Nucleolar Morphology and Nuclear Actin

Confocal image stacks of S10B follicles stained for fibrillarin or EU were genotypically blinded and each follicle was assigned a nucleolar defect severity score according to the number of nurse cell nucleoli with morphological changes; the four posterior nurse cells were excluded from the analysis as their morphology is distinct. The nucleolar morphology categories included: normal (0-1 disrupted nucleoli), mild (2-4 disrupted nucleoli), and severe (5 or more disrupted nucleoli). Morphology defects included nucleoli that are pebbled (several smaller, disconnected portions of nucleolus) or rounded (single, nearly spherical nucleolus). In the context of NLS Actin overexpression (Figure 6), we assessed nucleolar morphology in relation to the level of nuclear actin. The level of nuclear actin was categorized as low which exhibits a nuclear actin haze, medium which exhibits a network of thin nuclear actin filaments, and high which exhibits thick nuclear actin filamentous structures termed rods. Quantification of nucleolar volume was performed using Imaris (Oxford, RRID:SCR_007370) on yw S10B follicles stained for EU. The single anterior-most nurse cell was compared to the average volume of four posterior nurse cells (from the third row of nurse cells).