The Greatwall kinase/Mastl is an essential gene that indirectly inhibits the phosphatase activity toward mitotic Cdk1 substrates. Here we show that although Mastl knockout (Mastl^NULL) MEFs enter mitosis, they progress through mitosis without completing cytokinesis despite the presence of misaligned chromosomes, which causes chromosome segregation defects. Furthermore, we uncover the requirement of Mastl for robust spindle assembly checkpoint (SAC) maintenance since the duration of mitotic arrest caused by microtubule poisons in Mastl^NULL MEFs is shortened, which correlates with premature disappearance of the essential SAC protein Mad1 at the kinetochores. Notably, Mastl^NULL MEFs display reduced phosphorylation of a number of proteins in mitosis, which include the essential SAC kinase MPS1. We further demonstrate that Mastl is required for multi-site phosphorylation of MPS1 as well as robust MPS1 kinase activity in mitosis. In contrast, treatment of Mastl^NULL cells with the phosphatase inhibitor okadaic acid (OKA) rescues the defects in MPS1 kinase activity, mislocalization of phospho-MPS1 as well as Mad1 at the kinetochore, and premature SAC silencing. Moreover, using in vitro dephosphorylation assays, we demonstrate that Mastl promotes persistent MPS1 phosphorylation by inhibiting PP2A/B55-mediated MPS1 dephosphorylation rather than affecting Cdk1 kinase activity. Our findings establish a key regulatory function of the Greatwall kinase/Mastl->PP2A/B55 pathway in preventing premature SAC silencing. Cdk1 phosphorylates many substrates in mitosis and simultaneoulsy reduces the activity of the corresponding phosphatase PP2A through the Greatwall kinase/Mastl. When Mastl is deleted, cells progress through mitosis with missegregated chromosomes, which become unraveled. However, the molecular mechansims by which Mastl promotes proper chromosome segregation and mitotic progression remain elusive. In this study, we show that the Cdk1->Greatwall kinase/Mastl->PP2A pathway plays a central role in regulating the spindle assembly checkpoint (SAC) by preventing premature SAC silencing. We further demonstrate that Mastl is required for multi-site phosphorylation of the essntial SAC protein MPS1 as well as robust MPS1 kinase activity in mitosis by inhibiting PP2A/B55-mediated MPS1 dephosphorylation. Our findings establish the requirement of Mastl for robust SAC maintenance.

Mastl is a mitotic kinase that is essential for error-free chromosome segregation. It is an atypical member of AGC kinase family, possessing a unique non-conserved middle region. The mechanism of Mastl activation has been studied extensively in vitro. Phosphorylation of several residues were identified to be crucial for activation. These sites correspond to T193 and T206 in the activation loop and S861 in the C-terminal tail of mouse Mastl. To date, the significance of these phosphosites was not confirmed in intact mammalian cells. Here, we utilize a genetic complementation approach to determine the essentials of mammalian Mastl kinase activation. We used tamoxifen-inducible conditional knockout mouse embryonic fibroblasts to delete endogenous Mastl and screened various mutants for their ability to complement its loss. S861A mutant was able to complement endogenous Mastl loss. In parallel, we performed computational molecular docking studies to evaluate the significance of this residue for kinase activation. Our in-depth sequence and structure analysis revealed that Mastl pS861 does not belong to a conformational state, where the phosphoresidue contributes to C-tail docking. C-tail of Mastl is relatively short and it lacks a hydrophobic (HF) motif that would otherwise help its anchoring over N-lobe, required for the final steps of kinase activation. Our results show that phosphorylation of Mastl C-tail turn motif (S861) is dispensable for kinase function in cellulo. Graphical Abstract Communicated by Ramaswamy H. Sarma

The linker histone H1 C‐terminal domain (CTD) plays a pivotal role in chromatin condensation. De novo frameshift mutations within the CTD coding region of H1.4 have recently been reported to be associated with Rahman syndrome, a neurological disease that causes intellectual disability and overgrowth. To investigate the mechanisms and pathogenesis of Rahman syndrome, we developed a cellular model using murine embryonic stem cells (mESCs) and CRISPR/Cas9 genome engineering. Our engineered mES cells facilitate detailed investigations, such as H1‐4 dynamics, immunoprecipitation, and nuclear localization; in addition, we tagged the mutant H1‐4 with a photoactivatable GFP (PA‐GFP) and an HA tag to facilitate pulldown assays. We anticipate that these engineered cells could also be used for the development of a mouse model to study the in vivo role of the H1‐4 protein.

Autophagy is a highly conserved eukaryotic cellular process whose dysfunction results in human pathologies including cancer and neurodegenerative disease. First identified in yeast, ATG genes are central players in autophagy. Although their roles in cancer and neurodegenerative disease are well known, Mendelian diseases associated with ATG genes are rare. Mutations in core autophagy genes ATG5 and ATG7 have been previously reported to cause rare genetic disorders with autosomal recessive inheritance pattern. Here we report, for the first time, a rare genetic disorder that results from a deletion/frameshift mutation in human ATG9B, the placenta specific homologue of yeast ATG9 in humans. The 11-nucleotide deletion causes a frameshift and addition of a premature stop codon, truncating the C-terminal cytosolic domain of the ATG9B protein. The pediatric patients carrying the mutant allele homozygous were children of a consanguineous marriage and displayed neurodevelopmental anomalies including mental retardation. We hypothesized that this phenotype originates during placental development. To characterize the effects of the mutation and gain insight on the specific functions of ATG9B in a physiological setting, we used mammalian cells and generated a knock-in mouse model. Truncated ATG9B was not stable when expressed in cells. It was localized to perinuclear vesicles like the WT protein, but not to peripheral vesicles. Homozygous knock-in mice were viable, fertile and displayed no gross phenotypical abnormalities. Histomorphometry analysis of the placenta layers did not reveal a significant difference between mutant and control embryos. The assessments of neurobehavioral tests were similar in wild-type and homozygous knock-in mice. However, knock-in mice had a reduced fear memory trend, which is an amygdala-involved response.