Protein phosphatase 4 is an essential enzyme required for organisation of microtubules at centrosomes in Drosophila embryos

Abstract

The protein serine/threonine phosphatase 4 (PP4), which localises to centrosomes/spindle pole bodies in human cells, is shown to exhibit a similar localisation in Drosophila cells and embryos and possess a highly conserved (91% identical) amino acid sequence from humans to invertebrates. A homozygous Drosophila melanogaster strain mutant in the PP4 gene at 19C1-2 has been produced using P element mutagenesis. This strain, termed centrosomes minus microtubules (cmm), has reduced amounts of PP4 mRNA, approximately 25% of normal PP4 protein in early embryos and exhibits a semi-lethal phenotype with only 10% viability in certain conditions. Reversion mutagenesis shows that the phenotype is due to the presence of the P element in the PP4 mRNA. In early cmm embryos, nuclear divisions become asynchronous and large regions containing centrosomes with no well defined radiating microtubules are visible. In such areas, most nuclei arrest during mitosis with condensed DNA, and mitotic spindle microtubules are either absent, or aberrant and unconnected to the centrosome. A reduction in the staining of gamma-tubulin at centrosomes in cmm embryos suggests a conformational change or relocation of this protein, which is known to be essential for initiation of microtubule growth. These findings indicate that PP4 is required for nucleation, growth and/or stabilisation of microtubules at centrosomes/spindle pole bodies.

Requirement of Hsp90 for centrosomal function reflects its regulation of Polo kinase stability

“…The following antibodies were used: mouse monoclonal MA294 anti‐Polo (Llamazares et al ., 1991); rabbit Rb1011 anti‐γ‐tubulin (Tavosanis et al ., 1997); mouse monoclonal GTU‐88 anti‐γ‐tubulin (Sigma); rabbit anti‐Cp60 (Kellogg et al ., 1995); rabbit Rb188 anti‐Cp190 (Whitfield et al ., 1988); rabbit anti‐Cnn (Li and Kaufman, 1996); mouse monoclonal N356 anti‐α‐tubulin (Amersham); rat monoclonal 16F1 anti‐Hsp90α (Stressgen); mouse monoclonal anti‐Hsp70 (Stressgen); rabbit anti‐LK6 (Kidd and Raff, 1997), anti‐centrin (Salisbury et al ., 1988); rabbit anti‐TCP‐1α (Stressgen); rabbit anti‐14‐3‐3β (Santa Cruz Technologies); rabbit MA8 anti‐D‐TACC (Gergely et al ., 2000); sheep anti‐PP4 (Helps et al ., 1998) and rabbit ANGT anti‐ Drosophila Nek2 (C.Gonzalez and C.E.Sunkel, unpublished).…”

Protein Phosphatase 4 Negatively Regulates the Immune Deficiency-NF-κB Pathway during the Drosophila Immune Response

“…PP4c is essential for centrosome maturation in the Drosophila embryo, and loss-of-function mutants exhibit a high lethality rate (88). Therefore, we took advantage of the yeast UAS-Gal4 system to selectively drive a restricted expression of a dsRNA targeting the PP4-19c transcript in the fat body of adult flies, the main immune organ of Drosophila, using either the c564-Gal4 (combined to Tub-Gal80 ts ) (83,84) or the yolk-Gal4 transgenes (33).…”

Centrosomes and microtubule organisation during Drosophila development

“…There are few other centrosomal proteins of Drosophila that have been characterised at the molecular level, like LK6 (Kidd and Raff, 1997) and PP4 (Helps et al, 1998), but they have not yet been studied in terms of their association with different MTOCs. Likewise, there are many mutants which disrupt microtubule organisation, but their molecular cloning is still in progress.…”