Functional Characterisation and Drug Target Validation of a Mitotic Kinesin-13 in Trypanosoma brucei

Mitotic kinesins are essential for faithful chromosome segregation and cell proliferation. Therefore, in humans, kinesin motor proteins have been identified as anti-cancer drug targets and small molecule inhibitors are now tested in clinical studies. Phylogenetic analyses have assigned five of the approximately fifty kinesin motor proteins coded by Trypanosoma brucei genome to the Kinesin-13 family. Kinesins of this family have unusual biochemical properties because they do not transport cargo along microtubules but are able to depolymerise microtubules at their ends, therefore contributing to the regulation of microtubule length. In other eukaryotic genomes sequenced to date, only between one and three Kinesin-13s are present. We have used immunolocalisation, RNAi-mediated protein depletion, biochemical in vitro assays and a mouse model of infection to study the single mitotic Kinesin-13 in T. brucei. Subcellular localisation of all five T. brucei Kinesin-13s revealed distinct distributions, indicating that the expansion of this kinesin family in kinetoplastids is accompanied by functional diversification. Only a single kinesin (TbKif13-1) has a nuclear localisation. Using active, recombinant TbKif13-1 in in vitro assays we experimentally confirm the depolymerising properties of this kinesin. We analyse the biological function of TbKif13-1 by RNAi-mediated protein depletion and show its central role in regulating spindle assembly during mitosis. Absence of the protein leads to abnormally long and bent mitotic spindles, causing chromosome mis-segregation and cell death. RNAi-depletion in a mouse model of infection completely prevents infection with the parasite. Given its essential role in mitosis, proliferation and survival of the parasite and the availability of a simple in vitro activity assay, TbKif13-1 has been identified as an excellent potential drug target

The characterisation of family-13 kinesins in Trypanosoma brucei

Kinesins are motor proteins involved in the movement of organelles and sub-organelles along microtubule tracks within the cell. Phylogenetic analysis of the 46 kinesin genes coded by the Trypanosoma brucei genome resulted in the grouping of seven kinesin sequences into the Kinesin-13 family. Members of this family have been characterised in a number of model organisms and, unlike most kinesins, do not exhibit microtubule processivity and are capable of depolymerising microtubules. They play important roles in bipolar spindle assembly and chromosome segregation. Of the six T. brucei Kinesin-13 proteins that were characterised during this study, only one was found to have a nuclear localisation, while the rest were found localised to the mitochondrion, cell body or flagellum. Attempts to probe the function of these kinesins using RNAi resulted in a reduction of cell growth in three of the six kinesins studied, but no gross changes in cellular morphology were observed. The distinct localisation of five Kinesin-13 family members outside the nucleus suggests that the functional diversity of the Kinesin-13 family is larger than previously thought

The characterisation of family-13 kinesins in Trypanosoma brucei

Kinesins are motor proteins involved in the movement of organelles and sub-organelles along microtubule tracks within the cell. Phylogenetic analysis of the 46 kinesin genes coded by the Trypanosoma brucei genome resulted in the grouping of seven kinesin sequences into the Kinesin-13 family. Members of this family have been characterised in a number of model organisms and, unlike most kinesins, do not exhibit microtubule processivity and are capable of depolymerising microtubules. They play important roles in bipolar spindle assembly and chromosome segregation. Of the six T. brucei Kinesin-13 proteins that were characterised during this study, only one was found to have a nuclear localisation, while the rest were found localised to the mitochondrion, cell body or flagellum. Attempts to probe the function of these kinesins using RNAi resulted in a reduction of cell growth in three of the six kinesins studied, but no gross changes in cellular morphology were observed. The distinct localisation of five Kinesin-13 family members outside the nucleus suggests that the functional diversity of the Kinesin-13 family is larger than previously thought

The characterisation of family-13 kinesins in Trypanosoma brucei

Kinesins are motor proteins involved in the movement of organelles and sub-organelles along microtubule tracks within the cell. Phylogenetic analysis of the 46 kinesin genes coded by the Trypanosoma brucei genome resulted in the grouping of seven kinesin sequences into the Kinesin-13 family. Members of this family have been characterised in a number of model organisms and, unlike most kinesins, do not exhibit microtubule processivity and are capable of depolymerising microtubules. They play important roles in bipolar spindle assembly and chromosome segregation. Of the six T. brucei Kinesin-13 proteins that were characterised during this study, only one was found to have a nuclear localisation, while the rest were found localised to the mitochondrion, cell body or flagellum. Attempts to probe the function of these kinesins using RNAi resulted in a reduction of cell growth in three of the six kinesins studied, but no gross changes in cellular morphology were observed. The distinct localisation of five Kinesin-13 family members outside the nucleus suggests that the functional diversity of the Kinesin-13 family is larger than previously thought.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Centrosomal MPF triggers the mitotic and morphogenetic switches of fission yeast

Activation of mitosis promoting factor (MPF) drives mitotic commitment(1). In human cells active MPF appears first on centrosomes(2). We show that local activation of MPF on the equivalent organelle of fission yeast, the spindle pole body (SPB), promotes Polo kinase activity at the SPBs long before global MPF activation drives mitotic commitment. Artificially promoting MPF or Polo activity at various locations revealed that this local control of Plo1 activity on G2 phase SPBs dictates the timing of mitotic commitment. Cytokinesis of the rod shaped fission yeast cell generates a naïve “new” cell end. Growth is restricted to the experienced old end until a point in G2 phase called “New End Take Off” (NETO) when bipolar growth is triggered(3). NETO coincided with MPF activation of Plo1 on G2 phase SPBs(4). Both MPF and Polo activities were required for NETO and both induced NETO when ectopically activated at interphase SPBs. NETO promotion by MPF required polo. Thus, local MPF activation on G2 SPBs directs polo kinase to control at least two distinct and temporally separated, cell cycle transitions at remote locations

Elevated basal AMP-activated protein kinase activity sensitizes colorectal cancer cells to growth inhibition by metformin

Expression and activity of the AMP-activated protein kinase (AMPK) α1 catalytic subunit of the heterotrimeric kinase significantly correlates with poor outcome for colorectal cancer patients. Hence there is considerable interest in uncovering signalling vulnerabilities arising from this oncogenic elevation of AMPKα1 signalling. We have therefore attenuated mammalian target of rapamycin (mTOR) control of AMPKα1 to generate a mutant colorectal cancer in which AMPKα1 signalling is elevated because AMPKα1 serine 347 cannot be phosphorylated by mTORC1. The elevated AMPKα1 signalling in this HCT116 α1.S347A cell line confers hypersensitivity to growth inhibition by metformin. Complementary chemical approaches confirmed this relationship in both HCT116 and the genetically distinct HT29 colorectal cells, as AMPK activators imposed vulnerability to growth inhibition by metformin in both lines. Growth inhibition by metformin was abolished when AMPKα1 kinase was deleted. We conclude that elevated AMPKα1 activity modifies the signalling architecture in such a way that metformin treatment compromises cell proliferation. Not only does this mutant HCT116 AMPKα1-S347A line offer an invaluable resource for future studies, but our findings suggest that a robust biomarker for chronic AMPKα1 activation for patient stratification could herald a place for the well-tolerated drug metformin in colorectal cancer therapy

Supplementary Figure 1; Supplementary Figure 2; Supplementary Figure 3; from Elevated basal AMP-activated protein kinase activity sensitizes colorectal cancer cells to growth inhibition by metformin

Validation of CRISPR-edited ATP1A1 HCT116 cell lines for use as control lines.; Genotypic validation of CRISPR-edited HCT116 cells lines; AMPK activation sensitises HCT116 AMPKα1 wild type but not AMPKα1-KO edited colorectal cancer cells to metformin

Dialogue between centrosomal entrance and exit scaffold pathways regulates mitotic commitment

The fission yeast scaffold molecule Sid4 anchors the septum initiation network to the spindle pole body (SPB, centrosome equivalent) to control mitotic exit events. A second SPB-associated scaffold, Cut12, promotes SPB-associated Cdk1-cyclin B to drive mitotic commitment. Signals emanating from each scaffold have been assumed to operate independently to promote two distinct outcomes. We now find that signals from Sid4 contribute to the Cut12 mitotic commitment switch. Specifically, phosphorylation of Sid4 by NIMAFin1 reduces Sid4 affinity for its SPB anchor, Ppc89, while also enhancing Sid4's affinity for casein kinase 1δ (CK1δ). The resulting phosphorylation of Sid4 by the newly docked CK1δ recruits Chk2Cds1 to Sid4. Chk2Cds1 then expels the Cdk1-cyclin B antagonistic phosphatase Flp1/Clp1 from the SPB. Flp1/Clp1 departure can then support mitotic commitment when Cdk1-cyclin B activation at the SPB is compromised by reduction of Cut12 function. Such integration of signals emanating from neighboring scaffolds shows how centrosomes/SPBs can integrate inputs from multiple pathways to control cell fate