An investigation into invaginated membrane system development, dynamics, and actin regulation throughout megakaryopoiesis

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O'Sullivan, Thomas James (2024). An investigation into invaginated membrane system development, dynamics, and actin regulation throughout megakaryopoiesis. University of Birmingham. Ph.D.

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Abstract

Thrombocytopenia is a condition defined by low blood platelet count, often linked to defects in platelet synthesis. Megakaryocytes are the precursors to platelets that undergo endomitosis and grow to up to 64n ploidy in the bone marrow. In the later stages of megakaryocyte development, a reservoir of membrane mass known as the invaginated membrane system (IMS) accumulates within megakaryocyte cytoplasm. Importantly, structural defects in the IMS can be seen in a range of conditions characterised by thrombocytopenia (\(\textit{Wiskott Aldrich syndrome}\), \(\textit{Bernard-Soulier syndrome}\) and \(\textit{filaminopathies}\)), linking defects in IMS maturation to a low platelet count. Furthermore, many thrombocytopenic conditions are caused by mutations in proteins linked to F-actin regulation \(\textit{FLNA}\), \(\textit{ACTB}\), \(\textit{MYH9}\), \(\textit{WAS}\)), whilst chemical inhibition of actin cytoskeleton during megakaryopoiesis leads to aberrated or halted IMS development, highlighting actin cytoskeleton as an essential component for functional IMS maturation.
During development, the IMS transverses through several distinctive morphological stages. Whilst IMS development has been broadly categorised: \(\textit{accumulation}\), \(\textit{expansion}\), and \(\textit{polarisation}\), there is a lack of definition surrounding IMS morphologies, or how IMS transitions through those morphologies during development. This study uses a range of microscopy techniques and bespoke image analysis pipelines to measure 4D IMS morphology in large populations of megakaryocytes. Here, we categorise megakaryocytes into 7 different stages of development: \(\textit{vacant}\), \(\textit{accumulation}\), \(\textit{compression}\), \(\textit{expansion}\), \(\textit{polarisation}\), \(\textit{destabilisation}\), and \(\textit{proplatelet formation}\) using qualitative morphometric data, and probe F-actin regulation of IMS maturation. Our data shows that:

• Megakaryocytes transition through polarisation of the IMS prior to proplatelet formation.
• A network of underlying invaginated tubules which prelude IMS compression and expansion.
• K-means cluster analysis can be used to perform unlabelled classification of morphometric datasets, and define rates of IMS maturation.

Furthermore, we provide evidence for:

• An Arp2/3 regulated actin cytoskeleton during IMS maturation
• An F-actin regulated polarisation and expulsion of the IMS.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):