RESUMO
Mitotic kinesin-like protein 2 (MKLP2; also known as KIF20A) is a motor protein with a well-established function in promoting cytokinesis. However, our results with siRNAs targeting MKLP2 and small-molecule inhibitors of MKLP2 (MKLP2i) suggest that it also has a function earlier in mitosis, prior to anaphase. In this study, we provide direct evidence that MKLP2 facilitates chromosome congression in prometaphase. We employed live imaging to observe HeLa cells with fluorescently tagged histones treated with MKLP2i and discovered a pronounced chromosome congression defect. We show that MKLP2 facilitates error correction, as inhibited cells have a significant increase in unstable, syntelic kinetochore-microtubule attachments. We find that the aberrant attachments are accompanied by elevated Aurora kinase (A and B) activity and phosphorylation of the downstream target HEC1 (also known as NDC80) at Ser55. Finally, we show that MKLP2 inhibition results in aneuploidy, confirming that MKLP2 safeguards cells against chromosomal instability. This article has an associated First Person interview with the first author of the paper.
Assuntos
Cinesinas/metabolismo , Mitose , Aurora Quinase B/genética , Aurora Quinase B/metabolismo , Segregação de Cromossomos , Cromossomos/metabolismo , Células HeLa , Humanos , Cinesinas/genética , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Mitose/genética , Fuso Acromático/metabolismoRESUMO
BACKGROUND: Sarcopenia, a group of muscle-related disorders, leads to the gradual decline and weakening of skeletal muscle over time. Recognizing the pivotal role of gastrointestinal conditions in maintaining metabolic homeostasis within skeletal muscle, we hypothesize that the effectiveness of the myogenic programme is influenced by the levels of gastrointestinal hormones in the bloodstream, and this connection is associated with the onset of sarcopenia. METHODS: We first categorized 145 individuals from the Emergency Room of Taipei Veterans General Hospital into sarcopenia and non-sarcopenia groups, following the criteria established by the Asian Working Group for Sarcopenia. A thorough examination of specific gastrointestinal hormone levels in plasma was conducted to identify the one most closely associated with sarcopenia. Techniques, including immunofluorescence, western blotting, glucose uptake assays, seahorse real-time cell metabolic analysis, flow cytometry analysis, kinesin-1 activity assays and qPCR analysis, were applied to investigate its impacts and mechanisms on myogenic differentiation. RESULTS: Individuals in the sarcopenia group exhibited elevated plasma levels of glucagon-like peptide 1 (GLP-1) at 1021.5 ± 313.5 pg/mL, in contrast to non-sarcopenic individuals with levels at 351.1 ± 39.0 pg/mL (P < 0.05). Although it is typical for GLP-1 levels to rise post-meal and subsequently drop naturally, detecting higher GLP-1 levels in starving individuals with sarcopenia raised the possibility of GLP-1 influencing myogenic differentiation in skeletal muscle. Further investigation using a cell model revealed that GLP-1 (1, 10 and 100 ng/mL) dose-dependently suppressed the expression of the myogenic marker, impeding myocyte fusion and the formation of polarized myotubes during differentiation. GLP-1 significantly inhibited the activity of the microtubule motor kinesin-1, interfering with the translocation of glucose transporter 4 (GLUT4) to the cell membrane and the dispersion of mitochondria. These impairments subsequently led to a reduction in glucose uptake to 0.81 ± 0.04 fold (P < 0.01) and mitochondrial adenosine triphosphate (ATP) production from 25.24 ± 1.57 pmol/min to 18.83 ± 1.11 pmol/min (P < 0.05). Continuous exposure to GLP-1, even under insulin induction, attenuated the elevated glucose uptake. CONCLUSIONS: The elevated GLP-1 levels observed in individuals with sarcopenia are associated with a reduction in myogenic differentiation. The impact of GLP-1 on both the membrane translocation of GLUT4 and the dispersion of mitochondria significantly hinders glucose uptake and the production of mitochondrial ATP necessary for the myogenic programme. These findings point us towards strategies to establish the muscle-gut axis, particularly in the context of sarcopenia. Additionally, these results present the potential of identifying relevant diagnostic biomarkers.
RESUMO
Leaf color is directly associated with plant photosynthesis. Here, we have isolated and identified a spontaneous rice mutant named yd1 that has yellowish leaves and dwarf stature. Map-based cloning reveals that YD1 encodes a previously reported kinesin protein from the kinesin-4 subfamily, BC12/GDD1. Arginine-328 is replaced by leucine in yd1, BC12328Leu. YD1 is mainly expressed in leaves and is involved in chlorophyll (Chl) synthesis. The yd1 mutant had less Chl and a reduced and disordered thylakoid ultrastructure. In yd1 plants, Chl biosynthesis and photosynthesis associated gene expression was decreased and Chl degradation gene expression was increased, thereby leading to a reduced photosynthesis rate and grain yield. In this study we reveal that the novel BC12328Leu allele of BC12 modulated plant leaf color in yd1 plants, which has not been previously reported in studies of BC12/GDD1/MTD1/SRG1. Gene knockout results indicated that YD1 regulates leaf color in the indica rice background, but not in the japonica rice background. Our study provides new insights into molecular regulation of rice growth by BC12/GDD1 in different genetic backgrounds.