Les microtubules (MTs) sont des éléments majeurs du cytosquelette, ils participent à un ensemble de fonctions cellulaires d’importance comme la migration, le trafic des vésicules et des organelles ainsi qu’{ la division cellulaire au cours de laquelle ils forment le fuseau mitotique, permettant une ségrégation correcte des chromosomes. Ce sont des polymères cylindriques composés de protofilaments parallèles faits d’hétérodimères de tubulines α/β. Pour exercer ces fonctions, les MTs nécessitent une dynamique d’assemblage et de désassemblage régulée.La stathmine est une phosphoprotéine cytosolique de 17 kDa qui séquestre la tubuline dans un complexe non polymérisable formé de deux hétérodimères de tubuline par molécule de stathmine (complexe T2S). Les autres protéines de la famille stathmine (SCG10, SCLIP, RB3 et ses deux variants d’épissage RB3' et RB3″) forment aussi des complexes de séquestration appelés T2-SLD (Stathmin Like Domain) ayant des stabilités variables.En s’appuyant sur ces observations, des travaux ont montré qu’une chimère SN-CR constituée de la partie N-terminale de la stathmine et de la partie C-terminale du SLD de la protéine RB3 forme un complexe T2S remarquablement stable avec la tubuline (Jourdain et al., 2004). En parallèle à ces travaux, Clément et collaborateurs ont découvert que de courts peptides issus de la région N-terminale des SLD peuvent à eux seuls inhiber la polymérisation de la tubuline. Le peptide le plus efficace est le peptide I19L issu de la stathmine (peptide I19L (IQVKELEKRASGQAFELIL)). Il correspond à la région repliée en «β-hairpin» observée dans les cristaux T2S-SLD formés avec le domaine RB3-SLD (Clement et al., 2005). Pour augmenter l'efficacité de l’interaction entre les peptides issus de la région N-terminale des SLD et la tubuline, différentes mutations ont été introduites au niveau du peptide I19L. Les expériences de polymérisation in vitro de tubuline en présence d’une série de mutants de peptide I19L montrent que les peptides I19L-K4R et I19L-K4R-A10R sont les plus efficaces pour l’inhibition de l’assemblage de la tubuline.L’objectif de mes travaux { été d’introduire ces mutations au niveau de la partie N-terminale de la protéine stathmine et de la chimère NS-CR, afin d’analyser leurs efficacités sur l’inhibition de l’assemblage des MTs et la dynamique des MTs dans des cellules en culture de type HeLa, et de voir leur impact sur le cycle cellulaire.Mes résultats montrent que la stathmine mutant 2 (possédant les mutations K9R-A15R homologues aux mutations K4R-A10R du peptide I19L) induit une dépolymérisation des MTs dans des cellules en mitose par formation de fagots de MTs, réduit la dynamique de MTs et induit une cytotoxicité. L’ensemble des résultats suggère que nous pouvons améliorer la stabilité de liaison de la stathmine avec la tubuline par l’introduction de ces mutations, ce qui aurait in vitro un impact dans certains aspects cellulaires comme la prolifération cellulaire et la dynamique des MTs. Cela pourrait conduire à développer de nouvelles stratégies anticancéreuses visant à perturber le fuseau mitotique. / Microtubules (MTs) are major constituents of the cytoskeleton and are involved in many cellular processes such as the determination of cell architecture, the formation of the mitotic spindle and intracellular trafficking. These tubular structures, composed by tubulin α/β heterodimers assemble and disassemble with a finely regulated dynamics.Stathmin is a cytosolic phosphoprotein that sequesters tubulin in a non polymerizable complex consisting of two tubulin heterodimers per stathmin molecule (T2S complex).The other stathmin family proteins (SCG10, SCLIP, RB3 and its two splice variants RB3 'and RB3 ") can also bind two tubulin heterodimers through their SLD (Stathmin Like Domain), but the different tubulin/SLD complexes display varying stabilities.Based on these observations, current works showed that one chimera protein consisting of the N-terminal domain of stathmin (NS) and of the C-terminal domain of RB3 (CR) forms a T2-SLD complex remarkably stable with tubulin (NS-CR chimera). Molecular simulation experiments in silico suggest that we can increase the affinity of the NS-CR chimera by introducing mutations in the NS subdomain (Jourdain et al., 2004).In parallel to this work, Clement and colleagues found that short peptides derived from the N-terminal region of SLD alone can inhibit tubulin polymerization. Among the N-terminal part of SLD, the most effective peptide is the peptide I19L (IQVKELEKRASGQAFELIL) derived from stathmin. It corresponds to region folded into a "β-hairpin" structure observed in the T2S crystals (Clement et al., 2005). To increase the efficiency of this interaction, different mutations were introduced at the level of the I19L peptide from the stathmin N-terminal domain and tested in vitro. The results showed that the peptides I19L-K4R and I19L-K4R-A10R are the most effective to in inhibit the assembly of MTs.In this work, we introduce these mutations in the N-terminal domains of stathmin and in the NS-CR chimera with the aim of analyzing their efficiencies on MTs disassembly in cancer cell cultures like HeLa, and the impact of the mutation on MTs dynamic and cell cycle.Results from overexpression experiments of WT and stathmin mutant 2 containing the double mutation (K9R-A15R similar to the mutations K4R-A10R for the I19L peptide), suggest that this mutant significantly induces depolymerization of mitotic MTs. In its less phosphorylated state on serine 16; this mutation also reduces MTs dynamics and induces cytotoxicity.We thus suggest that we can ameliorate the binding and efficiency of the N-terminal stathmin part by including this double mutation. This variation has an impact in vitro and in some cellular aspects such as cell proliferation and MTs dynamics. These findings may lead to a targeted therapy for this type of cancer.

La holoenzima proteína quinasa A (PKA) es un tetrámero que consiste en dos subunidades catalíticas y dos subunidades reguladoras, se encuentra en forma ubicua fosforilando un gran número de proteínas celulares y participa en la regulación de vías metabólicas y otros procesos (transporte de membrana, motilidad celular, expresión génica, apoptosis, aprendizaje y memoria). Las proteínas quinasas no solamente se encuentran en el interior de las células, sino además se observa la "exportación" hacia la superficie de la cara externa de la membrana plasmática de las células (Redegeld, F.A et al., 1999). Este fenómeno de exportación de las proteína quinasas hacia la superficie de la cara externa de las células en cultivo se denomina "ectoquinasas." El objetivo del presente trabajo es estudiar la exportación de la proteína quinasa dependiente del AMPc sobrexpresada en células humanas en cultivo a nivel intracelular o endógeno, a nivel ectópico o en la membrana plasmática, y libre en el líquido extracelular o proteína exógeno. En la presente Tesis se utilizó dos líneas celulares llamadas HEK293T y HELA, realizándose la técnica de transfección por lipofectamina para introducir en estas células la proteína PKA, específicamente el vector (pcDNA3-HA) con el inserto que contiene el gen de la subunidad catalítica con el epítope anti-HA.

TNF-alpha can stimulate a variety of kinases with the ability to activate non-muscle myosin II. As a result, increases in actin filament formation and actomyosin contractility (AMC) have been reported in response to TNF-alpha. These events are thought to play an important role in mediating TNF-alpha induced apoptosis but how they do so is unclear. In this study we prevented non-muscle myosin II activation in response to TNF-alpha by treating cells with the myosin light chain kinase (MLCK) inhibitor ML-7 or through isoform specific siRNA knockdown of myosin IIA and IIB. We found that treatment with ML-7 or knockdown of myosin IIB, but not IIA, impaired the cleavage of caspase 3 and caspase 8 as well as nuclear condensation in response to TNF-alpha. During this cell death process myosin II seemed to function independent of AMC since treatment of cells with blebbistatin or cytochalasin D failed to inhibit TNF-alpha induced caspase cleavage. Immunoprecipitation studies revealed associations of myosin IIB with clathrin and FADD in response to TNF-alpha suggesting a role for myosin IIB in TNFR1 endocytosis and DISC formation. Taken together these findings suggest that myosin IIB activation promotes TNF-alpha cell death signaling in a manner independent of its force generating property.

The objective of the project is to use the RICS analysis technique in complement with confocal microscopy to determine the diffusion coefficient of the selectively labeled green fluorescent protein (GFP), GFP-EGFR and GFP-p53 in cervical cancer cells. This is a collaboration work with MD Anderson Cancer Center. The application of the study is to lay the foundation for further study in understanding the cell metabolism, subcellular morphologic and dynamic biochemical processes to aid in the diagnosis and to potentially screen cancers. Fluorescence microscopy techniques have been developed for the study of cellular processes and molecular signal pathway. However, the spatial resolution to distinguish and resolve the interactions of single molecular complexes or molecule in cells is limited by the wavelength. Hence, indirect image correlation methods complementary to the imaging techniques were developed to obtain the dynamic information within the cell. RICS is one such mathematical image processing method to determine the dynamics of the cell. HeLa cells are transfected with GFP to highlight the protein of interest. These samples were imaged with confocal microscope, Olympus FV1000 with a 60 x 1.2 NA water objective in the pseudo photon counting mode with an excitation of 488 nm argon ion laser. About 100 frames of scan area 256x256 pixels were collected from each sample at scan speed of 12.5 seconds per pixel. The stacks of images were processed with SimFCS software. The images were subjected to immobile subtraction algorithm to remove the immobile features. Consequently, each frame in the stack is subjected to 2D-correlation; then, the average 2D-spatial correlation is calculated. This 2D-spatial correlated data constitutes as RICS data which is then displayed and analyzed by fitting it to specific models. This generates a spatial temporal map of the molecular dynamics of fluorescently labeled probes within the cell. In summary, we apply RICS techniques based on correlation spectroscopy to the image data and quantify diffusion coefficient of protein of interest in cancerous cells with different treatments. This is expected to better understand cellular dynamics of cancerous cells and build better diagnostic biosensor devices for early screening.

La holoenzima proteína quinasa A (PKA) es un tetrámero que consiste en dos subunidades catalíticas y dos subunidades reguladoras, se encuentra en forma ubicua fosforilando un gran número de proteínas celulares y participa en la regulación de vías metabólicas y otros procesos (transporte de membrana, motilidad celular, expresión génica, apoptosis, aprendizaje y memoria). Las proteínas quinasas no solamente se encuentran en el interior de las células, sino además se observa la "exportación" hacia la superficie de la cara externa de la membrana plasmática de las células (Redegeld, F.A et al., 1999). Este fenómeno de exportación de las proteína quinasas hacia la superficie de la cara externa de las células en cultivo se denomina "ectoquinasas." El objetivo del presente trabajo es estudiar la exportación de la proteína quinasa dependiente del AMPc sobrexpresada en células humanas en cultivo a nivel intracelular o endógeno, a nivel ectópico o en la membrana plasmática, y libre en el líquido extracelular o proteína exógeno. En la presente Tesis se utilizó dos líneas celulares llamadas HEK293T y HELA, realizándose la técnica de transfección por lipofectamina para introducir en estas células la proteína PKA, específicamente el vector (pcDNA3-HA) con el inserto que contiene el gen de la subunidad catalítica con el epítope anti-HA.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1984. / Typescript. Vita. Description based on print version record. Includes bibliographical references.

The reversible acetylation of histones is thought to play a role in chromatin processing, including transcription, replication and repair. Studies on the acetyltransferases, responsible for acetylating the nucleosomal core histones, have resulted in characterization of these enzymes. However, very little is known about the properties and distribution of histone deacetylase. The reversible inhibition of histone deacetylase by butyrate was employed to permit studies on the chromatin-bound histone deacetylase of HeLa cells using endogenous [3H]-acetyl labelled polynucleosomes containing the enzyme. These were prepared in the presence of 50mM butyrate and histone deacetylase was assayed upon removal of the inhibitor. It was found that active enzyme is present only in association with a high molecular weight complex. This deacetylase-containing complex is relatively resistant to digestion with micrococcal nuclease. No activity is found on mononucleosomes or oligonucleosomes. Up to 90% of labelled acetyl groups are removed from histone deacetylase complexes incubated in the absence of butyrate, indicating that denaturation of the histone deacetylase is kept to a minimum using the techniques developed in this study. Free histones are a poor substrate under these conditions, but histones in mononucleosomes are deacetylated when they are incubated with histone deacetylase complex. Histone deacetylase remains bound to this complex in 1-2 M NaCl and does not dissociate from it during its reaction with acetylated core hisones. Under typical nuclease digestion conditions, the histone deacetylase complex contains DNA with a size distribution of 5-11 kilobase pairs and a variety of nonhistone proteins. Comparison of the protein composition of histone deacetylase complexes with that of nuclear matrix preparations shows some similarities. Taken together, the results on the chromatographic behaviour, the DNA fragment sizes, and the protein composition of the deacetylase complex suggest that protein-protein interactions may be important in maintaining its structure and also in the binding of the deacetylase itself to the complex Later research efforts were concerned with characterization of the histone deacetylase complex. The effect of J3-mercaptoethanol and neocuproine on histone deacetylase was examined in view of the fact that these reagents are known to disrupt chromosome scaffolds. HeLa cell histone deacetylase complex partially dissociates in 10 mM B-mercaptoethanol, resulting in a loss of non-histone proteins. The presence of 10 mM J3-mercaptoethanol during the partial micrococcal nuclease digestion of HeLa cell nuclei, results in a very low yield of histone deacetylase complex, with a correspondingly large increase in the production of small oligonucleosomes and mononucleosomes. Histone deacetylase activity on endogenous labelled histone is strongly inhibited by either 1 or 10 mM J3-mercaptoethanol or 3 mM neocuproine. The loss of histone deacetylase activities is not due to an inactivation of the enzyme, but appears to be a consequence of the disruption of the structure of the histone deacetylase complex. Histone H4 in histone deacetylase complex prepared from HeLa cell nuclei by micrococcal nuclease digestion was more highly acetylated than H4 in bulk nucleosomes. Restriction enzyme analysis of the DNA associated with the histone deacetylase complex revealed neither an enrichment nor depletion of major satellite sequences in this material. In view of these findings, histone deacetylase appears to be associated with a high molecular weight chromatin complex which may be a site of rapid acetyl group turnover. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Apurinic endonuclease activity in human fibroblasts had been previously resolved into aflow-through and a high-salt eluate species by phosphocellulose chromatography (Kuhnlein, U. et al., Nucl. Acid. Res. 5: 951-960, 1978). Enzyme activity in the flow-through species amounted to 20-30% that of the high-salt eluate species. The flow—through enzyme species was not found in-eel I lines of xeroderma pigmentosum complementation group D. In this thesis, apurinic endonuclease activity was analysed in Hela ceflls. Specific enzyme activity in crude extracts "of Hela cells was in the range of 400-800 units/mg protein, similar to that of , human fibroblasts which was between 380-680 units/mg protein. Three species of endonuclease activity for apurinic DNA were resolved by phosphocellulose chromatography. They were designated as Peak I, Peak If, and Peak III. Peak I did not adsorb to the phosphoceIIuIose column at' 10 mM KP04 (pH 7.4) (flow-through activity), Peak II eluted from the column at about 210 mM KP0₄ (pH 7.4) and Peak I I I at 260 mM KPO₄ (pH 7.4). Based on their affinity to phosphoceIIuIose, we presumed Peak I and Peak III corresponded to the flow-through and high-salt eluate species in human fibroblasts respectively. Under our experimental conditions, the flow-through enzyme activity in both Hela cells and normaS human fibroblasts was only 2-4% of the activity of high-salt eluate species. We suspect that tissue culture conditions may affect the cellular level of the flow-through species of apurinic endonuclease. Peaks l-lII were optimally active at pH 7.5-8.0 and 5-10 mM MgCI, They were 'inhibited by increasing concentrations of KCI and NaCI except Peak III which was slightly stimulated by 20-40 mM KCI. The three species were distinguished by their thermosensitivities in a 50 mM KPO. buffer. Peak I was stable at 45°C. Peak III was heat-labile, having a half-life of 2-3 min at 45°C. Peak II seemed to contain two components, one with a ha If-life of 2-3 min at 45°C, and the other with a half-life of 25 min. In human fibroblasts, both the flow-through and high-salt eluate species of apu-rinic endonuclease were reported to be stimulated to 2.5-fold by 10 mM KCI. They had a ha If-life of 6 min at 45°C in a 230 mM KP0₄ (pH 7.4) buffer. Thus, Peaks I-lI I and enzyme species from human fibroblasts had a similar pH optimum, and Mg²⁺ requirement, but they differed in their thermosensitivities and inhibition by higher salt concentration. We do not know as yet whether these differences reflect the neoplastic nature of Hela cells or the different tissue origins of Hela cells and human fibroblasts. When either Peak I or Peak I I I was rechromatographed on the phosphoceIIulose column, activity was recovered in both the flow-through and high-salt eluate fractions. The result suggested an interconversion phenomenon between the flow-through and high-salt eluate species of apurinic endonuclease, This was further supported by molecular weight determinations of the apurinic endonucI eases In Peaks l-lII. Apurinic endonuclease activity in Peak III and Peak II had a molecular weight of 35,000-40,000 and 22,000-25,000 respectively. Peak I had two components with molecular weights similar to those of Peak II and Peak III. An understanding of the conversion between the different apurinic endonuclease species may help in elucidating the molecular defects of xeroderma pigmentosum complementation group D. Apurinic endonuclease activity in Peaks l-lII was found to be associated with a high molecular weight complex. The complex could be dissociated by high salt treatment. The possible biological significance of the high molecular weight complex Is discussed. We also found that apurinic endonuclease could adsorb to the Sephadex gel. The adsorption would lead to an aberrant estimation of molecular weight of the protein. The problem was solved with an elution buffer of high ionic strength. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

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