No description available.

Protein trafficking between the endoplasmic reticulum (ER) and Golgi complex is regulated by the activity of ADP-ribosylation factors (Arfs). Arf activation by guanine nucleotide exchange factors (GEFs) leads to the recruitment of the coatomer protein COPI and vesicle formation. By using fluorescently-tagged proteins in live cells, we have been able to identify novel functions for Arfs and the Arf-GEF GBF1 at the ER-Golgi intermediate compartment (ERGIC) and mitotic Golgi clusters. We first focused on Arf function at the ERGIC after observing both class I (Arf1) and class II (Arfs 4 and 5) Arfs at this structure. We discovered that class II Arfs remain bound to ERGIC membranes independently of GBF1 activity following treatment with brefeldin A (BFA). Further characterization of the class II Arfs using additional pharmacological agents such as Exo1 and inactive mutant forms of Arf4 demonstrated that the class II Arfs associate with the ERGIC membrane via receptors distinct from GBF1. Our work suggests that GBF1 accumulation on membranes in the presence of BFA is due to loss of Arfs from the membrane rather than the formation of an abortive complex with Arf and GBF1. Next, while studying GBF1 in live cells, we unexpectedly observed GBF1 localizing to large fragmented structures during mitosis. We identified these structures as mitotic Golgi fragments that are positive for GBF1 and COPI throughout mitosis. Again using live cells treated with BFA and Exo1, we demonstrated that GBF1 concentrates on these mitotic fragments suggesting that they are derived from Golgi membranes. By colocalization studies and fluorescence recovery after photobleaching, we demonstrated that these mitotic fragments maintain a cis-to-trans subcompartmental Golgi polarization and membrane dynamics of GBF1 similar to interphase cells. Interestingly, inactivation of GBF1 and loss of COPI from the membranes of the mitotic Golgi fragments did not delay progressing through mitosis. Our results from our second project indicate for the first time that the mitotic Golgi clusters are bona fide Golgi structures that exist throughout mitosis with a functional COPI machinery.

No description available.

Abstract The epithelial cells form barriers that compartmentalize the organs. Carcinomas are cancers stemming from epithelial cells and are the most common cancer type. The aim of this study was to understand the differentiation and malignant transformation of epithelial Madin-Darby canine kidney (MDCK) cells and to analyse the electrophysiological parameters which regulate their transport capacity. Emphasis was placed on comparing different culture environments, both in 2D and 3D. First, the effects of drugs or basal extracellular fluid composition on MDCK cell, cyst and lumen volumes were analysed using time-lapse microscopy. The results showed that MDCK cells were capable of both water secretion and reabsorption. The cells were able to perform these functions in a hyperpolarizing or depolarizing environment; change in osmolality of basal fluid was not required. Taken together, these results validate MDCK cells as a good basic model for studying kidney function. Next, the aim was to analyse the effect of 2D and 3D culture environments on the gene expression of untransformed MDCK and temperature sensitive ts-Src -transformed MDCK cells and the changes a single oncogene can induce. Microarray analysis revealed a decrease in the expression of survivin, an inhibitor of apoptosis protein, when switching the untransformed cells from 2D environment to 3D. This downregulation of survivin occurs in adult tissues as well, indicating that the cells grown in 3D are closer to the in vivo state than 2D cells. Src oncogene induced disintegration of cell junctions, but did not downregulate E-cadherin expression. The last part was to study further the factors controlling survivin expression and its significance to cell survival. MDCK cells grown in 3D did not suffer apoptosis if the cells remained in contact with the extracellular matrix. If MDCK cells were denied of ECM contacts they were more susceptible to apoptosis than survivin-expressing ts-Src MDCK cells. Finally, if cells were denied of cell-cell junctions, cells lacking survivin suffered apoptosis even though they had proper cell-matrix contacts. Taken together, these results highlighted the importance of cellular contacts to the cells: MDCK cells needed ECM contacts to differentiate and cell-cell contacts to avoid apoptosis. / Tiivistelmä Epiteelisolut ovat erikoistuneet toimimaan rajapintana elimen ja ympäristön välillä. Ihmisten yleisin syöpä on epiteelisoluista alkunsa saanut karsinooma. Tämän tutkimuksen tarkoituksena oli ymmärtää Madin-Darby-koiran munuaisen solujen (MDCK) erilaistumista ja pahanlaatuistumista sekä analysoida sähköfysiologisia tekijöitä, jotka säätelevät näiden solujen kuljetustoimintaa. Erityisenä kiinnostuksen kohteena oli erilaisten kasvuympäristöjen vertailu. Farmakologisten aineiden tai basaalisen, solunulkopuolisen nesteen koostumuksen vaikutusta MDCK-solujen, -kystan sekä luumenin kokoon tutkittiin valomikroskooppisten aikasarjojen avulla. Tulokset osoittivat MDCK-solujen olevan kykeneviä sekä veden eritykseen että absorptioon, niin hyperpolarisoivassa kuin depolarisoivassakin ympäristössä. Basaalisen nesteen osmolaliteetin muutosta ei tarvittu. Nämä tulokset osoittavat MDCK-solujen olevan hyvä munuaisen tutkimuksen perusmalli. Seuraavaksi analysoitiin kaksi- ja kolmiulotteisten (2D ja 3D) viljely-ympäristöjen vaikutusta ei-transformoitujen MDCK-solujen ja lämpötilaherkkien ts-Src-transformoitujen MDCK-solujen geenien ilmentymiseen sekä yhden onkogeenin aktivoimisen aikaansaamia muutoksia. Microarray-analyysi osoitti apoptoosin estäjän, surviviinin, ilmentymisen vähenemisen, kun kasvuympäristö vaihdettiin 2D-ympäristöstä 3D-ympäristöön. Koska surviviinin väheneminen on normaali tapahtuma aikuisissa kudoksissa, voitiin todeta, että 3D-ympäristössä kasvatetut solut ovat lähempänä luonnonmukaista olotilaa kuin 2D-ympäristössä kasvaneet. Src-onkogeeni sai aikaan soluliitosten hajoamisen, mutta ei vähentänyt E-kadheriinin ilmentymistä. Tutkimuksen viimeinen osa keskittyi surviviinin ilmentymistä säätelevien tekijöiden analysoimiseen ja surviviinin merkitykseen solujen eloonjäämiselle. 3D-ympäristössä kasvaneet MDCK-solut eivät kärsineet apoptoosista edellyttäen, että solut pysyivät kosketuksissa soluväliaineeseen. Jos solut irtautuivat soluväliaineesta, ne päätyivät herkemmin apoptoosiin kuin surviviinia ilmentävät ts-Src MDCK-solut. Mikäli solujen väliset liitokset pakotettiin avautumaan, solut joutuivat apoptoosiin, vaikka ne olivat kosketuksissa soluväliaineeseen. Yhteenvetona nämä tulokset korostavat solujen kontaktien merkitystä: MDCK-solut tarvitsevat soluväliainekontakteja erilaistumiseen ja solujen välisiä kontakteja välttyäkseen apoptoosilta.

Control of cultivation conditions in the~live cell imaging extends the possibilities of biological experiments and makes the experimental results more reliable. In order to change the~cultivation conditions in a controlled manner and increase the reproducibility of the experiments, it is necessary to reduce the amount of manual operations and replace them with automated procedures. Therefore, the concept of a new automated culture device (bioreactor) was created. This device controls the exchange of medium in the observation chamber, ensures the circulation and exchange of the atmosphere and controls its composition. The bioreactor is intended for use in the Laboratory of Experimental Biophotonics. This laboratory is equipped with coherence-controlled holographic microscope (CCHM), which uses quantitative phase imaging (QPI) method. Thus, the bioreactor is adapted to the current requirements of this laboratory and optical elements of the bioreactor meet the requirements of the QPI method. This text specifies the cultivation conditions of the living cells and summarizes, how the conditions could be controlled in the live cell microscopy. Next some commercially available culture devices are described and assessed, whether they are convenient for the~use in Laboratory of Experimental Biophotonics. The crucial part of the thesis is the~design, construction and testing of the new bioreactor.

This diploma thesis deals with the issue of motility analysis in leukemia cells. An accurate description of the cell movement and the detection of differences in motility under experimental conditions can be obtained by quantitative analysis of cell motility using time-lapse recording. The first part of this work describes various types of tumor cell migraton. The second part focuses on methods of analysis of cell motility in tissue culture using time-lapse recording, which include image acquisition and processing. Part of this chapter describes a coherence-controlled holographic microscope, which was used in the practical part and for which an insert was designed to ensure the exact and stable position of the individual chambers. The last part is focused on the research of leukemic cell motility, which is concluded by a discussion of the obtained results. The appendix contains a published study included acknowledgement to the author of this diploma thesis for participation in the project.

Chlamydien sind obligat intrazelluläre Bakterien, die sich in einem membranumschlossenen Kompartiment namens Inklusion vermehren. Nach Infektion fragmentiert der Golgi-Apparat der Wirtszelle in kleine Membranstapel. Dies verbessert die Aufnahme von Sphingolipiden und ist deshalb für die chlamydiale Vermehrung essentiell. Die infektionsinduzierte Golgi-Fragmentierung geschieht nach Spaltung des Golgi-Matrix-Proteins Golgin-84. In dieser Arbeit konnte, durch den Vergleich mit bekannten Substraten und Inhibitorstudien, die chlamydiale Protease CPAF (Chlamydia protease-like activity factor) als das Enzym identifiziert werden, das diese Spaltung induziert, abhängig von der Anwesenheit zweier Rab-Proteine, Rab6 und Rab11, die den zellulären Vesikeltransport kontrollieren und zur Inklusion rekrutiert werden. Die Fragmentierung des Golgi-Apparates verhinderte dessen Relokalisierung während der Zellpolarisierung nach Einbringen eines migratorischen Stimulus. Sowohl infizierte als auch Golgin-84-depletierte Zellen migrierten langsamer und randomisiert in einem Motilitätsassay. Die Relokalisierung des Golgi-Apparates konnte durch seine Stabilisierung mittels WEHD oder Rab-Depletion wieder gewonnen werden, was die Zellmotilität teilweise wieder herstellte. Darüber hinaus konnte gezeigt werden, dass die Infektion außer der Golgi-Reorientierung die Signaltransduktion durch GTPasen beeinflusst. Die Aktivität von Cdc42 in infizierten Zellen war erhöht und die Interaktionen mit vielen ihrer Effektoren laut quantitativer Massenspektrometrie stark verändert. Die Ergebnisse dieser Arbeit zeigen, dass CPAF die für Chlamydien lebenswichtige Golgin-84 Prozessierung und Fragmentierung des Golgi-Apparates auslöst. Dies verringert die Mobilität der Wirtszelle, vor allem da der Golgi-Apparat während der Polarisierung nicht mehr ausgerichtet werden kann, des Weiteren durch Modulierung der Protein-Protein-Interaktionen von Cdc42. / Chlamydia are obligate intracellular human pathogens that proliferate inside a membrane-bound compartment called the inclusion. In infected cells, the Golgi apparatus is fragmented into small ministacks that are aligned around the inclusion. This facilitates uptake of host cell sphingolipids and is essential for chlamydial development. Infection-induced Golgi fragmentation happens after processing of the Golgi matrix protein golgin-84. This work could, via comparison with well-known substrates and inhibitor studies, identify the chlamydial protease CPAF (Chlamydia protease-like activity factor) as the enzyme accountable for this cleavage. Golgi Fragmentation depended on two Rab proteins, Rab6 and Rab11, which control vesicle transport and are recruited to the Chlamydia inclusion. As a consequence of Golgi fragmentation, cells lost the capacity to reorient the Golgi apparatus during polarization after a migratory stimulus. Both infected and golgin-84 depleted cells with a permanently fragmented Golgi apparatus displayed decelerated and furthermore randomized migration in a motility assay. Relocalization of the Golgi apparatus could be restored via stabilizing WEHD treatment or Rab depletion which partly rescued cell motility. Moreover, it could be shown that migration signaling via small GTPases was influenced by Chlamydia infection. Infected cells exhibited activation of the small polarity GTPase Cdc42. Numerous interactions with downstream effectors were strongly altered in infected cells according to quantitative mass spectrometry. Particularly, the binding of Cdc42 to migration-associated effectors was decreased. The results of this work show that CPAF, by processing of golgin-84, induces Golgi fragmentation which is vitally important for Chlamydia. This disturbs host cell motility because the Golgi apparatus cannot be reoriented during polarization and, additionally, via the modulation of protein-protein-interactions of Cdc42.