Biology subjects

Kanemaki, M. T.

Publications and source records attributed to Kanemaki, M. T..

23 records · Page 2Linked to original sources

bioRxiv2019

Generation of conditional auxin-inducible degron (AID) cells and tight control of degron-fused proteins using the degradation inhibitor auxinole

Controlling protein expression using a degron is drawing more attention because the protein of interest can be rapidly depleted in a reversible manner. We pioneered the development of the auxin-inducible degron (AID) technology by transplanting a plant-specific degradation pathway to non-plant cells. In human cells expressing an E3 ligase component, OsTIR1, it is possible to degrade a degron-fused protein with a half-life of 15-45 min in the presence of the phytohormone auxin. We reported previously the generation of human HCT116 mutants in which the C terminus of endogenous proteins was fused with the degron by CRISPR-Cas9-based knock-in. Here, we show new plasmids for N-terminal tagging and describe a detailed protocol for the generation of AID mutants of human HCT116 and DLD1 cells. Moreover, we report the use of an OsTIR1 inhibitor, auxinole, to suppress leaky degradation of degron-fused proteins. The addition of auxinole is also useful for rapid re-expression after depletion of degron-fused proteins. These improvements enhance the utility of AID technology for studying protein function in living human cells.

bioengineering↗

bioRxiv2018

Cyclin A triggers Mitosis either via Greatwall or Cyclin B

Two mitotic Cyclins, A and B, exist in higher eukaryotes, but their specialised functions in mitosis are poorly understood. Using degron tags we analyse how acute depletion of these proteins affects mitosis. Loss of Cyclin A in G2-phase prevents the initial activation of Cdk1. Cells lacking Cyclin B can enter mitosis and phosphorylate most mitotic proteins, because of parallel PP2A:B55 phos-phatase inactivation by Greatwall kinase. The final barrier to mitotic establishment corresponds to nuclear envelope breakdown that requires a decisive shift in the balance of Cdk1 and PP2A:B55 activity. Beyond this point Cyclin B/Cdk1 is essential to phosphorylate a distinct subset mitotic Cdk1 substrates that are essential to complete cell division. Our results identify how Cyclin A, B and Greatwall coordinate mitotic progression by increasing levels of Cdk1-dependent substrate phos-phorylation.

cell biology↗

bioRxiv2018

Optogenetic reconstitution reveals that Dynein-Dynactin-NuMA clusters generate cortical spindle-pulling forces as a multi-arm ensemble

To position the mitotic spindle within the cell, dynamic plus ends of astral microtubules are pulled by membrane-associated cortical force-generating machinery. However, in contrast to the chromosome-bound kinetochore structure, how the diffusion-prone cortical machinery is organized to generate large spindle-pulling forces remains poorly understood. Here, we develop a light-induced reconstitution system in human cells. We find that induced cortical targeting of NuMA, but not dynein, is sufficient for spindle pulling. This spindle-pulling activity requires dynein-dynactin recruitment/activation by NuMAs N-terminal long arm, and NuMAs direct microtubule-binding activities to achieve a multiplicity of microtubule interactions. Importantly, we demonstrate that cortical NuMA assembles specialized focal structures that cluster multiple force-generating modules to generate cooperative spindle-pulling forces. This clustering activity of NuMA is required for spindle positioning, but not for spindle-pole focusing. We propose that cortical Dynein-Dynactin-NuMA (DDN) clusters act as the core force-generating machinery that organizes a multi-arm ensemble reminiscent of the kinetochore.

cell biology↗

bioRxiv2017

Ki-67 and condensins support the integrity of mitotic chromosomes through distinct mechanisms

Although condensins play essential roles in mitotic chromosome assembly, Ki-67, a protein localizing to the periphery of mitotic chromosomes, had also been shown to make a contribution to the process. To examine their respective roles, we generated a set of HCT116-based cell lines expressing Ki-67 and/or condensin subunits that were fused with an auxin-inducible degron for their conditional degradation. Both the localization and the dynamic behavior of Ki-67 on mitotic chromosomes were not largely affected upon depletion of condensin subunits, and vice versa. When both Ki-67 and SMC2 (a core subunit of condensins) were depleted, ball-like chromosome clusters with no sign of discernible thread-like structures were observed. This severe defective phenotype was distinct from that observed in cells depleted of either Ki-67 or SMC2 alone. Our results show that Ki-67 and condensins, which localize to the external surface and the central axis of mitotic chromosomes, respectively, have independent yet cooperative functions in supporting the structural integrity of mitotic chromosomes.\n\nList of Abbreviations used

cell biology↗

bioRxiv2017

Rapid degradation and 3D CLEM of condensin reveal chromatin compaction uncoupled from chromosome architecture in mitosis

The requirement for condensin in chromosome formation in somatic cells remains unclear as imperfectly condensed chromosomes do form in conventional condensindepleted cells. Here we have dissected the role of condensin at different stages of vertebrate mitosis by combining auxin-mediated rapid depletion of condensin subunit SMC2 with chemical genetics to obtain near-synchronous mitotic entry of chicken DT40 cells. We analysed the outcomes by live and fixed-cell microscopy methods, including 3D correlative light and serial block face scanning electron microscopy. Following rapid depletion of condensin, chromosomal defects were obvious. The chromatin was compacted normally, but formed a single mass of mitotic chromosomes clustered at one side of a bent mitotic spindle. Cultures arrest at prometaphase, eventually exiting mitosis without segregating chromosomes. Experiments titrating the auxin concentration suggest a previously unsuspected dual role of condensin, as different condensin levels are required for anaphase chromosome segregation and formation of a normal chromosome architecture.

cell biology↗