title: Investigating Calcium binding protein 7 (CaBP7), phosphoinositide signalling and lysosomes during mammalian cell mitosis
Creators: name: Rajamanoharan, Dayani
Publisher: name: University of Liverpool Repository
Year: 2016

description: Calcium binding proteins (CaBPs) are a subfamily of the calmodulin related superfamily of EF hand containing proteins. CaBPs can be further divided into two subgroups (CaBPs 1-5 and CaBP7 and 8) due to differing cation binding properties and because CaBP7 and 8 have a distinct trans- membrane domain at the C-terminal that is essential for determining their subcellular location. CaBP7 and 8 have been shown to interact with Golgi resident Phosphatidylinositol 4-Kinase-IIIbeta (PI4KIIIβ) and to be involved in calcium (Ca2+) regulated Golgi to plasma membrane trafficking pathway. At resting Ca2+ levels CaBP7 and 8 interact with PI4KIIIβ and inhibit its enzymatic function, to prevent phosphatidylinositol 4-phosphate (PI4P) synthesis and vesicular trafficking. At high Ca2+ levels another Golgi resident Ca2+-binding protein, neuronal calcium sensor-1 (NCS-1), displaces CaBP7 and 8 from PI4KIIIβ, stimulating PI4P production and thereby coupling local Ca2+ signals to vesicular transport. In addition to this documented trafficking function, a high- throughput RNAi screen identified CaBP7 as an essential factor for successful completion of cytokinesis in HeLa cells. Mitotic cell division is a fundamental biological process required for normal cellular growth, development and aging. Mitotic failure can lead to a state of aneuploidy, which is an accepted driver of cellular transformation and tumorigenesis. Therefore, this thesis specifically focused on CaBP7 with an aim to understand its unique role during mammalian cell mitosis. When the subcellular localisation of CaBP7 was examined it was found to be present on both the Golgi complex and, unexpectedly, lysosomes. A recent study identified a previously uncharacterised lysosomal pool of PI4KIIIβ, ! i! cellular depletion of which disrupted lysosome trafficking and ultimately led to distinctive lysosomal clustering. In an effort to connect these findings, analyses were designed to reveal whether CaBP7 was involved in regulation lysosomal PI4KIIIβ. CaBP7 overexpression (inhibition of PI4KIIIβ) increased clustering of lysosomes in a similar manner to that observed on cellular depletion of PI4KIIIβ. This result provides evidence to suggest a role for CaBP7 in lysosomal PI4KIIIβ regulation and lysosome trafficking, which will require further research to fully delineate. In order to further understand CaBP7 involvement in mitosis, CaBP7 was depleted from cells using shRNAi, which resulted in a 3-fold increase in binucleate cells compared to control cells. Binucleate cells form as a direct consequence of cytokinesis failure implying a functional requirement for CaBP7 during this process. This data replicated findings from the previous large scale RNAi screen and was extended upon significantly in this study through an analysis of a range of PI4KIIIβ effectors and their influence on mitosis. The same binucleate phenotype was observed with PI4KIIIβ overexpression suggesting a role for CaBP7 in regulating PI4KIIIβ during cytokinesis. Localisation studies revealed that CaBP7, PI4KIIIβ and lysosomes re-distributed together extensively during mitosis implying a link between all three in this process. In particular, at cytokinesis, all three components were localised in discrete clumps flanking either side of the nucleus. Intriguingly this marked re-distribution was lost upon CaBP7 depletion, possibly revealing a mechanistic link to cytokinesis failure. Collectively, data acquired regarding CaBP7, PI4KIIIβ and lysosomes inferred a role for lysosome positioning during mitosis and to test this ! ii! hypothesis experiments were designed to examine a requirement for specific lysosomal activities during cytokinesis. Lysosomes have emerged as Ca2+ signalling platforms and this function was assessed using novel genetically encoded Ca2+ sensors targeted specifically to these organelles. No Ca2+ signals originating from lysosomes during mitotic cell division were detected in these analyses. The other known functions of lysosomes were also examined in these studies. Inhibition of lysosomal catabolism failed to influence mitosis however disruption of lysosomal membrane fusion with the agents GPN and vacuolin-1 induced a significant increase of binucleate cell numbers. Collectively these functional assays suggest a potential requirement for lysosomal membrane fusion during cytokinesis, which would be consistent with a documented function for endosomes during this process. This thesis provides new insights into the role of a Ca2+ binding proteins, phosphoinositide signalling and, uniquely, lysosomal compartments, during mammalian cell mitosis. It describes an outline for a potentially new regulatory input into mitosis and provides a platform for future detailed examinations of the mechanistic links between CaBP7, lysosomes, lysosomal PI4KIIIβ activity and PI4P levels during normal cytokinesis in mammalian cells.