Periphery Of Cytoplasm Research Articles

Proteasome inhibitor ‐induced protein aggregation is regulated via HSP90, HDACs and microtubulus stability in ARPE‐19 cells

Abstract Purpose Impaired degradation of cellular proteins is implicated in aged RPE cells. In addition to lysosomal protein clearance, many cellular proteins are degradated in proteasomes. Heat shock proteins (HSPs) tend to prevent the accumulation of cytotoxic protein aggregates. Regulatory role of HSPs, histone deacetylases (HDACs) and tubulin stability state in proteasome inhibitor ‐induced protein aggregation in ARPE–19 cells were evaluated. Methods HSP90, HSP70, HSC70, HSP27, ubiquitin and acetylated tubulin expression levels were analyzed by Western blotting. Phase contrast and transmission electron microscopy and immunofluorescence analysis were used to detect cellular organelles and to evaluate morphological changes. Results Western blotting analysis showed increased HSP70 expression levels in response to HSP90 inhibitor geldanamycin (GA) and proteasome inhibitor MG‐132. Trichostatin A(HDAC inhibitor) and taxol evoked increased acetylation level of tubulin. Interestingly, MG‐132 ‐induced juxtranuclear protein aggregates were not formed in response to GA, while during taxol treatment aggregation process was conventional. During trichostatin A and nocodazole treatment the aggregates were localized to periphery of cytoplasm rather than to juxtanuclear position. The HSP90 could not be seen together with the aggregates, while the HSP70 stained strongly with the observed juxtanuclear aggregates. Conclusion HSP90, HDACs and tubulin polymerization state are critical regulators of proteasome inhibitor –induced protein aggregates in ARPE‐19 cells. However, total acetylation level of tubulin does not seem to be essential in the aggregation process.

MPP+ impairs autophagic clearance of alpha-synuclein by impairing the activity of dynein

Increasing evidence suggests that dynein has an important role in the clearance of misfolded proteins by autophagy. Here we show that treatment of cells with 1-methyl-4-phenylpyridinium (MPP) cause alpha-synuclein overexpression and aggregation, leading to the accumulation of autophagic vacuoles and the recruitment of LC3-II to these vacuoles in the cytoplasm. After MPP treatment, dynein expression decreased and was mainly aggregated at the periphery of cytoplasm and lost its colocalization with alpha-synuclein and lamp1, indicating that dynein lost its function in the aggresome formation and failed to return autophagosome and lysosomes to the center of the cell for degradation. We consider that dynein plays an important role in the autophagic clearance of aggregate-prone proteins.

A Role for Survivin in Enucleation of Erythroid Progenitors.

Enucleation is the final step in the development of reticulocytes and this process is unique to mammalian red blood cells among vertebrates. Even though enucleation of erythroblasts was structurally studied by electron microscopy half a century ago, little is known about the underlying mechanism. Initially it was thought that enucleation involved degradation of DNA and other apoptotic processes. More recently it has been shown that enucleation of erythroid progenitors does not require major apoptotic machinery including caspase-3 activation (Krauss et al, 2005; Yoshida et al, 2005). Alternatively, it has been suggested that enucleation in erythroblasts is a result of asymmetric cytokinesis. In support of this later model, survivin, a regulator of chromosomal segregation and cytokinesis, which also has an anti apoptotic role, is highly expressed in orthochromatic erythroblasts, cells at the stage of differentiation just prior to enucleation (Gurbuxani et al, 2005). Moreover conditional heterozygous deletion of survivin in hematopoietic cells caused a decrease in enucleated cells in the spleen (Leung et al, 2007). This has led us to hypothesize that survivin has a role in enucleation of erythroblasts. To test this prediction, we first over expressed survivin in MEL (murine erythroleukemia) cells and then subjected them to DMSO induced differentiation. Normally MEL cells decrease their survivin expression level upon differentiation and at the same time undergo limited enucleation (<4%) even after 5 days of differentiation. In contrast, survivin overexpressing clones consistently enucleated 3–5 fold more than control cells, suggesting that levels of survivin are likely to be important for enucleation. We are currently harnessing the power of survivin antisense nanoparticle agents to assess the specific requirement of survivin in enucleation of primary human orthochromatic erythroblasts. Separately, we evaluated the localization of survivin and its known partners in enucleating primary human erythroid cells by confocal microscopy. Survivin is predominantly localized to the cytoplasmic compartment and to our surprise did not colocalize with Aurora B kinase and INCENP, two of its partners in the chromosomal passenger complex, which is essential for mitosis. On the contrary, we noticed that survivin partially colocalized with actin at the junction between the extruding nucleus and cytoplasm, as well as in the periphery of cytoplasm in these enucleating cells. Moreover enucleation assays performed in the presence of various inhibitors of cell cycle and cytokinesis did not prevent enucleation in erythroblasts. Taken together, these findings demonstrate that enucleation is not a form of cytokinesis and further suggest that survivin participates in enucleation by a novel mechanism.

Inhibition of U snRNP assembly by a virus-encoded proteinase

It has been proposed that defects in the assembly of spliceosomal uridine-rich small nuclear ribonucleoprotein (U snRNP) complexes could account for the death of motor neurons in spinal muscular atrophy (SMA). We discovered that infection of cultured cells with poliovirus results in the specific cleavage of the host factor Gemin3 by a virus-encoded proteinase, 2A(pro). Gemin3 is a component of the macromolecular SMN complex that mediates assembly of U snRNP complexes by aiding the heptameric oligomerization of Sm proteins onto U snRNAs. Using in vitro Sm core assembly assays, we found that lowering the intracellular amounts of Gemin3 by either poliovirus infection or small interfering RNA (siRNA)-mediated knockdown of Gemin3 resulted in reduced assembly of U snRNPs. Immunofluorescence analyses revealed a specific redistribution of Sm proteins from the nucleoplasm to the cytoplasmic periphery of the nucleus in poliovirus-infected cells. We propose that defects in U snRNP assembly may be shared features of SMA and poliomyelitis.

Autographa californica Multiple Nucleopolyhedrovirus Nucleocapsid Assembly Is Interrupted upon Deletion of the 38K Gene

38K (ac98) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved baculovirus gene whose function is unknown. To determine the role of 38K in the baculovirus life cycle, a 38K knockout bacmid containing the AcMNPV genome was generated through homologous recombination in Escherichia coli. Furthermore, a 38K repair bacmid was constructed by transposing the 38K open reading frame with its native promoter region into the polyhedrin locus of the 38K knockout bacmid. After transfection of these viruses into Spodoptera frugiperda cells, the 38K knockout bacmid led to a defect in production of infectious budded virus, while the 38K repair bacmid rescued this defect, allowing budded-virus titers to reach wild-type levels. Slot blot analysis indicated that 38K deletion did not affect the levels of viral DNA replication. Subsequent immunoelectron-microscopic analysis revealed that masses of electron-lucent tubular structures containing the capsid protein VP39 were present in cells transfected with 38K knockout bacmids, suggesting that nucleocapsid assembly was interrupted. In contrast, the production of normal nucleocapsids was restored when the 38K knockout bacmid was rescued with a copy of 38K. Recombinant virus that expresses 38K fused to green fluorescent protein as a visual marker was constructed to monitor protein transport and localization within the nucleus during infection. Fluorescence was first detected along the cytoplasmic periphery of the nucleus and subsequently localized to the center of the nucleus. These results demonstrate that 38K plays a role in nucleocapsid assembly and is essential for viral replication in the AcMNPV life cycle.

Intracellular localization of small heat shock protein, hsp30, in A6 kidney epithelial cells

Small heat shock proteins (shsps) are molecular chaperones that are inducible by environmental stress. In this study, immunocytochemical analysis and laser scanning confocal microscopy revealed that the shsp family, hsp30, was localized primarily in the cytoplasm of Xenopus A6 kidney epithelial cells after heat shock or sodium arsenite treatment. Heat shock-induced hsp30 was enriched in the perinuclear region with some immunostaining in the nucleus but not in the nucleolus. In sodium arsenite-treated cells hsp30 was enriched towards the cytoplasmic periphery as well as showing some immunostaining in the nucleus. At higher heat shock temperatures (35 degrees C) or after 10 microM sodium arsenite treatment, the actin cytoskeleton displayed some disorganization that co-localized with areas of hsp30 enrichment. Treatment of A6 cells with 50 microM sodium arsenite induced a collapse of the cytoskeleton around the nucleus. These results coupled with previous studies suggest that stress-inducible hsp30 acts as a molecular chaperone primarily in the cytoplasm and may interact with cytoskeletal proteins.

The ubiquitin-proteasome system is enhanced in degenerating cardiomyocytes of dilated cardiomyopathy

To maintain intracellular homeostasis against cellular stresses, proteins are not only synthesized but also degraded by proteolytic mechanisms. The ubiquitin (Ub)-proteasome system is the most important mechanism to select and digest damaged intracellular proteins. However, it is still unclear how the Ub-proteasome system plays a role in the failing heart. To elucidate the expressions of Ub and proteasomes in the myocardium of dilated cardiomyopathy (DCM), myocardial tissue obtained from nineteen DCM patients at the left ventriculoplasty was studied. Four normal autopsied hearts served as control. Immunohistochemistry revealed that both Ub and proteasomes were very weak in controls and in less degenerated cardiomyocytes of DCM hearts. On the other hand, their expressions were markedly enhanced in vesicles accumulated adjacent to nuclei, periphery of large vacuoles, hyaline materials and disorganized cytoplasm of degenerated cardiomyocytes. The quantitative analysis of percent area showed that expression of Ub and proteasome were statistically higher in DCM hearts than in normal controls. We propose that the Ub-proteasome system may be protectively enhanced to disorganize damaged proteins and reduce their toxicity. However, it is also possible that the Ub-proteasome system, when its expression is massive, may associate with cytoplasmic degeneration preceding cell death. In conclusion, proteolysis by the Ub-proteasome system is enhanced under the condition where cardiomyocyte degeneration is processing in DCM hearts.

Subcellular Localization Determines the Protective Effects of Activated ERK2 against Distinct Apoptogenic Stimuli in Myeloid Leukemia Cells

ERKs, mitogen-activated protein kinases, are well characterized as key mediators in the conveyance of signals that promote cell survival in cells of hemopoietic origin, a key factor in the upbringing of leukemogenesis. It is also well known that ERKs phosphorylate a wide array of substrates distributed throughout distinct cellular locations such as the nucleus, cytoplasm, and cell periphery, but the relative contribution of these compartmentalized signal components to the overall survival signal generated by activation of ERKs has yet to be established. To this end, we have utilized constitutively activated forms of ERK2, whose expression is restricted to the nucleus or to the cytoplasm, to investigate the consequences of compartmentalized activation of ERK in the survival of chronic myelogenous leukemia cells subjected to distinct apoptogenic stimuli. We show that cytoplasmic ERK2 activity protected against apoptosis caused by prolonged serum starvation, whereas ERK2 activation restricted to the nucleus antagonized apoptosis induced by the Bcr-Abl inhibitor STI571. On the other hand, neither cytoplasmic nor nuclear ERK2 activities were effective in counteracting apoptosis induced by UV light. These results demonstrate that the protective effects of ERK2 against defined apoptogenic stimuli are strictly dependent on the cellular localization where ERK activation takes place. Furthermore, we present evidence suggesting that the complex I kappa B-NF kappa B participates on ERK2-mediated survival mechanisms, in a fashion dependent on the cellular location where ERK2 is active and on the causative apoptogenic stimulus.

Requirement for Bbp1p in the Proper Mitotic Functions of Cdc5p inSaccharomyces cerevisiae

The polo-box domain of the budding yeast polo kinase Cdc5p plays an essential role for targeting the catalytic activity of Cdc5p to spindle pole bodies (SPBs) and cytokinetic neck-filaments. Here, we report the isolation of Bbp1p as a polo-box interacting protein by a yeast two-hybrid screen. Bbp1p localizes to the periphery of the central plaque of the SPB and plays an important role in SPB duplication. Similarly, Cdc5p localized to the cytoplasmic periphery of the SPB. In vitro binding studies showed that Cdc5p interacted with the N-terminal domain of Bbp1p (Bbp1pDeltaC), but apparently not with Mps2p, a component shown to form a stable complex with Bbp1p. In addition, Bbp1p, but likely not Mps2p, was required for proper localization of Cdc5p to the SPB. The C-terminal coiled-coil domain of Bbp1p (Bbp1p(243-385)), which is crucial for both the homodimerization and the SPB localization, could target the localization-defective Cdc5pDeltaC to the SPB and induce the release of Cdc14p from the nucleolus. Consistent with this observation, expression of CDC5DeltaC-BBP1(243-385) under CDC5 promoter control partially complemented the cdc5Delta defect. These data suggest that Bbp1pDeltaC interacts with the polo-box domain of Cdc5p, and this interaction is critical for the subcellular localization and mitotic functions of Cdc5p.

Incorporation of mammalian actin into microfilaments in plant cell nucleus

BackgroundActin is an ancient molecule that shows more than 90% amino acid homology between mammalian and plant actins. The regions of the actin molecule that are involved in F-actin assembly are largely conserved, and it is likely that mammalian actin is able to incorporate into microfilaments in plant cells but there is no experimental evidence until now.ResultsVisualization of microfilaments in onion bulb scale epidermis cells by different techniques revealed that rhodamine-phalloidin stained F-actin besides cytoplasm also in the nuclei whereas GFP-mouse talin hybrid protein did not enter the nuclei. Microinjection of fluorescently labeled actin was applied to study the presence of nuclear microfilaments in plant cells. Ratio imaging of injected fluorescent rabbit skeletal muscle actin and phalloidin staining of the microinjected cells showed that mammalian actin was able to incorporate into plant F-actin. The incorporation occurred preferentially in the nucleus and in the perinuclear region of plant cells whereas part of plant microfilaments, mostly in the periphery of cytoplasm, did not incorporate mammalian actin.ConclusionsMicroinjected mammalian actin is able to enter plant cell's nucleus, whereas incorporation of mammalian actin into plant F-actin occurs preferentially in the nucleus and perinuclear area.

Cathepsin L plays an active role in involution of the mouse mammary gland.

Involution of the mammary gland after weaning occurs in two stages. The first stage is reversible, whereas the second stage is characterized by the irreversible collapse of the alveolar structure. A differential display analysis using cDNAs from tissues obtained at various times after forced weaning of pups identified cathepsin L as up-regulated during early involution. Levels of cathepsin L mRNA were dramatically increased within 24 hr after weaning. Cathepsin L protein detected by immunoblot was also increased during involution, reaching near maximal levels by 36 hr after weaning. In situ immunohistochemistry detected pronounced cathepsin L protein in the cytoplasm and cell periphery. Mice treated with a specific inhibitor of cathepsin L exhibited substantially reduced numbers of apoptotic cells at times up to 72 hr after weaning when compared with untreated animals. The cathepsin L inhibitor did not alter levels of cathepsin L detected in immunoblots or influence molecular weight of the cathepsin L species detected. These data suggest that cathepsin L plays a regulatory role early in the process of mammary gland involution.

In vitro analysis of nuclear transport mediated by the C-terminal shuttle domain of Tap.

The Tap protein of higher eukaryotes is implicated in the nuclear export of type D retroviral mRNA and some cellular mRNAs. Here we have developed an in vitro assay to study nuclear export mediated by the C-terminal shuttle domain of Tap involving the rapamycin-induced attachment of this transport domain to a nuclear green fluorescent protein-containing reporter. We found that export by the Tap transport domain does not involve cytosolic transport factors including the GTPase Ran. The transport domain directly binds to several nucleoporins positioned in different regions of the nuclear pore complex. These results argue that a direct interaction of the Tap transport domain with nucleoporins is responsible for its nucleocytoplasmic translocation. We found that the karyopherin beta-related export receptor CRM1 competes with the Tap transport domain for binding to Nup214 but not for binding to Nup62 or Nup153, suggesting that the Tap and CRM1 nuclear export pathways converge at the cytoplasmic periphery of the nuclear pore complex. Because the rates of in vitro nuclear import and export by the Tap transport domain are very similar, the directionality of mRNA export mediated by Tap probably is determined by mechanisms other than simple binding of the Tap transport domain to nucleoporins.

A role for RanBP1 in the release of CRM1 from the nuclear pore complex in a terminal step of nuclear export.

We recently developed an assay in which nuclear export of the shuttling transcription factor NFAT (nuclear factor of activated T cells) can be reconstituted in permeabilized cells with the GTPase Ran and the nuclear export receptor CRM1. We have now used this assay to identify another export factor. After preincubation of permeabilized cells with a Ran mutant that cannot hydrolyze GTP (RanQ69L), cytosol supports NFAT export, but CRM1 and Ran alone do not. The RanQ69L preincubation leads to accumulation of CRM1 at the cytoplasmic periphery of the nuclear pore complex (NPC) in association with the p62 complex and Can/Nup214. RanGTP-dependent association of CRM1 with these nucleoporins was reconstituted in vitro. By biochemical fractionation and reconstitution, we showed that RanBP1 restores nuclear export after the RanQ69L preincubation. It also stimulates nuclear export in cells that have not been preincubated with RanQ69L. RanBP1 as well as Ran-binding domains of the cytoplasmic nucleoporin RanBP2 promote the release of CRM1 from the NPC. Taken together, our results indicate that RanGTP is important for the targeting of export complexes to the cytoplasmic side of the NPC and that RanBP1 and probably RanBP2 are involved in the dissociation of nuclear export complexes from the NPC in a terminal step of transport.

Cytoskeletal proteins connecting intermediate filaments to cytoplasmic and nuclear periphery.

Intermediate filaments (IFs), together with microtubules and microfilaments build up the cytoskeleton of most eukaryotic cells. Cytoplasmic IFs form a dense filament network radiating from the nucleus and extending to the plasma membrane. The association between the cytoplasmic and nuclear surfaces appears to provide a continuous link important for the organisation of the cytoplasm, for cellular communication, and possibly for the transport into and out of the nucleus. Cytoplasmic IFs approach the nuclear surface, thin fibrils seem to connect the IFs with the nuclear pore complexes and a direct interaction of cytoplasmic IFs with the nuclear lamin B has been observed by in vitro binding studies. However, none of the components that cross-link IFs to the nucleus has been unambiguously identified. Furthermore, if a direct interaction between cytoplasmic IFs and the nuclear lamin B occurs in vivo, the question of how cytoplasmic IFs get access to the nuclear interior remains to be resolved. The association of IFs with the plasma membranes involves different components, some of which are cell type specific. Two specialised complexes in epithelial cells: the desmosome and the hemidesmosome, serve as attachment sites for keratin filaments. Desmoplakin is considered as the cross-linking component of IFs to the desmosomal plaque, whereas BPAG1 (bullous pemphigoid antigen) would cross-link IFs at the hemidesmosomal plaque. In other cell types the modality of how IFs are anchored to the plasma membrane is less well understood. It involves different components such as the spectrin based membrane skeleton, ankyrin, myosin, plectin and certainly many other still unravelled partners. Association between the IFs and cellular membranes plays an important role in determining cell shape and tissue integrity. Thus, the identification and characterisation of the components involved in these interactions will be crucial for understanding the function of intermediate filaments.

Human ubiquitin-protein ligase Nedd4: expression, subcellular localization and selective interaction with ubiquitin-conjugating enzymes.

Nedd4 is a ubiquitin-protein ligase containing a calcium/lipid-binding domain, multiple WW domains and a C-terminal Hect domain, which is required for both the ubiquitin transfer and the association with E2 ubiquitin-conjugating enzymes. Nedd4 has been reported to be involved in the selective ubiquitination of some regulatory proteins in transcription and membrane transport. Three mRNA species for human Nedd4 were found to be 6.4-, 7.8- and 9.5-kb in size, and their expression patterns varied among normal tissues and cancer cell lines, indicating the tissue- and cell-specificities of Nedd4 expression. The Nedd4 protein, approximately 120 kDa in weight, was found in the cytoplasm, mainly in the perinuclear region and cytoplasmic periphery, of human cultured cells. Neural differentiation induced not only the down-regulation of Nedd4 but also the localization of the protein to both the cytoplasm and neurites. To identify the ubiquitination pathway that is linked to Nedd4, we demonstrated that specific E2 enzymes, including human Ubc4, UbcH5B, UbcH5C, UbcH6 and UbcH7, could transfer ubiquitin molecules to Nedd4 at the active cysteine residue, whereas E6AP accepted ubiquitins from Ubc4, UbcH5B, UbcH5C and UbcH7. Furthermore, nuclear localization of N-terminal deletion mutant Nedd4 enabled us to investigate the interaction between Nedd4 and E2 enzyme (Ubc4 or UbcH7) in the cell. The simultaneous expression of the full-length Nedd4 and E2 enzyme revealed the both proteins mostly colocalized in the cytoplasmic periphery, while the N-terminal deleted Nedd4 induced the nuclear and perinuclear colocalization with E2 enzyme. Our findings suggested that Nedd4 plays an important role in the cell regulation, including neural differentiation through cooperation with specific E2 ubiquitination pathways.

Nuclear Pore Transport: Insight in Situ

Abstract The nuclear envelope (NE) physically separates the genetic machinery residing in the nucleus from protein synthesis taking place in the cytoplasm. Bidirectional transport of proteins, RNAs and RNP particles between these two compartments is mediated by the nuclear pore complexes (NPCs), large (-120 MDa) supramolecular assemblies embedded in the NE and being built of -100 different polypeptides, called nucleoporins. Extensive structural studies have revealed the 3D architecture of NPCs, and epitopes of several nucleoporins have been localized within their 3D structure. In an attempt to go beyond the current consensus model of the NPC (Fig. lc, inset), we have embarked on a more systematic structural analysis of the molecular architecture of native NPCs. This is achieved by field-emission (FETEM; Fig. la) or energy-filtering (EFTEM; Fig. lb) TEM of completely unfixed and unstained Xenopus oocyte NEs embedded in thick (i.e. -200 nm) amorphous ice so that the 3D organization of the cytoplasmic and nuclear periphery of the NPCs (i.e. the cytoplasmic fibrils and nuclear baskets) is fully preserved (Fig. lb).

Changes in H+-translocating vacuolar-type ATPase in the anterior silk gland cell of Bombyx mori during metamorphosis.

A proton-translocating vacuolar-type ATPase (V-ATPase) was identified and characterized in the anterior silk gland of Bombyx mori. By incubating the intact tissue with the fluorescent dye Acridine Orange, the acidified compartment was detected at the apical pole of the epithelial cells. This was observed throughout the feeding period of the fifth-instar larva until the onset of spinning. Acidification was prevented completely and reversibly by 0.8 micromol l-1 bafilomycin A1, a specific inhibitor of V-ATPase. The presence of V-ATPase in a microsomal fraction was verified by immunoblots using an antiserum to the V-ATPase holoenzyme from Manduca sexta midgut. The antiserum localized the V-ATPase to the apical plasma membrane of the anterior silk gland cells, suggesting that the enzyme is functionally active in pumping protons out of the cell towards the glandular lumen of feeding silkworm larvae. In spinning larvae, the acidification produced by the V-ATPase appears to cease, because acidic compartments were seen rarely and only in the periphery of basal cytoplasm, and because immunocytochemical staining for the V-ATPase was greatly reduced at the apical surface. The metamorphic changes in relation to the occurrence of V-ATPase corresponded well with the ultrastructural changes in the anterior silk gland cell of Bombyx mori larvae.

RanGTP targets p97 to RanBP2, a filamentous protein localized at the cytoplasmic periphery of the nuclear pore complex.

RanBP2, a protein containing FG repeat motifs and four binding sites for the guanosine triphosphatase Ran, is localized at the cytoplasmic periphery of the nuclear pore complex (NPC) and is believed to play a critical role in nuclear protein import. We purified RanBP2 from rat liver nuclear envelopes and examined its structural and biochemical properties. Electron microscopy showed that RanBP2 forms a flexible filamentous molecule with a length of approximately 36 nm, suggesting that it comprises a major portion of the cytoplasmic fibrils implicated in initial binding of import substrates to the NPC. Using in vitro assays, we characterized the ability of RanBP2 to bind p97, a cytosolic factor implicated in the association of the nuclear localization signal receptor with the NPC. We found that RanGTP promotes the binding of p97 to RanBP2, whereas it inhibits the binding of p97 to other FG repeat nucleoporins. These data suggest that RanGTP acts to specifically target p97 to RanBP2, where p97 may support the binding of an nuclear localization signal receptor/substrate complex to RanBP2 in an early step of nuclear import.

N-terminal sequences from Autographa californica nuclear polyhedrosis virus envelope proteins ODV-E66 and ODV-E25 are sufficient to direct reporter proteins to the nuclear envelope, intranuclear microvesicles and the envelope of occlusion derived virus.

Baculovirus occlusion-derived virus (ODV) derives its envelope from an intranuclear membrane source. N-terminal amino acid sequences of the Autographa californica nuclear polyhedrosis virus (AcMNPV) envelope proteins, ODV-E66 and ODV-E25 (23 and 24 amino acids, respectively) are highly hydrophobic. Recombinant viruses that express the two N-terminal amino acid sequences fused to green fluorescent protein (23GFP or 24GFP) provided visual markers to follow protein transport and localization within the nucleus during infection. Autoflourescence was first detected along the cytoplasmic periphery of the nucleus and subsequently localized as foci to discrete locations within the nucleus. Immunoelectron microscopy confirmed that these foci predominantly contained intranuclear microvesicles and the reporter fusion proteins were also detected in cytoplasmic membranes near the nucleus, and the outer and inner nuclear membrane. Therefore, these defined hydrophobic domains are sufficient to direct native and fusion proteins to induced membrane microvesicles within a baculovirus-infected cell nucleus and the viral envelope. In addition, these data suggest that movement of these proteins into the nuclear envelope may initiate through cytoplasmic membranes, such as endoplasmic reticulum, and that transport into the nucleus may be mediated through the outer and inner nuclear membrane.

Process of dispersion and fragmentation of Golgi complex by microtubule bundles formed in taxol treated HeLa cells.

By means of a monoclonal antibody (mAbG3A5) against Golgi membrane glycoprotein, we have visualized the relative position of cytoplasmic polymerized microtubule bundles to Golgi stack cisternae in taxol treated HeLa cells, and found extensive fragmentation of the Golgi stack cisternae brought about by microtubule bundles. Within a 1 h period of taxol treatment, polymerization of cytoplasmic microtubules increased rapidly to form microtubule bundles, while the Golgi complex dispersed slightly along with the polymerized microtubule bundles. After 2 to 3 h taxol treatment the dispersal of the Golgi complex from the microtubule-organizing center (MTOC) to cytoplasmic periphery rapidly progressed in the direction which the microtubules ran. At early dispersal, the microtubule bundles were oriented apart from the stretched end of the Golgi stack cisternae and exhibited little direct contact with the Golgi stack cisternae membrane. The Golgi stack cisternae then began to wind around the microtubule bundles, followed by the beginning of fragmentation of the Golgi stack cisternae. At this step, some of the microtubules seemed to attach to a part of the Golgi stack cisternae. After prolonged exposure of cells to taxol (25 h) the microtubule bundles were highly developed throughout the cells and most of the Golgi fragments were trapped at their termini. In these cells, extensive fragmentation of Golgi stack cisternae occurred, resulting in small Golgi vesicles bound to the microtubule bundle.