LAUSR

Ribosome biogenesis is initiated in the nucleolus, a cell condensate essential to gene expression, whose morphology informs cancer pathologists on the health status of a cell. Here, we describe a protocol for assessing, both qualitatively and quantitatively, the involvement of trans-acting factors in the nucleolar structure. The protocol involves use of siRNAs to deplete cells of factors of interest, fluorescence imaging of nucleoli in an automated high-throughput platform, and use of dedicated software to determine an index of nucleolar disruption, the iNo score. This scoring system is unique in that it integrates the five most discriminant shape and textural features of the nucleolus into a parametric equation. Determining the iNo score enables both qualitative and quantitative factor classification with prediction of function (functional clustering), which to our knowledge is not achieved by competing approaches, as well as stratification of their effect (severity of defects) on nucleolar structure. The iNo score has the potential to be useful in basic cell biology (nucleolar structure–function relationships, mitosis, and senescence), developmental and/or organismal biology (aging), and clinical practice (cancer, viral infection, and reproduction). The entire protocol can be completed within 1 week.This protocol qualitatively and quantitatively assesses the involvement of factors in the nucleolar structure. After siRNA-mediated depletion of factors of interest, dedicated software is used to analyze images of nucleoli to determine an index of nucleolar disruption, the iNo score.