Investigating Calcium binding protein 7 (CaBP7), phosphoinositide signalling and lysosomes during mammalian cell mitosis

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

Modulation of phosphatidylinositol 4-phosphate levels by CaBP7 controls cytokinesis in mammalian cells

Calcium and phosphoinositide signaling regulate cell division in model systems, but their significance in mammalian cells is unclear. Calcium-binding protein-7 (CaBP7) is a phosphatidylinositol 4-kinaseIIIβ (PI4KIIIβ) inhibitor required during cytokinesis in mammalian cells, hinting at a link between these pathways. Here we characterize a novel association of CaBP7 with lysosomes that cluster at the intercellular bridge during cytokinesis in HeLa cells. We show that CaBP7 regulates lysosome clustering and that PI4KIIIβ is essential for normal cytokinesis. CaBP7 depletion induces lysosome mislocalization, extension of intercellular bridge lifetime, and cytokinesis failure. These data connect phosphoinositide and calcium pathways to lysosome localization and normal cytokinesis in mammalian cells

A centrosome-localized calcium signal is essential for mammalian cell mitosis

Mitosis defects can lead to premature ageing and cancer. Understanding mitosis regulation therefore has important implications for human disease. Early data suggested that calcium (Ca2+) signals could influence mitosis, but these have hitherto not been observed in mammalian cells. Here, we reveal a prolonged yet spatially restricted Ca2+ signal at the centrosomes of actively dividing cells. Local buffering of the centrosomal Ca2+ signals, by flash photolysis of the caged Ca2+ chelator diazo‐2‐acetoxymethyl ester, arrests mitosis. We also provide evidence that this Ca2+ signal emanates from the endoplasmic reticulum. In summary, we characterize a unique centrosomal Ca2+ signal as a functionally essential input into mitosis.—Helassa, N., Nugues, C., Rajamanoharan, D., Burgoyne, R. D., Haynes, L. P. A centrosome‐localized calcium signal is essential for mammalian cell mitosis

Lysosome exocytosis is required for mitosis

Mitosis, the accurate segregation of duplicated genetic material into what will become two new daughter cells, is accompanied by extensive membrane remodelling and membrane trafficking activities. Early in mitosis, adherent cells partially detach from the substratum, round up and their surface area decreases. This likely results from an endocytic uptake of plasma membrane material. As cells enter cytokinesis they re-adhere, flatten and exhibit an associated increase in surface area. The identity of the membrane donor for this phase of mitosis remains unclear. Here we show by biochemical and imaging approaches that lysosomes undergo exocytosis at telophase and that this requires the activity of phosphatidylinositol 4-kinase-IIIβ. Inhibition of lysosome exocytosis resulted in mitotic failure in a significant proportion of cells suggesting that this facet of lysosome physiology is essential and represents a new regulatory mechanism in mitosis

Small Heat Shock Proteins Are Novel Common Determinants of Alcohol and Nicotine Sensitivity in Caenorhabditis elegans

Addiction to drugs is strongly determined by multiple genetic factors. Alcohol and nicotine produce distinct pharmacological effects within the nervous system through discrete molecular targets; yet, data from family and twin analyses support the existence of common genetic factors for addiction in general. The mechanisms underlying addiction, however, are poorly described and common genetic factors for alcohol and nicotine remain unidentified. We investigated the role that the heat shock transcription factor, HSF-1, and its downstream effectors played as common genetic modulators of sensitivity to addictive substances. Using Caenorhabditis elegans, an exemplary model organism with substance dose-dependent responses similar to mammals, we demonstrate that HSF-1 altered sensitivity to both alcohol and nicotine. Using a combination of a targeted RNAi screen of downstream factors and transgenic approaches we identified that these effects were contingent upon the constitutive neuronal expression of HSP-16.48, a small heat shock protein (HSP) homolog of human α-crystallin. Furthermore we demonstrated that the function of HSP-16.48 in drug sensitivity surprisingly was independent of chaperone activity during the heat shock stress response. Instead we identified a distinct domain within the N-terminal region of the HSP-16.48 protein that specified its function in comparison to related small HSPs. Our findings establish and characterize a novel genetic determinant underlying sensitivity to diverse addictive substances