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.

Regulation of condensin II by self-suppression and release mechanisms.

In vertebrates, two distinct condensin complexes, condensin I and condensin II, cooperate to drive mitotic chromosome assembly. It remains largely unknown how the two complexes differentially contribute to this process at a mechanistic level. We have previously dissected the role of individual subunits of condensin II by introducing recombinant complexes into egg extracts.

Monitoring the compaction of single DNA molecules in egg extract in real time.

DNA compaction is required for the condensation and resolution of chromosomes during mitosis, but the relative contribution of individual chromatin factors to this process is poorly understood. We developed a physiological, cell-free system using high-speed egg extracts and optical tweezers to investigate real-time mitotic chromatin fiber formation and force-induced disassembly on single DNA molecules. Compared to interphase extract, which compacted DNA by ~60%, metaphase extract reduced DNA length by over 90%, reflecting differences in whole-chromosome morphology under these two conditions.

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.

Solubilization of Mouse Sperm Chromatin for Sequencing Analyses Using a Chaperon Protein.

Sperm chromatin compaction is physiologically essential for sperm to acquire the fertility. However, this unique structure composed of protamines makes us unable to solubilize the chromatin due to its resistance to sonication and enzymes usually used for chromatin fragmentation in somatic cells. Even when intense enzymatic treatment is applied, it appears to solubilize only certain portions of sperm chromatin presumably because of the heterogeneous properties.

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. Although the mechanism of action and regulation of condensin I have been studied extensively, our corresponding knowledge of condensin II remains very limited. By introducing recombinant condensin II complexes into egg extracts, we dissect the roles of its individual subunits in chromosome assembly.

Making Mitotic Chromosomes in a Test Tube.

Mitotic chromosome assembly is an essential preparatory step for accurate transmission of the genome during cell division. During the past decades, biochemical approaches have uncovered the molecular basis of mitotic chromosomes. For example, by using cell-free assays of frog egg extracts, the condensin I complex central for the chromosome assembly process was first identified, and its functions have been intensively studied.

A loop extrusion-independent mechanism contributes to condensin I-mediated chromosome shaping.

Condensin I is a five-subunit protein complex that is central to mitotic chromosome assembly in eukaryotic cells. Despite recent progress, its molecular mechanisms of action remain to be fully elucidated. By using Xenopus egg extracts as a functional assay, we find that condensin I complexes harboring mutations in its kleisin subunit CAP-H produce chromosomes with confined axes in the presence of topoisomerase IIα (topo IIα) and highly compact structures (termed "beans") with condensin-positive central cores in its absence.

Guiding functions of the C-terminal domain of topoisomerase IIα advance mitotic chromosome assembly.

Topoisomerase II (topo II) is one of the six proteins essential for mitotic chromatid reconstitution in vitro. It is not fully understood, however, mechanistically how this enzyme regulates this process. In an attempt to further refine the reconstitution assay, we have found that chromosomal binding of Xenopus laevis topo IIα is sensitive to buffer conditions and depends on its C-terminal domain (CTD).

Reconstitution of Mitotic Chromatids In Vitro.

The mitotic chromosome, which is composed of a pair of sister chromatids, is a large macromolecular assembly that ensures faithful transmission of genetic information into daughter cells. Despite its fundamental importance, how a nucleosome fiber is folded and assembled into a large-scale chromatid structure remains poorly understood. To address this question, we have established a biochemically tractable system in which mitotic chromatids can be reconstituted in vitro by mixing a simple substrate (sperm nucleus) and a limited number of purified factors.

Mitotic Chromosome Assembly In Vitro: Functional Cross Talk between Nucleosomes and Condensins.

The mitotic chromosome is a macromolecular assembly that ensures error-free transmission of the genome during cell division. It has long been a big mystery how long stretches of DNA might be folded into rod-shaped chromosomes or how such an elaborate process might be accomplished at a mechanistic level. Cell-free extracts made from frog eggs offer a unique opportunity to address these questions by enabling mitotic chromosomes to be assembled in a test tube.

Mitotic chromosome assembly despite nucleosome depletion in egg extracts.

The nucleosome is the fundamental structural unit of eukaryotic chromatin. During mitosis, duplicated nucleosome fibers are organized into a pair of rod-shaped structures (chromatids) within a mitotic chromosome. However, it remains unclear whether nucleosome assembly is indeed an essential prerequisite for mitotic chromosome assembly.

A Sister Chromatid Cohesion Assay Using Xenopus Egg Extracts.

Cell-free extracts made from Xenopus laevis eggs enable us to recapitulate many chromosomal events associated with cell cycle progression in a test tube. When sperm chromatin is incubated with these extracts, it is first duplicated within an assembled nucleus, and is then transformed into mitotic chromosomes, in each of which sister chromatids are juxtaposed with each other in a cohesin-dependent manner. Here we describe our protocols for assembling duplicated chromosomes using egg extracts, along with cytological and biochemical assays for addressing the molecular mechanisms of sister chromatid cohesion.

Reconstitution of mitotic chromatids with a minimum set of purified factors.

The assembly of mitotic chromosomes, each composed of a pair of rod-shaped chromatids, is an essential prerequisite for accurate transmission of the genome during cell division. It remains poorly understood, however, how this fundamental process might be achieved and regulated in the cell. Here we report an in vitro system in which mitotic chromatids can be reconstituted by mixing a simple substrate with only six purified factors: core histones, three histone chaperones (nucleoplasmin, Nap1 and FACT), topoisomerase II (topo II) and condensin I.

MCPH1 regulates chromosome condensation and shaping as a composite modulator of condensin II.

Mutations in human MCPH1 (hMCPH1) cause primary microcephaly, which is characterized by a marked reduction of brain size. Interestingly, hMCPH1 mutant patient cells display unique cellular phenotypes, including premature chromosome condensation (PCC), in G2 phase. To test whether hMCPH1 might directly participate in the regulation of chromosome condensation and, if so, how, we developed a cell-free assay using Xenopus laevis egg extracts.

The relative ratio of condensin I to II determines chromosome shapes.

To understand how chromosome shapes are determined by actions of condensins and cohesin, we devised a series of protocols in which their levels are precisely changed in Xenopus egg extracts. When the relative ratio of condensin I to II is forced to be smaller, embryonic chromosomes become shorter and thicker, being reminiscent of somatic chromosomes. Further depletion of condensin II unveils its contribution to axial shortening of chromosomes.

Sister chromatid resolution: a cohesin releasing network and beyond.

When chromosomes start to assemble in mitotic prophase, duplicated chromatids are not discernible within each chromosome. As condensation proceeds, they gradually show up, culminating in two rod-shaped structures apposed along their entire length within a metaphase chromosome. This process, known as sister chromatid resolution, is thought to be a prerequisite for rapid and synchronous separation of sister chromatids in anaphase.

Releasing cohesin from chromosome arms in early mitosis: opposing actions of Wapl-Pds5 and Sgo1.

The cohesin complex establishes sister chromatid cohesion during S phase. In metazoan cells, most if not all cohesin dissociates from chromatin during mitotic prophase, leading to the formation of metaphase chromosomes with two cytologically discernible chromatids. This process, known as sister chromatid resolution, is believed to be a prerequisite for synchronous separation of sister chromatids in subsequent anaphase.

How are cohesin rings opened and closed?

The cohesin complex is proposed to embrace sister chromatids within its ring-like structure, in which two ATP-binding 'head' domains of an SMC (structural maintenance of chromosomes) heterodimer are linked by a kleisin subunit. Recent studies shed new light on the crucial functions of the 'hinge' domain of the SMC dimer, which is located approximately 50 nm from the head domains. An emerging idea is that the hinge and head domains cooperatively modulate cohesin-DNA interactions by opening and closing the ring in a highly regulated manner.

Nucleosome assembly protein-1 is a linker histone chaperone in Xenopus eggs.

In eukaryotic cells, genomic DNA is primarily packaged into nucleosomes through sequential ordered binding of the core and linker histone proteins. The acidic proteins termed histone chaperones are known to bind to core histones to neutralize their positive charges, thereby facilitating their proper deposition onto DNA to assemble the core of nucleosomes. For linker histones, however, little has been known about the regulatory mechanism for deposition of linker histones onto the linker DNA.