Biology subjects

Shiozaki, M.

Publications and source records attributed to Shiozaki, M..

8 recordsLinked to original sources

bioRxiv2026

In situ molecular architecture of the mammalian sperm nuclear vacuole

Direct identification of macromolecular complexes in their native context remains a major barrier to unbiased biological discovery. This challenge is particularly acute in mammalian sperm nuclei, in which condensed chromatin is interspersed with poorly understood phase-separated compartments termed nuclear vacuoles. Vacuoles are associated with reduced fertilization efficiency, yet their composition remains unclear. Here we combine high-resolution in situ cryo-electron tomography (cryo-ET) with AlphaFold docking to identify vacuole components as proteasomes, the proteasome activator PA200, and ferritin. In situ structures at resolutions up to 3.8 [A] reveal distinct proteasome-PA200 associations and gating states, consistent with a stepwise activation mechanism. Ferritin assemblies exhibit heterogeneous mineralization states and directly contact chromatin. Together, these findings establish the molecular organization of sperm nuclear vacuoles and implicate protein turnover and metal homeostasis in shaping the nuclear landscape, while demonstrating the power of in situ cryo-ET to resolve protein identity and conformational dynamics in native cellular environments.

biochemistry↗

bioRxiv2025

Multi-scale structure of chromatin condensates rationalizes phase separation and material properties

Biomolecular condensates, compartments that concentrate molecules without surrounding membranes, are integral to numerous cellular processes. The structure and interaction networks of molecules within condensates remain poorly understood. Using cryo-electron tomography and molecular dynamics simulations we elucidated the structure of phase separated chromatin condensates across scales, from individual amino acids to network architecture. We found that internucleosomal DNA linker length controls nucleosome arrangement and histone tail interactions, shaping the structure of individual chromatin molecules both within and outside condensates. This structural modulation determines the balance between intra- and intermolecular interactions, which in turn governs the molecular network, thermodynamic stability, and material properties of chromatin condensates. Mammalian nuclei contain dense clusters of nucleosomes whose non-random organization is mirrored by the reconstituted condensates. Our work explains how the structure of individual chromatin molecules ultimately determines physical properties of chromatin condensates, with implications for cellular chromatin organization.

biophysics↗

bioRxiv2024

Quantitative Spatial Analysis of Chromatin Biomolecular Condensates using Cryo-Electron Tomography

Phase separation is an important mechanism to generate certain biomolecular condensates and organize the cell interior. Condensate formation and function remain incompletely understood due to difficulties in visualizing the condensate interior at high resolution. Here we analyzed the structure of biochemically reconstituted chromatin condensates through cryo-electron tomography. We found that traditional blotting methods of sample preparation were inadequate, and high-pressure freezing plus focused ion beam milling was essential to maintain condensate integrity. To identify densely packed molecules within the condensate, we integrated deep learning-based segmentation with novel context-aware template matching. Our approaches were developed on chromatin condensates, and were also effective on condensed regions of in situ native chromatin. Using these methods, we determined the average structure of nucleosomes to 6.1 and 12 [A] resolution in reconstituted and native systems, respectively, and found that nucleosomes form heterogeneous interaction networks in both cases. Our methods should be applicable to diverse biochemically reconstituted biomolecular condensates and to some condensates in cells.

biophysics↗

bioRxiv2024

Cryo-electron tomographic investigation of native hippocampal glutamatergic synapses

Chemical synapses are the major sites of communication between neurons in the nervous system and mediate either excitatory or inhibitory signaling [1]. At excitatory synapses, glutamate is the primary neurotransmitter and upon release from presynaptic vesicles, is detected by postsynaptic glutamate receptors, which include ionotropic AMPA and NMDA receptors [2, 3]. Here we have developed methods to identify glutamatergic synapses in brain tissue slices, label AMPA receptors with small gold nanoparticles (AuNPs), and prepare lamella for cryo-electron tomography studies. The targeted imaging of glutamatergic synapses in the lamella is facilitated by fluorescent pre- and postsynaptic signatures, and the subsequent tomograms allow for identification of key features of chemical synapses, including synaptic vesicles, the synaptic cleft and AuNP-labeled AMPA receptors. These methods pave the way for imaging brain regions at high resolution, using unstained, unfixed samples preserved under near-native conditions.

biochemistry↗

bioRxiv2022

De novo protein identification in mammalian sperm using high-resolution in situ cryo-electron tomography

Understanding molecular mechanisms of cellular pathways requires knowledge of the identities of participating proteins, their cellular localization and their 3D structures. Contemporary workflows typically require multiple techniques to identify target proteins, track their localization using fluorescence microscopy, followed by in vitro structure determination. To identify mammal-specific sperm proteins and understand their functions, we developed a visual proteomics workflow to directly address these challenges. Our in situ cryo-electron tomography and subtomogram averaging provided 6.0 [A] resolution reconstructions of axonemal microtubules and their associated proteins. The well-resolved secondary and tertiary structures allowed us to computationally match, in an unbiased manner, novel densities in our 3D reconstruction maps with 21,615 AlphaFold2-predicted protein models of the mouse proteome. We identified Tektin 5, CCDC105 and SPACA9 as novel microtubule inner proteins that form an extensive network crosslinking the lumen of microtubule and existing proteins. Additional biochemical and mass spectrometry analyses helped validate potential candidates. The novel axonemal sperm structures identified by this approach form an extensive interaction network within the lumen of microtubules, suggesting they have a role in the mechanical and elastic properties of the microtubule filaments required for the vigorous beating motions of flagella.

biophysics↗

bioRxiv2022

In situ cryo-electron tomography reveals the asymmetric architecture of mammalian sperm axonemes

The flagella of mammalian sperm display non-planar, asymmetric beating, in contrast to the planar, symmetric beating of flagella from sea urchin sperm and unicellular organisms. The molecular basis of this difference is unclear. Here, we perform in situ cryo-electron tomography of mouse and human sperm axonemes, providing the highest resolution structural information to date. Our subtomogram averages reveal mammalian sperm- specific protein complexes within the outer microtubule doublets, the radial spokes and nexin-dynein regulatory complexes. The locations and structures of these complexes suggest potential roles in enhancing the mechanical strength of mammalian sperm axonemes and regulating dynein-based axonemal bending. Intriguingly, we find that each of the nine outer microtubule doublets is decorated with a distinct combination of sperm- specific complexes. We propose that this asymmetric distribution of proteins differentially regulates the sliding of each microtubule doublet and may underlie the asymmetric beating of mammalian sperm.

biophysics↗

bioRxiv2021

Structure and RNA template requirements of Arabidopsis RNA-DEPENDENT RNA POLYMERASE 2

RNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In Arabidopsis, RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and their activities are tightly coupled, with Pol IV transcriptional arrest or termination, involving the nontemplate DNA strand, somehow enabling RDR2 to engage Pol IV transcripts and generate double-stranded RNAs. The dsRNAs are then released from the Pol IV-RDR2 complex and diced into siRNAs that guide RNA-directed DNA methylation and silencing. Here we report the structure of full-length RDR2, at an overall resolution of 3.1 [A], determined by cryo-electron microscopy. The N-terminal region contains an RNA-recognition motif (RRM) adjacent to a positively charged channel that leads to a catalytic center with striking structural homology to the catalytic centers of multisubunit DNA-dependent RNA polymerases. We show that RDR2 initiates 1-2 nucleotides (nt) internal to the 3 ends of its templates and can transcribe the RNA of an RNA-DNA hybrid provided that 9 or more nucleotides at the RNAs 3 end is unpaired. Using a nucleic acid configuration that mimics the arrangement of RNA and DNA strands upon Pol IV transcriptional arrest, we show that displacement of the RNA 3 end occurs as the DNA template and non-template strands reanneal, enabling RDR2 transcription. These results suggest a model in which Pol IV arrest and backtracking displaces the RNA 3 end as the DNA strands reanneal, allowing RDR2 to engage the RNA and transcribe the second strand. SignificanceRDR2 is critical for siRNA-directed DNA methylation in Arabidopsis, functioning in physical association with DNA-dependent Pol IV to synthesize the second strands of double-stranded siRNA precursors. Basepairing between the DNA template strand transcribed by Pol IV and the nontemplate DNA strand is known to induce Pol IV arrest and Pol IV-RDR2 transcriptional coupling, but how this occurs is unknown. We report the structure of RDR2 and experimental evidence for how RDR2 engages its RNA templates and initiates transcription. RDR2 engages the ends of RNAs displaced from RNA-DNA hybrids, suggesting a model in which Pol IV arrest and backtracking, accompanied by DNA strand reannealing, extrudes the 3 end of the Pol IV transcript, allowing RNA engagement and second-strand synthesis.

molecular biology↗

bioRxiv2018

The Claustrum Coordinates Cortical Slow-Wave Activity

During sleep and awake rest, the neocortex generates large-scale slow-wave activity. Here we report that the claustrum, a poorly understood subcortical neural structure, coordinates neocortical slow-wave generation. We established a transgenic mouse line allowing genetic and electrophysiological interrogation of a subpopulation of claustral glutamatergic neurons. These claustral excitatory neurons received inputs from glutamatergic neurons in a large neocortical network. Optogenetic activation of claustral neurons in vitro induced excitatory post-synaptic responses in most neocortical neurons, but elicited action potentials primarily in inhibitory interneurons. Optogenetic activation of claustral neurons in vivo induced a Down-state featuring a prolonged silencing of neural acticity in all layers of many cortical areas, followed by a globally synchronized Down-to-Up state transition. These results demonstrate a crucial role of the claustrum in synchronizing inhibitory interneurons across the neocortex for spatiotemporal coordination of brain state. Thus, the claustrum is a major subcortical hub for the synchronization of neocortical slow-wave activity.

neuroscience↗