Kid-mediated chromosome compaction ensures proper nuclear envelope formation.

Toward the end of mitosis, neighboring chromosomes gather closely to form a compact cluster. This is important for reassembling the nuclear envelope around the entire chromosome mass but not individual chromosomes. By analyzing mice and cultured cells lacking the expression of chromokinesin Kid/kinesin-10, we show that Kid localizes to the boundaries of anaphase and telophase chromosomes and contributes to the shortening of the anaphase chromosome mass along the spindle axis. Loss of Kid-mediated anaphase chromosome compaction often causes the formation of multinucleated cells, specifically at oocyte meiosis II and the first couple of mitoses leading to embryonic death. In contrast, neither male meiosis nor somatic mitosis after the morula-stage is affected by Kid deficiency. These data suggest that Kid-mediated anaphase/telophase chromosome compaction prevents formation of multinucleated cells. This protection is especially important during the very early stages of development, when the embryonic cells are rich in ooplasm.

Stress granule formation inhibits stress-induced apoptosis by selectively sequestering executioner caspases.

Cytoplasmic stress granules (SGs) are phase-separated membrane-less organelles that form in response to various stress stimuli. SGs are mainly composed of non-canonical stalled 48S preinitiation complexes. In addition, many other proteins also accumulate into SGs, but the list is still incomplete. SG assembly suppresses apoptosis and promotes cell survival under stress. Furthermore, hyperformation of SGs is frequently observed in various human cancers and accelerates tumor development and progression by reducing stress-induced damage of cancer cells. Therefore, they are of clinical importance. However, the precise mechanism underlying SG-mediated inhibition of apoptosis remains ill-defined. Here, using a proximity-labeling proteomic approach, we comprehensively analyzed SG-resident proteins and identified the executioner caspases, caspase-3 and -7, as SG components. We demonstrate that accumulation of caspase-3/7 into SGs is mediated by evolutionarily conserved amino acid residues within their large catalytic domains and inhibits caspase activities and consequent apoptosis induced by various stresses. Expression of an SG-localization-deficient caspase-3 mutant in cells largely counteracted the anti-apoptotic effect of SGs, whereas enforced relocalization of the caspase-3 mutant to SGs restored it. Thus, SG-mediated sequestration of executioner caspases is a mechanism underlying the broad cytoprotective function of SGs. Furthermore, using a mouse xenograft tumor model, we show that this mechanism prevents cancer cells from apoptosis in tumor tissues, thereby promoting cancer progression. Our results reveal the functional crosstalk between SG-mediated cell survival and caspase-mediated cell death signaling pathways and delineate a molecular mechanism that dictates cell-fate decisions under stress and promotes tumorigenesis.

Involvement of CNOT3 in mitotic progression through inhibition of MAD1 expression.

The stability of mRNA influences the dynamics of gene expression. The CCR4-NOT complex, the major deadenylase in mammalian cells, shortens the mRNA poly(A) tail and contributes to the destabilization of mRNAs. The CCR4-NOT complex plays pivotal roles in various physiological functions, including cell proliferation, apoptosis, and metabolism. Here, we show that CNOT3, a subunit of the CCR4-NOT complex, is involved in the regulation of the spindle assembly checkpoint, suggesting that the CCR4-NOT complex also plays a part in the regulation of mitosis. CNOT3 depletion increases the population of mitotic-arrested cells and specifically increases the expression of MAD1 mRNA and its protein product that plays a part in the spindle assembly checkpoint. We showed that CNOT3 depletion stabilizes the MAD1 mRNA, and that MAD1 knockdown attenuates the CNOT3 depletion-induced increase of the mitotic index. Basing on these observations, we propose that CNOT3 is involved in the regulation of the spindle assembly checkpoint through its ability to regulate the stability of MAD1 mRNA.

Microtubule stabilization triggers the plus-end accumulation of Kif18A/kinesin-8.

The precise control of spindle microtubule (MT) dynamics is essential for chromosome capture and alignment. Kif18A/kinesin-8, an essential regulator of kinetochore MT dynamics, accumulates at its plus-ends in metaphase but not prometaphase cells. The underlying mechanism of time-dependent and kinetochore MT-specific plus-end accumulation of Kif18A is unknown. Here, we examined the factors required for the MT plus-end accumulation of Kif18A. In Eg5 inhibitor-treated cells, Kif18A localized along the MTs in the monopolar spindle and rarely accumulated at their plus-ends, indicating that MT-kinetochore association was not sufficient to induce Kif18A accumulation. In contrast, taxol treatment triggered the rapid MT plus-end accumulation of Kif18A regardless of kinetochore association. Furthermore, Aurora B inhibitor-induced stabilization of the plus-ends of kinetochore MTs promoted the plus-end accumulation of Kif18A. In the absence of Kif18A, treatment with taxol but not Eg5 inhibitor causes highly elongated mitotic MTs, suggesting the importance of plus-end accumulation for the MT length-controlling activity of Kif18A. Taken together, we propose that there is a mutual regulation of kinetochore MT plus-end dynamics and Kif18A accumulation, which may contribute to the highly regulated and ordered changes in kinetochore MT dynamics during chromosome congression and oscillation.

The chromokinesin Kid is required for maintenance of proper metaphase spindle size.

The human chromokinesin Kid/kinesin-10, a plus end-directed microtubule (MT)-based motor with both microtubule- and DNA-binding domains, is required for proper chromosome alignment at the metaphase plate. Here, we performed RNA interference experiments to deplete endogenous Kid from HeLa cells and confirmed defects in metaphase chromosome arm alignment in Kid-depleted cells. In addition, we noted a shortening of the spindle length, resulting in a pole-to-pole distance only 80% of wild type. The spindle microtubule-bundles with which Kid normally colocalize became less robust. Rescue of the two Kid deficiency phenotypes-imprecise chromosome alignment at metaphase and shortened spindles- exhibited distinct requirements. Mutants lacking either the DNA-binding domain or the MT motor ATPase failed to rescue the former defect, whereas rescue of the shortened spindle phenotype required neither activity. Kid also exhibits microtubule bundling activity in vitro, and rescue of the shortened spindle phenotype and the bundling activity displayed similar domain requirements, except that rescue required a coiled-coil domain not needed for bundling. These results suggest that distinct from its role in chromosome movement, Kid contributes to spindle morphogenesis by mediating spindle microtubules stabilization.

ESCRT-III mediates budding across the inner nuclear membrane and regulates its integrity.

Vesicle-mediated nucleocytoplasmic transport is a nuclear pore-independent mechanism for the nuclear export of macromolecular complexes, but the molecular basis for this transport remains largely unknown. Here we show that endosomal sorting complex required for transport-III (ESCRT-III) is recruited to the inner nuclear membrane (INM) during the nuclear export of herpes simplex virus 1 (HSV-1). Scission during HSV-1 budding through the INM is prevented by depletion of ESCRT-III proteins. Interestingly, in uninfected human cells, the depletion of ESCRT-III proteins induces aberrant INM proliferation. Our results show that HSV-1 expropriates the ESCRT-III machinery in infected cells for scission of the INM to produce vesicles containing progeny virus nucleocapsids. In uninfected cells, ESCRT-III regulates INM integrity by downregulating excess INM.

Importin-beta and the small guanosine triphosphatase Ran mediate chromosome loading of the human chromokinesin Kid.

Nucleocytoplasmic transport factors mediate various cellular processes, including nuclear transport, spindle assembly, and nuclear envelope/pore formation. In this paper, we identify the chromokinesin human kinesin-like DNA binding protein (hKid) as an import cargo of the importin-alpha/beta transport pathway and determine its nuclear localization signals (NLSs). Upon the loss of its functional NLSs, hKid exhibited reduced interactions with the mitotic chromosomes of living cells. In digitonin-permeabilized mitotic cells, hKid was bound only to the spindle and not to the chromosomes themselves. Surprisingly, hKid bound to importin-alpha/beta was efficiently targeted to mitotic chromosomes. The addition of Ran-guanosine diphosphate and an energy source, which generates Ran-guanosine triphosphate (GTP) locally at mitotic chromosomes, enhanced the importin-beta-mediated chromosome loading of hKid. Our results indicate that the association of importin-beta and -alpha with hKid triggers the initial targeting of hKid to mitotic chromosomes and that local Ran-GTP-mediated cargo release promotes the accumulation of hKid on chromosomes. Thus, this study demonstrates a novel nucleocytoplasmic transport factor-mediated mechanism for targeting proteins to mitotic chromosomes.

The human chromokinesin Kid is a plus end-directed microtubule-based motor.

Kid is a kinesin-like DNA-binding protein known to be involved in chromosome movement during mitosis, although its actual motor function has not been demonstrated. Here, we describe the initial characterization of Kid as a microtubule-based motor using optical trapping microscopy. A bacterially expressed fusion protein consisting of a truncated Kid fragment (amino acids 1-388 or 1-439) is indeed an active microtubule motor with an average speed of approximately 160 nm/s, and the polarity of movement is plus end directed. We could not detect processive movement of either monomeric Kid or dimerizing chimeric Kid; however, low levels of processivity (a few steps) cannot be detected with our method. These results are consistent with Kid having a role in chromosome congression in vivo, where it would be responsible for the polar ejection forces acting on the chromosome arms.

Cdc2-mediated phosphorylation of Kid controls its distribution to spindle and chromosomes.

The chromokinesin Kid is important in chromosome alignment at the metaphase plate. Here, we report that Kid function is regulated by phosphorylation. We identify Ser427 and Thr463 as M phase-specific phosphorylation sites and Cdc2-cyclin B as a Thr463 kinase. Kid with a Thr463 to alanine mutation fails to be localized on chromosomes and is only detected along spindles, although it retains the ability to bind DNA or chromosomes. Localization of rigor-type mutant Kid, which shows nucleotide-independent microtubule association, is also confined to the spindle, implying that strong association of Kid with the spindle can sequester it from chromosomes. T463A substitution in DNA-binding domain-truncated Kid consistently enhances its spindle localization. At physiological ionic strength, unphosphorylated Kid shows ATP-independent microtubule association, whereas Thr463-phosphorylated Kid shows ATP dependency. Moreover, the stalk region of unphosphorylated Kid interacts with microtubules and the interaction is weakened when Thr463 is phosphorylated. Our data suggest that phosphorylation on Thr463 of Kid downregulates its affinity for microtubules to ensure reversible association with spindles, allowing Kid to bind chromosomes and exhibit its function.