siRNA-Mediated BmAurora B Depletion Impedes the Formation of Holocentric Square Spindles in Silkworm Metaphase BmN4 Cells

Silkworm ovary-derived BmN4 cells rely on chromatin-induced spindle assembly to form microtubule-based square mitotic spindles that ensure accurate segregation of holocentric chromosomes during cell division. The chromosome passenger protein Aurora B regulates chromosomal condensation and segregation, spindle assembly checkpoint activation, and cytokinesis; however, its role in holocentric organisms needs further clarification. This study examined the architecture and dynamics of spindle microtubules during prophase and metaphase in BmN4 cells and those with siRNA-mediated BmAurora B knockdown using immunofluorescence labeling. Anti-alpha-tubulin and anti-gamma-tubulin antibodies revealed faint gamma-tubulin signals colocalized with alpha-tubulin in early prophase during nuclear membrane rupture, which intensified as prophase progressed. At this stage, bright regions of alpha-tubulin around and on the nuclear membrane surrounding the chromatin suggested the start of microtubules assembling in the microtubule-organizing centers (MTOCs). In metaphase, fewer but larger gamma-tubulin foci were detected on both sides of the chromosomes. This resulted in a distinctive multipolar square spindle with holocentric chromosomes aligned at the metaphase plate. siRNA-mediated BmAurora B knockdown significantly reduced the gamma-tubulin foci during prophase, impacting microtubule nucleation and spindle structure in metaphase. Spatiotemporal BmAurora B expression analysis provided new insights into the regulation of this mitotic kinase in silkworm larval gonads during gametogenesis. Our results suggest that BmAurora B is crucial for the formation of multipolar square spindles in holocentric insects, possibly through the activation of gamma-tubulin ring complexes in multiple centrosome-like MTOCs.