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Journal of Virology Aug 2018Nuclear egress of herpesvirus capsids is mediated by the conserved nuclear egress complex (NEC), composed of the membrane-anchored pUL34 and its nucleoplasmic...
Nuclear egress of herpesvirus capsids is mediated by the conserved nuclear egress complex (NEC), composed of the membrane-anchored pUL34 and its nucleoplasmic interaction partner, pUL31. The recently solved crystal structures of the NECs from different herpesviruses show a high structural similarity, with the pUL34 homologs building a platform recruiting pUL31 to the inner nuclear membrane. Both proteins possess a central globular fold, while the conserved N-terminal portion of pUL31 forms an extension reaching around the core of pUL34. However, the extreme N terminus of the pUL31 homologs, which is highly variable in length and amino acid composition, had to be removed for crystallization. Several pUL31 homologs contain a classical nuclear localization signal (NLS) within this part mediating efficient nuclear import. In addition, membrane-binding activity, blocking premature interaction with pUL34, nucleocapsid trafficking, and regulation of NEC assembly and disassembly via phosphorylation were assigned to the extreme pUL31 N terminus. To test the functional importance in the alphaherpesvirus pseudorabies virus (PrV) pUL31, N-terminal truncations and site-specific mutations were generated, and the resulting proteins were tested for intracellular localization, interaction with pUL34, and functional complementation of PrV-ΔUL31. Our data show that neither the bipartite NLS nor the predicted phosphorylation sites are essential for pUL31 function during nuclear egress. Moreover, nearly the complete variable N-terminal part was dispensable for function as long as a stretch of basic amino acids was retained. Phosphorylation of this domain controls efficient nucleocapsid release from the perinuclear space. Nuclear egress of herpesvirus capsids is a unique vesicle-mediated nucleocytoplasmic transport. Crystal structures of the heterodimeric NECs from different herpesviruses provided important details of this viral nuclear membrane deformation and scission machinery but excluded the highly variable N terminus of the pUL31 component. We present here a detailed mutagenesis study of this important portion of pUL31 and show that basic amino acid residues within this domain play an essential role for proper targeting, complex formation, and function during nuclear egress, while phosphorylation modulates efficient release from the perinuclear space. Thus, our data complement previous structure-function assignments of the nucleocapsid-interacting component of the NEC.
Topics: Animals; Cell Line; Crystallography, X-Ray; Herpesvirus 1, Suid; Humans; Phosphorylation; Protein Domains; Viral Proteins; Virus Release
PubMed: 29793954
DOI: 10.1128/JVI.00566-18 -
PloS One 2012Interferons (IFNs) play a crucial role in the antiviral immune response. Whereas the C proteins of wild-type human parainfluenza virus type 1 (WT HPIV1) inhibit both...
Interferons (IFNs) play a crucial role in the antiviral immune response. Whereas the C proteins of wild-type human parainfluenza virus type 1 (WT HPIV1) inhibit both IFN-β induction and signaling, a HPIV1 mutant encoding a single amino acid substitution (F170S) in the C proteins is unable to block either host response. Here, signaling downstream of the type 1 IFN receptor was examined in Vero cells to define at what stage WT HPIV1 can block, and F170S HPIV1 fails to block, IFN signaling. WT HPIV1 inhibited phosphorylation of both Stat1 and Stat2, and this inhibition was only slightly reduced for F170S HPIV1. Degradation of Stat1 or Stat2 was not observed. The HPIV1 C proteins were found to accumulate in the perinuclear space, often forming large granules, and co-localized with Stat1 and the cation-independent mannose 6-phosphate receptor (M6PR) that is a marker for late endosomes. Upon stimulation with IFN-β, both the WT and F170S C proteins remained in the perinuclear space, but only the WT C proteins prevented Stat1 translocation to the nucleus. In addition, WT HPIV1 C proteins, but not F170S C proteins, co-immunoprecipitated both phosphorylated and unphosphorylated Stat1. Our findings suggest that the WT HPIV1 C proteins form a stable complex with Stat1 in perinuclear granules that co-localize with M6PR, and that this direct interaction between the WT HPIV1 C proteins and Stat1 is the basis for the ability of HPIV1 to inhibit IFN signaling. The F170S mutation in HPIV1 C did not prevent perinuclear co-localization with Stat1, but apparently weakened this interaction such that, upon IFN stimulation, Stat1 was translocated to the nucleus to induce an antiviral response.
Topics: Animals; Blotting, Western; Cell Nucleus; Chlorocebus aethiops; Endosomes; Humans; Immunoprecipitation; Interferons; Phosphorylation; Protein Transport; STAT1 Transcription Factor; STAT2 Transcription Factor; Signal Transduction; Vero Cells; Viral Proteins; Virus Replication
PubMed: 22355301
DOI: 10.1371/journal.pone.0028382 -
Journal of Molecular and Cellular... Nov 2016Phospholamban (PLB) regulates the cardiac Ca-ATPase (SERCA2a) in sarcoplasmic reticulum (SR). However, the localization of PLB at subcellular sites outside the SR and...
AIMS
Phospholamban (PLB) regulates the cardiac Ca-ATPase (SERCA2a) in sarcoplasmic reticulum (SR). However, the localization of PLB at subcellular sites outside the SR and possible contributions to Ca cycling remain unknown. We examined the intracellular distribution of PLB and tested whether a pool of PLB exists in the nuclear envelope (NE) that might regulate perinuclear/nuclear Ca (nCa) handling in cardiomyocytes (CMs).
METHODS AND RESULTS
Using confocal immunofluorescence microscopy and immunoblot analyses of CMs and CM nuclei, we discovered that PLB was highly concentrated in NE. Moreover, the ratio of PLB levels to SERCA levels was greater in NE than in SR. The increased levels of PLB in NE were a consistent finding using a range of antibodies, tissue samples, and species. To address a possible role in affecting Ca handling, we used Fluo-4 based confocal Ca imaging, with scan-lines across cytosol and nuclei, and evaluated the effects of PLB on cytosolic and nCa uptake and release in mouse CMs. In intact CMs, isoproterenol increased amplitude and decreased the decay time of Ca transients not only in cytosol but also in nuclear regions. In saponin-permeabilized mouse CMs ([Ca]=400nM), we measured spontaneous Ca waves after specific reversal of PLB activity by addition of the Fab fragment of an anti-PLB monoclonal antibody (100μg/ml). This highly selective immunological reagent enhanced Ca uptake (faster decay times) and Ca release (greater intensity) in both cytosol and across the nuclear regions.
CONCLUSIONS
Besides SR, PLB is concentrated in NE of CMs, and may be involved in modulation of nCa dynamics.
Topics: Animals; Biological Transport; Calcium; Calcium Signaling; Calcium-Binding Proteins; Cell Nucleolus; Humans; Intracellular Space; Isoproterenol; Mice; Microscopy, Fluorescence; Molecular Imaging; Myocytes, Cardiac; Nuclear Envelope; Rabbits; Species Specificity
PubMed: 27642167
DOI: 10.1016/j.yjmcc.2016.09.008 -
Cell Jun 2016Through a network of progressively maturing vesicles, the endosomal system connects the cell's interior with extracellular space. Intriguingly, this network exhibits a...
Through a network of progressively maturing vesicles, the endosomal system connects the cell's interior with extracellular space. Intriguingly, this network exhibits a bilateral architecture, comprised of a relatively immobile perinuclear vesicle "cloud" and a highly dynamic peripheral contingent. How this spatiotemporal organization is achieved and what function(s) it curates is unclear. Here, we reveal the endoplasmic reticulum (ER)-located ubiquitin ligase Ring finger protein 26 (RNF26) as the global architect of the entire endosomal system, including the trans-Golgi network (TGN). To specify perinuclear vesicle coordinates, catalytically competent RNF26 recruits and ubiquitinates the scaffold p62/sequestosome 1 (p62/SQSTM1), in turn attracting ubiquitin-binding domains (UBDs) of various vesicle adaptors. Consequently, RNF26 restrains fast transport of diverse vesicles through a common molecular mechanism operating at the ER membrane, until the deubiquitinating enzyme USP15 opposes RNF26 activity to allow vesicle release into the cell's periphery. By drawing the endosomal system's architecture, RNF26 orchestrates endosomal maturation and trafficking of cargoes, including signaling receptors, in space and time.
Topics: Cell Line, Tumor; Dendritic Cells; Endoplasmic Reticulum; Endosomes; Humans; Intracellular Membranes; Macrophages; Neoplasm Proteins; Sequestosome-1 Protein; Transport Vesicles; Ubiquitin-Specific Proteases
PubMed: 27368102
DOI: 10.1016/j.cell.2016.05.078 -
Communicative & Integrative Biology Sep 2009The biosynthetic pathway allows the transport of newly synthesized proteins to the TGN via the reticulum endoplasmic and Golgi apparatus. However, many large particles...
The biosynthetic pathway allows the transport of newly synthesized proteins to the TGN via the reticulum endoplasmic and Golgi apparatus. However, many large particles reach the TGN by unconventional means. For instance, Herpes simplex virus type 1(HSV-1) capsids assemble within the nucleus, bud into the perinuclear space, fuse with the outer nuclear membrane and finally travel unenveloped towards the TGN. Given the central role of protein kinase D in the transport of small cargo from the TGN to the cell surface, we probed its potential contribution in HSV-1 egress, as a model for studying large cargo exiting from the TGN. Using a synchronized infection, we show that inactivation of protein kinase D with pharmacological inhibitors, a kinase dead mutant or siRNA all causes the retention of HSV-1 at the TGN. This highlights the role of PKD in viral exit and a dependence of the virus on the classical host cell machinery to leave the TGN, unlike its previous transport steps. Conceptually, this supports a model in which the TGN is a meeting point where conventional and unconventional routes encounter.
PubMed: 19907711
DOI: 10.4161/cib.2.5.9217 -
The Journal of Cell Biology Sep 1987The nuclei of bovine spermatids and spermatozoa are surrounded by dense cytoplasmic webs sandwiched between the nuclear envelope and the acrosome and plasma membrane,...
The nuclei of bovine spermatids and spermatozoa are surrounded by dense cytoplasmic webs sandwiched between the nuclear envelope and the acrosome and plasma membrane, respectively, filling most of the cytoplasmic space of the sperm head. This web contains a complex structure, the perinuclear theca, which is characterized by resistance to extractions in nondenaturing detergents and high salt buffers, and can be divided into two major subcomponents, the subacrosomal layer and the postacrosomal calyx. Using calyces isolated from bull and rat spermatozoa we have identified two kinds of basic proteins as major constituents of the thecal structure and have localized them by specific antibodies at the light and electron microscopic level. These are an Mr 60,000 protein, termed calicin, localized almost exclusively to the calyx, and a group of multiple-band polypeptides (MBP; Mr 56,000-74,000), which occur in both the calyx and the subacrosomal layer. The polypeptides of the MBP group are immunologically related to each other, but unrelated, by antibody reactions and peptide maps, to calicin. We show that these basic cytoskeletal proteins are first detectable in the round spermatid stage. As we have not detected any intermediate filament proteins and proteins related to nuclear lamins of somatic cells in sperm heads, we conclude that the perinuclear theca and its constituents, calicin and MBP proteins, are the predominant cytoskeletal elements of the sperm head. Immunologically cross-reacting polypeptides with similar properties have been identified in the heads of rat and human spermatozoa. We speculate that these insoluble basic proteins contribute, during spermiogenesis, to the formation of the perinuclear theca as an architectural element involved in the shape changes and the intimate association of the nucleus with the acrosome and the plasma membrane.
Topics: Animals; Antibodies; Cattle; Cytoskeletal Proteins; Fluorescent Antibody Technique; Male; Microscopy, Electron; Molecular Weight; Nuclear Proteins; Rats; Rats, Inbred Strains; Spermatids; Spermatozoa
PubMed: 3308904
DOI: 10.1083/jcb.105.3.1105 -
Biophysics of Structure and Mechanism 1982In innervated and denervated sternohyoid muscles of adult mice the AChE with a pH optimum at 7.2 was shown to occur in all three fiber types in two separate structural...
In innervated and denervated sternohyoid muscles of adult mice the AChE with a pH optimum at 7.2 was shown to occur in all three fiber types in two separate structural areas located: extrafibrillarly (synaptic cleft, postsynaptic folds, subsarcolemmal vesicles, T-tubules, interfibrillar space) and intrafibrillarly (perinuclear cisternae, SR including SR cisternae). There is not a stable connection between the two areas. The functional significance of the intrafibrillar AChE, in particular, is unknown. After muscle denervation, intrafibrillar AChE of the A and B fibers disappears more quickly than that of C fibers. This phenomenon not only suggests a general, but possibly also a fiber-specific neurotrophic effect.
Topics: Acetylcholinesterase; Animals; Histocytochemistry; Male; Mice; Mice, Inbred Strains; Microscopy, Electron; Motor Endplate; Muscle Denervation; Neuromuscular Junction
PubMed: 7150700
DOI: 10.1007/BF00539110 -
Methods in Molecular Biology (Clifton,... 2017Eicosanoids are bioactive lipids derived from enzymatic metabolism of arachidonic acid via the cyclooxygenase (COX) and lipoxygenase (LOX) pathways. These lipids are...
Eicosanoids are bioactive lipids derived from enzymatic metabolism of arachidonic acid via the cyclooxygenase (COX) and lipoxygenase (LOX) pathways. These lipids are newly formed and nonstorable molecules that have important roles in physiological and pathological processes. The particular interest to determine intracellular compartmentalization of eicosanoid-synthetic machinery has emerged as a key component in the regulation of eicosanoid synthesis and in delineating functional intracellular and extracellular actions of eicosanoids. In this chapter, we discuss the EicosaCell protocol, an assay that enables the intracellular detection and localization of eicosanoid lipid mediator-synthesizing compartments by means of a strategy to covalently cross-link and immobilize eicosanoids at their sites of synthesis followed by immunofluorescent-based localization of the targeted eicosanoid. EicosaCell assays have been successfully used to identify different intracellular compartments of synthesis of prostaglandins and leukotrienes upon cellular activation. This chapter covers basics of EicosaCell assay including its selection of reagents, immunodetection design as well as some troubleshooting recommendations.
Topics: Animals; Biological Assay; Eicosanoids; Fluorescent Antibody Technique; Image Processing, Computer-Assisted; Intracellular Space; Lipid Metabolism; Mice; Microscopy, Fluorescence; Molecular Imaging; Optical Imaging; Phagosomes; Software; Staining and Labeling
PubMed: 28185186
DOI: 10.1007/978-1-4939-6759-9_6 -
Micron (Oxford, England : 1993) Sep 2022The ovaries of Sander lucioperca (Actinopterygii, Perciformes) are made up of the germinal epithelium and ovarian follicles, in which primary oocytes grow. Each follicle...
The ovaries of Sander lucioperca (Actinopterygii, Perciformes) are made up of the germinal epithelium and ovarian follicles, in which primary oocytes grow. Each follicle is composed of an oocyte surrounded by flattened follicular cells, the basal lamina, and thecal cells. The early stages of oocyte development (primary growth = previtellogenesis) are not fully explained in this species. The results of research with the use of stereoscopic, light, fluorescence, and transmission electron microscopes on ovarian follicles containing developing primary oocytes of S. lucioperca are presented. The polarization and ultrastructure of oocytes are described and discussed. The deposition of egg envelopes during the primary growth and the ultrastructure of the eggshell in maturing follicles of S. lucioperca are also presented. Nuclei in primary oocytes comprise lampbrush chromosomes, nuclear bodies, and nucleoli. Numerous additional nucleoli arise in the nucleoplasm during primary growth and locate close to the nuclear envelope. The Balbiani body in the cytoplasm of oocytes (ooplasm) is composed of nuage aggregations of nuclear origin and mitochondria, endoplasmic reticulum (ER), and Golgi apparatus. The presence of the Balbiani body was reported in oocytes of numerous species of Actinopterygii; however, its ultrastructure was investigated in a limited number of species. In primary oocytes of S. lucioperca, the Balbiani body is initially located in the perinuclear ooplasm on one side of the nucleus. Next, it surrounds the nucleus, expands toward the plasma membrane of oocytes (oolemma), and becomes fragmented. Within the Balbiani body, the granular nuage condenses in the form of threads, locates near the oolemma, at the vegetal oocyte pole, and then dissolves. Mitochondria and cisternae of the rough endoplasmic reticulum (RER) are present between the threads. During primary growth micropylar cells differentiate in the follicular epithelium. They contain cisternae and vesicles of the RER and Golgi apparatus as well as numerous dense vesicles suggesting high synthetic and secretory activity. During the final step of primary growth several follicular cells delaminate from the follicular epithelium, migrate toward the oocyte and submerge in the most external egg envelope. In the ooplasm, three regions are distinguished: perinuclear, endoplasm, and periplasm. Cortical alveoli arise in the perinuclear ooplasm and in the endoplasm as a result of the fusion of RER vesicles with Golgi ones. They are evenly distributed. Lamellar bodies in the periplasm store the plasma membrane and release it into a space between the follicular cells and the oocyte. The developing eggshell in this space is made up of two egg envelopes (the internal one and the external) that are pierced by canals formed around the microvilli of oocytes and the processes of follicular cells. In the deposition of egg envelopes the oocyte itself and follicular cells are engaged. In maturing ovarian follicles the eggshell is solid and the internal egg envelope is covered with protuberances.
Topics: Animals; Cell Nucleus; Female; Oocytes; Ovarian Follicle; Perches; Perciformes
PubMed: 35759902
DOI: 10.1016/j.micron.2022.103318 -
The Journal of Biophysical and... May 1955An electron microscope study of thin sections of interphase cells has revealed the following:- Circular pores are formed in the double nuclear envelope by continuities...
An electron microscope study of thin sections of interphase cells has revealed the following:- Circular pores are formed in the double nuclear envelope by continuities between the inner and outer membranes which permit contact between the nucleoplasm and the cytoplasm unmediated by a well defined membrane. The pores, seen in sections normal to the nuclear envelope, are profiles of the ring-shaped structures described by others and seen in tangential section. The inner and outer nuclear membranes are continuous with one another and enclose the perinuclear space. The pores contain a diffuse, faintly particulate material. A survey of cells of the rat derived from the embryonic ectoderm, mesoderm, and endoderm, and of a protozoan and an alga has revealed pores in all tissues examined, without exception. It is concluded that pores in the nuclear envelope are a fundamental feature of all resting cells. In certain cells, the outer nuclear membrane is continuous with membranes of the endoplasmic reticulum, hence the perinuclear space is continuous with cavities enclosed by those membranes. There are indications that this is true for all resting cells, at least in a transitory way. On the basis of these observations, the hypothesis is made that two pathways of exchange exist between the nucleus and the cytoplasm; by way of the perinuclear space and cavities of the endoplasmic reticulum and by way of the pores in the nuclear envelope.
Topics: Animals; Cell Nucleus; Cytoplasm; Endoplasmic Reticulum; Invertebrates; Microscopy, Electron; Nuclear Envelope; Plants; Rats
PubMed: 13242591
DOI: 10.1083/jcb.1.3.257