Biology subjects

Shindo, Y.

Publications and source records attributed to Shindo, Y..

6 recordsLinked to original sources

bioRxiv2026

H2A.Z levels control the timing of major events at the maternal-zygotic transition

Across animals, early embryonic events are temporally tightly coordinated but the underlying mechanisms remain incompletely understood. In Drosophila, both global and nuclear levels of the histone variant H2Av rise progressively during the maternal-zygotic transition (MZT), but whether this increase is functionally important is unknown. We find that increased H2Av dosage expedites specific MZT events: the transition from nuclear cycle (NC) 13 to 14 occurs prematurely as does the turnover of thousands of maternal transcripts; in addition, a subset of genes is precociously expressed in the zygote. Reduced H2Av dosage has reciprocal effects. Comparable transcriptional shifts are observed in zebrafish embryos overexpressing the H2Av ortholog H2A.Z, suggesting H2Av/H2A.Z dosage as a conserved timer of early development. We also examined mutants with impaired H2Av sequestration on lipid droplets which exhibit increased nuclear H2Av levels but reduced cytoplasmic levels. Unexpectedly, nuclear H2Av abundance influences the timing of NC 13 but is not the main driver of transcriptome remodeling. In summary, we find that H2Av/H2A.Z levels are critical timers of early embryogenesis and that H2Av can act in part via a non-nuclear mechanism. Author summaryIn animals, the earliest stages of embryogenesis are initially driven by proteins and RNAs that the mother provides via the egg; later development is controlled by the embryos own genes. The switch from maternal to zygotic control is called the maternal-zygotic transition (MZT). This conserved process involves degradation of maternal mRNAs, activation of zygotic genes, cell cycle lengthening, and morphological remodeling. All these events require precise control, but how they are coordinated remains incompletely understood. Here we show that the levels of a specific histone, H2A.Z, provide a timer for the MZT. In Drosophila, H2A.Z levels - both in the embryo overall and in nuclei - increase during the MZT. When we ectopically increased H2A.Z levels, specific MZT events occurred prematurely. Reducing H2A.Z levels had the opposite effect. Using mutant flies in which H2A.Z levels are ectopically increased in the nucleus but reduced in the embryo as a whole, we uncovered both nuclear and non-nuclear roles for H2A.Z in the establishment of developmental timing. Moreover, we observed similar outcomes in genetically manipulated zebrafish embryos. Our results are consistent with an ancient mechanism in which H2A.Z abundance functions as a timer of early embryogenesis.

developmental 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↗

bioRxiv2022

Genetically encoded sensors for analysing neurotransmission among synaptically-connected neurons

Anatomical connectome mapping in small areas of the nervous system as well as large-scale detection of neuronal activity patterns have been respectively achieved; however, it is still challenging to evaluate the functional connections among anatomically-connected neurons in a large-scale nervous system. We have developed a novel method to visualize neurotransmission named Split Protein HEmispheres for REconstitution (Sphere). By splitting a sensor into two fragments and expressing them in pre- and postsynaptic neurons separately, functional neurotransmitter sensors can be reconstituted only at the synapses between those neurons. We developed a Sphere-SF-iGluSnFR to measure glutamate levels, and further demonstrated that this system is functional in cultured cells, worms, and mouse brains. Moreover, this system is applicable to sensors other than glutamate, and colour variants have also been developed. This could allow for brain-wide imaging of functional synaptic transmission among particular neurons and identification of important neuronal circuits in the nervous system.

neuroscience↗

bioRxiv2020

An infectivity-enhancing site on the SARS-CoV-2 spike protein is targeted by COVID-19 patient antibodies

SARS-CoV-2 infection causes severe symptoms in a subset of patients, suggesting the presence of certain unknown risk factors. Although antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike have been shown prevent SARS-CoV-2 infection, the effects of antibodies against other spike protein domains are largely unknown. Here, we screened a series of anti-spike monoclonal antibodies from COVID-19 patients, and found that some of antibodies against the N-terminal domain (NTD) dramatically enhanced the binding capacity of the spike protein to ACE2, and thus increased SARS-CoV2 infectivity. Surprisingly, mutational analysis revealed that all the infectivity-enhancing antibodies recognized a specific site on the surface of the NTD. The antibodies against this infectivity-enhancing site were detected in all samples of hospitalized COVID-19 patients in the study. However, the ratio of infectivity-enhancing antibodies to neutralizing antibodies differed among patients. Furthermore, the antibodies against the infectivity-enhancing site were detected in 3 out of 48 uninfected donors, albeit at low levels. These findings suggest that the production of antibodies against SARS-CoV-2 infectivity-enhancing site could be considered as a possible exacerbating factors for COVID-19 and that a spike protein lacking such antibody epitopes may be required for safe vaccine development, especially for individuals with pre-existing enhancing antibodies.

microbiology↗

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↗