The vesicle-mediated degradation of low-density lipoprotein (LDL) is an essential cellular function due to its role in cellular biosynthesis of membranes and steroids. Using multi-color single particle tracking fluorescence microscopy, the intracellular degradation of LDL was probed in live, intact cells. Unique to these experiments is the direct observation of LDL degradation using an LDL-based probe that increases fluorescence intensity upon degradation. Specifically, individual LDL particles were labeled with multiple fluorophores resulting in a quenched fluorescent signal. The characteristics of the vesicle responsible for degradation were determined and the vesicle dynamics involved in LDL degradation were quantified. Visualization of early endosomes, late endosomes and lysosomes was accomplished by fluorescently labeling vesicles with variants of GFP. Transient colocalization of LDL with specific vesicles and the intensity of the LDL particle were measured simultaneously. These studies, which are the first to directly observe the degradation of LDL within a cell, strive to completely describe the endo-lysosomal pathway and quantify the dynamics of LDL degradation in cells.

Το επιστημονικό ενδιαφέρον της παρούσας διδακτορικής διατριβής εστιάζεται στο ρόλο του κυτταροσκελετού της δεσμίνης (desmin), της μυοειδικής πρωτεΐνης, μέλος της οικογένειας των ενδιάμεσων ινιδίων, στη διατήρηση και τη λειτουργία του καρδιακού μυός. H καταστολή της έκφρασης της δεσμίνης σε μύες με τη χρησιμοποίηση της γονιδιακής στόχευσης, έδειξε πως η έκφραση της δεσμίνης είναι απαραίτητη για τη διατήρηση της λειτουργικής και δομικής ακεραιότητας του καρδιακού μυός. Μύες που δεν εκφράζουν δεσμίνη αναπτύσσουν διατατική καρδιομυοπάθεια, που χαρακτηρίζεται από ανωμαλίες σε υποκυτταρικά οργανίδια, όπως είναι τα μιτοχόνδρια, εκτεταμένη ίνωση και εναπόθεση ασβεστίου, με επακόλουθο τον κυτταρικό θάνατο, και τελικά την καρδιακή ανεπάρκεια. Πιο συγκεκριμένα σε μια προσπάθεια διερεύνησης της βιολογικής δράσης της δεσμίνης, μελετήθηκαν οι πρωτεϊνικές αλληλεπιδράσεις αυτού του μορίου. Η ανεύρεση μορίων με τα οποία η δεσμίνη αλληλεπιδρά μπορεί να δώσει σημαντικές πληροφορίες για τη βιολογικής της δράση και να βοηθήσει στην κατανόηση της παθολογίας που παρατηρείται σε επίμυς όπου έχει κατασταλεί η έκφρασή της (διαγονιδιακό μοντέλο διατατικής καρδιομυοπάθειας που έχει δημιουργηθεί από το εργαστήριό μας, des-/-) ή σε ασθενείς με καρδιομυοπάθειες εξαιτίας μεταλλαγών στο μόριο της δεσμίνης. Για το σκοπό αυτό εφαρμόστηκε η μέθοδος του διυβριδικού συστήματος ζύμης (Yeast two-hybrid), όπου χρησιμοποιήθηκε τμήμα του μορίου της δεσμίνης (το αμινο-τελικό άκρο), με το οποίο ‘σαρώθηκε’ (screening) μια cDNA καρδιακή βιβλιοθήκη, και βρέθηκε ένας αριθμός δυνητικών αλληλεπιδράσεων. Η σάρωση αποκάλυψε την αλληλεπίδραση της δεσμίνης με ένα νέο μόριο, τη Myospryn, μια μυοειδική πρωτεΐνη 413 kDa, μέλος της οικογένειας των TRIM-like πρωτεϊνών. Η πρωτεΐνη myospryn βρέθηκε αρχικά να αλληλεπιδρά με την πρωτεΐνη dysbindin, η οποία εμπλέκεται σε διαδικασίες διαμετακίνησης πρωτεϊνών, κυστιδίων και βιοσύνθεσης οργανιδίων, και αποτελεί συστατικό ενός πρωτεϊνικού συμπλόκου υπεύθυνου για τη βιογένεση λυσοσωμάτων και λυσοσωματικά συσχετιζόμενων οργανιδίων, γνωστό ως Biogenesis of Lysosome Related Organelles (BLOC-1). Η πρόσδεση της δεσμίνης με τη myospryn επαληθεύτηκε με in vitro μεθόδους (GST pull down assay, Co-immunoprecipitation). H ανάλυση ανοσοαποτύπωσης (western blot) απεκάλυψε ότι το ανοσολογικό σύμπλοκο που κατακρημνίζεται με χρήση αντισωμάτων δεσμίνης περιέχει, εκτός της myospryn, και δυο τουλάχιστον υπομονάδες του πρωτεϊνικού συμπλόκου BLOC-1, dysbindin και pallidin. Πειράματα με το δι-υβριδικό σύστημα ζύμης απεκάλυψαν την ακριβή περιοχή αλληλεπίδρασης των δύο πρωτεϊνών, και η οποία περιλαμβάνει τα αα58-103 του αμινο-τελικού άκρου της δεσμίνης και τα τελευταία 24αα του καρβόξυ-τελικού άκρου του μορίου της myospryn, που αντιστοιχούν στο SPRY τμήμα του TRIM-like μοτίβου της οικογένειας των πρωτεϊνών στην οποία ανήκει η myospryn. Πειράματα ανοσοφθορισμού σε απομονωμένα καρδιομυοκύτταρα απεκάλυψαν ότι η myospryn τοπολογείται στην περιφέρεια του πυρήνα, σε στενή γειτνίαση με τις μεμβράνες του ενδοπλασματικού δικτύου, και στο σημείο αυτό αλληλο-επικαλύπτεται με τη δεσμίνη, η οποία σχηματίζει ένα δίκτυο ινιδίων που διατρέχει όλο το μήκος του κυττάρου. Στον ενήλικο καρδιακό ιστό, η myospryn βρίσκεται κυρίως στους εμβόλιμους δίσκους, καθώς και στο επίπεδο της πλασματικής μεμβράνης, του σαρκολείματος, όπου και αλληλοεπικαλύπτεται με τη δεσμίνη. Η αλληλο-επικάλυψη των δυο πρωτεϊνών είναι έντονη στο επίπεδο των εμβόλιμων δίσκων. Δείχθηκε πως η myospryn άλληλο-επικαλύπτεται με τα λυσοσώματα καθώς και ότι η δεσμίνη είναι απαραίτητη τόσο για τη σωστή τοπολόγηση της myospryn, όσο και για αυτήν των λυσοσωμάτων. Τέλος, με πειράματα καταστολής τηw έκφρασης της myospryn, αποκαλύφθηκε πως η παρουσία της myospryn είναι απαραίτητη προκειμένου η δεσμίνη να μπορεί να ανοσοκατακρημνίσει τις υπομονάδες του BLOC-1, dysbindin και pallidin Τέλος, στην προσπάθεια χαρακτηρισμού του λειτουργικού ρόλου της myospryn συμπεριλήφθηκε η χρησιμοποίηση του μοντέλου του D. rerio (zebrafish). Χρησιμοποιώντας την τεχνολογία των antisense morpholinos, ήταν δυνατή η παρεμπόδιση της έκφρασης της myospryn και η μελέτη των επιπτώσεων στην ανάπτυξη και λειτουργία του καρδιακού ιστού. Η καταστολή της έκφρασης της myospryn είχε ως αποτέλεσμα σοβαρές δομικές και λειτουργικές ανωμαλίες στο καρδιακό σύστημα ατόμων Danio rerio, καταδεικνύοντας την σπουδαιότητα αυτού του μορίου στην ανάπτυξη του καρδιακού συστήματος του D.rerio. Συμπερασματικά η παρούσα διδακτορική διατριβή διαπραγματεύεται τις πρωτεϊνικές αλληλεπιδράσεις της μυοειδικής πρωτεΐνης δεσμίνης, αποκαλύπτοντας νέους ρυθμιστικούς ρόλους για το μόριο αυτό. / The scientific interest of this PhD thesis is mainly the role of desmin cytoskeleton (the muscle-specific intermediate filament) in myocyte maintenance and function. The ablation of desmin expression in mice by gene targeting demonstrated that desmin expression is crucial for maintanance of healthy muscle. Mice null for desmin develop dilated cardiomyopathy characterized by mitochondrial abnormalities, extensive cardiomyocyte death, fibrosis, calcification and eventually heart failure. Study of desmin protein interactions could facilitate our understanding of the molecular mechanisms responsible for the observed pathology. To that end, we have performed a yeast two-hybrid screening of a cardiac cDNA library We showed that the desmin amino-terminal domain [Naa(1 -103)] binds to a 413kDa TRIM-like protein, myospryn, originally identified as the muscle-specific partner of dysbindin, a component of the Biogenesis of Lysosome Related Organelles Complex 1 (BLOC-1). Binding of desmin with myospryn was confirmed with GST pull down assays and co-immunoprecipitation experiments. Western blot analysis revealed that the complex immunoprecipitated by desmin antibodies, in addition to myospryn contained the BLOC-1 components dysbindin and pallidin. Deletion analysis revealed that only the [Naa(1 -103)] fragment of desmin binds to myospryn carboxyl terminus and that this association takes place through the 24aa long C-terminal end of the SPRY domain of myospryn. Using an antibody against the COOH terminus of myospryn, we demonstrated that myospryn co-localizes with desmin at the periphery of the nucleus, in close proximity to the endoplasmic reticulum, of mouse neonatal cardiomyocytes. In adult heart muscle, the two proteins co localize, predominantly, at intercalated discs and costameres. We also showed that myospryn colocalizes with lysosomes. Using desmin null hearts, we demonstrated that desmin is required for both the proper perinuclear localization of myospryn, as well as the proper positioning of lysosomes, thus suggesting a potential role of desmin IFs in lysosomes and lysosome-related organelle biogenesis and/or positioning. siRNAs experiments for myospryn knock down revealed that when myospryn is absent, desmin is not able to immunoprecipitate BLOC-1 subunits. Additionally, the in vivo role of myospryn was examined using the model of D. rerio. Using a splice morpholino for knocking down myospryn transcript, we examined the effects on zebrafish heart development and function caused by the absence of myospryn, where it was shown that myospryn is essential for the early heart development in D.rerio. In summary, this work reveals the association of desmin with a novel molecule, myospryn, and it sheds light on novel regulatory role for this member of intermediate filament family.

Thesis (Ph. D.)--University of Adelaide, Dept. of Paediatrics, 1991. / Copies of author's previously published articles inserted. Includes bibliographical references.

Thesis (Ph.D.)-- University of Adelaide, Dept. of Paediatrics, 2001. / Addendum inserted at back. Includes bibliographical references (leaves 266-287).

Thesis (M.S.) -- Mississippi State University. Department of Chemistry. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 152-161).

Thesis (Ph.D.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 123 pages. Includes Vita. Includes bibliographical references.

A flagelina é um agonista natural do sensor TLR5 e do inflamassoma NAIP/NLRC4 que é responsável pela secreção de IL-1β e IL-18 e pela indução de morte celular necrótica, via ativação da caspase-1. Entretanto, nós observamos que a inserção da flagelina de B. subtilis no citosol celular por meio de vesículas lipídicas, induz um processo atípico de morte nos macrófagos peritoneais (PMs) deficientes em NLRC4, ASC e caspase-1/11. A morte dos PMs manteve seu resultado antimicrobiano, sendo acompanhada da liberação de IL-1α. A morte celular e a secreção das citocinas IL-1α e IL-1β, foi mediada por catepsinas lisossomais, sugerindo uma cooperação entre a via lisossomal e os inflamassomas nas respostas induzidas pela flagelina. Além disso, a flagelina de S. typhimurium foi capaz de induzir dano lisossomal e secreção de IL-1α e IL-1β mediada pelo eixo caspase-catepsinas, na ausência de carreadores, e estas citocinas tiveram um impacto na imunidade adaptativa induzida pela flagelina, no modelo de ativação de linfócitos T específicos por células dendríticas, in vitro. / Flagellin is a natural agonist of TLR5 and NAIP/NLRC4 inflammasome that is responsible for IL-1β and IL-18 secretion and for the induction of a necrotic cell death, both mediated by caspase-1. However, we observed that flagellin from B. subtilis inserted into lipid vesicles, induced an atypical cell death in peritoneal macrophages (PMs) in the absence of NLRC4, ASC and caspase-1/11. This inflammasome-independent cell death retained its antimicrobial outcome, being accompanied with IL-1α secretion. Importantly, cell death and caspase-1-dependent IL-1α and IL-1β secretion were regulated by lysosomal cathepsins, suggesting a cooperation between the inflammasome and lysosomal pathway in response to flagellin. We also observed that flagellin from S. typhimurium is able to induce lysosomal damage and IL-1α and IL-1β secretion by PMs in the absence of a carrier, through a caspase-catepsins-dependent manner, and that cytokines were important to the ability of flagellin in to induce adaptive immune response by antigen-specific T cells.

Le cancer est une maladie multifactorielle définie par un développement rapide de cellules anormales. Les cellules malignes acquièrent des avantages compétitifs qui permettent une croissance et prolifération anormales, grâce à un large spectre de changements génétiques et épigénétiques conduisant à des changements majeurs dans les profils de transcriptome et protéome et ainsi des modifications dans des voies de signalisation, le trafic intracellulaire et le métabolisme. Des nombreuses voies cellulaires ont été étudiées dans le contexte du cancer, y compris la signalisation, la migration, la perte de la polarité cellulaire apico-basale et l'adhésion cellulaire, cependant très peu est connu sur les altérations au niveau des organelles. Cette thèse a comme objectif d'identifier des altérations dans les compartiments intracellulaires et d'étudier leurs corrélations avec la progression du cancer. Dans la culture cellulaire classique, l'étude systématique de l'organisation du positionnement relatif des organelles est difficile en raison des fortes hétérogénéités morphologiques des cellules. Pour contourner ce problème, nous avons utilisé l’innovante technique des micro-patrons combinée à des cartes de densité des organelles. Après une analyse systématique de différentes lignées cellulaires représentant différents grades du cancer de la vessie, nous avons identifié des changements dans le positionnement de plusieurs organelles. Le changement de position le plus important a été observé pour les lysosomes, dont la distribution était plus périphérique dans les cellules représentant des grades plus avancées du cancer de la vessie. Ceci suggère que le positionnement des lysosomes pourrait être potentiellement important dans la progression du cancer. Par conséquent, nous avons cherché à caractériser l'impact de l’altération des lysosomes sur le comportement des cellules transformées. Nous avons constaté que les changements dans le positionnement des lysosomes jouent un rôle dans l'invasion des cellules cancéreuses de la vessie. En effet, le transport antérograde des lysosomes est en corrélation avec l’invasion 3D, contrairement au transport rétrograde qui corrèle avec une diminution de l’invasion cellulaire. Enfin, nous avons étudié les mécanismes moléculaires par lesquels les altérations du lysosome ont un impact sur l'invasion cellulaire. / Cancer is a multifactorial disease defined by a rapid development of abnormal cells. Malignant cells acquire competitive advantages for growth and proliferation through a big spectrum of genetic and epigenetic changes leading to major changes in the transcriptome and proteome profiles and thus to alterations in multiple signaling pathways, intracellular trafficking and metabolism. Although many cellular pathways have been studied in the context of cancer, including signaling, migration, loss of apical-basal cell-polarity and cell adhesion, little is known about cancer-related alterations on the sub-cellular, organelle level. This PhD thesis aimed to identify alterations in intracellular compartments and to study how these changes correlate with cancer progression. In classical culture, the systematic study on the organization and relative positioning of organelles is challenging because of the strong morphological cell-to-cell variations. To overcome this problem, we used innovative micro-patterning technique in combination with quantitative, probabilistic mapping of cell organelles. Using a systematic analysis of different cell lines representing different stages of bladder cancer, we identified several changes in the positioning of organelles. The most striking phenotype was revealed by lysosomes, whose distribution was more peripheral in cells representing higher grades of bladder cancer. This suggested that lysosome positioning could be potentially relevant in cancer progression. Therefore, we aimed to characterize the impact of lysosome alteration on cell behavior in transformed cells. We found that changes in lysosome positioning played a role on bladder cancer cell invasion. Indeed, anterograde transport of lysosomes correlate with 3D invasion behavior, contrary to retrograde transport that correlated with decreased cell invasion. Finally, we studied about the molecular mechanisms by which lysosome alterations impact cell invasion.

In the last decade, a tremendous gain in knowledge concerning the molecular events of apoptosis signaling and execution has been achieved. The aim of this thesis was to clarify the role of lysosomal membrane permeabilization and lysosomal proteases, cathepsins, in signaling for apoptosis. We identified cathepsin D as an important factor in staurosporine-induced human fibroblast cell death. After release to the cytosol, cathepsin D promoted mitochondrial release of cytochrome c by proteolytic activation of Bid. Cathepsin D-mediated cleavage of Bid generated two fragments with the apparent molecular mass of 15 and 19 kDa. By sequence analysis, three cathepsin D-specific cleavage sites, Phe24, Trp48, and Phe183, were identified. Moreover, we investigated the mechanism by which cathepsins escape the lysosomal compartment, and found that Bax is translocated from the cytosol to lysosomes upon staurosporine treatment. In agreement with these data, recombinant Bax triggered release of cathepsins from isolated rat liver lysosomes. Conceivably, the Bcl-2 family of proteins may govern release of pro-apoptotic factors from both lysosomes and mitochondria. The importance of lysosomal cathepsins in apoptosis signaling was studied also in oral squamous cell carcinoma cells following exposure to the redox-cycling drug naphthazarin or agonistic anti-Fas antibodies. In this experimental system, cathepsins were released to the cytosol, however, inhibition of neither cathepsin D, nor cysteine cathepsin activity suppressed cell death. Interestingly, cysteine cathepsins still appeared to be involved in activation of the caspase cascade. Cathepsins are often overexpressed and secreted by cancer cells, and it has been reported that extracellular cathepsins promote tumor growth and metastasis. Here, we propose that cathepsin B secreted from cancer cells may suppress cancer cell death by shedding of the Fas death receptor. Defects in the regulation of apoptosis contribute to a wide variety of diseases, such as cancer, neurodegeneration and autoimmunity. Increased knowledge of the molecular details of apoptosis could lead to novel, more effective, treatments for these illnesses. This thesis emphasizes the importance of the lysosomal death pathway, which is a promising target for future therapeutic intervention. / In the last decade, a tremendous gain in knowledge concerning the molecular events of apoptosis signaling and execution has been achieved. The aim of this thesis was to clarify the role of lysosomal membrane permeabilization and lysosomal proteases, cathepsins, in signaling for apoptosis. We identified cathepsin D as an important factor in staurosporine-induced human fibroblast cell death. After release to the cytosol, cathepsin D promoted mitochondrial release of cytochrome c by proteolytic activation of Bid. Cathepsin D-mediated cleavage of Bid generated two fragments with the apparent molecular mass of 15 and 19 kDa. By sequence analysis, three cathepsin D-specific cleavage sites, Phe24, Trp48, and Phe183, were identified. Moreover, we investigated the mechanism by which cathepsins escape the lysosomal compartment, and found that Bax is translocated from the cytosol to lysosomes upon staurosporine treatment. In agreement with these data, recombinant Bax triggered release of cathepsins from isolated rat liver lysosomes. Conceivably, the Bcl-2 family of proteins may govern release of pro-apoptotic factors from both lysosomes and mitochondria. The importance of lysosomal cathepsins in apoptosis signaling was studied also in oral squamous cell carcinoma cells following exposure to the redox-cycling drug naphthazarin or agonistic anti-Fas antibodies. In this experimental system, cathepsins were released to the cytosol, however, inhibition of neither cathepsin D, nor cysteine cathepsin activity suppressed cell death. Interestingly, cysteine cathepsins still appeared to be involved in activation of the caspase cascade. Cathepsins are often overexpressed and secreted by cancer cells, and it has been reported that extracellular cathepsins promote tumor growth and metastasis. Here, we propose that cathepsin B secreted from cancer cells may suppress cancer cell death by shedding of the Fas death receptor. Defects in the regulation of apoptosis contribute to a wide variety of diseases, such as cancer, neurodegeneration and autoimmunity. Increased knowledge of the molecular details of apoptosis could lead to novel, more effective, treatments for these illnesses. This thesis emphasizes the importance of the lysosomal death pathway, which is a promising target for future therapeutic intervention.