611 Menschliche Anatomie, Zytologie, Histologie
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- Megakaryozyt (2)
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- RhoA (1)
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- fluorescence microscopy (1)
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- megakaryocyte (1)
- megakaryopoiesis (1)
- object segmentation (1)
- podosome formation (1)
- polarization (1)
Institute
Die MP4-induzierte experimentelle autoimmune Encephalomyelitis (EAE) erlaubt eine fokussierte Betrachtung von B-Zellen, die eine wichtige Rolle bei der Pathogenese der Multiplen Sklerose (MS) spielen. Es konnte zum Beispiel gezeigt werden, dass das Vorhandensein von B-Zell-Aggregaten im zentralen Nervensystem (ZNS) von MS-Patienten mit einem aggravierten Krankheitsverlauf assoziiert war. Diese Follikel könnten dabei als ektope lymphatische Strukturen den Immunprozess aktiv gestalten und somit ein therapeutisches Ziel darstellen. In der vorliegenden Studie wurde der Effekt des Sphingosin-1-Phosphat-Rezeptor-Modulators Fingolimod (FTY720) auf die autoreaktive B-Zell-Antwort und speziell die Bildung von B-Zell-Aggregaten im Kleinhirn der MP4-EAE-Mäuse untersucht.
Functional analysis of polarization and podosome formation of murine and human megakaryocytes
(2019)
In mammals, blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MK) that extend polarized cell protrusions (proplateles) into BM sinusoids. Proplatelet formation (PPF) requires substantial cytoskeletal rearrangements that have been shown to involve the formation of podosomes, filamentous actin (F-actin) and integrin-rich structures. However, the exact molecular mechanisms regulating MK podosome formation, polarization and migration within the BM are poorly defined. According to current knowledge obtained from studies with other cell types, these processes are regulated by Rho GTPase proteins like RhoA and Cdc42.
In this thesis, polarization and podosome formation were investigated in MKs from genetically modified mice, as well as the cell lines K562 and Meg01 by pharmacological modulation of signaling pathways.
The first part of this thesis describes establishment of the basic assays for investigation of MK polarization. Initial data on polarization of the MK-like erythroleukemia cell line K562 revealed first insights into actin and tubulin dynamics of wild type (WT) and RhoA knock-out (RhoA-/-) K562 cells. Phorbol 12-myristate 13-acetate (PMA)-induction of K562 cells led to the expected MK-receptor upregulation but also RhoA depletion and altered polarization patterns.
The second part of this thesis focuses on podosome formation of MKs. RhoA is shown to be dispensable for podosome formation. Cdc42 is revealed as an important, but not essential regulator of MK spreading and podosome formation. Studies of signaling pathways of podosome formation reveal the importance of the tyrosine kinases Src, Syk, as well as glycoprotein (GP)VI in MK spreading and podosome formation.
This thesis provides novel insights into the mechanisms underlying polarization and podosome formation of MKs and reveals new, important information about cytoskeletal dynamics of MKs and potentially also platelets.
The thesis provides insights in reconstruction and analysis pipelines for processing of
three-dimensional cell and vessel images of megakaryopoiesis in intact murine bone.
The images were captured in a Light Sheet Fluorescence Microscope. The work
presented here is part of Collaborative Research Centre (CRC) 688 (project B07) of
the University of Würzburg, performed at the Rudolf-Virchow Center. Despite ongoing
research within the field of megakaryopoiesis, its spatio-temporal pattern of
megakaryopoiesis is largely unknown. Deeper insight to this field is highly desirable to
promote development of new therapeutic strategies for conditions related to
thrombocytopathy as well as thrombocytopenia. The current concept of
megakaryopoiesis is largely based on data from cryosectioning or in vitro studies
indicating the existence of spatial niches within the bone marrow where specific stages
of megakaryopoiesis take place. Since classic imaging of bone sections is typically
limited to selective two-dimensional views and prone to cutting artefacts, imaging of
intact murine bone is highly desired. However, this has its own challenges to meet,
particularly in image reconstruction. Here, I worked on processing pipelines to account
for irregular specimen staining or attenuation as well as the extreme heterogeneity of
megakaryocyte morphology. Specific challenges for imaging and image reconstruction
are tackled and solution strategies as well as remaining limitations are presented and
discussed. Fortunately, modern image processing and segmentation strongly benefits
from continuous advances in hardware as well as software-development. This thesis
exemplifies how a combined effort in biomedicine, computer vision, data processing
and image technology leads to deeper understanding of megakaryopoiesis. Tailored
imaging pipelines significantly helped elucidating that the large megakaryocytes are
broadly distributed throughout the bone marrow facing a surprisingly dense vessel
network. No evidence was found for spatial niches in the bone marrow, eventually
resulting in a revised model of megakaryopoiesis.