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. 2012 Apr 10;109(15):5729-34.
doi: 10.1073/pnas.1108537109. Epub 2012 Mar 26.

Drosophila Mgr, a Prefoldin subunit cooperating with von Hippel Lindau to regulate tubulin stability

Nathalie Delgehyr  1 , Uta Wieland, Hélène Rangone, Xavier Pinson, Guojie Mao, Nikola S Dzhindzhev, Doris McLean, Maria G Riparbelli, Salud Llamazares, Giuliano Callaini, Cayetano Gonzalez, David M Glover
Affiliations

Drosophila Mgr, a Prefoldin subunit cooperating with von Hippel Lindau to regulate tubulin stability

Nathalie Delgehyr et al. Proc Natl Acad Sci U S A. .

Erratum in

Abstract

Mutations in Drosophila merry-go-round (mgr) have been known for over two decades to lead to circular mitotic figures and loss of meiotic spindle integrity. However, the identity of its gene product has remained undiscovered. We now show that mgr encodes the Prefoldin subunit counterpart of human von Hippel Lindau binding-protein 1. Depletion of Mgr from cultured cells also leads to formation of monopolar and abnormal spindles and centrosome loss. These phenotypes are associated with reductions of tubulin levels in both mgr flies and mgr RNAi-treated cultured cells. Moreover, mgr spindle defects can be phenocopied by depleting β-tubulin, suggesting Mgr function is required for tubulin stability. Instability of β-tubulin in the mgr larval brain is less pronounced than in either mgr testes or in cultured cells. However, expression of transgenic β-tubulin in the larval brain leads to increased tubulin instability, indicating that Prefoldin might only be required when tubulins are synthesized at high levels. Mgr interacts with Drosophila von Hippel Lindau protein (Vhl). Both proteins interact with unpolymerized tubulins, suggesting they cooperate in regulating tubulin functions. Accordingly, codepletion of Vhl with Mgr gives partial rescue of tubulin instability, monopolar spindle formation, and loss of centrosomes, leading us to propose a requirement for Vhl to promote degradation of incorrectly folded tubulin in the absence of functional Prefoldin. Thus, Vhl may play a pivotal role: promoting microtubule stabilization when tubulins are correctly folded by Prefoldin and tubulin destruction when they are not.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The mgr mutant flies have microtubule-based abnormalities. (A) Representative mitotic spindles from squashed preparations of wild-type (Oregon R) and mgr (mgr5/mgrl4) mutant third-instar larval brains stained to reveal microtubules (β-tubulin, green), centrosome (Spd-2, red), and DNA (Dapi, blue). (Right column, Left and Right) Images of mgr neuroblasts show examples of monopolar spindles; (Center) a bipolar spindle of abnormal morphology. (Scale bar, 5 μm.) (B) Table indicating the mitotic defects observed in wild-type, mgr5/mgrl4 (compared with wild-type, mitotic defects P = 0.001; monopolar spindles P = 0.021), and mgrl4/Df (compared with wild-type P < 0.0001 for both mitotic defects and monopolar spindles); P values from χ2 analysis. Mitotic defects comprised monopolar and disorganized spindles (at least five independent brains scored). (C) Testes from wild-type and mgr (mgr5/mgrl4) mutant flies stained to reveal microtubules (β-tubulin, green) and DNA (Dapi, blue). (Scale bar, 10 μm.) (Upper) Young cysts (spermatogonia) in the apical region: arrowheads indicate mitotic cysts, shown in the Inset at 3× magnification; (Lower) Late primary spermatocytes with impaired microtubule network particularly in meiosis (compare outlined cysts). (D) Meiosis I spindles from wild-type and mgr stained to reveal microtubules (β-tubulin, green), centrosomes (Spd-2, red), and DNA (Dapi, blue). (Scale bar, 10 μm.) Of the meiotic spindles observed in mgr mutant testes, 100% were abnormal compared with wild-type where no abnormalities were observed (at least five testes scored and >208 complete cysts observed, P value from Student t test < 0.0001). (E) Western blots of the ubiquitous α- and β1-tubulin and the testes specific β2-tubulin isoform in testes protein extracts from wild-type and mgr mutant flies, showing that all three tubulin levels are reduced in mgr mutants. Amido black staining is the loading control (Ctrl). (F) Western blot of β-tubulin and Mgr in wild-type and mgr CNS and testes protein extracts, showing the absence of Mgr and the differential depletion of tubulin according to the tissue. Amido black staining is the loading control (Ctrl).
Fig. 2.
Fig. 2.
Mgr or partial β-tubulin depletion result in similar microtubule-based abnormalities. DMEL-2 cells transfected with control (Ctrl) or mgr dsRNA for 3-d intervals up to a maximum of 12 d (A–F). (A) Percentage of prometaphase and metaphase cells with monopolar or disorganized spindles scored following immunostaining, as in B. Error bars = SEMs for more than three independent experiments; n > 150 metaphase cells. (B) Cells immunostained to reveal microtubules (α-tubulin) 6 d after transfection. (Scale bar, 10 μm.) (C) Percentage of cells without centrosomes scored after immunostaining, as in D. Error bars = SEMs of more than five independent experiments; n > 1,000 cells. (D) Cells immunostained to reveal centrosomes (Dplp) 6 d after transfection. (Scale bar, 10 μm.) (E) Electron micrographs of centrioles in control cells (Ctrl RNAi) and following 9–12 d of mgr RNAi. Twenty-percent of the centrioles showed an abnormal structure after Mgr depletion, whereas none were observed in the control depletion (n = 10). (Scale bar, 0.1 μm.) (F) Western blot of β-tubulin, Mgr and H2A (loading control, Ctrl) 6 d after transfection with mgr, pfdn4, or control dsRNAs. (G–K) DMEL-2 cells treated with a range of concentrations (3, 10, and 25 ng/mL) of β-tubulin dsRNA for 6 d. (G) Western blot of β-tubulin and H2A (loading control, Ctrl) following such treatment. (H) Proportion of prometaphase and metaphase cells with monopolar or disorganized spindles in relation to β-tubulin dsRNA treatment. Error bars = SEMs of more than two independent experiments; n > 100 metaphase cells. (I) Cells labeled with an anti-α-tubulin to reveal spindle microtubules in control and β-tubulin dsRNA treated cells. (Scale bar 10 μm.) (J) Percentage of cells without centrosomes in relation to β-tubulin dsRNA treament. Error bars = SEMs of more than two independent experiments; n > 200 cells. (K) Cells labeled to reveal centrosomes (Dplp) following control dsRNA and β-tubulin dsRNA treatment. (Scale bar, 10 μm.)
Fig. 3.
Fig. 3.
Mgr is a sensor of a free pool of tubulin. (A) Western blot of GFP or endogenous β-tubulin in extracts of testes of wild-type or mgrl4/mgr5 flies in presence or absence of an exogenous GFP-tagged β1-tubulin transgene. Note the β1-tubulin-GFP is not detected by the β-tubulin antibody due to low levels of expression. Amido black staining is loading control (Ctrl). (B) Quantitation of the endogenous β-tubulin levels relative to the wild-type control on Western blots. Error bars = SEMs for two independent experiments. (C) Wild-type field images of brain squashes from indicated genotypes stained to reveal microtubules (β-tubulin). (Scale bar, 5 μm.)
Fig. 4.
Fig. 4.
Mgr and Vhl cooperate in regulating tubulin destruction. (A) MBP, MBP-Mgr, and MBP-Vhl, affinity purified from Escherichia coli extracts (Coomassie stain) tested for binding 35S-Methionine labeled Mgr and Vhl synthesized by coupled transcription-translation in vitro (Autoradiography). (B) MBP and MBP-Mgr, affinity-purified from E. coli extracts (Coomassie stain) tested for binding purified αβ-tubulin (Western blot). (C) MBP and MBP-Vhl, affinity-purified from E. coli extracts (Coomassie stain, Right) tested for binding-purified αβ-tubulin (Coomassie stain, Left). (D) MBP-Vhl, affinity purified from E. coli extracts, and tested for binding 35S-Mgr (as in A). Excess of purified αβ-tubulin is insufficient to release the Vhl:Mgr interaction. (E–J) DMEL-2 cells treated with Control, mgr, Vhl, or mgr and Vhl dsRNA for 6 or 9 d. (E) Levels of β-tubulin in three independent experiments 9 d after transfection. (F) Western blot of β-tubulin and Mgr after such treatment. H2A is used as loading control (Ctrl). (G) Percentage of prometaphase and metaphase cells with monopolar or disorganized spindles after indicated dsRNA treatment. Error bars = SEMs of three independent experiments. n > 300 metaphase cells; (H) Mitotic cells immunostained to reveal microtubules (α-tubulin). (Scale bar, 10 μm.) (I) Percentage of cells without centrosome 9 d after indicated transfections. Error bars = SEM of three independent experiments. n > 600 cells. (J) Cells immunostained to reveal centrosomes (Dplp). (Scale bar, 10 μm.) All P values are from Student t tests.
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