Developmentally regulated H2Av buffering via dynamic sequestration to lipid droplets in Drosophila embryos
- University of Rochester, United States
Figures
Figure 1
H2Av can translocate from lipid droplets (LDs) to nearby nuclei.
In NC13 embryos expressing H2Av-paGFP, a region enriched for LDs was photoactivated by exposure to 405 nm light (Dashed box, A’). (A) Prior to photoactivation, (A’) ~30 s post-photoactivation, (A’') …
Figure 2
A photoswitchable H2Av-Dendra2 for in-vivo H2Av tracking.
(A) Generation of a transgene containing H2Av-Dendra2 under endogenous regulation. The endogenous H2Av genomic region (~4 Kb) was amplified via PCR and the Dendra2 protein coding sequence (Evrogen) …
Figure 3 with 1 supplement
H2Av is rapidly lost from LDs throughout early embryogenesis.
(A) Scheme of experimental design. (Left) Photoswitching of LD-enriched regions is achieved by focusing just below the embryo surface. Dashed line represents plane of view. (Right) When viewing the …
Figure 3—figure supplement 1
H2Av is dynamically associated with LDs.
(A) After photoswitching, H2Av-Dendra2 signal is rapidly lost from the photoswitched region, appearing to spread throughout the embryo. Shown are images from the red channel as in Figure 3B, but …
Figure 4 with 1 supplement
H2Av exchanges between LDs via a cytoplasmic route.
(A–C) Fluorescence Recovery After Photobleaching (FRAP) in cleavage stage embryos expressing LSD2-YFP (green), a LD marker, and H2Av-mRFP. Photobleaching was induced in LD enriched regions near the …
Figure 4—figure supplement 1
Observed H2Av dynamics do not result from cytoplasmic streaming.
FRAP experiments in cleavage stage embryos occasionally show large-scale movement within the cytoplasm, likely due to cytoplasmic streaming. (A-C) FRAP experiment in a cleavage stage embryo …
Figure 5 with 1 supplement
Model: LDs constitute the major regulator of H2Av protein levels both free in the cytoplasm and in the nucleus.
(A) In early Drosophila embryos, LDs are the main H2Av regulator. H2Av mRNA levels are stable; these represent maternal mRNAs, and there is no zygotic contribution (red dashed line). H2Av …
Figure 5—figure supplement 1
In most cells, histones are regulated at multiple steps.
Such regulation of histone abundance/deposition onto chromatin occurs transcriptionally, post-transcriptionally (e.g. degradation of mRNA), and post-translationally (e.g. protein degradation, …
Figure 6
Nuclear H2Av levels are driven by nuclear import.
(A,B) A region encompassing several nuclei was photobleached in NC13 embryos expressing H2Av-GFP, and nuclear signal was monitored over time. (A) Representative images of a FRAP experiment in NC13 …
Figure 7
Nuclear H2Av is chromosome associated and scales with interphase length.
(A) NC11-NC13 embryos expressing H2Av-GFP in interphase (A) or metaphase (A’). Scale bars represent 10 and 2 μm (inlay). (B) Quantitation of total nuclear H2Av-GFP fluorescent signal per nucleus in …
Figure 8
Total and nuclear H2Av levels are determined by H2Av gene dosage.
(A) Global H2Av protein levels scale with H2Av gene dosage. Total protein from equal numbers of NC14 embryos laid by mothers with either 1, 2, or 4 copies of the H2Av gene were separated by SDS PAGE …
Figure 9 with 1 supplement
Proposed kinetic model for LD-mediated H2Av buffering.
(A) Proposed kinetic model for the buffering effects of LDs on nuclear incorporation of H2Av. (Top) H2Av free in the cytosol binds LDs with on rate k1on and off rate k1off and binds to DNA with on …
Figure 9—figure supplement 1
Effects of H2Av levels and Jabba dosage on nuclear H2Av accumulation as predicted by the model in Figure 9.
(A) As total H2Av levels increase, the relative levels of DNA-bound H2Av increase proportionally at all time points. (B) As levels of H2Av-binding sites on LDs increase (i.e. with increased Jabba …
Figure 10
Reduction in buffering capacity increases nuclear H2Av.
(A) Jabba protein levels scale with Jabba gene dosage. Total protein from equal numbers of NC14 embryos laid by mothers with either 0, 1, or 2 copies of the Jabba gene were separated by SDS PAGE and …
Figure 11 with 3 supplements
H2Av buffering by LDs is developmentally regulated.
(A) FRAP experiments show reduced nuclear import of H2Av in NC14 compared to NC13. FRAP was performed near the surface of NC13 and NC14 embryos expressing H2Av-GFP, and nuclear recovery was …
Figure 11—figure supplement 1
Nuclear export of H2Av is minimal in NC14.
(A) Photoswitching was induced in single nuclei (white asterisks) in mid-stage NC14 embryos and signal within nuclei was monitored over time. After 5 min, no significant loss of H2Av-Dendra2 signal …
Figure 11—figure supplement 2
H2Av-Dendra2 loss from LDs is reduced in mid-NC14.
In mid-NC14, loss of H2Av-Dendra2 signal from LDs is drastically reduced. Experiment/images are the same as in Figure 11D but pixels have been inverted and brightness increased linearly across the …
Figure 11—figure supplement 3
Zygotic transcription is not required for transition in H2Av dynamics.
Embryos laid by H2Av-Dendra2 expressing mothers were injected with α-amanitin and photoswitching was induced. (A) Images from a photoswitching experiment in a mid-NC14 embryo that had been injected …
Videos
Video 1
LDs show little movement and minimal LD-LD contact during cleavage stages.
Video shows a composite of two sequences from the same embryo. Images were taken every 15 s. Playback speed is 7 frames/s. Left: video showing a FRAP experiment in embryos expressing the LD marker …
Tables
Key resources table
| Reagent type | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (D. melanogaster) | His2Av | NA | FBgn0001197 | |
| Gene (D. melanogaster) | Jabba | NA | FBgn0259682 | |
| Gene (D. melanogaster) | halo | NA | FBgn0001174 | |
| Gene (D. melanogaster) | LSD-2 | NA | FBgn0030608 | |
| Genetic reagent (D. melanogaster) | H2Av810 | Bloomington Drosophila Stock Center | BDSC:9264; FLYB: FBst0009264 | |
| Genetic reagent (D. melanogaster) | H2Av-GFP | Bloomington Drosophila Stock Center | BDSC:24163; FLYB: FBst0024163 | |
| Genetic reagent (D. melanogaster) | H2Av-mRFP | Bloomington Drosophila Stock Center | BDSC:23650; FLYB: FBst0023650 | |
| Genetic reagent (D. melanogaster) | ms(3)K81 | Bloomington Drosophila Stock Center | BDSC:53252; FLYB: FBst0005352 | |
| Genetic reagent (D. melanogaster) | H2Av-paGFP | other | FLYB: FBtp0020089 | Described in (Post et al., 2005) |
| Genetic reagent (D. melanogaster) | LSD2-YFP | Kyoto Stock Center | DGRC:115301; FLYB: FBti0143786 | |
| Genetic reagent (D. melanogaster) | JabbaDL, Jabbazl01 | other | FLYB: FBal0280317; FBal0280318 | Previously generated Jabba null alleles. Described in (Li et al., 2012). |
| Genetic reagent (D. melanogaster) | Df(2L)ΔhaloAJ | other | FLYB: FBab0047638 | Small deletion encompassing the halo gene. Described in (Arora et al., 2016). |
| Genetic reagent (D. melanogaster) | H2Av-Dendra2 | this paper | genomic H2Av region (~4 kb), Dendra2 inserted downstream of exon 4, cloned into pattB, genomic insertion site 68A4 | |
| Genetic reagent (D. melanogaster) | gH2Av | this paper | genomic H2Av region (~4 kb), cloned into pattB, genomic insertion site 68A4 | |
| genetic reagent (D. melanogaster) | gJabba | this paper | genomic Jabba region (~5.3 kb). Insertion site 68A4 | |
| Antibody | anti-H2AvD (rabbit polyclonal) | Active Motif | Cat. No. 39715 | (1:1000 immunostain) (1:2500 WB) |
| Antibody | anti-Jabba (rabbit polyclonal) | this paper | raised against in-vitro synthesized peptide encoded in exon 5 of Jabba followed by affinity purification | |
| Antibody | anti-alpha tubulin (mouse monoclonal) | Cell Signaling | Cat. No. #3873 | (1:10,000) |
| Antibody | IRDye secondaries 800CW or 680RD | Li-COR | (1:10,000) | |
| Antibody | Alexa 594 secondary | ThermoFisher | Cat. No. A-11012 | (1:1000) |
| Commercial kit | mMESSAGE mMACHINE T7 | Ambion Inc. | Cat. No. AM1344 (Fisherscientific) | |
| Drug | α-amanitin | Sigma | Cat. No. A2263 | (500 µg/mL) |
| Drug | cycloheximide | Sigma | Cat. No. C7698 | (1 mg/mL) |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.36021.021
