Räumlich und zeitlich aufgelöste Untersuchung der Mechanik der Phagozytose
Biophysik
Zusammenfassung der Projektergebnisse
Living cells are able to take up external objects by a process called phagocytosis. The basic principles of this process developed already about two billion years ago and they are considered an important evolutionary step towards the development of complex multicellular organisms. Phagocytosis plays an important role in various biological functions including food intake in single cellular organisms and the degradation of bacteria as part of the immune system of multicellular organisms. Phagocytosis starts when an external object like a bacterium binds to the membrane of an immune cell such as a macrophage. The binding is often mediated by antibodies, which are proteins of the immune system and which attach to the bacteria to enable their binding to specific receptors in the membrane of the macrophage. An important type of antibody in this process is immunoglobulin G (IgG), which binds to Fcγ-receptors. These phagocytic receptors initiate intracellular signaling, which finally leads to the engulfment of the bacteria mediated by a restructuring of the actin cytoskeleton. Although a large number of the molecules that are involved in phagocytosis are already identified, a quantitative understanding of the mechanics of this process is still lacking. The goal of this project was therefore to investigate the mechanics of phagocytosis to contribute to a more fundamental and comprehensive understanding of this important biological process. We used microparticles coated with IgG antibodies as phagocytic targets for single macrophage cells. We trapped the particles with highly focused laser beams, so called optical tweezers, to bring them in contact with the membrane of the cells. In these experiments, we used the optical tweezers not only to initiate contact between the particles and the cells, but also to measure the mechanical properties of the cells at the location of the particle uptake and as a function of time. By using blinking tweezers, i.e. by turning them off and on sequentially, we were able the measure viscoelastic parameters like the local compliance and the viscoelastic power law exponent within the framework of a creep compliance model. We found that while the contact area between the particles and the cells increases, the actin cortex of the cells appears to get harder due to stress stiffening and it also becomes more elastic, i.e. less fluid as time progresses. Furthermore, we found that a partial disruption of the actin cytoskeleton leads to locally more compliant and more fluid cells. From these findings, we conclude that previously suggested local and transient fluidizations of the actin cortex during the early stages of phagocytosis can likely only occur on length scales that are smaller than the phagocytic targets used here. In addition, we also tracked the intracellular transport of the microparticles after their internalization. It was already known that biochemical factors influence this transport, which is an important part of the intracellular degradation of internalized bacteria. However, it was not known whether the size of the phagosome has also an influence on the transport. To investigate this, we tracked the transport of small (1 µm), medium-sized (2 µm) and large phagosomes (3 µm) by time-lapse microscopy and found that the size of phagosomes has a strong influence on their transport: Large phagosomes are transported very persistently and effectively to the nucleus, whereas small phagosomes show strong bidirectional transport with a low persistency and a small effective velocity. We show that dynein motors play a larger role in the transport of large phagosomes, whereas actin filament-based motility plays a larger role in the transport of small phagosomes. Furthermore, we investigated the spatial distribution of dyneins and microtubules around phagosomes and hypothesize that the spatial organization of the motors and the cytoskeleton can explain part of the observed transport characteristics. Our findings regarding the size-dependent phagosomal transport and its possible underlying mechanisms might contribute to optimizing particle-based drug carriers that are targeted to either the perinuclear region or the cell periphery. Furthermore, our findings suggest that a basic size-dependent cellular sorting mechanism might exist that increases transport of large phagocytosed bacteria towards the nucleus to support their digestion, and that simultaneously increases backwards transport of small bacterial fragments for example for antigen presentation. If such a size-dependent intracellular sorting leads to faster degradation of larger phagosomes, drugs that cause a clustering of pathogens could potentially lead to more efficient clearing by macrophages.
Projektbezogene Publikationen (Auswahl)
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Application of optical tweezers for biochemical and thermal cell stimulation. in: Light Robotics - Structure-mediated Nanobiophotonics, 385-410 (2017)
K. Berghoff, S. Keller, W. Gross, L. Gebhardt and H. Kress
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Phagosomal transport depends strongly on phagosome size. Scientific Reports 7, 17068 (2017)
S. Keller, K. Berghoff and H. Kress
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Entwicklung einer Arbeitsstation zur optischen Mikromanipulation und Charakterisierung des Einflusses von Aktin auf die Zellmechanik der Phagozytose. PhD Thesis, University of Bayreuth (2018)
K. Berghoff
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Abhängigkeit des phagosomalen Transportes von der Größe des Phagosomes. PhD Thesis, University of Bayreuth (2019)
S. Keller
