Augmin deficiency in neural stem cells causes p53-dependent apoptosis and aborts brain development

Microtubules that assemble the mitotic spindle are generated by centrosomal nucleation, chromatin-mediated nucleation, and nucleation from the surface of other microtubules mediated by the augmin complex. Impairment of centrosomal nucleation in apical progenitors of the developing mouse brain induces p53-dependent apoptosis and causes non-lethal microcephaly. Whether disruption of non-centrosomal nucleation has similar effects is unclear. Here we show, using mouse embryos, that conditional knockout of the augmin subunit Haus6 in apical progenitors led to spindle defects and mitotic delay. This triggered massive apoptosis and abortion of brain development. Co-deletion of Trp53 rescued cell death, but surviving progenitors failed to organize a pseudostratified epithelium, and brain development still failed. This could be explained by exacerbated mitotic errors and resulting chromosomal defects including increased DNA damage. Thus, in contrast to centrosomes, augmin is crucial for apical progenitor mitosis, and, even in the absence of p53, for progression of brain development.

A brain-penetrant microtubule-targeting agent that disrupts hallmarks of glioma tumorigenesis

Background Glioma is sensitive to microtubule-targeting agents (MTAs), but most MTAs do not cross the blood brain barrier (BBB). To address this limitation, we developed the new chemical entity, ST-401, a brain-penetrant MTA. Methods Synthesis of ST-401. Measures of MT assembly and dynamics. Cell proliferation and viability of patient-derived (PD) glioma in culture. Measure of tumor microtube (TM) parameters using immunofluorescence analysis and machine learning-based workflow. Pharmacokinetics (PK) and experimental toxicity in mice. In vivo antitumor activity in the RCAS/tv-a PDGFB-driven glioma (PDGFB-glioma) mouse model. Results We discovered that ST-401 disrupts microtubule (MT) function through gentle and reverisible reduction in MT assembly that triggers mitotic delay and cell death in interphase. ST-401 inhibits the formation of TMs, MT-rich structures that connect glioma to a network that promotes resistance to DNA damage. PK analysis of ST-401 in mice shows brain penetration reaching antitumor concentrations, and in vivo testing of ST-401 in a xenograft flank tumor mouse model demonstrates significant antitumor activity and no over toxicity in mice. In the PDGFB-glioma mouse model, ST-401 enhances the therapeutic efficacies of temozolomide (TMZ) and radiation therapy (RT). Conclusion Our study identifies hallmarks of glioma tumorigenesis that are sensitive to MTAs and reports ST-401 as a promising chemical scaffold to develop brain-penetrant MTAs.

Disruption of augmin-mediated microtubule nucleation in neural stem cells causes p53-dependent apoptosis and aborts brain development

SummaryMicrotubules that assemble the mitotic spindle are generated by three different mechanisms: centrosomal nucleation, chromatin-mediated nucleation, and nucleation from the surface of other microtubules mediated by the augmin complex. Impairment of centrosomal nucleation in apical progenitors of the developing mouse brain induces p53-dependent apoptosis and causes non-lethal microcephaly. Whether disruption of non-centrosomal nucleation has similar effects is unclear. Here we show, using mouse embryos, that conditional knockout of the augmin subunit Haus6 in apical progenitors led to spindle defects and mitotic delay. This triggered massive apoptosis and complete abortion of brain development. Co-deletion of p53 rescued cell death, but brain development was still aborted. This could be explained by exacerbated mitotic errors and resulting chromosomal defects including increased DNA damage. Surviving progenitors had lost apico-basal polarity and failed to organize a pseudostratified epithelium. Thus, in contrast to the centrosomal nucleation pathway, augmin is crucial for apical progenitor mitosis, and, even in the absence of p53, for progression of brain development.

The centriole protein CEP76 negatively regulates PLK1 activity in the cytoplasm for proper mitotic progression

ABSTRACTPolo-like kinase 1 (PLK1) dynamically changes its localization and plays important roles in proper mitotic progression. In particular, strict control of cytoplasmic PLK1 is needed to prevent mitotic defects. However, the regulation of cytoplasmic PLK1 is not fully understood. In this study, we show that CEP76, a centriolar protein, physically interacts with PLK1 and tightly controls the activation of cytoplasmic PLK1 during mitosis in human cells. We found that removal of centrosomes induced ectopic aggregation of PLK1, which is highly phosphorylated, in the cytoplasm during mitosis. Importantly, a targeted RNAi screen revealed that depletion of CEP76 resulted in a similar phenotype. In addition, depletion of CEP76 caused defective spindle orientation and mitotic delay. Moreover, the formation of ectopic PLK1 aggregates and defective spindle orientation were significantly suppressed by the inhibition of PLK1 kinase activity. Overall, these results demonstrate that CEP76 suppresses the aberrant activation of cytoplasmic PLK1 for proper mitotic progression.This article has an associated First Person interview with the first author of the paper.

Growth-Dependent Activation of Protein Kinases Suggests a Mechanism for Measuring Cell Growth

In all cells, progression through the cell cycle occurs only when sufficient growth has occurred. Thus, cells must translate growth into a proportional signal that can be used to measure and transmit information about growth. Previous genetic studies in budding yeast suggested that related kinases called Gin4 and Hsl1 could function in mechanisms that measure bud growth; however, interpretation of the data was complicated by the use of gene deletions that cause complex terminal phenotypes. Here, we used the first conditional alleles of Gin4 and Hsl1 to more precisely define their functions. We show that excessive bud growth during a prolonged mitotic delay is an immediate consequence of inactivating Gin4 and Hsl1. Thus, acute loss of Gin4 and Hsl1 causes cells to behave as though they cannot detect that bud growth has occurred. We further show that Gin4 and Hsl1 undergo gradual hyperphosphorylation during bud growth that is dependent upon growth and correlated with the extent of growth. Moreover, gradual hyperphosphorylation of Gin4 during bud growth requires binding to anionic phospholipids that are delivered to the growing bud. While alternative models are possible, the data suggest that signaling lipids delivered to the growing bud generate a growth-dependent signal that could be used to measure bud growth.

A new approach to automated CBMN scoring following high doses

In recent years we have automated the CBMN assay using microvolumes of blood, processed in multiwell plates. We have seen that at doses above 6 Gy the detected yield of micronuclei actually declines with dose, likely because of mitotic delay, preventing cells from forming micronuclei and also, when using one color imaging, resulting in many false binucleated cells, consisting of two randomly-adjacent nuclei. By using the inverse mitotic index (the ratio of mononuclear to binuclear cells) to adjust the micronucleus yield we were able to obtain a monotonic increasing dose response curve at doses of up to at least 10 Gy from the same samples which generated dose-response curve with a peak near 6 Gy, when scored using the traditional micronucleus yield.

Growth-dependent activation of protein kinases suggests a mechanism for measuring cell growth

In all cells, progression through the cell cycle occurs only when sufficient growth has occurred. Thus, cells must translate growth into a proportional signal that can be used to measure and transmit information about growth. Previous genetic studies in budding yeast suggested that related kinases called Gin4 and Hsl1 could play roles in mechanisms that measure bud growth; however, interpretation of the data was complicated by the use of gene deletions that cause complex terminal phenotypes. Here, we used the first conditional alleles of Gin4 and Hsl1 to more precisely define their functions. We show that excessive bud growth during a prolonged mitotic delay is an immediate consequence of inactivating Gin4 and Hsl1. Thus, acute loss of Gin4 and Hsl1 causes cells to behave as though they cannot detect that bud growth has occurred. We further show that Gin4 and Hsl1 undergo gradual hyperphosphorylation during bud growth that is dependent upon growth and correlated with the extent of growth. Moreover, gradual hyperphosphorylation of Gin4 during bud growth requires binding to anionic phospholipids that are delivered to the growing bud. While alternative models are possible, the data suggest that signaling lipids delivered to the growing bud generate a growth-dependent signal that could be used to measure bud growth.

A Nodal/Eph signalling relay drives the transition from apical constriction to apico-basal shortening in ascidian endoderm invagination

Gastrulation is the first major morphogenetic event during animal embryogenesis. Ascidian gastrulation starts with the invagination of 10 endodermal precursor cells between the 64- and late 112-cell stages. This process occurs in the absence of endodermal cell division and in two steps, driven by myosin-dependent contractions of the acto-myosin network. First, endoderm precursors constrict their apex. Second, they shorten apico-basally, while retaining small apical surfaces, thereby causing invagination. The mechanisms controlling the endoderm mitotic delay, the step 1 to step 2 transition, and apico-basal shortening have remained elusive. Here, we demonstrate the conserved role during invagination of Nodal and Eph signalling in two distantly related ascidian species (Phallusia mammillata and Ciona intestinalis). We show that the transition to step 2 is controlled by Nodal relayed by Eph signalling and that Eph signalling has a Nodal-independent role in mitotic delay. Interestingly, both Nodal and Eph signals are dispensable for endodermal germ layer fate specification.Summary statementIdentification of a regulatory developmental signalling sub-network driving endoderm cell shape changes during ascidian endoderm invagination, not involved in cell fate specification.