Cellular Uptake and Nuclear Delivery of Recombinant Adenovirus Penton Base

AbstractAn Ad2 capsid component, the penton base, expressed as recombinant protein, was found to be capable of affecting the entire entry pathway of adenovirion in HeLa cells, i.e., cell attachment, endocytosis, vesicular escape, intracytoplasmic movement, and translocation through the nuclear pore complex. Data with pentamerization-defective mutants suggested that none of these successive steps depended upon penton base pentamer status, indicating that the peptide domains responsible for these functions were carried by the monomer. Observations performed with wild-type (WT) and an integrin-binding-site double-mutant (K288E340) suggested that the penton base could enter the cell via an alternative, RGD- and LDV-independent, pathway. Of three mutants that were found to be defective in nuclear addressing in insect cells, only one, W165H, was also altered in nuclear transport in HeLa cells. The other two, W119H and RRR547EQQ, showed a WT pattern of nuclear localization in HeLa cells, suggesting that the region including tryptophan-119 and the basic signal at position 547 did not act as a nuclear localization signal in the human cell context. The integrity of cellular structures and the cytoskeleton seemed to be required for the vectorial movement and nuclear import of WT penton base, as suggested by experiments using permeabilized HeLa cells, isolated nuclear membranes, and cytoskeleton-targeted drugs

High mobility group proteins of amphibian oocytes: a large storage pool of a soluble high mobility group-1-like protein and involvement in transcriptional events

No abstract availabl

Expression and localization of nuclear proteins in autosomal-dominant Emery-Dreifuss muscular dystrophy with LMNA R377H mutation

BACKGROUND: The autosomal dominant form of Emery-Dreifuss muscular dystrophy (AD-EDMD) is caused by mutations in the gene encoding for the lamins A and C (LMNA). Lamins are intermediate filament proteins which form the nuclear lamina underlying the inner nuclear membrane. We have studied the expression and the localization of nuclear envelope proteins in three different cell types and muscle tissue of an AD-EDMD patient carrying a point mutation R377H in the lamin A/C gene. RESULTS: Lymphoblastoid cells, skin fibroblasts, primary myoblasts and muscle thin sections were studied by immunocytochemistry and electron microscopy. Cellular levels of A-type lamins were reduced compared to control cells. In contrast, the amount of emerin and lamin B appeared unaltered. Cell synchronization experiments showed that the reduction of the cellular level of A-type lamin was due to instability of lamin A. By electron microscopy, we identified a proportion of nuclei with morphological alterations in lymphoblastoid cells, fibroblasts and mature muscle fibres. Immunofluorescence microscopy showed that a major population of the lamin B receptor (LBR), an inner nuclear membrane protein, was recovered in the cytoplasm in association with the ER. In addition, the intranuclear organization of the active form of RNA polymerase II was markedly different in cells of this AD-EDMD patient. This aberrant intranuclear distribution was specifically observed in muscle cells where the pathology of EDMD predominates. CONCLUSIONS: From our results we conclude: Firstly, that structural alterations of the nuclei which are found only in a minor fraction of lymphoblastoid cells and mature muscle fibres are not sufficient to explain the clinical pathology of EDMD; Secondly, that wild type lamin A is required not only for the retention of LBR in the inner nuclear membrane but also for a correct localization of the transcriptionally active RNA pol II in muscle cells. We speculate that a rearrangement of the internal chromatin could lead to muscle-specific disease symptoms by interference with proper mRNA transcription

Temporal differences in the appearance of NEP-B78 and an LBR-like protein during Xenopus nuclear envelope reassembly reflect the ordered recruitment of functionally discrete vesicle types

In this work, we have used novel mAbs against two proteins of the endoplasmic reticulum and outer nuclear membrane, termed NEP-B78 and p65, in addition to a polyclonal antibody against the inner nuclear membrane protein LBR (lamin B receptor), to study the order and dynamics of NE reassembly in the Xenopus cell-free system. Using these reagents, we demonstrate differences in the timing of recruitment of their cognate membrane proteins to the surface of decondensing chromatin in both the cell-free system and XLK-2 cells. We show unequivocally that, in the cell-free system, two functionally and biochemically distinct vesicle types are necessary for NE assembly. We find that the process of distinct vesicle recruitment to chromatin is an ordered one and that NEP-B78 defines a vesicle population involved in the earliest events of reassembly in this system. Finally, we present evidence that NEP-B78 may be required for the targeting of these vesicles to the surface of decondensing chromatin in this system. The results have important implications for the understanding of the mechanisms of nuclear envelope disassembly and reassembly during mitosis and for the development of systems to identify novel molecules that control these processes

DNA Methyltransferase Is Actively Retained in the Cytoplasm during Early Development

The overall DNA methylation level sharply decreases from the zygote to the blastocyst stage despite the presence of high levels of DNA methyltransferase (Dnmt1). Surprisingly, the enzyme is localized in the cytoplasm of early embryos despite the presence of several functional nuclear localization signals. We mapped a region in the NH2-terminal, regulatory domain of Dnmt1 that is necessary and sufficient for cytoplasmic retention during early development. Altogether, our results suggest that Dnmt1 is actively retained in the cytoplasm, which prevents binding to its DNA substrate in the nucleus and thereby contributes to the erasure of gamete-specific epigenetic information during early mammalian development

Pre-M Phase-promoting Factor Associates with Annulate Lamellae in Xenopus Oocytes and Egg Extracts

We have used complementary biochemical and in vivo approaches to study the compartmentalization of M phase-promoting factor (MPF) in prophase Xenopus eggs and oocytes. We first examined the distribution of MPF (Cdc2/CyclinB2) and membranous organelles in high-speed extracts of Xenopus eggs made during mitotic prophase. These extracts were found to lack mitochondria, Golgi membranes, and most endoplasmic reticulum (ER) but to contain the bulk of the pre-MPF pool. This pre-MPF could be pelleted by further centrifugation along with components necessary to activate it. On activation, Cdc2/CyclinB2 moved into the soluble fraction. Electron microscopy and Western blot analysis showed that the pre-MPF pellet contained a specific ER subdomain comprising "annulate lamellae" (AL): stacked ER membranes highly enriched in nuclear pores. Colocalization of pre-MPF with AL was demonstrated by anti-CyclinB2 immunofluorescence in prophase oocytes, in which AL are positioned close to the vegetal surface. Green fluorescent protein-CyclinB2 expressed in oocytes also localized at AL. These data suggest that inactive MPF associates with nuclear envelope components just before activation. This association may explain why nuclei and centrosomes stimulate MPF activation and provide a mechanism for targeting of MPF to some of its key substrates