Effect of αB-Crystallin on Protein Aggregation in Drosophila

Disorganisation and aggregation of proteins containing expanded polyglutamine (polyQ) repeats, or ectopic expression of α-synuclein, underlie neurodegenerative diseases including Alzheimer's, Parkinson, Huntington, Creutzfeldt diseases. Small heat-shock proteins, such as αB-crystallin, act as chaperones to prevent protein aggregation and play a key role in the prevention of such protein disorganisation diseases. In this study, we have explored the potential for chaperone activity of αB-crystallin to suppress the formation of protein aggregates. We tested the ability of αB-crystallin to suppress the aggregation of a polyQ protein and α-synuclein in Drosophila. We found that αB-crystallin suppresses both the compound eye degeneration induced by polyQ and the α-synuclein-induced rough eye phenotype. Furthermore, by using histochemical staining we have determined that αB-crystallin inhibits the aggregation of polyQ in vivo. These data provide a clue for the development of therapeutics for neurodegenerative diseases

Dynamics of Endoreplication during Drosophila Posterior Scutellar Macrochaete Development

Endoreplication is a variant type of DNA replication, consisting only of alternating G1 and S phases. Many types of Drosophila tissues undergo endoreplication. However, the timing and the extent to which a single endocycling macrochaete undergoes temporally programmed endoreplication during development are unclear. Here, we focused on the dynamics of endoreplication during posterior scutellar (pSC) macrochaete development. Quantitative analyses of C values in shaft cells and socket cells revealed a gradual rise from 8C and 4C at 8 hours after pupal formation (APF) to 72C and 24C at 29 hours APF, respectively. The validity of the values was further confirmed by the measurement of DNA content with a confocal laser microscope. BrdU incorporation assays demonstrated that shaft cells undergo four rounds of endoreplication from 18 to 29.5 hours APF. In contrast, socket cells undergo two rounds of endoreplication during the same period. Statistical analyses showed that the theoretical C values, based on BrdU assays, nearly coincide with the actually measured C values in socket cells, but not in shaft cells after 22 hours APF. These analyses suggest that socket cells undergo two rounds of endoreplication. However, the mechanism of endoreplication in the shaft cells may change from 22 hours APF, suggesting the possibility that shaft cells undergo two or four rounds of endoreplication during the periods. We also found that the timing of endoreplication differs, depending on the type of macrochaete. Moreover, endocycling in shaft cells of both the left and right sides of pSC bristle lineages occurs in the same pattern, indicating that the process is synchronized for specific types of macrochaete. Our findings suggest that endocycling in macrochaete cell lineages can be a model for understanding mechanisms of endoreplication at the single-cell level

Dynamics of BrdU incorporation in pSC shaft and socket cells from 17.5 to 29 hours APF.

<p>Pupae with the +/+ genotype were grown until particular times of development and BrdU incorporation assays were performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038714#s4" target="_blank">Materials and Methods</a>. Images show pSC shaft and socket cells stained with DAPI and anti-BrdU antibodies (A–H). Arrows indicate socket cell nuclei. Arrowheads indicate shaft cell nuclei. Note the BrdU incorporation observed in A, C, F, G in shaft cells and in C, D, H in socket cells. (I) Pattern of BrdU incorporation in shaft cells from 17.5 to 29 hours APF. The Y axis indicates the % of BrdU incorporation in shaft and socket cells. Sample numbers and detailed data are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038714#pone.0038714.s002" target="_blank">Table S1</a>.</p

Dynamics of nuclear growth in pSC shaft cells from 8 to 29 hours APF.

<p>(A–O) Images of pSC shaft cells from 8 to 29 hours APF. Pupae with the <i>A101-lacZ /+</i> genotype were grown till 8(n = 6), 12(n = 3), 18(n = 8), 20(n = 11), 21(n = 8), 22(n = 8), 23(n = 3), 24(n = 7), 25(n = 5), 26(n = 11), 27(n = 6), 29 hours APF (n = 7) and dissected thoraxes were stained with anti-lacZ and DAPI. Arrows indicate socket cell nuclei and arrowheads shaft cell nuclei. (P) Dynamics of nuclear growth in a pSC shaft cell from 8 to 29 hours APF. The Y axis indicates the section area of the shaft cell nucleus. Significant differences in mean values at each time point to those of adjacent time points were set at *P<0.05, **P<0.01 and ***P<0.001. (Q) Illustration of 22 macrochaetes on left and right sides of an adult thorax. pSC, posterior scutellar; aSC, anterior scutellar; pDC, posterior dorsocentral; aDC, anterior dorsocentral; pPA, posterior postalar; aPA, anterior postalar; pSA, posterior supraalar; aSA, anterior supraalar; pNP, posterior notopleural;, aNP, anterior notopleural; PC, presutural.</p

Dynamic changes in the C values of pSC shaft and socket cells from 8 to 29 hours APF.

<p>Pupae with the <i>A101-lacZ /+</i> genotype were grown till 8, 12, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 hours APF and dissected thoraxes were stained with anti-lacZ and DAPI. n = 7, 3, 12, 8, 9, 7, 5, 8, 5, 7, 8, 11, 7 or n = 4, 2, 10, 6, 4, 6, 5, 3, 2, 7, 6, 10, 6 corresponds to sample numbers at 8, 12, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 hours APF for shaft cells (A) or socket cells (B), respectively. The Y axis indicates the C value of each nucleus. Brackets indicate the time points where significant increases in C values were observed. Significant differences in mean values at each time point to those of adjacent time points were set at *P<0.05, **P<0.01 and ***P<0.001.</p

Model of dynamics of endoreplication during the development of pSC shaft and socket cells.

<p>Pattern of endoreplication and dynamic changes in C values in shaft and socket cells from 4 to 30 hours APF. Black boxes indicate S phases. Gray boxes indicate possible G1 phases. Patterns of BrdU incorporation in the periods indicated by Gray boxes with question marks were not determined.</p

The C values of pSC shaft and socket cells at 29 hours APF as measured with a confocal laser microscope.

<p>Wild type female pupae were grown till 29 hours APF and dissected thoraxes were stained with PI. (A) The white arrowhead indicates a shaft cell nucleus. Open arrowheads indicate epidermal cell nuclei. (B) The arrow indicates a socket cell nucleus. (C) Quantification of C values in shaft or socket cell nuclei at 29 hours APF. The Y axis indicates C values.</p

Dynamics of endoreplication in aSC shaft and socket cells from 26.5 to 29 hours APF.

<p>(A) Dynamics of BrdU incorporation in aSC shaft and socket cells from 26.5 to 29 hours APF. Wild type pupae were grown until 26.5, 27, 27.5, 28, 29, 29.5 hours APF and BrdU assays were carried out. BrdU signals were counted in shaft and socket cells of aSC bristle lineages in both the left and right sides of a thorax. The Y axis indicates the % of BrdU incorporation in shaft and socket cells in left and right sides of a thorax. n = 23, 17, 18, 17, 4, 11 or n = , 23, 21, 21, 14, 5, 10 or n = 23, 16, 18, 16, 5, 11 or n = 23, 19, 20, 14, 5, 12 corresponds to sample numbers at 26.5, 27, 27.5, 28, 29, 29.5 hours APF for left aSC shaft, right aSC shaft, left aSC socket cells or right aSC socket cells, respectively. (B) Endoreplication is synchronized in aSC shaft cells. The Y axis indicates % of pupae in which BrdU signals were simultaneously detected in aSC shaft cells of both left and right sides of a thorax.</p

The endocycle is synchronized in pSC macrochaetes.

<p>Wild type pupae were grown until particular times of development and BrdU incorporation assays were performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038714#s4" target="_blank">Materials and Methods</a>. The Y axis indicates % of pupae in which BrdU signals were simultaneously detected in pSC shaft cells of both left and right sides of a thorax. Sample numbers are shown at the top of each bar.</p