8 research outputs found
Facilitating trypanosome imaging.
Research on trypanosomes as a model organism has provided a substantial contribution to a detailed understanding of basic cellular processes within the last few years. At the same time, major advances in super-resolution microscopy have been achieved, facilitating the resolution of biological structures in living cells at a scale of a few nm. However, the motility of trypanosomes has prevented access to high resolution microscopy of live cells. Here, we present a hydrogel based on poly(ethylene glycol) functionalized with either norbornene or thiol moieties for UV induced thiol-ene crosslinking for the embedding and imaging of live trypanosomes. The resulting gel exhibits low autofluorescence properties, immobilizes the cells efficiently on the nanometer scale and is compatible with cell viability for up to one hour at 24 °C. We applied super-resolution imaging to the inner plasma membrane leaflet using lipid-anchored eYFP as a probe. We find specific domains within the membrane where the fluorescence either accumulates or appears diluted rather than being homogenously distributed. Based on a Ripley's analysis, the size of the domains was determined to be raccumulated=170±5 nm and rdilute>115±15 nm. We hypothesize that this structuring of the membrane is associated with the underlying cytoskeleton.ME is supported by DFG grants EN305 and SPP1726. ME is a member of the Wilhelm Conrad Röntgen Center for Complex Material Systems (RCCM). Work in MC's lab was funded by the a Wellcome Trust award (093008/Z10/Z)
Einzelmolekül-Fluoreszenzmikroskopie in lebenden und Modellmembranen
Der eukaryotische Parasit Trypanosoma brucei hat komplexe Strategien entwickelt um der
Immunantwort eines Wirtes zu entkommen und eine persistente Infektion innerhalb dessen
aufrechtzuerhalten. Ein zentrales Element seiner Verteidigungsstrategie stützt sich auf die
Schutzfunktion seines Proteinmantels auf der Zelloberfläche. Dieser Mantel besteht aus
einer dichten Schicht aus identischen, Glykosylphosphatidylinositol (GPI)-verankerten
variablen Oberflächenglykoproteinen (VSG). Der VSG Mantel verhindert die Erkennung
der darunterliegenden, invarianten Epitope durch das Immunsystem. Obwohl es notwendig
ist die Funktionsweise des VSG Mantels zu verstehen, vor allem um ihn als mögliches Angriffsziel
gegen den Parasiten zu verwenden, sind seine biophysikalischen Eigenschaften
bisher nur unzureichend verstanden. Dies ist vor allem der Tatsache geschuldet, dass die
hohe Motilität der Parasiten mikroskopische Studien in lebenden Zellen bisher weitestgehend
verhinderten.
In der vorliegenden Arbeit wird nun hochmoderne Einzelmolekül-Fluoreszenzmikroskopie
(EMFM) als Möglichkeit für mikroskopische Untersuchungen im Forschungsbereich der
Trypanosomen vorgestellt. Die Arbeit umfasst Untersuchungen der VSG Dynamik unter
definierten Bedingungen künstlicher Membransysteme. Es wurde zuerst der Einfluss der
lateralen Proteindichte auf die VSG Diffusion untersucht. Experimente mittels Fluoreszenz-
Wiederkehr nach irreversiblem Photobleichen und komplementäre Einzelmolekül-
Verfolgungs Experimente offenbarten, dass ein molekularer Diffusionsschwellenwert
existiert. Über diesem Schwellenwert wurde eine dichteabhänige Reduzierung des
Diffusionskoeffizienten gemessen. Eine relative Quantifizierung der rekonstituierten VSGs
verdeutlichte, dass der Oberflächenmantel der Trypanosomen sehr nahe an diesem
Schwellenwert agiert. Der VSG Mantel ist optimiert um eine hohe Proteindichte bei gleichzeitiger
hoher Mobilität der VSGs zu gewährleisten. Des Weiteren wurde der Einfluss
der VSG N-Glykosylierung auf die Diffusion des Proteins quantitativ untersucht. Die
Messungen ergaben, dass die N-Glykosylierung dazu beiträgt eine hohe Mobilität bei
hohen Proteindichten aufrechtzuerhalten. Eine detaillierte Analyse von VSG Trajektorien
offenbarte, dass zwei unterschiedliche Populationen frei diffundierender VSGs in der
künstlichen Membran vorlagen. Kürzlich wurde entdeckt, dass VSGs zwei strukturell unterschiedliche Konformationen annehmen können. Die Messungen in der Arbeit stimmen
mit diesen Beschreibungen überein.
Die Ergebnisse der EMFM in künstlichen Membranen wurden durch VSG Einzelmolekül-
Verfolgungs Experimente auf lebenden Zellen ergänzt. Es wurde eine hohe Mobilität
und Dynamik einzelner VSGs gemessen, was die allgemein dynamische Natur des VSG
Mantels verdeutlicht. Dies führte zu der Schlussfolgerung, dass der VSG Mantel auf
lebenden Trypanosomen ein dichter und dennoch dynamischer Schutzmantel ist. Die
Fähigkeit der VSGs ihre Konformation flexibel anzupassen, unterstützt das Erhalten der Fluidität bei variablen Dichten. Diese Eigenschaften des VSG Mantels sind elementar für
die Aufrechterhaltung einer presistenden Infektion eines Wirtes.
In dieser Arbeit werden des Weiteren verschiedene, auf Hydrogel basierende Einbettungsmethoden
vorgestellt. Diese ermöglichten die Zellimmobilisierung und erlaubten
EMFM in lebenden Trypanosomen. Die Hydrogele wiesen eine hohe Zytokompatibilität
auf. Die Zellen überlebten in den Gelen für eine Stunde nach Beginn der Immobilisierung.
Die Hydrogele erfüllten die Anforderungen der Superresolution Mikroskopie (SRM) da
sie eine geringe Autofluoreszenz im Spektralbereich der verwendeten Fluorophore besaßen.
Mittels SRM konnte nachgewiesen werden, dass die Hydrogele die Zellen effizient
immobilisierten. Als erstes Anwendungsbeispiel der Methode wurde die Organisation
der Plasmamembran in lebenden Trypanosomen untersucht. Die Untersuchung eines
fluoreszenten Tracers in der inneren Membranschicht ergab, dass dessen Verteilung nicht
homogen war. Es wurden spezifische Membrandomänen gefunden, in denen das Molekül
entweder vermehrt oder vermindert auftrat. Dies führte zu der Schlussfolgerung, dass diese
Verteilung durch eine Interaktion des Tracers mit Proteinen des zellulären Zytoskeletts
zustande kam.
Die in dieser Arbeit präsentierten Ergebnisse zeigen, dass EMFM erfolgreich für verschiedene
biologische Untersuchungen im Forschungsfeld der Trypanosomen angewendet
werden kann. Dies gilt zum Beispiel für die Untersuchung von der VSG Dynamik in künstlichen
Membransystemen, aber auch für Studien in lebenden Zellen unter Verwendung
der auf Hydrogelen basierenden Zelleinbettung.The eukaryotic parasite Trypanosoma brucei has evolved sophisticated strategies to escape
the host immune response and maintain a persistent infection inside a host. One central
feature of the parasite’s defense mechanism relies on the shielding function of their surface
protein coat. This coat is composed of a dense arrangement of one type of glycosylphosphatidylinositol
(GPI)-anchored variant surface glycoproteins (VSGs) which impair the
identification of epitopes of invariant surface proteins by the immune system. In addition
to the importance of understanding the function of the VSG coat and use it as a potential
target to efficiently fight the parasite, it is also crucial to study its biophysical properties as it is not yet understood sufficiently. This is due to the fact that microscopic investigations
on living trypanosomes are limited to a great extent by the intrinsic motility of the parasite.
In the present study, state-of-the-art single-molecule fluorescence microscopy (SMFM)
is introduced as a tool for biophysical investigations in the field of trypanosome research.
The work encompasses studies of VSG dynamics under the defined conditions of an
artificial supported lipid bilayer (SLB). First, the impact of the lateral protein density on
VSG diffusion was systematically studied in SLBs. Ensemble fluorescence after photobleaching
(FRAP) and complementary single-particle tracking experiments revealed that a
molecular crowding threshold (MCT) exists, above which a density dependent decrease
of the diffusion coefficient is measured. A relative quantification of reconstituted VSGs
illustrated that the VSG coat of living trypanosomes operates very close to its MCT and
is optimized for high density while maintaining fluidity. Second, the impact of VSG
N-glycosylation on VSG diffusion was quantitatively investigated. N-glycosylation was
shown to contribute to preserving protein mobility at high protein concentrations. Third,
a detailed analysis of VSG trajectories revealed that two distinct populations of freely
diffusing VSGs were present in a SLB, which is in agreement with the recent finding, that
VSGs are able to adopt two main structurally distinct conformations. The results from
SLBs were further complemented by single-particle tracking experiments of surface VSGs
on living trypanosomes. A high mobility and free diffusion were measured on the cell
surface, illustrating the overall dynamic nature of the VSG coat. It was concluded that
the VSG coat on living trypanosomes is a protective structure that combines density and
mobility, which is supported by the conformational flexibility of VSGs. These features are
elementary for the persistence of a stable infection in the host.
Different hydrogel embedding methods are presented, that facilitated SMFM in immobilized,
living trypanosomes. The hydrogels were found to be highly cytocompatible for one
hour after cross-linking. They exhibited low autofluorescence properties in the spectral
range of the investigations, making them suitable for super-resolution microscopy (SRM).
Exemplary SRM on living trypanosomes illustrated that the hydrogels efficiently immobilized
the cells on the nanometer lever. Furthermore, the plasma membrane organization was studied in living trypanosomes. A statistical analysis of a tracer molecule inside the
inner leaflet of the plasma membrane revealed that specific membrane domains exist, in
which the tracer appeared accumulated or diluted. It was suggested that this distribution
was caused by the interaction with proteins of the underlying cytoskeleton.
In conclusion, SMFM has been successfully introduced as a tool in the field of trypanosome
research. Measurements in model membranes facilitated systematic studies of VSG dynamics
on the single-molecule level. The implementation of hydrogel immobilization
allowed for the study of static structures and dynamic processes with high spatial and
temporal resolution in living, embedded trypanosomes for the first time
Multi-color, bleaching-resistant super-resolution optical fluctuation imaging with oligonucleotide-based exchangeable fluorophores
Super-resolution optical fluctuation imaging (SOFI) is a super-resolution microscopy technique that overcomes the diffraction limit by analyzing intensity fluctuations of statistically independent emitters in a time series of images. The final images are background-free and show confocality and enhanced spatial resolution (super-resolution). Fluorophore photobleaching, however, is a key limitation for recording long time series of images that will allow for the calculation of higher order SOFI results with correspondingly increased resolution. Here, we demonstrate that photobleaching can be circumvented by using fluorophore labels that reversibly and transiently bind to a target, and which are being replenished from a buffer which serves as a reservoir. Using fluorophore-labeled short DNA oligonucleotides, we labeled cellular structures with target-specific antibodies that contain complementary DNA sequences and record the fluctuation events caused by transient emitter binding. We show that this concept bypasses extensive photobleaching and facilitates two-color imaging of cellular structures with SOFI
Serine-ubiquitination regulates Golgi morphology and the secretory pathway upon Legionella infection
SidE family of Legionella effectors catalyze non-canonical phosphoribosyl-linked ubiquitination (PR-ubiquitination) of host proteins during bacterial infection. SdeA localizes predominantly to ER and partially to the Golgi apparatus, and mediates serine ubiquitination of multiple ER and Golgi proteins. Here we show that SdeA causes disruption of Golgi integrity due to its ubiquitin ligase activity. The Golgi linking proteins GRASP55 and GRASP65 are PR-ubiquitinated on multiple serine residues, thus preventing their ability to cluster and form oligomeric structures. In addition, we found that the functional consequence of Golgi disruption is not linked to the recruitment of Golgi membranes to the growing Legionella-containing vacuoles. Instead, it affects the host secretory pathway. Taken together, our study sheds light on the Golgi manipulation strategy by which Legionella hijacks the secretory pathway and promotes bacterial infection
N-glycosylation enables high lateral mobility of GPI-anchored proteins at a molecular crowding threshold
The protein density in biological membranes can be extraordinarily high, but the impact of molecular crowding on the diffusion of membrane proteins has not been studied systematically in a natural system. The diversity of the membrane proteome of most cells may preclude systematic studies. African trypanosomes, however, feature a uniform surface coat that is dominated by a single type of variant surface glycoprotein (VSG). Here we study the density-dependence of the diffusion of different glycosylphosphatidylinositol-anchored VSG-types on living cells and in artificial membranes. Our results suggest that a specific molecular crowding threshold (MCT) limits diffusion and hence affects protein function. Obstacles in the form of heterologous proteins compromise the diffusion coefficient and the MCT. The trypanosome VSG-coat operates very close to its MCT. Importantly, our experiments show that N-linked glycans act as molecular insulators that reduce retarding intermolecular interactions allowing membrane proteins to function correctly even when densely packed
Exchangeable HaloTag Ligands for Super-Resolution Fluorescence Microscopy
The specific and
covalent labeling of the protein HaloTag with
fluorescent probes in living cells makes it a powerful tool for bioimaging.
However, the irreversible attachment of the probe to HaloTag precludes
imaging applications that require transient binding of the probe and
comes with the risk of irreversible photobleaching. Here, we introduce
exchangeable ligands for fluorescence labeling of HaloTag (xHTLs)
that reversibly bind to HaloTag and that can be coupled to rhodamines
of different colors. In stimulated emission depletion (STED) microscopy,
probe exchange of xHTLs allows imaging with reduced photobleaching
as compared to covalent HaloTag labeling. Transient binding of fluorogenic
xHTLs to HaloTag fusion proteins enables points accumulation for imaging
in nanoscale topography (PAINT) and MINFLUX microscopy. We furthermore
introduce pairs of xHTLs and HaloTag mutants for dual-color PAINT
and STED microscopy. xHTLs thus open up new possibilities in imaging
across microscopy platforms for a widely used labeling approach
Exchangeable HaloTag Ligands for Super-Resolution Fluorescence Microscopy
The specific and
covalent labeling of the protein HaloTag with
fluorescent probes in living cells makes it a powerful tool for bioimaging.
However, the irreversible attachment of the probe to HaloTag precludes
imaging applications that require transient binding of the probe and
comes with the risk of irreversible photobleaching. Here, we introduce
exchangeable ligands for fluorescence labeling of HaloTag (xHTLs)
that reversibly bind to HaloTag and that can be coupled to rhodamines
of different colors. In stimulated emission depletion (STED) microscopy,
probe exchange of xHTLs allows imaging with reduced photobleaching
as compared to covalent HaloTag labeling. Transient binding of fluorogenic
xHTLs to HaloTag fusion proteins enables points accumulation for imaging
in nanoscale topography (PAINT) and MINFLUX microscopy. We furthermore
introduce pairs of xHTLs and HaloTag mutants for dual-color PAINT
and STED microscopy. xHTLs thus open up new possibilities in imaging
across microscopy platforms for a widely used labeling approach