Figure 6

Thd14 resides in mitochondria and nucleoli during growth. a Line drawing schematic of the construct engineered on an rDNA vector for expression of GFP-Thd14 fusion protein from the metallothioneine promoter (MTT1 pr). During conjugation, the vector is processed and amplified as a small linear rDNA chromosome. b Fluorescence microscopy image of a live cell showing GFP-Thd14 localizing to mitochondria (stained with Mitotracker) near the surface of the cell. Inlays show magnification of one mitochondrial-dense region. The larger region of intense staining is rarely observed in other cells and thus considered an artifact. c Differential interference contrast (DIC) imaging of a live cell in vegetative growth shows locations of nuclear structures, including the micronucleus (m) and multiple nucleoli (nu) positioned around the macronuclear (M) periphery. Fluorescence microscopy shows a merge of DAPI imaging with GFP-Thd14 in nucleoli (center panel). "GFP con/DAPI" is the GFP only control (no fusion) with a DAPI image overlay. Green spheres outside the nucleus are probably food vacuoles, a common artifact observed with high GFP-protein expression. Live cells were concentrated and mounted in 2% methylcellulose for observation. d Fluorescence microscopy on a paraformaldehyde-fixed cell nucleus (bounded by dashed circle) showing co-localization of GFP-Thd14 and Nop52 by immunofluorescence in regions staining poorly with DAPI (arrow indicates an example).