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. 2024 Jul 1;223(10):e202310022. doi: 10.1083/jcb.202310022

Figure 2.

Figure 2.

The novel mutation S3372C selectively affects embryonic development. (A) Hatching frequency of eggs laid by mated females of the indicated genotypes. Columns show mean values per egg collection; error bars represent SD; circles are values for individual egg collections. Number of collections per genotype (from three independent crosses) is shown above columns (114–575 eggs per collection). S3372C/− are trans-heterozygous for S3372C and a Dhc null allele. Control genotype: yw. (B and C) Images showing (B) normal bristle length in adults and (C) lack of synaptic vesicle accumulations in L3 segmental nerves (proximal to the ventral ganglion; anterior to the top; Z-projection) in S3372C homozygotes (for comparisons with controls, see Fig. 1, D and E). The arrowhead in B points to posterior scutellar macrochaetae. Images in B and C are representative of >160 flies and three larvae analyzed, respectively. (D) Analysis of mitochondrial motility in wing nerve axons of 2-day-old control, F579Y, and S3372C adult flies. Left, cartoon of wing region imaged (magenta box). Top images, example stills from 3-min time series (single focal plane) of fluorescent mitochondria (expression of mito::GFP with a pan-neuronal driver). Bottom images, traces of motile mitochondria in corresponding time series. Blue, retrograde tracks; orange, anterograde tracks. (E) Percentages of mitochondria transported in the retrograde or anterograde directions, or moving bidirectionally, during the 3 min of data acquisition. Columns show mean values per movie; error bars represent SD; circles are values for individual movies (each from a different wing). Number of wings analyzed shown above bars. Note that we observed an increased frequency of retrograde transport in S3372C homozygotes, but this was not recapitulated in S3372C/− animals. (F) Analysis of mitotic duration in neuroblasts (NBs) in control and S3372C/− L3 larval brains. Left: Stills from image series of NBs with fluorescently labeled spindles (expression of RFP-tagged α-tubulin with an NB-specific driver). NBs and daughter cells highlighted with dashed lines. Timestamps are min:s after nuclear envelope breakdown (NEBD). Right: Quantification of mitotic duration (NEBD to anaphase onset). Lines show medians; circles are values for individual NBs. Number of NBs analyzed (from four wild-type or five S3372C/− larvae) shown above plot. (G) Summary of in vivo effects of homozygosity for F579Y and S3372C. SV, synaptic vesicle; Mito., mitochondrial. (H) Immunoblot of extracts of embryos from control and S3372C mothers (0–160-min collections), probed with antibodies to Dhc and α1-tubulin (loading control). The position of the molecular weight (Mw) marker is shown for α1-tubulin blot region; as there is no marker where Dhc migrates, the predicted Mw of Dhc is shown in parentheses. Evaluation of statistical significance (compared to control) was performed with a one-way ANOVA with Dunnett’s multiple comparisons test (A and E) or a Mann-Whitney test (F): ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, not significant. Scale bars: B, 500 µm; C, 50 µm; D, 10 µm; F, 5 µm. Source data are available for this figure: SourceData F2.