La localisation des ARNm dans différents compartiments subcellulaires est conservée dans un large éventail d'espèces et de divers types cellulaires. Le trafic est médié par l'interaction entre les protéines de liaison à l'ARN (RBP) et l'ARNm. Les RBP reconnaissent les éléments cis-régulateurs de l'ARNm, également appelés éléments de localisation. Ceux-ci sont définis par leur séquence et/ou leurs caractéristiques structurelles résidant dans la molécule d'ARNm. La localisation des ARNm est essentielle pour la résolution subcellulaire et temporelle. De plus, les ARNm se sont avérés enrichis dans de nombreux compartiments cellulaires, notamment les mitochondries, l'appareil mitotique, et le réticulum endoplasmique. En outre, des études ont démontré que les RBP et les ARNm sont associés aux structures de l'appareil mitotique. Cependant, le rôle que joue la localisation de l'ARNm au cours de la mitose reste largement inexploré. Ma thèse de doctorat vise à comprendre comment le trafic d'ARNm est impliqué lors de la mitose. La première partie de cette thèse porte sur l'interaction post-transcriptionnelle qui se produit entre les deux ARNm, cen et ik2. Les gènes qui se chevauchent sont une caractéristique frappante de la plupart des génomes. En fait, il a été constaté que le chevauchement des séquences génomiques module différents aspects de la régulation des gènes tels que l'empreinte génomique, la transcription, l'édition et la traduction de l'ARN. Cependant, la mesure dans laquelle cette organisation influence les événements réglementaires opérant au niveau post-transcriptionnel reste incertaine. En étudiant les gènes cen et ik2 de Drosophila melanogaster, qui sont transcrits de manière convergente avec des régions 3' non traduites qui se chevauchent, nous avons constaté que la liaison physique de ces gènes est un déterminant clé dans la co-localisation de leurs ARNm aux centrosomes cytoplasmiques. Le ciblage du transcrit ik2 dépend de la présence et de l'association physique avec l'ARNm de cen, qui est le principal moteur de la co-localisation centrosomale. En interrogeant les ensembles de données de séquençage de fractionnement, nous constatons que les ARNm codés par des gènes qui se chevauchent en 3' sont plus souvent co-localisés par rapport aux paires de transcrits aléatoires. Ce travail suggère que les interactions post-transcriptionnelles des ARNm avec des séquences complémentaires peuvent dicter leur destin de localisation dans le cytoplasme. La deuxième partie de cette thèse consiste à étudier le rôle que jouent les RBP au cours de la mitose. Auparavant, les RBP se sont avérés être associés au fuseau et aux centrosomes. Cependant, leur rôle fonctionnel au niveau de ces structures reste à étudier. Grâce à un criblage par imagerie avec plus de 300 anticorps, nous avons identifié 30 RBP localisés dans les structures mitotiques des cellules HeLa. Ensuite, pour évaluer les rôles fonctionnels de ces RBP, nous avons utilisé l'interférence ARN (ARNi) pour évaluer si la fidélité du cycle cellulaire était compromise dans les cellules HeLa et les embryons de Drosophila melanogaster. Fait intéressant, nous avons identifié plusieurs candidats RBP pour lesquels le knockdown perturbe la mitose et la localisation de l'ARNm dans les cellules HeLa. De plus, la perte des orthologues a entraîné des défauts de développement chez l'embryon de mouche. Grâce à ce travail, nous avons démontré que les RBP sont impliquées pour assurer une mitose sans erreur. En résumé, les travaux que j'ai menés mettent en lumière l'implication de la régulation post-transcriptionnelle au cours de la mitose. En définissant les fonctions et le mécanisme de localisation des ARNm en mitose, ce travail permettra de définir de nouvelles voies moléculaires impliquées dans la régulation de la mitose. Puisque la division cellulaire non contrôlée peut mener à des maladies tel le cancer, étudier le contrôle du cycle cellulaire sous cet angle « centré sur l'ARN » peut aider à développer de nouvelles approches thérapeutiques pour trouver des solutions aux problèmes de santé. / The localization of mRNAs to different subcellular compartments is conserved in a wide range of species and diverse cell types. Trafficking is mediated by the interaction between RNA binding proteins (RBPs) and mRNA. RBPs recognize mRNA cis regulatory motifs, otherwise known as localization elements. These are defined by their sequence and/or structural features residing within the mRNA molecule. Localization of mRNAs is essential for subcellular and temporal resolution. Furthermore, mRNAs have been found to be enriched in many cellular compartments including the mitochondria, mitotic apparatus, and endoplasmic reticulum. Moreover, studies have demonstrated that RBPs and mRNAs are associated with mitotic apparatus structures. However, the role that mRNA localization plays during mitosis remains largely unexplored. My PhD thesis aims to understand how the trafficking of mRNAs is implicated during mitosis. The first part of this thesis encompasses the post-transcriptional interaction that occurs between the two mRNAs, cen and ik2. Overlapping genes are a striking feature of most genomes. In fact, genomic sequence overlap has been found to modulate different aspects of gene regulation such as genomic imprinting, transcription, RNA editing and translation. However, the extent to which this organization influences regulatory events operating at the post-transcriptional level remains unclear. By studying the cen and ik2 genes of Drosophila melanogaster, which are convergently transcribed with overlapping 3’untranslated regions, we found that the physical linkage of these genes is a key determinant in co-localizing their mRNAs to cytoplasmic centrosomes. Targeting of the ik2 transcript is dependent on the presence and physical association with cen mRNA, which serves as the main driver of centrosomal colocalization. By interrogating global fractionation-sequencing datasets, we find that mRNAs encoded by 3’overlapping genes are more often co-localized as compared to random transcript pairs. This work suggests that post-transcriptional interactions of mRNAs with complementary sequences can dictate their localization fate in the cytoplasm. The second part of this thesis involves investigating the role that RBPs play during mitosis. Previously, RBPs have been found to be associated with the spindle and centrosomes. However, their functional role at these structures was yet to be investigated. Through an imaging screen with >300 antibodies, we identified 30 RBPs localized to mitotic structures in HeLa cells. Then, to assess the functional roles of these RBPs, we used RNA interference (RNAi) to assess whether cell cycle fidelity was compromised in HeLa cells and Drosophila melanogaster embryos. Interestingly, we identified several RBP candidates for which the knockdown disrupted mitosis and mRNA localization in HeLa cells. Furthermore, loss of the orthologs led to developmental defects in the fly embryo. Through this work, we demonstrated that RBPs are involved in ensuring an error-free mitosis. In summary, the work that I have conducted sheds light on the involvement of post-transcriptional regulation during mitosis. By defining the functions and mechanism of mRNA localization in mitosis, this work will help define new molecular pathways involved in mitosis regulation. As uncontrolled cell division can lead to diseases such as cancer, studying cell cycle control from this ‘RNA-centric’ angle may help to develop new therapeutic approaches to find solutions to health problems.

No description available.

Collagen is one of the most abundant human proteins. It exists as a right-handed superhelix called the triple helix. The triple helix consists of three left-handed polyproline type II (PPII helices) that intertwine around a common axis. Each PPII helix has the repeating peptide sequence (Gly–Xaa–Yaa)n with a high content of (2S)-proline (Pro) in the Xaa position (ca. 28%) and (2S,4R)-hydroxyproline (Hyp) in the Yaa position (ca. 38%). Unique to the prolyl amide is the ease of cis-trans isomerization. Since the triple helix necessitates that all peptide bonds be in the trans conformation, isomerization is the rate-limiting step in collagen folding. However, eliminating isomerization with a trans-locked alkene isostere destabilizes collagen-like peptides. Collagen is stabilized by electronic interactions, namely the n→π* interaction. Halo-alkene isosteres may be used to recapture these electronic interactions and stabilize a collagen-like peptide. An in-depth conformational analysis was conducted at the MP2/6-311+G(2d,p) level of theory to determine the viability of conformationally-locked halo-alkene isosteres. Fluoro-alkenes and chloro-alkenes were modeled at both the Gly–Pro and Pro–Pro (as a Pro–Hyp mimic) amide positions. Compared to the collagen crystal structure PDB ID: 1K6F, we found the fluoro-alkenes were closer geometric matches to both Gly–Pro and Pro–Pro than the corresponding chloro-alkenes. The chloro-alkene was predicted to have stronger n→π* interactions. The trans-locked proteo-alkene was also analyzed to understand why it destabilized the triple helix. We found that these models had other local minima close to the desired PPII geometry, likely leading to enhanced backbone flexibility. This deleterious flexibility was not predicted for either fluoro-alkene or chloro-alkene models. The conformationally-locked halo-alkene isostere Fmoc–Gly–Ψ[(Z)CF=C]-Pro–Hyp(tBu)–OH was designed and synthesized as a (Z)-fluoro-alkene Gly–Pro isostere. We used the chiral catalyst, L-Thr, for asymmetric aldol addition to cyclopentanone, which inadvertently enhanced the yield of the wrong enantiomer, in contrast with aldol addition to cyclohexanone. A Mg2+-promoted Horner-Wadsworth-Emmons reaction afforded the (Z)-fluoro-alkene over the (E)-fluoro-alkene in about a 2:1 ratio. The two diastereomers, Fmoc–Gly–Ψ[(Z)CF=C]-L-Pro–Hyp(tBu)–OH and Fmoc–Gly–Ψ[(Z)CF=C]-D-Pro–Hyp(tBu)–OH were separated by supercritical CO2 chromatography. The collagen-like peptides Ac–(Gly–Pro–Hyp)3–Gly–Ψ[(Z)CF=C]-L-Pro–Hyp–(Gly–Pro–Hyp)4–Gly–Gly–Tyr–NH2, Ac–(Gly–Pro–Hyp)3–Gly–Ψ[(Z)CF=C]-D-Pro–Hyp–(Gly–Pro–Hyp)4–Gly–Gly–Tyr–NH2, and the control peptide Ac–(Gly–Pro–Hyp)8–Gly–Gly–Tyr–NH2 were synthesized on solid-phase resin. The CD spectra of all three peptides showed the characteristic collagen triple-helix signature. The folding stability was determined by thermal melting (Tm). The peptide with the fluoro-alkene guest, Gly–Ψ[(Z)CF=C]-L-Pro–Hyp, was found to have a Tm value of 42.2 °C. The Tm of the control peptide was found to be 49.0 °C, a difference in stability of only ΔTm –6.8. Thus, the (Z)-fluoro-alkene as a Gly–Pro isostere forms a relatively stable triple helix. The peptide with the Gly–Ψ[(Z)CF=C]-D-Pro–Hyp guest was shown to have a linear relationship between ellipticity and temperature, indicating that a stable triple helix did not form. The enhanced stability of the (Z)-fluoro-alkene compared to the (E)-alkene Gly–Pro isostere (Tm = 28.3 °C) may be due to a stabilizing n→π* interaction, as determined by NMR deshielding of the 19F nucleus in the collagen-like peptide. In biological systems, isomerization of the prolyl amide is catalyzed by enzymes called PPIases. The PPIase Pin1 specifically catalyzes isomerization of the pSer–Pro sequence from the cis-conformation to the trans-conformation. Pin1 plays a crucial role in the G2→M transition of the cell cycle, implying the importance of cis-trans isomerization. The dipeptides H–Ser–Ψ[(Z)CH=C]-Pro–OH, H–Ser–Ψ[(E)CH=C]-Pro–OH and native H–Ser–Pro–OH were synthesized by literature methods, and activated for aminoacylation of tRNACUA for in vitro transcription-translation. Aminoacylation by chemical methods required the synthesis of a pdCpA dinucleotide. Formation of the dipeptide-dinucleotide complex was not completed because protection of the Ser side chain was problematic. On the other hand, conversion of the dipeptide into the 3,5-dinitrobenzyl ester conjugate allowed for enzymatic aminoacylation using the dFx flexizyme, an RNA enzyme. The native dipeptide was successfully coupled to tRNACUA and is ready for incorporation into a full-length Bora protein by in vitro transcription-translation. Both cis- and trans-locked alkene mimics have been converted to their respective 3,5-dinitrobenzyl ester conjugates. / Doctor of Philosophy / The proline amide (Xaa–Pro) in peptides and proteins is unique in that it allows for cis-trans isomerization. The triple-helix region of human collagen consists mostly of the repeating sequence (Gly–Pro–Hyp)n. Xaa–Pro amide-bond isomerization is rate-limiting for triple-helix formation. We eliminated isomerization at one position in a collagen-like peptide with a locked alkene mimic of Gly–Pro to attempt to stablize the triple-helix. Our computational results predicted that a fluoro-alkene Gly–Pro isostere would be a close geometric match for the native amide. Experimental results showed that a collagen-like peptide with a fluoro-alkene Gly–Pro isostere has an unfolding temperature that is 6.9 °C lower than the native control peptide. 19F NMR data of the collagen-like peptide shows a surprising deshielding of the fluorine nucleus, suggesting its participation in a stabilizing n→π* electronic interaction, similar to the native amide. Isomerization also plays a key role in proper cell division. We followed established methods to synthesize the cis- and trans-locked alkene mimics of Boc–Ser–Pro–OH and converted them into the 3,5-dinitrobenzyl ester conjugates. The 3,5-dinitrobenzyl ester is recognized by the dinitrobenzyl flexizyme (dFx) for enzymatic aminoacylation of tRNA. Once the alkene isosteres are aminoacylated, they will be incorporated into a full-length cell cycle regulatory protein called Bora to determine whether the cis- or trans-Pro state is necessary for healthy human mitosis, and which results in cancerous human mitosis.

Die Metaphasenspindel ist eine selbstorganisierende molekulare Maschine, die die entscheidende Funktion erfüllt, das Genom während der Zellteilung gleichmäßig zu trennen. Spindellänge und -form sind emergente Eigenschaften, die durch komplexe Wechselwirkungsnetzwerke zwischen Molekülen hervorgerufen werden. Obwohl erhebliche Fortschritte beim Verständnis der einzelnen molekularen Akteure erzielt wurden, die ihre Länge und Form beeinflussen, haben wir erst kürzlich damit begonnen, die Zusammenhänge zwischen Spindelmorphologie, Dynamik und Materialeigenschaften zu untersuchen. In dieser Arbeit untersuchte ich zunächst quantitativ die Rolle zweier molekularer Kraftgeneratoren - Kinesin-5 und Dynein - bei der Regulierung der Spindelform von Xenopus-Eiextrakt. Eine Störung ihrer Aktivität veränderte die Spindelmorphologie, ohne die Gesamtmasse der Mikrotubuli zu beeinflussen. Um die Spindelform physikalisch zu stören, wurde ein Optical Stretcher (OS) -Aufbau entwickelt. Obwohl das OS Vesikel in Extrakten verformen könnte, konnte keine Kraft auf Spindeln ausgeübt werden. Die Untersuchung des Brechungsindex der Struktur mittels optischer Beugungstomographie (ODT) ergab, dass es keinen Unterschied zwischen Spindel und Zytoplasma gab. Korrelative Fluoreszenz- und ODT-Bildgebung zeigten, wie sich die Materialeigenschaften innerhalb verschiedener Biomoleküle räumlich unterschieden. Die Gesamttrockenmasse der Spindel skalierte mit der Länge, während die Gesamtdichte konstant blieb. Interessanterweise waren die Spindeln in HeLa-Zellen dichter als das Zytoplasma. Schließlich deckte eine störende Mikrotubulusdichte auf, wie die Gesamttubulinkonzentration die Spindelgröße, die Gesamtmasse und die Materialeigenschaften regulierte. Insgesamt bietet diese Studie eine grundlegende Charakterisierung der physikalischen Eigenschaften der Spindel und hilft dabei, Zusammenhänge zwischen der Biochemie und der Biophysik einer aktiven Form weicher Materie zu beleuchten. / The metaphase spindle is a self-organising molecular machine that performs the critical function of segregating the genome equally during cell division. Spindle length and shape are emergent properties brought about by complex networks of interactions between molecules. Although significant progress has been made in understanding the individual molecular players influencing its length and shape, we have only recently started exploring the links between spindle morphology, dynamics, and material properties. A thorough analysis of spindle material properties is essential if we are to comprehend how such a dynamic structure responds to forces, and maintains its steady-state length and shape. In this work, I first quantitatively investigated the role of two molecular force generators– Kinesin-5 and Dynein in regulating Xenopus egg extract spindle shape. Perturbing their activity altered spindle morphology without impacting total microtubule mass. To physically perturb spindle shape, an Optical Stretcher (OS) setup was developed. Although the OS could deform vesicles in extracts, force could not be exerted on spindles. Investigating the structure’s refractive index using Optical Diffraction Tomography (ODT) revealed that there was no difference between the spindle and cytoplasm. Correlative fluorescence and ODT imaging revealed how material properties varied spatially within different biomolecules. Additionally, spindle mass density and the microtubule density were correlated. The total dry mass of the spindle scaled with length while overall density remained constant. Interestingly, spindles in HeLa cells were denser than the cytoplasm. Finally, perturbing microtubule density uncovered how total tubulin concentration regulated spindle size, overall mass and material properties. Overall, this study provides a fundamental characterisation of the spindle’s physical properties and helps illuminate links between the biochemistry and biophysics of an active form of soft matter.

During mitosis, adherent animal cells undergo a drastic shape change, from essentially flat to round, in a process known as mitotic cell rounding (MCR). The aim of this thesis was to critically examine the physical and biological basis of MCR. The experimental part of this thesis employed a combined optical microscope-atomic force microscope (AFM) setup in conjunction with flat tipless cantilevers to analyze cell mechanics, shape and volume. To this end, two AFM assays were developed: the constant force assay (CFA), which applies constant force to cells and measures the resultant height, and the constant height assay (CHA), which confines cell height and measures the resultant force. These assays were deployed to analyze the shape and mechanical properties of single cells trans-mitosis. The CFA results showed that cells progressing through mitosis could increase their height against forces as high as 50 nN, and that higher forces can delay mitosis in HeLa cells. The CHA results showed that mitotic cells confined to ~50% of their normal height can generate forces around 50-100 nN without disturbing mitotic progression. Such forces represent intracellular pressures of at least 200 Pascals and cell surface tensions of around 10 nN/µm. Using the CHA to compare mitotic cell rounding with induced cell rounding, it was observed that the intracellular pressure of mitotic cells is at least 3-fold higher than rounded interphase cells. To investigate the molecular basis of the mechanical changes inherent in mitotic cell rounding, inhibitors and toxins were used to pharmacologically dissect the role of candidate cellular processes. These results implicated the actomyosin cortex and osmolyte transporters, the most prominent of which is the Na+/H+ exchanger, in the maintenance of mechanical properties and intracellular hydrostatic pressure. Observations on blebbing cells under the cantilever supported the idea that the actomyosin cortex is required to sustain hydrostatic pressure and direct this pressure into cell shape changes. To gain further insight into the relationship between actomyosin activity and intracellular pressure, dynamic perturbation experiments were conducted. To this end, the CHA was used to evaluate the pressure and volume of mitotic cells before, during and after dynamic perturbations that included tonic shocks, influx of specific inhibitors, and exposure to pore-forming toxins. When osmotic pressure gradients were depleted, pressure and volume decreased. When the actomyosin cytoskeleton was abolished, cell volume increased while rounding pressure decreased. Conversely, stimulation of actomyosin cortex contraction triggered an increase in rounding pressure and a decrease in volume. Taken together, the dynamic perturbation results demonstrated that the actomyosin cortex contracts against an opposing intracellular pressure and that this relationship sets the surface tension, pressure and volume of the cell. The discussion section of this thesis provides a comprehensive overview of the physical basis of MCR by amalgamating the experimental results of this thesis with the literature. Additionally, the biochemal signaling pathways and proteins that drive MCR are collated and discussed. An exhaustive and unprecedented synthesis of the literature on cell rounding (approx. 750 papers as pubmed search hits on “cell rounding”, April 2012) reveals that the spread-to-round transition can be thought of in terms of a surface tension versus adhesion paradigm, and that cell rounding can be physically classified into four main modes, of which one is an MCR-like category characterized by increased actomyosin cortex tension and diminution of focal adhesions. The biochemical pathways and signaling patterns that correspond with these four rounding modes are catalogued and expounded upon in the context of the relevant physiology. This analysis reveals cell rounding as a pertinent topic that can be leveraged to yield insight into core principles of cell biophysics and tissue organization. It furthermore highlights MCR as a model problem to understand the adhesion versus cell surface tension paradigm in cells and its fundamentality to cell shape, mechanics and physiology.

During mitosis, adherent animal cells undergo a drastic shape change, from essentially flat to round, in a process known as mitotic cell rounding (MCR). The aim of this thesis was to critically examine the physical and biological basis of MCR. The experimental part of this thesis employed a combined optical microscope-atomic force microscope (AFM) setup in conjunction with flat tipless cantilevers to analyze cell mechanics, shape and volume. To this end, two AFM assays were developed: the constant force assay (CFA), which applies constant force to cells and measures the resultant height, and the constant height assay (CHA), which confines cell height and measures the resultant force. These assays were deployed to analyze the shape and mechanical properties of single cells trans-mitosis. The CFA results showed that cells progressing through mitosis could increase their height against forces as high as 50 nN, and that higher forces can delay mitosis in HeLa cells. The CHA results showed that mitotic cells confined to ~50% of their normal height can generate forces around 50-100 nN without disturbing mitotic progression. Such forces represent intracellular pressures of at least 200 Pascals and cell surface tensions of around 10 nN/µm. Using the CHA to compare mitotic cell rounding with induced cell rounding, it was observed that the intracellular pressure of mitotic cells is at least 3-fold higher than rounded interphase cells. To investigate the molecular basis of the mechanical changes inherent in mitotic cell rounding, inhibitors and toxins were used to pharmacologically dissect the role of candidate cellular processes. These results implicated the actomyosin cortex and osmolyte transporters, the most prominent of which is the Na+/H+ exchanger, in the maintenance of mechanical properties and intracellular hydrostatic pressure. Observations on blebbing cells under the cantilever supported the idea that the actomyosin cortex is required to sustain hydrostatic pressure and direct this pressure into cell shape changes. To gain further insight into the relationship between actomyosin activity and intracellular pressure, dynamic perturbation experiments were conducted. To this end, the CHA was used to evaluate the pressure and volume of mitotic cells before, during and after dynamic perturbations that included tonic shocks, influx of specific inhibitors, and exposure to pore-forming toxins. When osmotic pressure gradients were depleted, pressure and volume decreased. When the actomyosin cytoskeleton was abolished, cell volume increased while rounding pressure decreased. Conversely, stimulation of actomyosin cortex contraction triggered an increase in rounding pressure and a decrease in volume. Taken together, the dynamic perturbation results demonstrated that the actomyosin cortex contracts against an opposing intracellular pressure and that this relationship sets the surface tension, pressure and volume of the cell. The discussion section of this thesis provides a comprehensive overview of the physical basis of MCR by amalgamating the experimental results of this thesis with the literature. Additionally, the biochemal signaling pathways and proteins that drive MCR are collated and discussed. An exhaustive and unprecedented synthesis of the literature on cell rounding (approx. 750 papers as pubmed search hits on “cell rounding”, April 2012) reveals that the spread-to-round transition can be thought of in terms of a surface tension versus adhesion paradigm, and that cell rounding can be physically classified into four main modes, of which one is an MCR-like category characterized by increased actomyosin cortex tension and diminution of focal adhesions. The biochemical pathways and signaling patterns that correspond with these four rounding modes are catalogued and expounded upon in the context of the relevant physiology. This analysis reveals cell rounding as a pertinent topic that can be leveraged to yield insight into core principles of cell biophysics and tissue organization. It furthermore highlights MCR as a model problem to understand the adhesion versus cell surface tension paradigm in cells and its fundamentality to cell shape, mechanics and physiology.

No description available.

Following the discovery of the one-dimensional sequence of human DNA, much focus has been directed on microscopy and molecular techniques to learn about the spatial organization of chromatin in a 3D cell. The development of these powerful tools has enabled high-resolution, genome-wide analysis of chromosome structure under many different conditions. In this thesis, I focus on how the organization of interphase chromatin is established and maintained following mitosis. Mitotic chromosomes are folded into helical loop arrays creating short and condensed chromosomes, while interphase chromosomes are decondensed and folded into a number of structures at different length scales ranging from loops between CTCF sites, enhancers and promoters to topologically associating domains (TADs), and larger compartments. While the chromatin organization at these two very different states is well defined, the transition from a mitotic to interphase chromatin state is not well understood. The aim of this thesis is to determine how interphase chromatin is organized following mitotic chromosome decondensation and to interrogate factors potentially responsible for driving the transition. First, I determine the temporal order with which CTCF-loops, TADs, and compartments reform as cells exit mitosis, revealing a unique structure at the anaphase-telophase transition never observed before. Second, I test the role of transcription in reformation of 3D chromosome structure and show that active transcription is not required for the formation of most interphase chromatin features; instead, I propose that transcription relies on the proper formation of these structures. Finally, I show that RNA in the interphase nucleus can be degraded with only slight consequences on the overall chromatin organization, suggesting that once interphase chromatin structures are achieved, the structures are stable and RNA is only required to reduce the mixing of active and inactive compartments. Together, these studies further our understanding of how interphase structures form, how these structures relate to functional activities of the interphase cell, and the stability of chromatin structures over time.

Indiana University-Purdue University Indianapolis (IUPUI) / Mammalian target of rapamycin (mTOR) is a key regulator of protein synthesis. Dysregulation of mTOR signaling occurs in many human cancers and its inhibition causes arrest at the G1 cell cycle stage. However, mTOR’s impact on mitosis (M-phase) is less clear. Here, suppressing mTOR activity impacted the G2-M transition and reduced levels of M-phase kinase, Aurora A. mTOR inhibitors did not affect Aurora A mRNA levels. However, translational reporter constructs showed that mRNA containing a short, simple 5’-untranslated region (UTR), rather than a complex structure, is more responsive to mTOR inhibition. mTOR inhibitors decreased Aurora A protein amount whereas blocking proteasomal degradation rescues this phenomenon, revealing that mTOR affects Aurora A protein stability. Inhibition of protein phosphatase, PP2A, a known mTOR substrate and Aurora A partner, restored mTOR-mediated Aurora A abundance. Finally, a non-phosphorylatable Aurora A mutant was more sensitive to destruction in the presence of mTOR inhibitor. These data strongly support the notion that mTOR controls Aurora A destruction by inactivating PP2A and elevating the phosphorylation level of Ser51 in the “activation-box” of Aurora A, which dictates its sensitivity to proteasomal degradation. In summary, this study is the first to demonstrate that mTOR signaling regulates Aurora-A protein expression and stability and provides a better understanding of how mTOR regulates mitotic kinase expression and coordinates cell cycle progression. The involvement of mTOR signaling in the regulation of cell migration by its upstream activator, Rheb, was also examined. Knockdown of Rheb was found to promote F-actin reorganization and was associated with Rac1 activation and increased migration of glioma cells. Suppression of Rheb promoted platelet-derived growth factor receptor (PDGFR) expression. Pharmacological inhibition of PDGFR blocked these events. Therefore, Rheb appears to suppress tumor cell migration by inhibiting expression of growth factor receptors that in turn drive Rac1-mediate actin polymerization.

Objetivo: Verificar resposta após debridamento epitelial de córneas humanas. Métodos: Córneas normais foram submetidas a debridamento antes da cirurgia de enucleação. Realizou-se histologia, TUNEL, Ki67, SMA e microscopia eletrônica. Resultados: Seis córneas foram debridadas e preservadas entre ½ e 65 horas, apresentando apoptose nos ceratócitos do estroma anterior. Células estromais em proliferação foram observadas apenas no tempo de 65 horas. Miofibroblastos não foram encontrados. Uma córnea serviu de controle. Conclusões: Os eventos observados em córneas humanas após debridamento epitelial, apoptose e proliferação dos ceratócitos, foram semelhantes aos descritos em animais de experimentação / Purpose: To examine the early wound healing response to epithelial scrape in human corneas. Methods: Normal corneas had epithelial scrape prior to enucleation. Histology, TUNEL assay, Ki67, SMA and transmission electron microscopy were performed. Results: Epithelial scrape was performed in six corneas from ½ to 65 hours prior to preservation. Keratocyte apoptosis was detected in the anterior stroma in all scraped corneas. Keratocyte proliferation was detected exclusively 65 hours after scrape. No myofibroblast was detected. One cornea was not scraped (control). Conclusion: Results obtained in human corneas (keratocyte apoptosis and proliferation) were similar to animal models