Caspase 8 plays an essential role in the execution of death receptor-mediated apoptosis. To determine the localization of endogenous caspase 8, we used a panel of subunit-specific anti-caspase 8 monoclonal antibodies in confocal immunofluorescence microscopy. In the human breast carcinoma cell line MCF7, caspase 8 predominantly colocalized with and bound to mitochondria. After induction of apoptosis through CD95 or tumor necrosis factor receptor I, active caspase 8 translocated to plectin, a major cross-linking protein of the three main cytoplasmic filament systems, whereas the caspase 8 prodomain remained bound to mitochondria. Plectin was quantitatively cleaved by caspase 8 at Asp 2395 in the center of the molecule in all cells tested. Cleavage of plectin clearly preceded that of other caspase substrates such as poly(ADP-ribose) polymerase, gelsolin, cytokeratins, or lamin B. In primary fibroblasts from plectin-deficient mice, apoptosis-induced reorganization of the actin cytoskeleton, as seen in wild-type cells, was severely impaired, suggesting that during apoptosis, plectin is required for the reorganization of the microfilament system. Apoptosis is essential for development and homeostasis of the organism (60). It is a morphologically and biochemically distinct form of cell death that can be triggered by a wide range of internal and external signals (for a review, see reference 70). Recent studies demonstrated that a subfamily of the tumor necrosis factor receptor (TNF-R) superfamily, the death receptors, constitute an important system which can induce apoptosis (for a review, see reference 48). Among this death receptor family, CD95 (also called APO-1 or Fas) is one of the best-characterized members, especially with regard to intracellular signaling events. Apoptosis mediated by CD95 involves activation of a cascade of cysteine proteases, the caspases (45). In the CD95 system, caspase 8 (also called FLICE, Mach, or Mch5) (4, 9, 43), the most receptor-proximal caspase, is recruited to CD95 through the adapter molecule FADD (Mort1) (5,8). This results in activation of caspase 8 by proteolytic cleavage into the prodomain containing two death effector domains (DEDs) and two active subunits, p18 and p10 (39, 56). We have recently shown that caspase 8 can be activated in two ways. Most of caspase 8 is activated at the CD95 receptor in type I cells and at the mitochondria in type II cells (55). Caspase 8 was also found to be essential for other death receptors such as TNF-RI, TRAIL-RI, and DR3 (25, 68).Activation of caspase 8 and other caspases located more downstream in the pathway results in cleavage of various death substrates. These protein targets include various intermediate filament (IF) proteins (7,16,29). Thereby, apoptosis signaling profoundly affects the integrity of the cytoskeleton and consequently the cellular structure as a whole. Activation of caspases is also responsible for the specific nuclear changes characteristic for apoptosis involving activation of the endonuclease CAD (DFF40) (33, 53) an...

The transcriptionally active rRNA genes have the remarkable ability to organize and integrate the biochemical pathway of ribosome production into a structural framework, the nucleolus. The past year has seen numerous advances in our understanding of the relationships between nucleolar substructures, the site of ribosomal RNA (rRNA) gene transcription and the pathway of ribosome maturation. Progress has also been made both in the molecular identification of nucleolar constituents and in our understanding of the interactions between these components and their assembly into higher order structures.

Abstract. When cells enter mitosis, RNA synthesisceases. Yet the RNA polymerase I (pol I) transcription machinery involved in the production of pre-rRNA remains bound to the nucleolus organizing region (NOR), the chromosome site harboring the tandemly repeated rRNA genes. Here we examine whether rDNA transcription units are transiently blocked or "frozen" during mitosis. By using fluorescent in situ hybridization we were unable to detect nascent prerRNA chains on the NORs of mouse 3T3 and rat kangaroo PtK2 cells. Appropriate controls showed that our approach was sensitive enough to visualize, at the light microscopic level, individual transcriptionally active rRNA genes both in situ after experimental unfolding of nucleoli and in chromatin spreads ("Miller spreads"). Analysis of the cell cycle-dependent redistribution of transcript-associated components also revealed that most transcripts are released from the rDNA at mitosis. Upon disintegration of the nucleolus during mitosis, U3 small nucleolar RNA (snoRNA) and the nucleolar proteins fibrillarin and nucleolin became dispersed throughout the cytoplasm and were excluded from the NORs. Together, our data rule out the presence of "frozen Christmas-trees" at the mitotic NORs but are compatible with the view that inactive pol I remains on the rDNA. We propose that expression of the rRNA genes is regulated during mitosis at the level of transcription elongation, similarly to what is known for a number of genes transcribed by pol II. Such a mechanism may explain the decondensed state of the NOR chromatin and the immediate transcriptional reactivation of the rRNA genes following mitosis.

The nucleolus: a paradigm for nuclear orderThe genes coding for ribosomal RNA (rRNA) are unique in that their activity leads to the formation of a