Endosomal interactions during root hair growth

The dynamic localization of endosomal compartments labeled with targeted fluorescent protein tags is routinely followed by time lapse fluorescence microscopy approaches and single particle tracking algorithms. In this way trajectories of individual endosomes can be mapped and linked to physiological processes as cell growth. However, other aspects of dynamic behavior including endosomal interactions are difficult to follow in this manner. Therefore, we characterized the localization and dynamic properties of early and late endosomes throughout the entire course of root hair formation by means of spinning disc time lapse imaging and post-acquisition automated multitracking and quantitative analysis. Our results show differential motile behavior of early and late endosomes and interactions of late endosomes that may be specified to particular root hair domains. Detailed data analysis revealed a particular transient interaction between late endosomes—termed herein as dancing-endosomes—which is not concluding to vesicular fusion. Endosomes preferentially located in the root hair tip interacted as dancing-endosomes and traveled short distances during this interaction. Finally, sizes of early and late endosomes were addressed by means of super-resolution structured illumination microscopy (SIM) to corroborate measurements on the spinning disc. This is a first study providing quantitative microscopic data on dynamic spatio-temporal interactions of endosomes during root hair tip growth

Motion Analysis of Live Objects by Super-Resolution Fluorescence Microscopy

Motion analysis plays an important role in studing activities or behaviors of live objects in medicine, biotechnology, chemistry, physics, spectroscopy, nanotechnology, enzymology, and biological engineering. This paper briefly reviews the developments in this area mostly in the recent three years, especially for cellular analysis in fluorescence microscopy. The topic has received much attention with the increasing demands in biomedical applications. The tasks of motion analysis include detection and tracking of objects, as well as analysis of motion behavior, living activity, events, motion statistics, and so forth. In the last decades, hundreds of papers have been published in this research topic. They cover a wide area, such as investigation of cell, cancer, virus, sperm, microbe, karyogram, and so forth. These contributions are summarized in this review. Developed methods and practical examples are also introduced. The review is useful to people in the related field for easy referral of the state of the art

Development of single molecule-sensitive, imaging probes targeting native RNA

The localization, trafficking and regulation of messenger ribonucleic acids (RNA) and viral RNA play crucial roles in cellular homeostasis and disease pathogenesis. In recent years biochemical and molecular biology methods used to study RNA function have made several important advances in the areas of RNA interference, expression of transgenes, and the sequencing of transcriptomes. In contrast, current technologies for imaging RNA in live cells remain in limited use. Previous studies of RNA localization and dynamics have relied primarily on the expression of a reporter RNA and a fluorescent protein fusion that binds to aptamer sequences in the expressed RNA. While these plasmid based systems offer methodological flexibility, there remains a need to develop methods to image native, non-engineered RNA as plasmid derived RNAs may not have the same regulatory elements (3'UTR and introns) or copy number as the native RNA. Additionally, viral pathogenesis is often sensitive to the size and sequence of their genomic RNA and may not be suitable for study using engineered systems. We sought to develop and validate a new method for imaging native, non-engineered RNA with single molecule-sensitivity. These probes have four important properties. They are modular, compatible with fixation and immunostaining, bind quickly and specifically to targets, and do not interfere with RNA function. We built upon the technique of delivering exogenous, linear probes that bind to their target by Watson-Crick base pairing. The probes are multiply labeled and tetramerized to increase their brightness. To validate the probes, targeting and utility was demonstrated in two model systems: beta-actin mRNA to show targeting of an endogenous target and the genomic RNA of human respiratory syncytial virus to show targeting of a viral RNA target. All video files are in QuickTime format.PhDCommittee Chair: Santangelo, Philip; Committee Member: Bassell, Gary; Committee Member: Bellamkonda, Ravi; Committee Member: Crowe, James; Committee Member: Le Doux, Josep

Targeted Nanodiamonds for Identification of Subcellular Protein Assemblies in Mammalian Cells

Transmission electron microscopy (TEM) can be used to successfully determine the structures of proteins. However, such studies are typically done ex situ after extraction of the protein from the cellular environment. Here we describe an application for nanodiamonds as targeted intensity contrast labels in biological TEM, using the nuclear pore complex (NPC) as a model macroassembly. We demonstrate that delivery of antibody-conjugated nanodiamonds to live mammalian cells using maltotriose-conjugated polypropylenimine dendrimers results in efficient localization of nanodiamonds to the intended cellular target. We further identify signatures of nanodiamonds under TEM that allow for unambiguous identification of individual nanodiamonds from a resin-embedded, OsO4-stained environment. This is the first demonstration of nanodiamonds as labels for nanoscale TEM-based identification of subcellular protein assemblies. These results, combined with the unique fluorescence properties and biocompatibility of nanodiamonds, represent an important step toward the use of nanodiamonds as markers for correlated optical/electron bioimaging.Comment: 38 pages, 6 figures, SI section with 3 figure

Discrete arginine topologies guide escape of miniature proteins from early endosomes to the cytoplasm

Polypeptides and peptide mimetics sample a wide chemical space with broad potential to modulate cellular function, but their application to cytoplasmic targets is limited because when added to cells their cytosolic concentration remains low. This limitation is due to a diffusion barrier (the plasma membrane) and absence of dedicated import machinery. Highly cationic peptides and proteins sometimes gain cytosolic access, but how they do so is not well understood. Using a small library of cationic miniature proteins, I probe the influence of positive charge number and orientation on the ability the miniature protein to access the cytoplasm. Using a novel assay, I identify a cationic miniature protein, which we called 5·3, that carries a discrete arginine motif and efficiently reaches the cytoplasm. Database searches find that the precise motif identified (an arginine present in positions i, i + 4, i + 7, i + 10, and i + 11 of an alpha-helix) is not present in nature, but that similar motifs are present in natural proteins that interact with cellular membranes. Finally, I examine the cellular pathway by which 5·3 reaches the cytoplasm. I find that this miniature protein enters the cell via a dynamin and cholesterol dependent endocytic mechanism and is delivered to Rab5+ early endosomes. In contrast to the shiga-like toxins, and many non-enveloped viruses (which escape to the cytoplasm from the endoplasmic reticulum) as well as other peptides previously identified as \u27cell penetrating\u27, only 5·3 escapes from early endosomes. These findings should enable the future dissection of the precise molecular events underlying cytoplasmic access of peptides and proteins, and may illuminate principles for the engineering of peptides and peptidomimetics that access cytoplasmic targets

A Patch-Based Method for Repetitive and Transient Event Detection in Fluorescence Imaging

Automatic detection and characterization of molecular behavior in large data sets obtained by fast imaging in advanced light microscopy become key issues to decipher the dynamic architectures and their coordination in the living cell. Automatic quantification of the number of sudden and transient events observed in fluorescence microscopy is discussed in this paper. We propose a calibrated method based on the comparison of image patches expected to distinguish sudden appearing/vanishing fluorescent spots from other motion behaviors such as lateral movements. We analyze the performances of two statistical control procedures and compare the proposed approach to a frame difference approach using the same controls on a benchmark of synthetic image sequences. We have then selected a molecular model related to membrane trafficking and considered real image sequences obtained in cells stably expressing an endocytic-recycling trans-membrane protein, the Langerin-YFP, for validation. With this model, we targeted the efficient detection of fast and transient local fluorescence concentration arising in image sequences from a data base provided by two different microscopy modalities, wide field (WF) video microscopy using maximum intensity projection along the axial direction and total internal reflection fluorescence microscopy. Finally, the proposed detection method is briefly used to statistically explore the effect of several perturbations on the rate of transient events detected on the pilot biological model

Cryoimmunoelectron microscopic localization of synaptic proteins in nerve terminals and astrocytic cells

In the present study the cryo-immunogold technique was used and optimized for investigating the ultrastructure and immunolabeling of synaptic proteins. It is evidently a suitable method for the localization of membrane proteins since the antigens are not treated with any chemical denaturation before immunolabeling except for the fixation and since the antigens are directly exposed to the surface of the cryo-ultrasections. The v-SNARE VAMP II and the vesicle-associated proteins SV2 and Rab3A were detected extensively at small vesicles in the mossy fiber terminals. The t-SNARE SNAP-25, and N-type and P/Q type Ca2+ channels were allocated to the plasma membrane both at the active zone and outside the active zone. SNAP-25 and N-type Ca2+ channels appeared also at synaptic vesicles. A significantly increased immunolabeling of VAMP II, SV2, Rab3A, SNAP-25 and N-type Ca2+ channels was found at the active zones of fast synapses, indicating a concentration of these proteins at sites of exocytosis. The widespread distribution of the t-SNARE SNAP-25 at the axonal plasma membrane reveals that membrane-targeting specificity cannot be determined solely by v/t-SNARE interactions. Additional control components are required to assure the docking and exocytosis of the synaptic vesicles at active zones. The novel protein Bassoon was only found at active zones of central synapses and showed the highest specific labeling among all proteins investigated. Its labeling pattern implies an association of Bassoon with the presynaptic dense projections, the structural guide for vesicle exocytosis. The involvement of Bassoon in the organization of the neurotransmitter release site suggests that Bassoon may play an important role in determining the specificity of vesicle docking and fusion. In the neurosecretory endings of neurohypophysis the synaptic proteins VAMP II, SNAP- 25, SV2, Rab3A, and the N-type Ca2+ channels showed a preferential labeling over microvesicles. Moreover, the immunolabeling intensity of these proteins over microvesicles corresponded closely to that over synaptic vesicles. This suggests that these synaptic proteins share an identical association with synaptic vesicle and microvesicles. A significant labeling of SNAP-25, the N-type Ca2+ channels and VAMP II was also detected at the plasma membrane near the clustered microvesicles, indicating the competence of microvesicles for docking and exocytosis along the plasma membrane in the absence of active zones. No significant labeling of VAMP II, SNAP-25, SV2 and N-type Ca2+ channel was observed at the membrane of neurosecretory granules. This is in agreement with the notion that synaptic vesicles and microvesicles possess regulatory mechanisms for exocytosis different from those of granules. In contrast, a/ß-SNAP and NSF were found on the granules, and Rab3A and the P/Q-type Ca2+ channels on granules in a subset of terminals. Rab3A is associated specifically with the oxytocin-containing granule population. Interestingly, some plasma membrane proteins, such as SNAP-25 and even N-type Ca2+ channels and P/Q-type Ca2+ channels, were observed not only at the plasma membrane but also at the vesicular organelles. This suggests that these vesicular organelles may be involved in transporting newly synthesized proteins from the soma to the plasma membrane of the terminal. Furthermore, the vesicular pool of the Ca2+ channels may serve in the stimulationinduced translocation into the plasma membrane when required. Using the conventional preembedding method with Epon and the post-embedding method with LR Gold, VAMP II was localized at vesicular organelles of varying size and on horseradish peroxidase filled endocytic organelles in cultured astrocytes, with and without stimulation in the presence of the horseradish peroxidase. This indicates that VAMP II is involved in the cycle of vesicular exocytosis and endocytosis in astrocytes. U373 cells are capable of expressing all three members of the synaptic SNARE complex (v-SNARE VAMP II, t-SNARE syntaxin I and SNAP25). This indicates the competence of U373 to carry out regulated exocytosis by means of the classical SNARE mechanism. In addition, the ubiquitous v-SNARE cellubrevin and the endosome-associated small GTPbinding protein Rab5 could be expressed in U373 cells. All recombinant synaptic proteins investigated in U373 cells revealed a punctuate cellular distribution under the fluorescence microscope, suggesting that they are mainly associated with intracellular compartments. The cryo-electron microscopy provided direct evidence for the association of all expressed proteins with electron-lucent vesicular organelles. It further supports the potential of U373 MG cells to release low molecular weight messengers by a regulated exocytosis mechanism. In addition, myc-VAMP II was found on dispersed granules. Probably, VAMP II also participates in the exocytosis event of granules in U373 cells. Gold labeling for the two presumptive t-SNAREs syntaxin I and SNAP-25 in U373 cells was confined to the vesicular organelles. At the ultrastructural level no significant labeling was identified at the plasma membrane. The high level of colocalization of the two SNARE proteins VAMP II and syntaxin I in the cell body and in cell processes suggests that the two proteins are mostly sorted into identical vesicular organelles. A partial colocalization of VAMP II and cellubrevin as well as of VAMP II and Rab5 was observed under the fluorescence microscope. At the ultrastructural level, a colocalization of VAMP II and cellubrevin as well as of VAMP II and Rab5 was found on some clustered vesicles. The partial colocalization of VAMP II and cellubrevin implies that they similarly function as v-SNAREs. The partial colocalization of Rab5 with VAMP II in U373 cells suggests that the endosomal protein Rab5 is associated with VAMP II-containing organelles during some stages of their life cycle.Die Freisetzung von Neurotransmittern aus kleinen synaptischen Vesikeln schneller Synapsen hängt von dem koordinierten Zusammenwirken spezifischer Vesikelproteine ab. Genetische, physiologische und biochemische Untersuchungen legen nahe, dass sich viele synaptische Proteine - einschließlich der Mitglieder der SNARE-Familie - an den Exozytose-Prozessen der synaptischen Vesikel beteiligen. Die genauen molekularen Mechanismen der Vesikel-Exozytose sind jedoch weitgehend unbekannt. Es fehlen außerdem eindeutige morphologische Nachweise der bevorzugten Lokalisation dieser Proteine an den aktiven Zonen des Synapsen, die ihre Assoziation mit der schnellen Neurotransmitter- Freisetzung in situ zeigen könnten. Die fundamentalen Mechanismen des "membrane traffic" gelten bei allen eukaryotischen Zellen als phylogenetisch konserviert. Dies bedeutet, dass ähnliche Proteine für die Kontrolle der Membranfusion sowohl bei Neuronen als auch bei anderen nicht-neuralen Zellen benutzt werden können. Wie zahlreiche jüngere Untersuchungen zeigen, können Astrozyten ihre Botenstoffe mittels eines Neuronenähnlichen sekretorischen Prozesses freisetzen. Dabei wird vermutlich eine regulierte Vesikel- Exozytose und besonders der klassische SNARE-Mechanismus in den Sekretionsprozess miteinbezogen. Allerdings gibt es für den Vesikelzyklus bei Astrozyten kaum morphologische Nachweise für die Funktion der Exozytose-relevanten Proteine. In der vorliegenden Arbeit wurden daher die Lokalisation von SNARE-Proteinen sowie anderen Vesikel-relevanten Proteinen an zwei unterschiedlichen Nerventerminalen sowie bei astrozytären Zellen mittels Immunfluoreszenz und Elektronenmikroskopie untersucht. Ziel dieser Arbeit ist es, ein besseres Verständnis der Funktion dieser Proteine bei der Freisetzung der Botenstoffe zu erhalten. 1. Lokalisation der synaptischen Proteine an zwei unterschiedlichen Typen von Nervenendigungen Zum morphologischen Nachweis der verschiedenen synaptischen Proteine wurde die Methode der Immungoldmarkierung an ultradünnen Kryoschnitten angewendet. Untersucht und analysiert wurden die Lokalisation und die Verteilung dieser synaptischen Proteine an (1) den großen Moosfaserendigungen mit vielen aktiven Zonen in der hippocampalen CA3 Region, und an (2) den neurosekretorischen Terminalen ohne spezielle Freisetzungsstellen in Neurohypophyse. Hier war besonders von Interesse, ob SNARE-Proteine vorzugsweise mit aktiven Zonen in den schnellen Synapsen verbunden sind, und inwieweit die Lokalisation dieser Proteine in solchen Endigungen, die keine definierten Freisetzungsstellen besitzen, von denjenigen in den synaptischen Terminalen verschieden sind. In den hippokampalen Moosfaserendigungen wurden das v-SNARE VAMP II und die weiteren Vesikel-gebundenen Proteine SV2 und Rab3A umfassend an den synaptischen Vesikeln lokalisiert. Das t-SNARE SNAP-25, und N- und P/Q-Typ Ca2+-Kanäle ließen sich an der Plasmamembran in und außerhalb der aktiven Zone nachweisen. SNAP-25 und N-Typ Ca2+-Kanäle wurden auch an synaptischen Vesikeln gefunden. Die quantitative Analyse zeigte eine deutlich erhöhte Immunmarkierung von VAMP II, SV2, Rab3A, SNAP-25, und N-Typ Ca2+-Kanäle an den aktiven Zonen. Dies deutet auf eine direkte Teilnahme solcher Proteine an der Exozytose von synaptischen Vesikeln in den schnellen zentralen Synapsen. Besonders die Markierung von VAMP II und SNAP-25 dicht an den angedockten synaptischen Vesikeln und an der Plasmamembran in aktiven Zonen weist auf eine Assoziation von diesen Proteinen mit dem präsynaptischen Docking-Komplex hin. Der Nachweis von N-Typ und P/Q-Typ Ca2+-Kanäle an den aktiven Zonen liefert einen morphologischen Beleg für ihre Beteiligung an der Regulierung der schnellen Neurotransmission. Er unterstützt auch die Hypothese, dass diese beiden Ca2+-Kanäle mit dem SNARE-Komplex interagieren können. Die breite Verteilung von SNAP-25 an der axonalen Plasmamembran weist darauf hin, dass seine Funktion nicht wesentlich für die Positionierung der Freisetzungsstelle von synaptischen Vesikeln sein dürfte. Die Docking-Spezifität der Vesikel wird nicht nur durch die v/t- SNARE-Interaktion definiert. Zusätzliche kontrollierende Faktoren sind folglich erforderlich, um "docking" und Exozytose der synaptische Vesikel an der aktiven Zone zu gewährleisten. Das hochmolekulare Protein Bassoon war hauptsächlich an den aktiven Zonen der zentralen Synapsen zu finden. Direkt auf der Plasmamembran waren jedoch keine Markierungen nachzuweisen. Das Verteilungsmuster deutet auf eine Assoziation von Bassoon mit den "presynaptic dense projections" hin. Die Einbeziehung von Bassoon in die Organisation des Freisetzungsortes von Neurotransmittern verdeutlicht, dass dieses Protein eine wichtige Rolle bei der Definition der Spezifität für "docking" und Fusion der synaptischen Vesikeln spielt. In den neurosekretorischen Endigungen der Neurohypophyse zeigten die synaptischen Proteine VAMP II, SV2, Rab3A, SNAP-25, und die N-Typ Ca2+-Kanäle eine bevorzugte Markierung auf Mikrovesikeln. Die Intensität der Immunmarkierung dieser Proteine auf den Mikrovesikeln entspricht derjenigen von synaptischen Vesikeln. Dieses deutet darauf hin, dass diese synaptischen Proteine eine identische funktionelle Bedeutung auf synaptischen Vesikeln und Mikrovesikeln besitzen. Die vorliegende Untersuchung stützt damit die Hypothese, dass die Mikrovesikel nicht nur bezüglich ihrer Größe und ihrer Morphologie denen typischer synaptischer Vesikel ähnlich sind, sondern auch ähnliche Eigenschaften wie diese haben. Eine starke Markierung von SNAP-25, N-Typ Ca2+-Kanäle sowie von VAMP II ließ sich auch an der Plasmamembran nahe der Mikrovesikel-Cluster nachweisen. Dies läßt vermuten, dass Mikrovesikel die Kompetenz zum "docking" und zur Exozytose entlang der Plasmamembran besitzen, auch wenn eine aktive Zone fehlt. Die aus den Mikrovesikeln freigesetzten Botenstoffe könnten als lokale regulierende Faktoren verwendet werden, um die Sekretion an neurohypophysären Endigungen zu koordinieren. An den neurosekretorischen Granula konnten Markierungen von VAMP II, SV2, SNAP-25, und N-Typ Ca2+-Kanäle nicht nachgewiesen werden. Dies entspricht der Vorstellung, dass Granula, verglichen mit Mikrovesikeln und synaptischen Vesikeln, unterschiedliche Mechanismen für die Exozytose besitzen. Allerdings wurden a/ß-SNAP und NSF auf den Granula nachgewiesen, und ebenso Rab3A und P/Q-Typ Ca2+-Kanäle auf den Granula einer Subpopulation von neurohypophysären Terminalen. Rab3A war mit Oxytocin-Granula spezifisch assoziiert, wie durch die Doppelmarkierung gezeigt werden konnte. Die Markierungen von a/ß-SNAP an der Plasmamembran und am Verbindungsstiel des Granulums mit der Plasmamembran lassen vermuten, dass a/ß-SNAP an die Regulierung der Exozytose der neurohypophysärer Granula mitbeteiligt ist. Die Markierungen für a/ß-SNAP und NSF auf den Granula unterstützen die Hypothese, dass vesikuläre Organellen beide Proteine zum Exozytoseort transportieren können. Möglicherweise können diese Proteine ihre Funktion bereits vor dem Vesikeldocking ausüben. Außerdem zeigte keine der untersuchten Proteine eine konzentrierte Markierung an spezifischen Domänen der Plasmamembran der neurohypophysären Endigungen. Auch diese Ergebnisse machen deutlich, dass in den neurohypophysären Endigungen ein definierter Freisetzungsort fehlt. Offensichtlich entsprechen die Unterschiede bezüglich der Verteilung der untersuchten Proteine in den beiden Nerventerminalen auch den spezifischen Funktionseigenschaften dieser Proteine, was wiederum in hohem Maße mit den spezifischen Funktionseigenschaften der beiden Nerventerminalen selbst korrespondiert. Interessanterweise konnten einige Plasmamembranproteine, wie SNAP-25, sogar N- und P/QTyp Ca2+-Kanäle, nicht nur auf der Plasmamembran sondern auch auf vesikulären Organellen lokalisiert werden. Vermutlich sind diese vesikulären Organellen am Transport von neu synthetisierten Proteinen vom Soma bis zur Plasmamembran in den Nervenendigungen beteiligt. Weiterhin wurde vorgeschlagen, dass SNAP-25 zusammen mit den synaptischen Vesikeln in den Terminalen rezirkuliert wird, um sie dann zu reaktivieren. Der vesikuläre Vorrat an Ca2+-Kanälen könnte wiederum der stimulationsabhängigen Translokation in die Plasmamembran dienen. 2. Lokalisation der Vesikel-relevanten Proteine in astrozytären Zellen Um die Funktion von synaptischen Proteinen in astrocytären Zellen zu verstehen, wurde die subzelluläre Lokalisation von den Proteinen auf vesikulären Organellen sowie auf den durch Stimulation induzierten endozytotischen Kompartimenten an kultivierten kortikalen Astrozyten von neonatalen Ratten nachgewiesen. Zusätzlich wurden die Expression und die Lokalisation vieler Proteine, die für eine regulierte Exozytose und die interzelluläre Membranfusion verantwortlich sind, an Zellen aus einer permanenten Astrozytoma-Zellinie (U373 MG) untersucht. Verschiedene Konstrukte, die für die Proteine und die relevanten zusätzlichen Tags kodieren, wurden transient in diese Zellen transfiziert. Die Lokalisation der exprimierten rekombinanten Proteine wurde anschließend mittels Immunfluoreszenz und Kryo-Immunelektronmikroskopie nachgewiesen. Das Ziel war es, die sekretorischen Charakteristika der U373 MG Zellen aufzuklären sowie zu prüfen, ob diese Zell-Linie als effektives experimentelles Modell zur weiteren Studie des astrozytären Exozytose- Mechanismus genutzt werden kann. In kultivierten Astrozyten konnte VAMP II an elektronenlichten vesikulären Organellen variierender Größe sowie an mit Meerrettich-Peroxidase (HRP) gefüllten endozytotischen Organellen - mit oder ohne Stimulation in Anwesenheit von HRP - sowohl mittels der Präembedding-Methode als auch mittels der LR Gold Postembedding-Methode nachgewiesen werden. Daraus folgt, dass in Astrozyten VAMP II am Zyklus der vesikulären Exo- und Endozytose beteiligt wird. U373 MG Zellen konnten alle drei Mitglieder des synaptischen SNARE-Komplexes (v- SNARE VAMP II, t-SNARE Syntaxin I, und SNAP-25) exprimieren. U373 MG Zellen sind folglich fähig, eine regulierte Exozytose mittels des klassischen vesikulären SNARE-Mechanismus durchzuführen. Zusätzlich konnten auch das ubiquitär vorhandene v-SNARE Cellubrevin und das Endosomen-assoziierte kleine GTP-bindende Protein Rab5 in den U373 MG Zellen exprimiert werden. Alle exprimierten rekombinanten Proteine zeigten in den U373 MG Zellen fluoreszenzmikroskopisch eine punktuelle zelluläre Verteilung. Vermutlich sind diese Proteine vor allem mit intrazellulären Kompartimenten assoziiert. Elektronenmikroskopisch zeigten sich in U373 MG Zellen zahlreiche vesikuläre Organellen. Den direkten Nachweis für eine Assoziation dieser Proteine mit den hellen Vesikeln lieferte der immunzytochemische Nachweis aller exprimierten Proteine an ultradünnen Kryoschnitten. Diese Daten weisen vor allem auf das Potential der U373 MG Zellen hin, niedermolekulare Botenstoffe mittels einer regulierten Exozytose freizusetzen. Die Kryoelektronen-Mikroskopie zeigte außerdem, dass myc-VAMP II in größerer Menge als die anderen untersuchten Proteine in U373 MG Zellen exprimiert wurden. Verschiedene Typen vesikulärer Organellen wurden intensiv mit myc-VAMP II markiert. Besonders auffallend sind dichte Markierungen auf zahlreichen kleinen Vesikeln, tubulovesikulären Organellen sowie der Plasmamembran. Vermutlich ist myc-VAMP II am Recycling von hellen vesikulären Organellen mitbeteiligt. Auch Granula sind mit myc-VAMP II markiert. Dies bedeutet, dass sich dieses Protein in den U373 MG Zellen auch an der Exozytose der Granula beteiligen kann. Die massive Expression von myc-VAMP II an multivesikulären Körpern und Lysosomen läßt sich wahrscheinlich auf überexprimiertes rekombinantes Protein zurückführen, das schließlich degradiert wird. Diese vielfältige VAMP II Markierung in U373 MG Zellen weist darauf hin, dass dieses Protein an den verschiedenen Stadien des Vesikelrecyclings beteiligt ist. Die hohe Kolokalisation der zwei SNARE-Proteine VAMP II und Syntaxin I im Zellkörper und in den Zellfortsätzen weist darauf hin, dass beide Proteine in der Regel in identische vesikuläre Organellen sortiert werden. Die teilweise Kolokalisation von VAMP II mit Cellubrevin an den hellen Vesikeln deutet auf eine ähnliche Funktion der beiden v-SNAREs hin. GFP-Rab5 ließ sich an hellen Vesikeln und Endosomen nachweisen. Vermutlich ist Rab5 sowohl in das Vesikelrecycling als auch in die Endosomenfusion der U373 MG Zellen involviert. Die teilweise Kolokalisation von Rab5 mit VAMP II an den hellen Vesikeln weist darauf hin, dass das endosomale Protein Rab5 während einiger Stadien seines Lebenszyklus mit den VAMP II-enthaltenen Organellen verbunden ist. Ähnlich wie bei den kultivierten Astrozyten konnten bei U373 MG Zellen keine eindeutigen Freisetzungsorte, wie eine neuronale aktive Zone, nachgewiesen werden. Auch kleine gedockte Vesikel sind in definierten Zellbereichen nicht zu finden. Überdies ähnelt das Expressionsmuster der untersuchten Proteine dem über lange Zeit kultivierter primärer Astrozyten. Die Markierung für die beiden mutmaßlichen t-SNARE Syntaxin I und SNAP-25 fand sich auf vesikulären Organellen. Die Plasmamembran der U373 MG Zellen zeigte keine signifikante Markierung. Diese Befunde legen nahe, dass U373 MG Zellen ihre Botenstoffe über einen ähnlichen Sekretionsmechanismus wie Astrozyten freisetzen. Sie können daher als geeignetes Model für weitere Untersuchungen des astrozytären Exozytosemechanismus eingesetzt werden

Immuno Magnetic Thermosensitive Liposomes For Cancer Therapy

The present work describes the encapsulation of the drug doxorubicin (DOX) in immuno paramagnetic thermosensitive liposomes. DOX is the most common chemotherapeutic agent for the treatment of a variety of carcinomas. However, the pure drug has high cytotoxicity and therefore requires a targeted and biocompatible delivery system. The introduction includes concepts, modalities, and functionalities of the project. First, a detailed description of the cell type (triple-negative breast cancer) is given. Furthermore, the importance of liposomal doxorubicin is explained and the current state of research is shown. The importance of modification to achieve thermosensitive properties and the procedure for co-encapsulation with Gd chelate to achieve paramagnetic properties is also discussed. In addition, the first part describes the surface modification with ADAM8 antibodies, which leads to improved targeting. The second part of the thesis covers the different materials and methods used in this paper. The production of the liposomes LipTS, LipTS-GD, LipTS-GD-CY, LipTS-GD-CY-MAB and the loading of DOX using an ammonium sulfate gradient method were described in detail. The results part deals with the physicochemical characterization using dynamic light scattering and laser Doppler velocimetry, which confirmed a uniform monodisperse distribution of the liposomes. These properties facilitate the approach of liposomes to target cancer cells. The influence of lipid composition of liposomes, co-encapsulation with Gd chelate and surface modification of liposomes was evaluated and described accordingly. The size and structure of the individual liposomal formulations were determined by atomic force microscopy and transmission electron microscopy. Morphological examination of the liposomes confirmed agreement with the sizes obtained by dynamic light scattering. Temperature-dependent AFM images showed an intact liposome structure at 37 °C, whereas heating by UHF-MRI led to a lipid film indicating the destruction of the lipid bilayer. Furthermore, TEM images showed the morphological properties of the liposomes and gave a more precise indication of how Gd-chelate accumulates within the liposomes. Liposomes with Gd-chelate showed well-separated vesicles, suggesting that Gd- chelate is deposited in the lipid bilayer of the liposomes. Gd was encapsulated in the hydrophilic core whereas chelate was extended into the lipid bilayer. By differential scanning calorimetry and drug release, the heat-sensitive functionality of the liposomes could be determined. Liposomes showed a beginning of phase transition temperature at about 38 °C, which can be achieved by UHF-MRI exposure. The maximum phase transition temperature in the case of LipTS-GD and LipTS-GD-CY-MAB was 42 °C and 40 °C, respectively. A proof of concept study for the thermosensitive properties of liposomes and a time-dependent DOX release profile in hyperthermia was performed. Gd-chelate is encapsulated in both LipTS-GD and LipTS-GD-CY-MAB and led to paramagnetic properties of the liposomes. This facilitates imaging mediated DOX delivery and diagnosis of the solid tumor and metastatic cells. The change in relaxation rate R1 of liposomes was quantified before and after heating above Tm (T> Tm). The relaxivity of the liposomes was obtained from the adapted slope of the relaxation rate against the Gd concentration. Remarkably, the relaxation rate and relaxivity increased after heating the liposomes above Tm (T> Tm), suggesting that the liposomes opened, released Gd chelate, and the exchange of water molecules became faster and more practicable. Toxicity studies describe the different mechanisms for induced DOX toxicity. The increased cytotoxic effect at elevated temperatures showed that the induced toxicity is thermally dependent, i.e. DOX was released from the liposomes. The high viability of the cells at 37 °C indicates that the liposomes were intact at normal physiological temperatures. Under UHF-MRI treatment, cell toxicity due to elevated temperature was observed. The cellular uptake of liposomes under UHF-MRI was followed by a confocal laser scanning microscope. An increase in fluorescence intensity was observed after UHF-MRI exposure. The study of the uptake pathway showed that the majority of liposomes were mainly uptake by clathrin-mediated endocytosis. In addition, the liposomes were modified with anti-ADAM8 antibodies (MAB 1031) to allow targeted delivery. The cellular binding capabilities of surface-modified and non-modified liposomes were tested on cells that had ADAM8 overexpression and on ADAM8 knockdown cells. Surface-modified liposomes showed a significant increase in binding ability, indicating significant targeting against cells that overexpress ADAM8 on their surface. In addition, cells with knockdown ADAM8 could not bind a significant amount of modified liposomes. The biocompatibility of liposomes was assessed using a hemolysis test, which showed neglected hemolytic potential and an activated thromboplastin time (aPTT), where liposomes showed minimal interference with blood clotting. Hemocompatibility studies may help to understand the correlation between in vitro and in vivo. The chorioallantois model was used in ovo to evaluate systematic biocompatibility in an alternative animal model. In the toxicity test, liposomes were injected intravenously into the chicken embryo. The liposomes showed a neglectable harmful effect on embryo survival. While free DOX has a detrimental effect on the survival of chicken embryos, this confirms the safety profile of liposomes compared to free DOX. LipTS-GD-CY-MAB were injected into the vascular system of the chicken embryo on egg development day 11 and scanned under UHF-MRI to evaluate the magnetic properties of the liposomes in a biological system with T2-weighted images (3D). The liposomal formulation had distinct magnetic properties under UHF MRI and the chick survived the scan. In summary, immunomagnetic heat-sensitive liposomes are a novel drug for the treatment of TNBC. It is used both for the diagnosis and therapy of solid and metastasizing tumors without side effects on the neighboring tissue. Furthermore, a tumor in the CAM model will be established. Thereafter, the selective targeting of the liposomes will be visualized and quantitated using fluorescence and UHF-MRI. Liposomes are yet to be tested on mice as a xenograft triple-negative breast cancer model, in which further investigation on the effect of DOX-LipTS-GD-CY-MAB is evaluated. On one hand, the liposomes will be evaluated regarding their targetability and their selective binding. On the other hand, the triggered release of DOX from the liposomes after UHF-MRI exposure will be quantitated, as well as evaluate the DOX-Liposomes therapeutic effect on the tumor

Rab-domain dynamics in endocytic membrane trafficking

Eukaryotic cells depend on cargo uptake into the endocytic membrane system, which comprises a functionally interconnected network of endosomal compartments. The establishment and maintenance of such diverse compartments in face of the high rates of exchange between them, poses a major challenge for obtaining a molecular understanding of the endocytic system. Rab-GTPases have emerged as architectural key element thereof: Individual family members localize selectively to endosomal compartments, where they recruit a multitude of cytoplasmic effector proteins and coordinate them into membrane sub-domains. Such "Rab-domains" constitute modules of molecular membrane identity, which pattern the endocytic membrane system into a mosaic of Rab-domains. The main objective of this thesis research was to link such "static" mosaic-view with the highly dynamic nature of the endosomal system. The following questions were addressed: How are neighbouring Rab-domains coordinated? Are Rab-domains stable or can they undergo assembly and disassembly? Are the dynamics of Rab-domains utilized in cargo transport? The first part of this thesis research focused on the organization of Rab-domains in the recycling pathway. Utilizing Total Internal Reflection (TIRF) microscopy, Rab11-, but neither Rab4- nor Rab5-positive vesicles were observed to fuse with the plasma membrane. Rab4-positive membranes, however, could be induced to fuse in presence of Brefeldin A. Thus, these experiments complete the view of the recycling pathway by the following steps: a) Rab11-carriers likely mediate the return of recycling cargo to the surface; b) such carriers are presumably generated in an Arf-dependent fission reaction from Rab4-positive compartments. Rab11-chromatography was subsequently carried out in the hope of identifying Rab11-effectors functioning at the Rab4-Rab11 domain interface. An as yet uncharacterized ubiquitin ligase was identified, which selectively interacts with both Rab4 and Rab11. Contrary to expectations, however, the protein (termed RUL for *R*ab interacting *U*biquitin *L*igase) does not function in recycling,but appears to mediate trafficking between Golgi/TGN and endosomes instead.In order to address the dynamics of Rab-domains, fluorescently tagged Rab-GTPases were imaged during cargo transport reactions in living cells. Herefore high-speed/long-term imaging procedures and novel computational image analysis tools were developed. The application of such methodology to the analysis of Rab5-positive early endosomes showed that a) The amount of Rab5 associated with individual endosomes fluctuates strongly over time; b) such fluctuations can lead to the "catastrophic" loss of the Rab5-machinery from membranes; c) Rab5 catastrophe is part of a functional cycle of early endosomes, involving net centripetal motility, continuous growth and increase in Rab5 density. Next, the relevance of Rab5 catastrophe with respect to cargo transfer into either the recycling- or degradative pathway was examined. Recycling cargo (transferrin) could be observed to exit Rab5-positive early endosomes via the frequent budding of tubular exit carriers. Exit of degradative cargo (LDL) from Rab5-positive endosomes did not involve budding, but the rapid loss of Rab5 from the limiting membrane.Rab5-loss was further coordinated with the concomitant acquisition of Rab7, suggesting "Rab conversion" as mechanism of transport between early- and late endosomes.Altogether, this thesis research has shown that first, Rab-machineries can be acquired and lost from membranes. Second, such dynamics provide a molecular mechanism for cargo exchange between endosomal compartments. Jointly, these findings lead to the concept of Rab-domain dynamics modulation in /trans/ between neighbouring domains as mechanistic principle behind the dynamic organization of membrane trafficking pathways