. In addition, a theory is presented which demonstrates that the resolution of STED microscopes can be further enhanced by using the arrival time of spontaneous emission by time-gating the detection.

Nuclear myosin I (NM1) is a nuclear isoform of the well-known "cytoplasmic" Myosin 1c protein (Myo1c). Located on the 11(th) chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes.

Chromatin is the packaged form of DNA in the eukaryotic nucleus, with the nucleosome as its basic unit. The nucleosome consists of DNA, wrapped around a histone octamer which is comprised of two copies of each of the four core histones H2A, H2B, H3 and H4. To allow DNA-related processes to occur, access to the DNA has to be regulated. One regulatory mechanism is the exchange of the canonical core histones by one of their replacement variants. Thus far, five members of the histone H3 family have been described: H3.1, H3.2, H3.3, testis-specific tH3 and centromere-specific CENP-A. Searching the database, we have recently identified two novel histone H3 variant genes on human chromosome 5, now termed H3.X and H3.Y. In my PhD thesis, I have analyzed their expression patterns, characterized their mRNA as well as their protein products and investigated their potential function(s). H3.X and H3.Y constitute primate-specific genes which have been found, in addition to humans, also in the chimpanzee and the macaque, but not in other mammals or even lower eukaryotes. H3.X and especially H3.Y mRNA is expressed at low but significant levels in the human osteosarcoma cell line U2OS and in some human bone, breast, lung and ovary tumor tissues, as well as in testis and certain areas of the brain. Tagged H3.X and H3.Y proteins behave similar to H3.1, H3.2 and H3.3 in their nuclear localization and stable incorporation into chromatin. However, in contrast to H3.1 and H3.3, novel H3 variants primarily form heterotypic nucleosomes. Endogenous H3.Y protein is expressed in a small number of U2OS cells (