@article{PuppeLeueSommeretal.2022, author = {Puppe, Daniel and Leue, Martin and Sommer, Michael and Schaller, J{\"o}rg and Kaczorek, Danuta}, title = {Auto-fluorescence in phytoliths}, series = {Frontiers in Environmental Science}, volume = {10}, journal = {Frontiers in Environmental Science}, publisher = {Frontiers Media}, address = {Lausanne}, issn = {2296-665X}, doi = {10.3389/fenvs.2022.915947}, pages = {14}, year = {2022}, abstract = {The detection of auto-fluorescence in phytogenic, hydrated amorphous silica depositions (phytoliths) has been found to be a promising approach to verify if phytoliths were burnt or not, especially in archaeological contexts. However, it is unknown so far at what temperature and how auto-fluorescence is induced in phytoliths. We used fluorescence microscopy, scanning electron microscope-energy dispersive X-ray spectroscopy (SEM-EDX), and Fourier transform infrared spectroscopy to analyze auto-fluorescence in modern phytoliths extracted from plant samples or in intact leaves of winter wheat. Leaves and extracted phytoliths were heated at different temperatures up to 600 degrees C. The aims of our experiments were i) to find out what temperature is needed to induce auto-fluorescence in phytoliths, ii) to detect temperature-dependent changes in the molecular structure of phytoliths related to auto-fluorescence, and iii) to derive a mechanistic understanding of auto-fluorescence in phytoliths. We found organic compounds associated with phytoliths to cause auto-fluorescence in phytoliths treated at temperatures below approx. 400 degrees C. In phytoliths treated at higher temperatures, i.e., 450 and 600 degrees C, phytolith auto-fluorescence was mainly caused by molecular changes of phytolith silica. Based on our results we propose that auto-fluorescence in phytoliths is caused by clusterization-triggered emissions, which are caused by overlapping electron clouds forming non-conventional chromophores. In phytoliths heated at temperatures above about 400 degrees C dihydroxylation and the formation of siloxanes result in oxygen clusters that serve as non-conventional chromophores in fluorescence events. Furthermore, SEM-EDX analyses revealed that extractable phytoliths were dominated by lumen phytoliths (62\%) compared to cell wall phytoliths (38\%). Our findings might be not only relevant in archaeological phytolith-based examinations, but also for studies on the temperature-dependent release of silicon from phytoliths and the potential of long-term carbon sequestration in phytoliths.}, language = {en} } @article{PetazziKoikkarahAjiTischleretal.2021, author = {Petazzi, Roberto Arturo and Koikkarah Aji, Amit and Tischler, Nicole D. and Chiantia, Salvatore}, title = {Detection of envelope glycoprotein assembly from old world hantaviruses in the Golgi apparatus of living cells}, series = {Journal of virology}, volume = {95}, journal = {Journal of virology}, number = {4}, publisher = {American Society for Microbiology}, address = {Baltimore, Md.}, issn = {1098-5514}, doi = {10.1128/JVI.01238-20}, pages = {18}, year = {2021}, abstract = {Hantaviruses are emerging pathogens that occasionally cause deadly outbreaks in the human population. While the structure of the viral envelope has been characterized with high precision, protein-protein interactions leading to the formation of new virions in infected cells are not fully understood. We used quantitative fluorescence microscopy (i.e., number and brightness analysis and fluorescence fluctuation spectroscopy) to monitor the interactions that lead to oligomeric spike complex formation in the physiological context of living cells. To this aim, we quantified protein-protein interactions for the glycoproteins Gn and Gc from Puumala and Hantaan orthohantaviruses in several cellular models. The oligomerization of each protein was analyzed in relation to subcellular localization, concentration, and the concentration of its interaction partner. Our results indicate that, when expressed separately, Gn and Gc form, respectively, homo-tetrameric and homo-dimeric complexes, in a concentration-dependent manner. Site-directed mutations or deletion mutants showed the specificity of their homotypic interactions. When both glycoproteins were coexpressed, we observed in the Golgi apparatus clear indication of GnGc interactions and the formation of Gn-Gc multimeric protein complexes of different sizes, while using various labeling schemes to minimize the influence of the fluorescent tags. Such large glycoprotein multimers may be identified as multiple Gn viral spikes interconnected via Gc-Gc contacts. This observation provides the possible first evidence for the initial assembly steps of the viral envelope within this organelle, and does so directly in living cells.
IMPORTANCE In this work, we investigate protein-protein interactions that drive the assembly of the hantavirus envelope. These emerging pathogens have the potential to cause deadly outbreaks in the human population. Therefore, it is important to improve our quantitative understanding of the viral assembly process in infected cells, from a molecular point of view. By applying advanced fluorescence microscopy methods, we monitored the formation of viral spike complexes in different cell types. Our data support a model for hantavirus assembly according to which viral spikes are formed via the clustering of hetero-dimers of the two viral glycoproteins Gn and Gc. Furthermore, the observation of large Gn-Gc hetero-multimers provide the possible first evidence for the initial assembly steps of the viral envelope, directly in the Golgi apparatus of living cells.}, language = {en} } @misc{TzonevaStoyanovaPetrichetal.2020, author = {Tzoneva, Rumiana and Stoyanova, Tihomira and Petrich, Annett and Popova, Desislava and Uzunova, Veselina and Albena, Momchilova and Chiantia, Salvatore}, title = {Effect of Erufosine on Membrane Lipid Order in Breast Cancer Cell Models}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {1000}, issn = {1866-8372}, doi = {10.25932/publishup-47705}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-477056}, pages = {19}, year = {2020}, abstract = {Alkylphospholipids are a novel class of antineoplastic drugs showing remarkable therapeutic potential. Among them, erufosine (EPC3) is a promising drug for the treatment of several types of tumors. While EPC3 is supposed to exert its function by interacting with lipid membranes, the exact molecular mechanisms involved are not known yet. In this work, we applied a combination of several fluorescence microscopy and analytical chemistry approaches (i.e., scanning fluorescence correlation spectroscopy, line-scan fluorescence correlation spectroscopy, generalized polarization imaging, as well as thin layer and gas chromatography) to quantify the effect of EPC3 in biophysical models of the plasma membrane, as well as in cancer cell lines. Our results indicate that EPC3 affects lipid-lipid interactions in cellular membranes by decreasing lipid packing and increasing membrane disorder and fluidity. As a consequence of these alterations in the lateral organization of lipid bilayers, the diffusive dynamics of membrane proteins are also significantly increased. Taken together, these findings suggest that the mechanism of action of EPC3 could be linked to its effects on fundamental biophysical properties of lipid membranes, as well as on lipid metabolism in cancer cells.}, language = {en} } @article{TzonevaStoyanovaPetrichetal.2020, author = {Tzoneva, Rumiana and Stoyanova, Tihomira and Petrich, Annett and Popova, Desislava and Uzunova, Veselina and Momchilova, Albena and Chiantia, Salvatore}, title = {Effect of Erufosine on Membrane Lipid Order in Breast Cancer Cell Models}, series = {Biomolecules}, volume = {10}, journal = {Biomolecules}, number = {5}, publisher = {MDPI}, address = {Basel}, issn = {2218-273X}, doi = {10.3390/biom10050802}, pages = {17}, year = {2020}, abstract = {Alkylphospholipids are a novel class of antineoplastic drugs showing remarkable therapeutic potential. Among them, erufosine (EPC3) is a promising drug for the treatment of several types of tumors. While EPC3 is supposed to exert its function by interacting with lipid membranes, the exact molecular mechanisms involved are not known yet. In this work, we applied a combination of several fluorescence microscopy and analytical chemistry approaches (i.e., scanning fluorescence correlation spectroscopy, line-scan fluorescence correlation spectroscopy, generalized polarization imaging, as well as thin layer and gas chromatography) to quantify the effect of EPC3 in biophysical models of the plasma membrane, as well as in cancer cell lines. Our results indicate that EPC3 affects lipid-lipid interactions in cellular membranes by decreasing lipid packing and increasing membrane disorder and fluidity. As a consequence of these alterations in the lateral organization of lipid bilayers, the diffusive dynamics of membrane proteins are also significantly increased. Taken together, these findings suggest that the mechanism of action of EPC3 could be linked to its effects on fundamental biophysical properties of lipid membranes, as well as on lipid metabolism in cancer cells.}, language = {en} } @phdthesis{Knigge2020, author = {Knigge, Xenia}, title = {Einzelmolek{\"u}l-Manipulation mittels Nano-Elektroden und Dielektrophorese}, doi = {10.25932/publishup-44313}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-443137}, school = {Universit{\"a}t Potsdam}, pages = {106, xxxii}, year = {2020}, abstract = {In dieser Arbeit wurden Nano-Elektroden-Arrays zur Einzel-Objekt-Immobilisierung mittels Dielektrophorese verwendet. Hierbei wurden fluoreszenzmarkierte Nano-Sph{\"a}ren als Modellsystem untersucht und die gewonnenen Ergebnisse auf biologische Proben {\"u}bertragen. Die Untersuchungen in Kombination mit verschiedenen Elektrodenlayouts f{\"u}hrten zu einer deterministischen Vereinzelung der Nano-Sph{\"a}ren ab einem festen Gr{\"o}ßenverh{\"a}ltnis zwischen Nano-Sph{\"a}re und Durchmesser der Elektrodenspitzen. An den Proteinen BSA und R-PE konnte eine dielektrophoretische Immobilisierung ebenfalls demonstriert und R-PE Molek{\"u}le zur Vereinzelung gebracht werden. Hierf{\"u}r war neben einem optimierten Elektrodenlayout, das durch Feldsimulationen den Feldgradienten betreffend gesucht wurde, eine Optimierung der Feldparameter, insbesondere von Spannung und Frequenz, erforderlich. Neben der Dielektrophorese erfolgten auch Beobachtungen anderer Effekte des elektrischen Feldes, wie z.B. Elektrolyse an Nano-Elektroden und Str{\"o}mungen {\"u}ber dem Elektroden-Array, hervorgerufen durch Joulesche W{\"a}rme und AC-elektroosmotischen Fluss. Zudem konnte Dielektrophorese an Silberpartikeln beobachtet werden und mittels Fluoreszenz-, Atom-Kraft-, Raster-Elektronen-Mikroskopie und energiedispersiver R{\"o}ntgenspektroskopie untersucht werden. Schließlich wurden die verwendeten Objektive und Kameras auf ihre Lichtempfindlichkeit hin analysiert, so dass die Vereinzelung von Biomolek{\"u}len an Nano-Elektroden nachweisbar war. Festzuhalten bleibt also, dass die Vereinzelung von Nano-Objekten und Biomolek{\"u}len an Nano-Elektroden-Arrays gelungen ist. Durch den parallelen Ansatz erlaubt dies, Aussagen {\"u}ber das Verhalten von Einzelmolek{\"u}len mit guter Statistik zu treffen.}, language = {de} } @article{PetrichChiantia2023, author = {Petrich, Annett and Chiantia, Salvatore}, title = {Influenza a virus infection alters lipid packing and surface electrostatic potential of the host plasma membrane}, series = {Viruses}, volume = {15}, journal = {Viruses}, number = {9}, publisher = {MDPI}, address = {Basel}, issn = {1999-4915}, doi = {10.3390/v15091830}, pages = {17}, year = {2023}, abstract = {The pathogenesis of influenza A viruses (IAVs) is influenced by several factors, including IAV strain origin and reassortment, tissue tropism and host type. While such factors were mostly investigated in the context of virus entry, fusion and replication, little is known about the viral-induced changes to the host lipid membranes which might be relevant in the context of virion assembly. In this work, we applied several biophysical fluorescence microscope techniques (i.e., F{\"o}rster energy resonance transfer, generalized polarization imaging and scanning fluorescence correlation spectroscopy) to quantify the effect of infection by two IAV strains of different origin on the plasma membrane (PM) of avian and human cell lines. We found that IAV infection affects the membrane charge of the inner leaflet of the PM. Moreover, we showed that IAV infection impacts lipid-lipid interactions by decreasing membrane fluidity and increasing lipid packing. Because of such alterations, diffusive dynamics of membrane-associated proteins are hindered. Taken together, our results indicate that the infection of avian and human cell lines with IAV strains of different origins had similar effects on the biophysical properties of the PM.}, language = {en} } @phdthesis{Roder2018, author = {Roder, Phillip}, title = {Kombination von Fluoreszenzmikroskopie und Rasterkraftmikroskopie zur Aufkl{\"a}rung physiologischer Prozesse in lebenden Zellen}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-419806}, school = {Universit{\"a}t Potsdam}, pages = {xvi, 113}, year = {2018}, abstract = {Innerhalb dieser Doktorarbeit wurde eine neuartige Mikromanipulationstechnik f{\"u}r die lokale Fl{\"u}ssigkeitsabgabe am komplexen Dr{\"u}sengewebe der Schabe P. americana charakterisiert und f{\"u}r die damit verbundene gezielte Manipulation von einzelnen Zellen in einem Zellkomplex (Gewebe) angewandt. Bei dieser Mikromanipulationstechnik handelt es sich um die seit 2009 bekannte nanofluidische Rasterkraftmikroskopie (FluidFM = fluidic force microscopy). Dabei werden sehr kleine mikrokan{\"a}lige Rasterkraftspitzen bzw. Mikro-/Nanopipetten mit einer {\"O}ffnung zwischen 300 nm und 2 µm verwendet, mit denen es m{\"o}glich ist, sehr kleine Volumina im Pikoliter- bis Femtoliter-Bereich (10-12 L - 10-15 L) gezielt und ortsgenau abzugeben. Das Ziel dieser Arbeit war die Analyse zellul{\"a}rer Prozesse, wie z. B. Zell-Zell-Kommunikation oder Signalweiterleitung, zwischen benachbarten Zellen unter Zuhilfenahme der Fluoreszenzmikroskopie. Mit dieser Methode k{\"o}nnen die Zellen und ihre Bestandteile mittels vorheriger Farbstoffbeladung unter einem Mikroskop mit hohem Kontrast optisch dargestellt werden. Mit Hilfe der Fluoreszenzmikroskopie sollten schlussendlich die zellul{\"a}ren Reaktionen innerhalb des Gewebes nach der lokalen Manipulation visualisiert werden. Zun{\"a}chst wurde die Anwendung des Systems an Luft und w{\"a}ssriger Umgebung beschrieben. In diesem Zusammenhang wurde eine Reinigungs- und Beladungsmethode entwickelt, mit der es m{\"o}glich war, die kostspieligen Mikro-/Nanopipetten zu reinigen und anschließend mehrmals wiederzuverwenden. Hierzu wurde eine alternative Methode getestet, mit der das Diffusionsverhalten von Farbstoffmolek{\"u}len in unterschiedlichen Medien untersucht werden kann. Des Weiteren wurden die Systemparameter optimiert, welche n{\"o}tig sind, um zwischen der Probenoberfl{\"a}che und der Pipette einen guten Pipetten{\"o}ffnungs-abschluss zu erhalten. Dieser Abschluss ist essentiell, damit die abgegebene Fl{\"u}ssigkeit ausschließlich in der Abgaberegion mit der Probe wechselwirkt und die darauffolgenden Reaktionen nur innerhalb des Gewebes erfolgen, da ansonsten die Zell-Zell-Signalweiterleitung zwischen den Zellen nicht eindeutig nachvollzogen werden kann. Diese interzellul{\"a}re Kommunikation wurde anhand zweier sekund{\"a}rer Botenstoffe (Ca2+ und NO) untersucht. Hierbei war es m{\"o}glich einzelne lokale Reaktionen zu detektieren, welche sich {\"u}ber weitere Zellen ausbreiteten. Schlussendlich wurde die Fertigung einer speziellen Injektionspipette beschrieben, welche an zwei biologischen Systemen getestet wurde.}, language = {de} } @phdthesis{Niedermayer2012, author = {Niedermayer, Thomas}, title = {On the depolymerization of actin filaments}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-63605}, school = {Universit{\"a}t Potsdam}, year = {2012}, abstract = {Actin is one of the most abundant and highly conserved proteins in eukaryotic cells. The globular protein assembles into long filaments, which form a variety of different networks within the cytoskeleton. The dynamic reorganization of these networks - which is pivotal for cell motility, cell adhesion, and cell division - is based on cycles of polymerization (assembly) and depolymerization (disassembly) of actin filaments. Actin binds ATP and within the filament, actin-bound ATP is hydrolyzed into ADP on a time scale of a few minutes. As ADP-actin dissociates faster from the filament ends than ATP-actin, the filament becomes less stable as it grows older. Recent single filament experiments, where abrupt dynamical changes during filament depolymerization have been observed, suggest the opposite behavior, however, namely that the actin filaments become increasingly stable with time. Several mechanisms for this stabilization have been proposed, ranging from structural transitions of the whole filament to surface attachment of the filament ends. The key issue of this thesis is to elucidate the unexpected interruptions of depolymerization by a combination of experimental and theoretical studies. In new depolymerization experiments on single filaments, we confirm that filaments cease to shrink in an abrupt manner and determine the time from the initiation of depolymerization until the occurrence of the first interruption. This duration differs from filament to filament and represents a stochastic variable. We consider various hypothetical mechanisms that may cause the observed interruptions. These mechanisms cannot be distinguished directly, but they give rise to distinct distributions of the time until the first interruption, which we compute by modeling the underlying stochastic processes. A comparison with the measured distribution reveals that the sudden truncation of the shrinkage process neither arises from blocking of the ends nor from a collective transition of the whole filament. Instead, we predict a local transition process occurring at random sites within the filament. The combination of additional experimental findings and our theoretical approach confirms the notion of a local transition mechanism and identifies the transition as the photo-induced formation of an actin dimer within the filaments. Unlabeled actin filaments do not exhibit pauses, which implies that, in vivo, older filaments become destabilized by ATP hydrolysis. This destabilization can be identified with an acceleration of the depolymerization prior to the interruption. In the final part of this thesis, we theoretically analyze this acceleration to infer the mechanism of ATP hydrolysis. We show that the rate of ATP hydrolysis is constant within the filament, corresponding to a random as opposed to a vectorial hydrolysis mechanism.}, language = {en} } @phdthesis{Breitenstein2012, author = {Breitenstein, Michael}, title = {Ortsaufgel{\"o}ster Aufbau von DNA-Nanostrukturen auf Glasoberfl{\"a}chen}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-61857}, school = {Universit{\"a}t Potsdam}, year = {2012}, abstract = {Im Fokus dieser Arbeit stand der Aufbau einer auf DNA basierenden Nanostruktur. Der universelle Vier-Buchstaben-Code der DNA erm{\"o}glicht es, Bindungen auf molekularer Ebene zu adressieren. Die chemischen und physikalischen Eigenschaften der DNA pr{\"a}destinieren dieses Makromolek{\"u}l f{\"u}r den Einsatz und die Verwendung als Konstruktionselement zum Aufbau von Nanostrukturen. Das Ziel dieser Arbeit war das Aufspannen eines DNA-Stranges zwischen zwei Fixpunkten. Hierf{\"u}r war es notwendig, eine Methode zu entwickeln, welche es erm{\"o}glicht, Funktionsmolek{\"u}le als Ankerelemente ortsaufgel{\"o}st auf eine Oberfl{\"a}che zu deponieren. Das Deponieren dieser Molek{\"u}le sollte dabei im unteren Mikrometermaßstab erfolgen, um den Abmaßen der DNA und der angestrebten Nanostruktur gerecht zu werden. Das eigens f{\"u}r diese Aufgabe entwickelte Verfahren zum ortsaufgel{\"o}sten Deponieren von Funktionsmolek{\"u}len nutzt das Bindungspaar Biotin-Neutravidin. Mit Hilfe eines Rasterkraftmikroskops (AFM) wurde eine zu einem „Stift" umfunktionierte Rasterkraftmikroskopspitze so mit der zu deponierenden „Tinte" beladen, dass das Absetzen von Neutravidin im unteren Mikrometermaßstab m{\"o}glich war. Dieses Neutravidinmolek{\"u}l {\"u}bernahm die Funktion als Bindeglied zwischen der biotinylierten Glasoberfl{\"a}che und dem eigentlichen Adressmolek{\"u}l. Das somit generierte Neutravidin-Feld konnte dann mit einem biotinylierten Adressmolek{\"u}l durch Inkubation funktionalisiert werden. Namensgebend f{\"u}r dieses Verfahren war die M{\"o}glichkeit, Neutravidin mehrmals zu deponieren und zu adressieren. Somit ließ sich sequenziell ein Mehrkomponenten-Feld aufbauen. Die Einschr{\"a}nkung, mit einem AFM nur eine Substanz deponieren zu k{\"o}nnen, wurde so umgangen. Ferner mußten Ankerelemente geschaffen werden, um die DNA an definierten Punkten immobilisieren zu k{\"o}nnen. Die Bearbeitung der DNA erfolgte mit molekularbiologischen Methoden und zielte darauf ab, einen DNA-Strang zu generieren, welcher an seinen beiden Enden komplement{\"a}re Adressequenzen enth{\"a}lt, um gezielt mit den oberfl{\"a}chenst{\"a}ndigen Ankerelementen binden zu k{\"o}nnen. Entsprechend der Geometrie der mit dem AFM erzeugten Fixpunkte und den oligonukleotidvermittelten Adressen kommt es zur Ausbildung einer definierten DNA-Struktur. Mit Hilfe von fluoreszenzmikroskopischen Methoden wurde die aufgebaute DNA-Nanostruktur nachgewiesen. Der Nachweis der nanoskaligen Interaktion von DNA-bindenden Molek{\"u}len mit der generierten DNA-Struktur wurde durch die Bindung von PNA (peptide nucleic acid) an den DNA-Doppelstrang erbracht. Diese PNA-Bindung stellt ihrerseits ein funktionales Strukturelement im Nanometermaßstab dar und wird als Nanostrukturbaustein verstanden.}, language = {de} } @phdthesis{KoikkarahAji2023, author = {Koikkarah Aji, Amit}, title = {Quantitative sub cellular characterization of Hantavirus structural proteins}, doi = {10.25932/publishup-58661}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-586612}, school = {Universit{\"a}t Potsdam}, pages = {101}, year = {2023}, abstract = {Hantaviruses (HVs) are a group of zoonotic viruses that infect human beings primarily through aerosol transmission of rodent excreta and urine samplings. HVs are classified geographically into: Old World HVs (OWHVs) that are found in Europe and Asia, and New World HVs (NWHVs) that are observed in the Americas. These different strains can cause severe hantavirus diseases with pronounced renal syndrome or severe cardiopulmonary system distress. HVs can be extremely lethal, with NWHV infections reaching up to 40 \% mortality rate. HVs are known to generate epidemic outbreaks in many parts of the world including Germany, which has seen periodic HV infections over the past decade. HV has a trisegmented genome. The small segment (S) encodes the nucleocapsid protein (NP), the middle segment (M) encodes the glycoproteins (GPs) Gn and Gc which forms up to tetramers and primarily monomers \\& dimers upon independent expression respectively and large segment (L) encodes RNA dependent RNA polymerase (RdRp). Interactions between these viral proteins are crucial in providing mechanistic insights into HV virion development. Despite best efforts, there continues to be lack of quantification of these associations in living cells. This is required in developing the mechanistic models for HV viral assembly. This dissertation focuses on three key questions pertaining to the initial steps of virion formation that primarily involves the GPs and NP. The research investigations in this work were completed using Fluorescence Correlation Spectroscopy (FCS) approaches. FCS is frequently used in assessing the biophysical features of bio-molecules including protein concentration and diffusion dynamics and circumvents the requirement of protein overexpression. FCS was primarily applied in this thesis to evaluate protein multimerization, at single cell resolution. The first question addressed which GP spike formation model proposed by Hepojoki et al.(2010) appropriately describes the evidence in living cells. A novel in cellulo assay was developed to evaluate the amount of fluorescently labelled and unlabeled GPs upon co-expression. The results clearly showed that Gn and Gc initially formed a heterodimeric Gn:Gc subunit. This sub-unit then multimerizes with congruent Gn:Gc subunits to generate the final GP spike. Based on these interactions, models describing the formation of GP complex (with multiple GP spike subunits) were additionally developed. HV GP assembly primarily takes place in the Golgi apparatus (GA) of infected cells. Interestingly, NWHV GPs are hypothesized to assemble at the plasma membrane (PM). This led to the second research question in this thesis, in which a systematic comparison between OWHV and NWHV GPs was conducted to validate this hypothesis. Surprisingly, GP localization at the PM was congruently observed with OWHV and NWHV GPs. Similar results were also discerned with OWHV and NWHV GP localization in the absence of cytoskeletal factors that regulate HV trafficking in cells. The final question focused on quantifying the NP-GP interactions and understanding their influence of NP and GP multimerization. Gc mutlimers were detected in the presence of NP and complimented by the presence of localized regions of high NP-Gc interactions in the perinuclear region of living cells. Gc-CT domain was shown to influence NP-Gc associations. Gn, on the other hand, formed up to tetrameric complexes, independent from the presence of NP. The results in this dissertation sheds light on the initial steps of HV virion formation by quantifying homo and heterotypic interactions involving NP and GPs, which otherwise are very difficult to perform. Finally, the in cellulo methodologies implemented in this work can be potentially extended to understand other key interactions involved in HV virus assembly.}, language = {en} }