- Picture from Kirkham M et al. (2003) Cell "SAS-4 is a C. elegans centriolar protein that controls centrosome ...."
Figure 4. SAS-4 Is Incorporated during Centriole Duplication and Remains Stably Associated Thereafter. Wild-type (N2) males were mated with hermaphrodites expressing GFP-SAS-4. Embryos were fixed at various times after fertilization and stained for microtubules (MTs) and DNA (red and blue in left images) and with antibodies to SAS-4 to detect both the labeled and unlabeled protein (total SAS-4) and to GFP to detect only the GFP-SAS-4 fusion (GFP-SAS-4). Centrosomes are shown in the insets (magnified 3). A schematic of our interpretation of the results is shown on the right.(A) In recently fertilized embryos, SAS-4 localizes to the centriole pair associated with the sperm nucleus. There is no staining in the GFP channel.(B) By prophase of the first mitotic division, GFP-SAS-4 has been incorporated into both centrosomes.(C) GFP-SAS-4 is also detected in a single focus in the center of each centrosome at metaphase.(D) When the centrosomes split in telophase, each daughter centrosome inherits one centriole. Both centrosomes have a focus of SAS-4 staining. However, only one of the centrosomes, likely the one containing the daughter centriole synthesized in the embryo, contains GFP-SAS-4.(E) By prophase of the two-cell stage the centrioles have duplicated again. All of the centrosomes contain at least one centriole that was synthesized in the oocyte cytoplasm and all contain the GFP-SAS-4 fusion protein. Scale bar is 10 μm.
- Picture from Kirkham M et al. (2003) Cell "SAS-4 is a C. elegans centriolar protein that controls centrosome ...."
Figure 3. SAS-4 Localizes to Centrioles Throughout the Cell Cycle. (A) Wild-type hermaphrodites were fixed and stained for DNA, gamma-tubulin, and SAS-4. SAS-4 colocalized with gamma-tubulin to centrosomes in sperm (arrow on left side of all images and insets) and to centrosomes associated with nuclei in meiotic prophase in the gonad arm (small arrows), but disappeared in oocytes (arrowheads). Insets were magnified 3.(B) Hermaphrodites expressing GFP-SAS-4 were examined by DIC (a, b) and for GFP fluorescence (a', b'). As was the case for the SAS-4 antibody staining, single punctate fluorescent foci were found in sperm and associated with meiotic nuclei in the gonad arm. Arrows in (a) and (a') indicate the location of a cluster of GFP-SAS-4 foci.(C) Wild-type embryos were fixed and stained to visualize microtubules (MTs) and DNA (green and red in left images), gamma-tubulin (middle images), and SAS-4 (right images). In recently fertilized embryos in which the oocyte nucleus was still completing meiosis (top images), SAS-4 was observed in a single punctate focus associated with the sperm pronucleus (arrows). No colocalizing gamma-tubulin staining was observed at these early time points. (C) At metaphase, two small foci of SAS-4 staining are seen in the center of the gamma-tubulin staining at each spindle pole.(D) Stills taken from a time-lapse recording of an embryo expressing GFP-SAS-4. Both DIC (a) and GFP fluorescence (a') are shown. In the DIC image, the mitotic spindle is visible as a region that excludes yolk granules. Punctate foci of GFP-SAS-4 are observed in the center of each spindle pole.(E) Higher magnification (6.4) view of the right centrosome in the metaphase image in (C). Scale bars in (A-D) are 10 μm.(F) Localization of SAS-4 by immunoelectron microscopy. Immunolabeling was found associated with centriole walls. Labeling was detected on both mother and daughter centrioles. Scale bar is 250 nm. Images in the right column show selected centrioles from the images on the left magnified 2.5.