Advantages and limitations of different p62-based assays for estimating autophagic activity in Drosophila.

Levels of the selective autophagy substrate p62 have been established in recent years as a specific readout for basal autophagic activity. Here we compared different experimental approaches for using this assay in Drosophila larvae. Similar to the more commonly used western blots, quantifying p62 dots in immunostained fat body cells of L3 stage larvae detected a strong accumulation of endogenous p62 aggregates in null mutants for Atg genes and S6K. Importantly, genes whose mutation or silencing results in early stage lethality can only be analyzed by microscopy using clonal analysis. The loss of numerous general housekeeping genes show a phenotype in large-scale screens including autophagy, and the p62 assay was potentially suitable for distinguishing bona fide autophagy regulators from silencing of a DNA polymerase subunit or a ribosomal gene that likely has a non-specific effect on autophagy. p62 accumulation upon RNAi silencing of known autophagy regulators was dependent on the duration of the knockdown effect, unlike in the case of starvation-induced autophagy. The endogenous p62 assay was more sensitive than a constitutively overexpressed p62-GFP reporter, which showed self-aggregation and large-scale accumulation even in control cells. We recommend western blots for following the conversion of overexpressed p62-GFP reporters to estimate autophagic activity if sample collection from mutant larvae or adults is possible. In addition, we also showed that overexpressed p62 or Atg8 reporters can strongly influence the phenotypes of each other, potentially giving rise to false or contradicting results. Overexpressed p62 aggregates also incorporated Atg8 reporter molecules that might lead to a wrong conclusion of strongly enhanced autophagy, whereas expression of an Atg8 reporter transgene rescued the inhibitory effect of a dominant-negative Atg4 mutant on basal and starvation-induced autophagy

Spatiotemporal dynamics of Spc105 regulates the assembly of the Drosophila kinetochore

The formation of kinetochores shortly before each cell division is a prerequisite for proper chromosome segregation. The synchronous mitoses of Drosophila syncytial embryos have provided an ideal in vivo system to follow kinetochore assembly kinetics and so address the question of how kinetochore formation is regulated. We found that the nuclear exclusion of the Spc105/KNL1 protein during interphase prevents precocious assembly of the Mis12 complex. The nuclear import of Spc105 in early prophase and its immediate association with the Mis12 complex on centromeres are thus the first steps in kinetochore assembly. The cumulative kinetochore levels of Spc105 and Mis12 complex then determine the rate of Ndc80 complex recruitment commencing only after nuclear envelope breakdown. The carboxy-terminal part of Spc105 directs its nuclear import and is sufficient for the assembly of all core kinetochore components and CENP-C, when localized ectopically to centrosomes. Super-resolution microscopy shows that carboxy-terminus of Spc105 lies at the junction of the Mis12 and Ndc80 complexes on stretched kinetochores. Our study thus indicates that physical accessibility of kinetochore components plays a crucial role in the regulation of Drosophila kinetochore assembly and leads us to a model in which Spc105 is a licensing factor for its onset

Atg17/FIP200 localizes to perilysosomal Ref(2)P aggregates and promotes autophagy by activation of Atg1 in Drosophila.

Phagophore-derived autophagosomes deliver cytoplasmic material to lysosomes for degradation and reuse. Autophagy mediated by the incompletely characterized actions of Atg proteins is involved in numerous physiological and pathological settings including stress resistance, immunity, aging, cancer, and neurodegenerative diseases. Here we characterized Atg17/FIP200, the Drosophila ortholog of mammalian RB1CC1/FIP200, a proposed functional equivalent of yeast Atg17. Atg17 disruption inhibits basal, starvation-induced and developmental autophagy, and interferes with the programmed elimination of larval salivary glands and midgut during metamorphosis. Upon starvation, Atg17-positive structures appear at aggregates of the selective cargo Ref(2)P/p62 near lysosomes. This location may be similar to the perivacuolar PAS (phagophore assembly site) described in yeast. Drosophila Atg17 is a member of the Atg1 kinase complex as in mammals, and we showed that it binds to the other subunits including Atg1, Atg13, and Atg101 (C12orf44 in humans, 9430023L20Rik in mice and RGD1359310 in rats). Atg17 is required for the kinase activity of endogenous Atg1 in vivo, as loss of Atg17 prevents the Atg1-dependent shift of endogenous Atg13 to hyperphosphorylated forms, and also blocks punctate Atg1 localization during starvation. Finally, we found that Atg1 overexpression induces autophagy and reduces cell size in Atg17-null mutant fat body cells, and that overexpression of Atg17 promotes endogenous Atg13 phosphorylation and enhances autophagy in an Atg1-dependent manner in the fat body. We propose a model according to which the relative activity of Atg1, estimated by the ratio of hyper- to hypophosphorylated Atg13, contributes to setting low (basal) vs. high (starvation-induced) autophagy levels in Drosophila

CDK-dependent phosphorylation and nuclear exclusion coordinately control kinetochore assembly state

Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex. Prior work has identified more than 100 different kinetochore components in human cells. However, little is known about the regulatory processes that specify their assembly upon mitotic entry and disassembly at mitotic exit. In this paper, we used a live-cell imaging–based assay to quantify kinetochore disassembly kinetics and systematically analyze the role of potential regulatory mechanisms in controlling kinetochore assembly state. We find that kinetochore assembly and disassembly was driven primarily by mitotic phosphorylation downstream of cyclin-dependent kinase (CDK). In addition, we demonstrate that nuclear exclusion of the Ndc80 complex helped restrict kinetochore formation to mitosis. Combining constitutive CDK-dependent phosphorylation of CENP-T and forced nuclear localization of the Ndc80 complex partially prevented kinetochore disassembly at mitotic exit and led to chromosome segregation defects in subsequent divisions. In total, we find that the coordinated temporal regulation of outer kinetochore assembly is essential for accurate cell division.Kinship Foundation. Searle Scholars ProgramLeukemia & Lymphoma Society of AmericaNational Institutes of Health (U.S.) (National Institute of General Medical Sciences (U.S.) Grant GM088313)Leukemia & Lymphoma Society of America (Special Fellows Award

Splicing factors Sf3A2 and Prp31 have direct roles in mitotic chromosome segregation

Several studies have shown that RNAi-mediated depletion of splicing factors (SFs) results in mitotic abnormalities. However, it is currently unclear whether these abnormalities reflect defective splicing of specific pre-mRNAs or a direct role of the SFs in mitosis. Here, we show that two highly conserved SFs, Sf3A2 and Prp31, are required for chromosome segregation in both Drosophila and human cells. Injections of anti-Sf3A2 and anti-Prp31 antibodies into Drosophila embryos disrupt mitotic division within 1 min, arguing strongly against a splicing-related mitotic function of these factors. We demonstrate that both SFs bind spindle microtubules (MTs) and the Ndc80 complex, which in Sf3A2- and Prp31-depleted cells is not tightly associated with the kinetochores; in HeLa cells the Ndc80/HEC1-SF interaction is restricted to the M phase. These results indicate that Sf3A2 and Prp31 directly regulate interactions among kinetochores, spindle microtubules and the Ndc80 complex in both Drosophila and human cells

A tumor suppressor role of the Bub3 spindle checkpoint protein after apoptosis inhibition.

Most solid tumors contain aneuploid cells, indicating that the mitotic checkpoint is permissive to the proliferation of chromosomally aberrant cells. However, mutated or altered expression of mitotic checkpoint genes accounts for a minor proportion of human tumors. We describe a Drosophila melanogaster tumorigenesis model derived from knocking down spindle assembly checkpoint (SAC) genes and preventing apoptosis in wing imaginal discs. Bub3-deficient tumors that were also deficient in apoptosis displayed neoplastic growth, chromosomal aneuploidy, and high proliferative potential after transplantation into adult flies. Inducing aneuploidy by knocking down CENP-E and preventing apoptosis does not induce tumorigenesis, indicating that aneuploidy is not sufficient for hyperplasia. In this system, the aneuploidy caused by a deficient SAC is not driving tumorigenesis because preventing Bub3 from binding to the kinetochore does not cause hyperproliferation. Our data suggest that Bub3 has a nonkinetochore-dependent function that is consistent with its role as a tumor suppressor

Orderly assembly underpinning built-in asymmetry in the yeast centrosome duplication cycle requires cyclin-dependent kinase

Funder: CSC Cambridge International ScholarshipAsymmetric astral microtubule organization drives the polarized orientation of the S. cerevisiae mitotic spindle and primes the invariant inheritance of the old spindle pole body (SPB, the yeast centrosome) by the bud. This model has anticipated analogous centrosome asymmetries featured in self-renewing stem cell divisions. We previously implicated Spc72, the cytoplasmic receptor for the gamma-tubulin nucleation complex, as the most upstream determinant linking SPB age, functional asymmetry and fate. Here we used structured illumination microscopy and biochemical analysis to explore the asymmetric landscape of nucleation sites inherently built into the spindle pathway and under the control of cyclin-dependent kinase (CDK). We show that CDK enforces Spc72 asymmetric docking by phosphorylating Nud1/centriolin. Furthermore, CDK-imposed order in the construction of the new SPB promotes the correct balance of nucleation sites between the nuclear and cytoplasmic faces of the SPB. Together these contributions by CDK inherently link correct SPB morphogenesis, age and fate