A first characterization of kinetochore proteins in the holocentric insect Spodoptera frugiperda

Abstract Insects with holocentric chromosomes have a centromere spread all over their length and are devoid of the proteins CENP-A and CENP-C, suggesting a different kinetochore assembly process compared to that in monocentrics. Very recently, the homolog of CENP-T was shown to play a key role in kinetochore assembly in Bombyx mori and to bind to silent chromatin regions. However, nothing is known in other holocentric insects. In this paper, we set up a protocol to silence the kinetochore genes cenp-L, cenp-S, cenp-X and ndc80 and searched for chromosome segregation defects in Spodoptera frugiperda cells. We also analyzed their expression pattern in the insect. As shown by immunofluorescence microscopy and RT-QPCR, we succeeded to silence target genes efficiently by transfection with dsRNA. In Sf9 cells depleted for CENP-L and NDC80, by immunofluorescence microscopy, we observed an increase of mitotic indices linked to metaphase arrest and, respectively unaligned chromosomes and multipolar spindles. In cells depleted for CENP-S and CENP-X, the mitotic indices were unchanged and no division defects were observed. Except cenp-S and X, kinetochore genes were overexpressed in gonadal versus somatic tissues. We conclude that CENP-L and NDC80 play a major role in chromosome segregation, while the function of CENP-S and -X are still unknown. We started to characterize the kinetochore proteins (CENP-L, CENP-S, CENP-X, NDC80), a prerequisite for holocentromere identification in S. frugiperda. We provide the first clues on CENP-L in Lepidoptera, a protein playing a central role in the structure of the Constitutive Centromere Associated Network in monocentrics