Biology subjects

Shintomi, K.

Publications and source records attributed to Shintomi, K..

8 recordsLinked to original sources

bioRxiv2025

Reconstitution of phospho-regulated mitotic chromatid assembly and disassembly

Exactly how cell cycle regulators control a sequential series of mitotic events is not fully understood. Here we report reconstitution assays that recapitulate the assembly and disassembly of mitotic chromatids in vitro using a minimal set of recombinant proteins, including condensin I. By incorporating cyclin B-Cdk1 and PP2A-B55, this system enables us to dissect the phospho-regulation of condensin I in these processes. We provide evidence that the terminal intrinsically disordered regions (tIDRs) of the non-SMC subunits suppress condensin I activity, and that this self-suppression is relieved by Cdk1 phosphorylation. Importantly, full activation of condensin I requires the phosphorylation of a conserved residue located in the central region of the kleisin subunit CAP-H. Conversely, PP2A-B55 induces dissociation of condensin I from reconstituted chromatids, leading to their disassembly. Complementary analyses using Xenopus egg extracts reveal that the tIDRs and the kleisin central region are phosphorylated and dephosphorylated with distinct kinetics during mitotic entry and exit. Together, these findings define an intricate regulatory network that coordinates chromatid assembly and disassembly with mitotic progression.

molecular biology↗

bioRxiv2025

CDK1-dependent N-terminal NuMA phosphorylation promotes dynein-dynactin-NuMA assembly for accurate chromosome segregation

The microtubule-based motor dynein and its cofactor dynactin fulfil essential functions throughout the cell cycle, including organelle transport and mitotic spindle assembly. To achieve these diverse functions, dynein-dynactin associates with different activating adaptors. Nuclear Mitotic Apparatus (NuMA) is a mitosis-specific adaptor that connects dynein-dynactin with microtubules to focus mitotic spindle poles. NuMAs C-terminal microtubule-binding activity is promoted by mitotic phosphorylation, but regulation of NuMAs N-terminal interaction with dynein-dynactin remains unclear. Here, we combine a membrane-tethering assay, quantitative proteomics, and live functional analyses in human cells to show that the interaction between NuMAs N-terminus and dynein-dynactin is cell cycle-regulated and driven by mitotic phosphorylation. We identify highly conserved CDK1 consensus sites proximal to NuMAs dynein heavy chain-binding site, which are phosphorylated by CDK1-Cyclin B1 in cells and in vitro. This CDK1-dependent phosphorylation, together with NuMAs Spindly-like motif, is crucial for stable dynein-dynactin-NuMA (DDN) complex formation. Replacement of endogenous NuMA with phosphorylation-deficient NuMA mutants leads to aberrant dynein distribution on mitotic spindles, resulting in chromosome mis-segregation and micronucleus formation. Together, our results highlight CDK1-dependent N-terminal NuMA phosphorylation as a crucial mitotic switch that constitutes a regulatable core of multivalent interactions with dynein-dynactin to assemble stable DDN complexes for accurate chromosome segregation.

cell biology↗

bioRxiv2025

Reorganization of DNA loops by competition between condensin I and a linker histone

Condensin-mediated loop extrusion is thought to be one of the primary mechanisms underlying mitotic chromosome assembly. However, how this process is affected by other chromosomal proteins, such as histones, is not well understood. Our previous study showed that in Xenopus egg extracts co-depleted of topoisomerase II and the histone chaperone Asf1, a highly characteristic chromatin structure called the "sparkler" is assembled. The sparkler is a compact structure assembled on nucleosome-free, entangled DNA in which multiple protrusions radiate from a core. Interestingly, condensin I is concentrated at the tips of the protrusions, whereas the linker histone H1.8 is enriched in the remaining regions of the structure. To understand the biophysical mechanisms underlying sparkler assembly, we construct a model predicting that DNA loops extruded from the entangled DNA undergo phase separation into two domains: loops enriched in condensin I remain as protrusions, whereas those enriched in H1.8 are reeled into the central region. We propose that H1.8 competes with condensin I for DNA binding, thereby reorganizing DNA loops formed by condensin I under this specialized condition.

biophysics↗

bioRxiv2025

Functional interplay between condensin I and topoisomerase IIα in single-molecule DNA compaction

Condensin I and topoisomerase II (topo II) are chromosomal ATPases essential for mitotic chromosome assembly. Mechanistically how the two ATPases cooperate to assemble mitotic chromosomes remains unknown. Here we use total internal reflection fluorescence microscopy to analyze the interplay between condensin I and topo II at single-molecule resolution. As observed in previous studies, condensin I alone predominantly forms DNA loops in an ATP-dependent manner. However, when topo II is included in the reaction, condensin I forms stable compact structures (termed "lumps") in a manner dependent on the C-terminal domain of topo II. Each of the stable lumps contains a single condensin I complex and a single topo II dimer. Remarkably, we find that topo II, when catalytically active, renders the lumps resistant to protease treatment. Several lines of evidence show that the protease-resistant lumps contain knotted DNA. A mutant condensin I complex defective in ATP hydrolysis, together with topo II, forms smaller lumps in which the probability of DNA knotting is greatly reduced. Our results demonstrate how topo II-mediated strand passage is coupled with condensin I-mediated loop extrusion to generate a compact DNA structure. Together with recent studies, we discuss the functional implications of these observations in mitotic chromosome assembly and stabilization.

molecular biology↗

bioRxiv2024

Nuclear assembly in giant unilamellar vesicles encapsulating Xenopus egg extract

The reconstitution of a cell nucleus in a lipid bilayer-enclosed synthetic cell makes great strides in bottom-up synthetic biology. In this study, we propose a method for assembling a nucleus in giant unilamellar vesicles (GUVs). To induce reconstitution of the nucleus, we utilize interphase egg extract of African clawed frogs Xenopus laevis, known as a biochemically controllable cell-free system capable of transforming an added sperm chromatin into a nucleus in vitro. We enhanced GUV formation efficiency by the inverted emulsion method through incorporating prolonged waiting time and adding chloroform into lipid-dispersed oil, facilitating subsequent nuclear assembly reactions in the GUVs.Characterization of nucleus-like structures formed in the GUVs revealed the presence of dense DNA and accumulated GFP-NLS in the structure, indicative of functional nuclear import. Immunostaining further validated the presence of nuclear pore complexes on the surfaces of these nucleus-like structures. Our approach offers a versatile platform for constructing artificial cellular systems that closely mimic eukaryotic cells. TeaserA cell nucleus is reconstituted in lipid bilayer-enclosed confinements using egg extract from African clawed frogs.

synthetic biology↗

bioRxiv2023

Recombinant cyclin B-Cdk1-Suc1 capable of multi-site mitotic phosphorylation in vitro

Cyclin-dependent kinase 1 (Cdk1) complexed with cyclin B phosphorylates multiple sites on hundreds of proteins during mitosis. However, it is not fully understood how multi-site mitotic phosphorylation by cyclin B-Cdk1 controls the structures and functions of individual substrates. Here we develop an easy-to-use protocol to express recombinant vertebrate cyclin B and Cdk1 in insect cells from a single baculovirus vector and to purify their complexes with excellent homogeneity. A series of in-vitro assays demonstrate that the recombinant cyclin B-Cdk1 can efficiently and specifically phosphorylate the SP and TP motifs in substrates. The addition of Suc1 (a Cks1 homolog in fission yeast) accelerates multi-site phosphorylation of an artificial substrate containing TP motifs. Importantly, we show that mitosis-specific multi-subunit and multi-site phosphorylation of the condensin I complex can be recapitulated in vitro using recombinant cyclin B-Cdk1-Suc1. The materials and protocols described here will pave the way for dissecting the biochemical basis of critical mitotic processes that accompany Cdk1-mediated large-scale phosphorylation.

cell biology↗

bioRxiv2022

Cell cycle-specific loading of condensin I is regulated by the N-terminal tail of its kleisin subunit

Condensin I is a pentameric protein complex that plays an essential role in mitotic chromosome assembly in eukaryotic cells. Although it has been shown that condensin I loading is mitosis-specific, it remains poorly understood how the robust cell cycle regulation of condensin I is achieved. Here we set up a panel of in vitro assays to demonstrate that cell cycle-specific loading of condensin I is regulated by the N-terminal tail (N-tail) of its kleisin subunit CAP-H. Deletion of the N-tail accelerates condensin I loading and chromosome assembly in Xenopus egg mitotic extracts. Phosphorylation-deficient and phosphorylation-mimetic mutations in the CAP-H N-tail decelerate and accelerate condensin I loading, respectively. Remarkably, deletion of the N-tail enables condensin I to assemble mitotic chromosome-like structures even in interphase extracts. Together with other extract-free functional assays in vitro, our results uncover one of the multilayered mechanisms that ensure cell cycle-specific loading of condensin I onto chromosomes.

cell biology↗

bioRxiv2022

Molecular dissection of condensin II-mediated chromosome assembly using in vitro assays

In vertebrates, condensin I and condensin II cooperate to assemble rod-shaped chromosomes during mitosis. Despite recent progress in our understanding of the mechanism of action of condensin I, how condensin II might work has remained largely unexplored. By introducing recombinant condensin II complexes into Xenopus egg extracts, we dissect the roles of its individual subunits in chromosome assembly. We show that two HEAT subunits, CAP-D3 and CAP-G2, play positive and negative roles, respectively, in condensin II-mediated chromosome assembly. The SMC ATPase and the basic amino acid clusters of the kleisin subunit are essential for this process. Deletion of the C-terminal tail of CAP-D3 increases the ability of condensin II to assemble chromosomes and further exposes a hidden function of CAP-G2 in the lateral compaction of chromosomes. Taken together, our results uncover a multilayered regulatory mechanism unique to condensin II, and provide profound implications in the evolution of condensin II.

cell biology↗