Dave G. Spiller

HP1α guides neuronal fate by timing E2F-targeted genes silencing during terminal differentiation

A critical step of neuronal terminal differentiation is the permanent withdrawal from the cell cycle that requires the silencing of genes that drive mitosis. Here, we describe that the α isoform of the heterochromatin protein 1 (HP1) protein family exerts such silencing on several E2F‐targeted genes. Among the different isoforms, HP1α levels progressively increase throughout differentiation and take over HP1γ binding on E2F sites in mature neurons. When overexpressed, only HP1α is able to ensure a timed repression of E2F genes. Specific inhibition of HP1α expression drives neuronal progenitors either towards death or cell cycle progression, yet preventing the expression of the neuronal marker microtubule‐associated protein 2. Furthermore, we provide evidence that this mechanism occurs in cerebellar granule neurons in vivo, during the postnatal development of the cerebellum. Finally, our results suggest that E2F‐targeted genes are packaged into higher‐order chromatin structures in mature neurons relative to neuroblasts, likely reflecting a transition from a ‘repressed’ versus ‘silenced’ status of these genes. Together, these data present new epigenetic regulations orchestrated by HP1 isoforms, critical for permanent cell cycle exit during neuronal differentiation.

p53-mediated delayed NF-κB activity enhances etoposide-induced cell death in medulloblastoma

Medulloblastoma (MB) is an embryonic brain tumour that arises in the cerebellum. Using several MB cell lines, we have demonstrated that the chemotherapeutic drug etoposide induces a p53- and caspase-dependent cell death. We have observed an additional caspase-independent cell death mechanism involving delayed nuclear factor κB (NF-κB) activity. The delayed induction was controlled by a p53-dependent transcription step and the production of death receptors (especially CD95/Fas). We further demonstrated that in both MB and glioblastoma (GM) cell lines, in which the p53 pathway was not functional, no p65 activation could be detected upon etoposide treatment. MB cell lines that have mutations in p53 or NF-κB are either less sensitive (NF-κB mutant) or even completely resistant (p53 mutant) to chemotherapeutic intervention. The optimal cell death was only achieved when both p53 and NF-κB were switched on. Taken together, our results shed light on the mechanism of NF-κB activation by etoposide in brain tumours and show that the genetic background of MB and GM cells determines their sensitivity to chemotherapy and has to be taken into account for efficient therapeutic intervention.

Unexpected intracellular localization of the AMD-associated cystatin C variant

Cystatin C is abundantly expressed by the retinal pigment epithelium (RPE) of the eye. Targeting of cystatin C to the Golgi apparatus and processing through the secretory pathway of RPE cells are dependent upon a 26‐amino acid signal sequence of precursor cystatin C. A variant with an alanine (A) to threonine (T) mutation in the penultimate amino acid of the signal sequence (A25T) was recently correlated with increased risk of developing exudative age‐related macular degeneration. The biochemical consequence of the A25T mutation upon targeting of the protein is reported here. Targeting and trafficking of full‐length mutant (A25T) precursor cystatin C–enhanced green fluorescent protein fusion protein were studied in living, cultured retinal pigment epithelial and HeLa cells. Confocal microscopy studies were substantiated by immunodetection. In striking contrast to wild‐type precursor cystatin C fusion protein conspicuously targeted to the Golgi apparatus, the threonine variant was associated principally with mitochondria. Some diffuse fluorescence was also observed throughout the cytoplasm and nucleus (but not nucleoli). Secretion of fusion protein derived from the threonine variant was reduced by approximately 50% compared with that of the wild‐type cystatin C fusion protein. Expression of the variant fusion protein did not appear to impair expression or secretion of endogenous cystatin C.

P53 apoptosis mediator PERP: localization, function and caspase activation in uveal melanoma

p53 apoptosis effector related to PMP‐22 (PERP) is a transcriptional target gene of p53 tumour suppressor that is specifically induced during apoptosis and not during cell cycle arrest. In primary uveal melanoma (UM), the most common intraocular malignancy in adults that has a reportedly unaffected signalling pathway upstream of and including p53, PERP expression is down‐regulated in the metastatic monosomy 3‐type tumours, compared with the less aggressive disomy 3‐type tumours. Here, we demonstrate experimentally, by the use of full‐length PERP‐green fluorescent protein (GFP) fusions and real‐time confocal microscopy, the intracellular targeting and plasma membrane localization of PERP in living UM cells and show that expression of PERP induces caspase‐mediated apoptosis in UM cells. Induction of PERP expression in GFP‐PERP‐transfected UM cells leads to increased levels of cleaved caspase‐8 forms, as well as to reduction of its full‐length substrate Bid, but not to detectable processing of caspase‐9. The levels of mature caspase‐8, ‐9 and ‐3 proteins significantly correlate with PERP expression levels in primary UMs. Transcriptional profiling of PERP and caspase‐8 in tumour specimens indicates that the positive association of PERP and caspase‐8 proteins is a consequence of post‐translational processing, most likely at the level of caspase‐8 cleavage, and not of increased transcription of pro‐caspase‐8. We conclude that PERP expression leads to activation of an extrinsic receptor‐mediated apoptotic pathway, with a possible subsequent engagement of the intrinsic apoptotic pathway. The findings underline the apoptotic pathway mediated by PERP as a critical mechanism employed by UM tumours to modulate susceptibility to apoptosis.

Spatio-temporal protein dynamics in single living cells

The development and application of single cell optical imaging has identified dynamic and oscillatory signalling processes in individual cells. This requires single cell analyses since the processes may otherwise be masked by the population average. These oscillations range in timing from seconds/minutes (e.g. calcium) to minutes/hours (e.g. NF-kappaB, Notch/Wnt and p53) and hours/days (e.g. circadian clock and cell cycle). Quantitative live cell measurement of the protein processes underlying these complex networks will allow characterisation of the core mechanisms that drive these signalling pathways and control cell function. Ultimately, such studies can be applied to develop predictive models of whole tissues and organisms.

Distinct roles for Caf1, Ccr4, Edc3 and CutA in the co-ordination of transcript deadenylation, decapping and P-body formation in Aspergillus nidulans

Transcript degradation is a key step in gene regulation. In eukaryotes, mRNA decay is generally initiated by removal of the poly(A) tail mediated by the Ccr4–Caf1–Not complex. Deadenylated transcripts are then rapidly degraded, primarily via the decapping‐dependent pathway. Components of this pathway can be localized into highly dynamic cytoplasmic foci, the mRNA processing (P)‐bodies. We have undertaken confocal fluorescence microscopy to monitor P‐bodies in Aspergillus nidulans. As in other organisms a dynamic shift in P‐body formation occurs in response to diverse physiological signals. Significantly, both this cellular response and the signalled degradation of specific transcripts are dependent on the nuclease activity of Caf1 but not Ccr4. P‐body formation is disrupted in A. nidulans strains deleted for Edc3, an enhancer of decapping, or CutA, which encodes a nucleotidyltransferase that triggers mRNA decapping by the addition of a CUCU tag to the poly(A) tail. As with ΔcutA, Δedc3 led to reduced rates of transcript degradation. These data link P‐bodies to both the optimization and regulation of transcript degradation.

Cell cycle phase regulates glucocorticoid receptor function.

The glucocorticoid receptor (GR) is a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors. In contrast to many other nuclear receptors, GR is thought to be exclusively cytoplasmic in quiescent cells, and only translocate to the nucleus on ligand binding. We now demonstrate significant nuclear GR in the absence of ligand, which requires nuclear localisation signal 1 (NLS1). Live cell imaging reveals dramatic GR import into the nucleus through interphase and rapid exclusion of the GR from the nucleus at the onset of mitosis, which persists into early G1. This suggests that the heterogeneity in GR distribution is reflective of cell cycle phase. The impact of cell cycle–driven GR trafficking on a panel of glucocorticoid actions was profiled. In G2/M-enriched cells there was marked prolongation of glucocorticoid-induced ERK activation. This was accompanied by DNA template-specific, ligand-independent GR transactivation. Using chimeric and domain-deleted receptors we demonstrate that this transactivation effect is mediated by the AF1 transactivation domain. AF-1 harbours multiple phosphorylation sites, which are consensus sequences for kinases including CDKs, whose activity changes during the cell cycle. In G2/M there was clear ligand independent induction of GR phosphorylation on residues 203 and 211, both of which are phosphorylated after ligand activation. Ligand-independent transactivation required induction of phospho-S211GR but not S203GR, thereby directly linking cell cycle driven GR modification with altered GR function. Cell cycle phase therefore regulates GR localisation and post-translational modification which selectively impacts GR activity. This suggests that cell cycle phase is an important determinant in the cellular response to Gc, and that mitotic index contributes to tissue Gc sensitivity.

PERP expression stabilizes active p53 via modulation of p53-MDM2 interaction in uveal melanoma cells

The activation and regulation of target genes by the tumour-suppressor p53 dictates the fate of a cell, with cell cycle arrest or apoptosis being two distinct outcomes. PERP (p53 apoptosis effector related to PMP-22), a p53 transcriptional target, is induced specifically during apoptosis but not cell cycle arrest. Downregulation of PERP is associated with the aggressive, monosomy 3-type of uveal melanoma (UM), the most common primary intraocular tumour in adults, and increased PERP expression has a pro-apoptotic effect in UM cells. Here, we identify a novel effect of PERP expression, as elevated PERP protein positively influences active levels of its own transcriptional regulator, p53. Using fluorescent fusion proteins of PERP, p53 and MDM2, we demonstrate in single living UM cells that PERP expression significantly enhances p53 activity and its nuclear localization, increases p53-dependent transcription (including that of MDM2) while allowing oscillatory nucleo-cytoplasmic shuttling of p53/MDM2 complexes. Phosphorylation of p53 serine residues that interfere with the interaction between p53 and its negative regulator MDM2 and enhance pro-apoptotic gene transcription also occurs subsequent to PERP expression. These results implicate a role for PERP in amplifying functional p53 levels that promote p53-dependent apoptosis, and reveal a potential target for exploitation in enhancing p53 activity.

No barrier to diffusion between cell soma and neurite membranes in sympathetic neurons for a GPI-anchored glycoprotein

As neurons extend their axons, it is thought that newly synthesised membrane components travel in vesicles along the axon, fuse with the growth cone membrane, and diffuse back along the axonal membrane. However, it is difficult to explain how axons continue to be populated with membrane proteins as they extend in length. To investigate this problem, we have used a CEPU-green fluorescent protein (GFP) chimeric protein to study the site of insertion of new glycosyl phosphatidyl inositol (GPI)-anchored glycoproteins and their subsequent behaviour in chick dorsal root ganglia (DRG) neurons. Infection of cultures grown for 24 h revealed rapid expression of CEPU-GFP over the whole surface of the neuron, more rapidly than could be accounted for by diffusion from the growth cone, and fluorescence intensity was uniform along the length of the neurite. Photobleaching experiments of neurite membrane revealed that recovery of fluorescence was due to diffusion from adjacent membranes and there was no evidence for membrane flow in either direction. Photobleaching of membrane adjacent to the cell body also showed rapid recovery, with chimera diffusing both from cell body membrane and the distal neurite membrane into the bleached area. These results suggest there is no barrier to diffusion between the cell body and neurite membrane in DRG and sympathetic neurons cultured for 1 or 2 days in vitro. We propose that the neurite is populated by newly synthesised chimera by diffusion from both regions. This situation may also occur in neurons in the early stages of extending axons in vivo prior to polarisation and the development of the dendritic field.