Biology subjects

Amodeo, A. A.

Publications and source records attributed to Amodeo, A. A..

6 recordsLinked to original sources

bioRxiv2025

NASP functions in the cytoplasm to prevent histone H3 aggregation during early embryogenesis

From their molecular birth until their incorporation into chromatin, histones are bound by specific chaperones that serve unique functions in histone trafficking, stability and chromatin deposition. The H3-specific chaperone NASP binds directly to H3 and is required to prevent degradation of soluble H3 in vivo. Where NASP functions and how NASP affects H3 dynamics and stability is unknown. Using the Drosophila early embryo as a model system to understand NASP function in vivo, we show that NASP does not directly affect H3 nuclear import or export rates. Rather, reduced H3 levels in NASP-deficient embryos indirectly affect nuclear import and the amount of H3 deposited into chromatin. Crucially, we find that cytoplasmic NASP prevents H3 aggregation in vivo and that H3 aggregation and degradation are developmentally separable events. Thus, we propose the main function of NASP in vivo is to prevent H3 aggregation, thereby indirectly protecting H3 from degradation.

molecular biology↗

bioRxiv2024

Visualizing developmental dynamics of nuclear morphology and transport machinery in Drosophila

Communication between the cytoplasm and the nucleus requires a continuous exchange of molecules across the nuclear envelope (NE). The nuclear pore complex (NPC) is the gateway embedded in the NE through which cargo moves, while transport receptors mediate the passage of macromolecules through the NPC. Although their essential role as the components of the nuclear transport machinery has been extensively studied, how these factors respond to developmental and environmental cues has been underexplored. Here we tag the nucleoporin Nup96 and the transport receptor Imp{beta} with mEGFP and mScarlet-I at their endogenous loci in Drosophila. We demonstrate the functionality of these markers in multiple tissues and offer new options for better visualization of nuclear morphology in densely packed, complex tissues. Then, we characterize the spatiotemporal dynamics of these markers in multiple developmental contexts. We find that Nup96 and Imp{beta} form cytoplasmic puncta, whose size, numbers, and co-localization patterns change dynamically during oogenesis and early embryogenesis. Moreover, we find that the abundance of NPCs per nucleus decreases during early embryogenesis, complementing the emerging model in which NPCs play a regulatory role in development. The tools and observations described here will be useful in understanding the dynamic regulation of nuclear morphology and transport machinery in development.

cell biology↗

bioRxiv2024

Local nuclear to cytoplasmic ratio regulates chaperone-dependent H3 variant incorporation during zygotic genome activation

Early embryos often have unique chromatin states prior to zygotic genome activation (ZGA). In Drosophila, ZGA occurs after 13 reductive nuclear divisions during which the nuclear to cytoplasmic (N/C) ratio grows exponentially. Previous work found that histone H3 chromatin incorporation decreases while its variant H3.3 increases leading up to ZGA. In other cell types, H3.3 is associated with sites of active transcription and heterochromatin, suggesting a link between H3.3 and ZGA. Here, we test what factors regulate H3.3 incorporation at ZGA. We find that H3 nuclear availability falls more rapidly than H3.3 leading up to ZGA. We generate H3/H3.3 chimeric proteins at the endogenous H3.3A locus and observe that chaperone binding, but not gene structure, regulates H3.3 behavior. We identify the N/C ratio as a major determinant of H3.3 incorporation. To isolate how the N/C ratio regulates H3.3 incorporation we test the roles of genomic content, zygotic transcription, and cell cycle state. We determine that cell cycle regulation, but not H3 availability or transcription, controls H3.3 incorporation. Overall, we propose that local N/C ratios control histone variant usage via cell cycle state during ZGA.

cell biology↗

bioRxiv2024

Bellymount-Pulsed Tracking: A Novel Approach for Real-Time In vivo Imaging of Drosophila Oogenesis

Quantitative live imaging is a valuable tool that offers insights into cellular dynamics. However, many fundamental biological processes are incompatible with current live imaging modalities. Drosophila oogenesis is a well-studied system that has provided molecular insights into a range of cellular and developmental processes. The length of the oogenesis coupled with the requirement for inputs from multiple tissues has made long-term culture challenging. Here, we have developed Bellymount-Pulsed Tracking (Bellymount-PT), which allows continuous, non-invasive live imaging of Drosophila oogenesis inside the female abdomen for up to 16 hours. Bellymount-PT improves upon the existing Bellymount technique by adding pulsed anesthesia with periods of feeding that support the long-term survival of flies during imaging. Using Bellymount-PT we measure key events of oogenesis including egg chamber growth, yolk uptake, and transfer of specific proteins to the oocyte during nurse cell dumping with high spatiotemporal precision within the abdomen of a live female. SUMMARY STATEMENTBellymount-Pulsed Tracking is a novel tool to capture in vivo dynamics for longer duration in Drosophila oogenesis.

developmental biology↗

bioRxiv2020

Excess histone H3 is a Chk1 inhibitor that controls embryonic cell cycle progression

The early embryos of many species undergo a switch from rapid, reductive cleavage divisions to slower, cell fate-specific division patterns at the Mid-Blastula Transition (MBT). The maternally loaded histone pool is used to measure the increasing ratio of nuclei to cytoplasm (N/C ratio) to control MBT onset, but the molecular mechanism of how histones regulate the cell cycle has remained elusive. Here, we show that excess histone H3 inhibits the DNA damage checkpoint kinase Chk1 to promote cell cycle progression in the Drosophila embryo. We find that excess H3-tail that cannot be incorporated into chromatin is sufficient to shorten the embryonic cell cycle and reduce the activity of Chk1 in vitro and in vivo. Removal of the Chk1 phosphosite in H3 abolishes its ability to regulate the cell cycle. Mathematical modeling quantitatively supports a mechanism where changes in H3 nuclear concentrations over the final cell cycles leading up to the MBT regulate Chk1-dependent cell cycle slowing. We provide a novel mechanism for Chk1 regulation by H3, which is crucial for proper cell cycle remodeling during early embryogenesis.

cell biology↗

bioRxiv2019

The nuclear to cytoplasmic ratio directly regulates zygotic transcription in Drosophila

Early embryos must rapidly generate large numbers of cells to form an organism. Many species accomplish this through a series of rapid, reductive, and transcriptionally silent cleavage divisions. Previous work has demonstrated that the number of divisions before both cell cycle elongation and zygotic genome activation (ZGA) is regulated by the ratio of nuclear content to cytoplasm (N/C). To understand how the N/C ratio affects the timing of ZGA, we directly assayed the behavior of several previously identified N/C-ratio-dependent genes using the MS2-MCP reporter system in living Drosophila embryos with altered ploidy and cell cycle durations. For every gene that we examined, we found that nascent RNA output per cycle is delayed in haploid embryos. Moreover, we found that the N/C ratio influences transcription through three separate modes of action. For some genes (knirps and snail) the effect of ploidy can be entirely accounted for by changes in cell cycle duration. However, for other genes (giant, bottleneck and fruhstart) the N/C ratio directly affects ZGA. For giant and bottleneck, the N/C ratio regulates the kinetics of transcription activation, while for fruhstart it controls the probability of transcription initiation. Our data demonstrate that the regulatory elements of N/C-ratio-dependent genes respond directly to the N/C ratio, through multiple modes of regulation, independent of interphase length.

developmental biology↗