The doublecortin-family kinase ZYG-8DCLK1 regulates microtubule dynamics and motor-driven forces to promote the stability of C. elegans acentrosomal spindles.

Although centrosomes help organize spindles in most cell types, oocytes of most species lack these structures. During acentrosomal spindle assembly in C. elegans oocytes, microtubule minus ends are sorted outwards away from the chromosomes where they form poles, but then these outward forces must be balanced to form a stable bipolar structure. Simultaneously, microtubule dynamics must be precisely controlled to maintain spindle length and organization. How forces and dynamics are tuned to create a stable bipolar structure is poorly understood. Here, we have gained insight into this question through studies of ZYG-8, a conserved doublecortin-family kinase; the mammalian homolog of this microtubule-associated protein is upregulated in many cancers and has been implicated in cell division, but the mechanisms by which it functions are poorly understood. We found that ZYG-8 depletion from oocytes resulted in overelongated spindles with pole and midspindle defects. Importantly, experiments with monopolar spindles revealed that ZYG-8 depletion led to excess outward forces within the spindle and suggested a potential role for this protein in regulating the force-generating motor BMK-1/kinesin-5. Further, we found that ZYG-8 is also required for proper microtubule dynamics within the oocyte spindle and that kinase activity is required for its function during both meiosis and mitosis. Altogether, our findings reveal new roles for ZYG-8 in oocytes and provide insights into how acentrosomal spindles are stabilized to promote faithful meiosis.

Identification of a novel linear B-cell epitope in porcine deltacoronavirus nucleocapsid protein.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that caused diarrhea and/or vomiting in neonatal piglets worldwide. Coronaviruses nucleocapsid (N) protein is the most conserved structural protein for viral replication and possesses good antigenicity. In this study, three monoclonal antibodies (mAbs), 3B4, 4D3, and 4E3 identified as subclass IgG2aκ were prepared using the lymphocytic hybridoma technology against PDCoV N protein. Furthermore, the B-cell epitope recognized by mAb 4D3 was mapped by dozens of overlapping truncated recombinant proteins based on the western blotting. The polypeptide 28QFRGNGVPLNSAIKPVE44 (EP-4D3) in the N-terminal of PDCoV N protein was identified as the minimal linear epitope for binding mAb 4D3. And the EP-4D3 epitope's amino acid sequence homology study revealed that PDCoV strains are substantially conserved, with the exception of the Alanine43 substitution Valine43 in the China lineage, the Early China lineage, and the Thailand, Vietnam, and Laos lineage. The epitope sequences shared high similarity (94.1%) with porcine coronavirus HKU15-155 (PorCoV HKU15), Asian leopard cats coronavirus (ALCCoV), sparrow coronavirus HKU17 (SpCoV HKU17), and sparrow deltacoronavirus. In contrast, the epitope sequences shared a very low homology (11.8 to 29.4%) with other porcine CoVs (PEDV, TGEV, PRCV, SADS-CoV, PHEV). Overall, the study will enrich the biological function of PDCoV N protein and provide foundational data for further development of diagnostic applications. KEY POINTS: • Three monoclonal antibodies against PDCoV N protein were prepared. • Discovery of a novel B-cell liner epitope (28QFRGNGVPLNSAIKPVE44) of PDCoV N protein. • The epitope EP-4D3 was conserved among PDCoV strains.