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. 2018 Jan 24;7:e29939. doi: 10.7554/eLife.29939

Figure 1. Miranda is cleared from the cortex before localizing in a basal crescent in mitosis.

(A) Larval brain NBs fixed and stained as labeled at the indicated cell cycle stage. (B) Selected frames from Video 1. NB in primary cell culture expressing Baz::GFP (green) and Mira::mCherry (red) in the transition from interphase to mitosis. Arrowheads point at Mira being cleared (−8) and at basal Mira crescent (+4). (B’) Quantification of cortical Mira::mCherry signal plotting the fluorescence intensities from the apical to the basal pole computationally straightening (Kocsis et al., 1991) the cortices of five NBs against the distance in percent. Fluorescence was background subtracted and normalized to background subtracted cytoplasmic signal (1, dotted line). Cortical signal (yellow dotted line) and signal after NEB (green dotted line). Error bars, standard deviation. (C) Schematic of Mira localization. BAC{mira::mcherry-MS2} was the source of Mira::mCherry. Scale bar 10 µm. Time stamp: minutes.

Figure 1.

Figure 1—figure supplement 1. Uniform cortical Prospero depends on Miranda in interphase larval NBs.

Figure 1—figure supplement 1.

In w1118 brains, Mira and Pros form basal crescents in mitosis and both are cortical in interphase (arrow). In an interphase miraL44 NB (MARCM clone, GFP+) cortical Mira and Pros are strongly reduced and Pros accumulates in the nucleus (yellow arrow). Transparent bars in merge interphase (control) and miraL44 indicate area used for plot profiles shown below. Pros and Mira are at the cortex (arrows) in the control and Pros is enriched in the nucleus in the mutant (yellow arrow). Arrows: outline of cell. Dotted line: nucleus (based on DAPI, control and GFP (MARCM clone). Scale bar: 10 µm.
Figure 1—figure supplement 2. BAC{mira::mcherry-MS2} rescues embryonic lethality of the loss of function allele miraL44 over the deficiency DF(3R)oraI9.

Figure 1—figure supplement 2.

However, animals die during puparium formation, when BAC{mira::mcherry-MS2} is the only source of Mira. (A) Brightfield images of fixed whole mount brain preparations. w1118 (control, n = 5) a BAC{mira::mcherry-MS2} brain over a wild type chromosome (1x BAC{mira::mcherry-MS2}, n = 12) and a brain from BAC{mira::mcherry-MS2} Df(3R)oraI9 over a unrecombined miraBACmCherry chromosome (2x BAC{mira::mcherry-MS2}, n = 12). 2x BAC{mira::mcherry-MS2} animals die as pharates. The ventral ganglion (VG) of these brains is frequently overgrown (arrows). Similar effects are seen with CrispR generated, homozygous mira::mCherry::HA larvae (not shown). (B) In fixed w1118 brains Mira as well as its cargo Brat are diffuse in the cytoplasm of NB daughter cells and Deadpan (Dpn) staining is restricted to NB nuclei. (B’) BAC{mira::mcherry-MS2}brains are not overgrown, Mira is sometimes more stable at the cortex in a daughter cell (arrowhead), but Dpn is normal. (C) In 2x BAC{mira::mcherry-MS2} animals, Mira is strongly cortical in several NB daughter cells and so is Brat (arrowheads). Dpn is no longer restricted to NB nuclei but frequently found in clusters of smaller nuclei close to NBs suggesting that Mira is stabilized at the cortex and fails to release its cargo, inducing fate changes. mCherry is fused to the C-terminus of Mira which was shown to be required for cargo release (Fuerstenberg et al., 1998; Matsuzaki et al., 1998). Scale bars: 10 µm.
Figure 1—figure supplement 3. Cortical Mira can be detected by antibody staining, in UAS-GFP-Mira overexpressing NBs and upon colcemid treatment, but not in interphase miraL44 loss of function clones.

Figure 1—figure supplement 3.

(A) Antibody staining against Mira performed on different fixed isolated NBs in primary cell culture. In this assay Mira (red) is cortical in an interphase NB (judged by DAPI, blue). In prophase NBs, differently sized Mira ‘crescents’ can be detected the ends of which are labeled by arrowheads. In a metaphase NBs Mira forms a crescent that appears larger than some of those seen in prophase NBs. (B) A living NB in primary cell culture expressing Mira::GFP driven by Mz1061. GFP signal is at the cortex in interphase, 29 min prior to NEB, GFP signal becomes cleared apically (arrowheads) until most of the cortex is cleared 1 min prior to NEB. 5 min after NEB a robust, larger crescent has formed. (C) Control brain (n = 5) treated with 50 µM colcemid for 30 min and stained for Mira. Over condensed chromatin in mitotic NBs demonstrates the effect of colcemid yet in all interphase NBs Mira remains at the cortex. (D) Fixed brain containing an interphase NB MARCM miraL44 clone surrounded by control NBs. In the clone, cortical Mira signal is gone (arrowhead) while present in a control interphase NB (arrow). Asterisks: mitotic NBs. Scale bar 10 µm.