The image contains multiple panels showing fluorescence micrographs and quantitative analysis of N o p1-m E o s in hyphae. Panel A shows fluorescence micrographs of a hypha expressing N o p 1-m E o s before and after photoconversion, with green indicating non-converted and magenta indicating photoconverted channels. two parallel, dashed horizontal lines appear in each micrograph. Panel B quantifies the fluorescence intensity of N o p 1-m E o s before and after photoconversion, with green and magenta signals measured within the same nucleolar region. In the graph, pink dots are connected by straight lines with an increasing trend, while the green dots are connected by straight lines with a decreasing trend. Panel C outlines the experimental workflow, including heat shock at 42 degree celsius, photoconversion at 405 nanometers, and imaging every 5 minutes. Heat shock shows a green dot, photoconversion shows a magenta dot, and imaging shows a union of a magenta and a green dot, with arrows labeled new and old pointing to the union on either side. Panel D presents time-lapse fluorescence micrographs of a hypha during recovery from heat shock, showing the remodeling of a single nucleolus over time with distinct nucleolar bud formation. The micrographs are divided into three sections labeled N o p 1 (magenta), N o p 1 (green), and Merged (white). Merged includes labels for old and new. Panel E shows a scatter plot, which quantifies the intensity of N o p 1-m E o s in new and old nucleolar compartments, with fluorescence ratios calculated for photoconverted and newly synthesized channels. Magenta N o p 1 shows low new to old nucleolar intensity ratio, while green N o p 1 shows higher ratio. Panel F continues the time-lapse sequence, highlighting the fate of the old nucleolus. It includes labels for extruded and daughter nucleus.
Nucleolar budding initiates from preexisting material but incorporates newly synthesized Nop1 during expansion. (A) Fluorescence micrographs of a hypha expressing Nop1-mEos before (“Pre”) and after (“Post”) photoconversion, shown as green (non-converted) and magenta (photoconverted) channels. Dashed lines indicate the outline of the hypha. (B) Quantification of Nop1-mEos fluorescence intensity before and after photoconversion. Green (non-converted) and magenta (photoconverted) signals were measured within the same nucleolar ROI in individual nuclei. Each pair of points represents measurements from a single nucleolus before and after photoconversion. P values (P = 0.0144 for the green channel and P = 0.0008 for the magenta channel) were calculated using two-tailed paired t tests comparing pre- and postphotoconversion fluorescence intensity for each channel independently. Statistical testing was used to confirm the consistency and magnitude of photoconversion across nucleoli. (C) Schematic outlining the experimental workflow: cells were heat shocked, photoconverted, and imaged every 5 min in both green and magenta channels during nucleolar remodeling and mitosis. (D) Time-lapse fluorescence micrographs of a hypha expressing Nop1-mEos during recovery from heat shock, showing remodeling of a single nucleolus over time. Photoconverted (magenta) and newly synthesized (green) Nop1 are shown separately and merged. A distinct nucleolar bud emerges from preexisting material and progressively incorporates new Nop1. (E) Quantification of Nop1-mEos intensity in the newly formed new versus preexisting old nucleolar compartments shown in D. Fluorescence ratios were calculated separately for photoconverted (magenta) and newly synthesized (green) channels. Each point represents a single nucleolus; P value (P = 0.0330) was calculated using a two-tailed unpaired t test. Data are presented as mean ± SD (n = 3 nucleoli). (F) Continuation of the time-lapse sequence shown in D, highlighting the fate of the old (photoconverted) nucleolus.