KIFC1 Is Essential for Bipolar Spindle Formation and Genomic Stability in the Primary Human Fibroblast IMR-90 Cell

Chromosomes are known to enhance spindle microtubule assembly in grasshopper spermatocytes, which suggested to us that chromosomes might play an essential role in the initiation of spindle formation. Chromosomes might, for example, activate other spindle components such as centrosomes and tubulin subunits upon the breakdown of the nuclear envelope. We tested this possibility in living grasshopper spermatocytes. We ruptured the nuclear envelope during prophase, which prematurely exposed the centrosomes to chromosomes and nuclear sap. Spindle assembly was promptly initiated. In contrast, assembly of the spindle was completely inhibited if the nucleus was mechanically removed from a late prophase cell. Other experiments showed that the trigger for spindle assembly is associated with the chromosomes; other constituents of the nucleus cannot initiate spindle assembly in the absence of the chromosomes. The initiation of spindle assembly required centrosomes as well as chromosomes. Extracting centrosomes from late prophase cells completely inhibited spindle assembly after dissolution of the nuclear envelope. We conclude that the normal formation of a bipolar spindle in grasshopper spermatocytes is regulated by chromosomes. A possible explanation is an activator, perhaps a chromosomal protein (Yeo, J.-P., F. Alderuccio, and B.-H. Toh. 1994a. Nature (Lond.). 367: 288-291), that promotes and stabilizes the assembly of astral microtubules and thus promotes assembly of the spindle.

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Bimodal Interaction of Mammalian Polo-Like Kinase 1 and a Centrosomal Scaffold, Cep192, in the Regulation of Bipolar Spindle Formation

Molecular and Cellular Biology ◽

10.1128/mcb.00068-15 ◽

2015 ◽

Vol 35 (15) ◽

pp. 2626-2640 ◽

Cited By ~ 23

Author(s):

Lingjun Meng ◽

Jung-Eun Park ◽

Tae-Sung Kim ◽

Eun Hye Lee ◽

Suk-Youl Park ◽

...

Keyword(s):

Xenopus Laevis ◽

Spindle Assembly ◽

Human Cells ◽

Mitotic Progression ◽

Spindle Formation ◽

Content Type ◽

Bipolar Spindle ◽

Egg Extracts ◽

Cultured Human Cells ◽

Bipolar Spindles

Serving as microtubule-organizing centers, centrosomes play a key role in forming bipolar spindles. The mechanism of how centrosomes promote bipolar spindle assembly in various organisms remains largely unknown. A recent study withXenopus laevisegg extracts suggested that the Plk1 ortholog Plx1 interacts with the phospho-T46 (p-T46) motif ofXenopusCep192 (xCep192) to form an xCep192-mediated xAurA-Plx1 cascade that is critical for bipolar spindle formation. Here, we demonstrated that in cultured human cells, Cep192 recruits AurA and Plk1 in a cooperative manner, and this event is important for the reciprocal activation of AurA and Plk1. Strikingly, Plk1 interacted with Cep192 through either the p-T44 (analogous toXenopusp-T46) or the newly identified p-S995 motif via its C-terminal noncatalytic polo-box domain. The interaction between Plk1 and the p-T44 motif was prevalent in the presence of Cep192-bound AurA, whereas the interaction of Plk1 with the p-T995 motif was preferred in the absence of AurA binding. Notably, the loss of p-T44- and p-S995-dependent Cep192-Plk1 interactions induced an additive defect in recruiting Plk1 and γ-tubulin to centrosomes, which ultimately led to a failure in proper bipolar spindle formation and mitotic progression. Thus, we propose that Plk1 promotes centrosome-based bipolar spindle formation by forming two functionally nonredundant complexes with Cep192.

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Heterogeneity of clinical isolates of chikungunya virus and its impact on the responses of primary human fibroblast-like synoviocytes

Journal of General Virology ◽

10.1099/jgv.0.001039 ◽

2018 ◽

Vol 99 (4) ◽

pp. 525-535 ◽

Cited By ~ 4

Author(s):

Apamas Sukkaew ◽

Montri Thanagith ◽

Tipparat Thongsakulprasert ◽

Margit Mutso ◽

Suresh Mahalingam ◽

...

Keyword(s):

Human Fibroblast ◽

Chikungunya Virus ◽

Clinical Isolates ◽

Primary Human Fibroblast ◽

Fibroblast Like Synoviocytes

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O-083 Non-invasive, label-free optical analysis to detect aneuploidy within the inner cell mass of the preimplantation embryo

Human Reproduction ◽

10.1093/humrep/deab125.013 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

C Y Tan ◽

S B Mahbub ◽

C A Campugan ◽

J Campbell ◽

A Habibalahi ◽

...

Keyword(s):

Hyperspectral Imaging ◽

Human Fibroblast ◽

Preimplantation Embryo ◽

Mouse Embryos ◽

Treated Mouse ◽

Fibroblast Cells ◽

Label Free ◽

Primary Human Fibroblast ◽

Non Invasive ◽

Human Fibroblast Cells

Abstract Study question Can we separate between control and reversine-treated cells within the inner cell mass (ICM) of the mouse preimplantation embryo by using label-free and non-invasive hyperspectral microscopy? Summary answer Hyperspectral microscopy is able to discern between control and reversine-treated cells using cellular autofluorescence in the complete absence of fluorescence tags. What is known already Embryo mosaicism (containing cells that are euploid (46 chromosomes) and aneuploid (deviation from the expected number of chromosomes)) affects up to 17.3% of human blastocyst embryos. Current diagnosis of aneuploidy in the IVF clinic involves a biopsy of trophectoderm (TE) cells or spent media followed by sequencing. In some blastocyst embryos these approaches will fail to diagnose of the proportion of aneuploid cells within the fetal lineage (ICM). Study design, size, duration The impact of aneuploidy on cellular metabolism was assessed by using cellular autofluoresence and hyperspectral microscopy (broad spectral profile). Two models were employed: (i) Primary human fibroblast cells with known karyotypes (4-6 independent replicates, euploid n = 467; aneuploid n = 969) and reversine induced aneuploidy in mouse embryos (5-8 independent replicates, 30-44 cells per group). Both models were subjected to hyperspectral imaging to quantify native cell fluorescence. Participants/materials, setting, methods The human model is comprised of euploid (male and female) and aneuploid (triploid and trisomies: 13, 18, 21, XXX, and XXY) primary human fibroblast cells. For the mouse model, we treated embryos with reversine, a reversible spindle assembly checkpoint inhibitor, during the 4- to 8-cell division. Individual blastomeres were dissociated from control and reversine treated 8-cell embryos. Blastomeres were either imaged directly or used to generate chimeric blastocysts with differing ratios of control:reversine-treated cells. Main results and the role of chance Following unsupervised linear unmixing, the relative abundance of metabolic cofactors was quantified: reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavins with the subsequent calculation of the optical redox ratio (ORR: Flavins/[NAD(P)H + Flavins]). Primary human fibroblast cells displayed an increase in the relative abundance of NAD(P)H with a decrease in flavins, leading to a significant reduction in the ORR for aneuploid cells (P < 0.05). The mouse embryos displayed an identical trend as the human model between control and reversine-treated embryos. Mathematical algorithms were applied and able to distinguish between (i) euploid and aneuploid primary human fibroblast cells, (ii) control and reversine-treated mouse blastomeres and (iii) chimeric blastocysts with differing ratios of control and reversine-treated cells. The accuracy of these separations was supported by receiver operating characteristic curves with areas under the curve. We also showed that hyperspectral imaging of the preimplantation embryo does not impact on embryo developmental competence, pregnancy outcome and offspring health in a mouse model. We believe the role of chance is low as both human somatic cells and mouse embryos showed a consistent shift in cellular metabolism in response to human fibroblast cells that are aneuploid and reversine treated mouse embryos. Limitations, reasons for caution Further validation of our approach could include sequencing of the ICM of individual blastocysts to determine the proportion of aneuploid cells in ICM and correlate this with the metabolic profile obtained through hyperspectral imaging. Wider implications of the findings With hyperspectral imaging able to discriminate between (i) euploid and aneuploid human fibroblast cells and (ii) control and reversine-treated mouse embryos, this could be an accurate, non-invasive and label-free optical imaging approach to assess mosaicism within the ICM of mouse embryos, potentially leading to a new diagnostic tool for embryos. Trial registration number Not applicable

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The association of Plk1 with the astrin–kinastrin complex promotes formation and maintenance of a metaphase plate

Journal of Cell Science ◽

10.1242/jcs.251025 ◽

2020 ◽

Vol 134 (1) ◽

pp. jcs251025

Author(s):

Zoë Geraghty ◽

Christina Barnard ◽

Pelin Uluocak ◽

Ulrike Gruneberg

Keyword(s):

Molecular Mechanisms ◽

Metaphase Plate ◽

Direct Interaction ◽

Mitotic Chromosomes ◽

Spindle Formation ◽

Bipolar Spindle ◽

Mitotic Kinase ◽

Spindle Poles ◽

Chromosome Congression ◽

Chromosome Alignment

ABSTRACTErrors in mitotic chromosome segregation can lead to DNA damage and aneuploidy, both hallmarks of cancer. To achieve synchronous error-free segregation, mitotic chromosomes must align at the metaphase plate with stable amphitelic attachments to microtubules emanating from opposing spindle poles. The astrin–kinastrin (astrin is also known as SPAG5 and kinastrin as SKAP) complex, also containing DYNLL1 and MYCBP, is a spindle and kinetochore protein complex with important roles in bipolar spindle formation, chromosome alignment and microtubule–kinetochore attachment. However, the molecular mechanisms by which astrin–kinastrin fulfils these diverse roles are not fully understood. Here, we characterise a direct interaction between astrin and the mitotic kinase Plk1. We identify the Plk1-binding site on astrin as well as four Plk1 phosphorylation sites on astrin. Regulation of astrin by Plk1 is dispensable for bipolar spindle formation and bulk chromosome congression, but promotes stable microtubule–kinetochore attachments and metaphase plate maintenance. It is known that Plk1 activity is required for effective microtubule–kinetochore attachment formation, and we suggest that astrin phosphorylation by Plk1 contributes to this process.

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