scholarly journals The SUN protein UNC-84 is required only in force-bearing cells to maintain nuclear envelope architecture

2014 ◽  
Vol 206 (2) ◽  
pp. 163-172 ◽  
Author(s):  
Natalie E. Cain ◽  
Erin C. Tapley ◽  
Kent L. McDonald ◽  
Benjamin M. Cain ◽  
Daniel A. Starr
Keyword(s):  
Nuclear Envelope ◽  
Hela Cells ◽  
Mechanical Strain ◽  
Mechanical Forces ◽  
The Sun ◽  
Observable Effect ◽  
Nuclear Membranes ◽  
Large Deletions ◽  
Perinuclear Space ◽  
In Cells

The nuclear envelope (NE) consists of two evenly spaced bilayers, the inner and outer nuclear membranes. The Sad1p and UNC-84 (SUN) proteins and Klarsicht, ANC-1, and Syne homology (KASH) proteins that interact to form LINC (linker of nucleoskeleton and cytoskeleton) complexes connecting the nucleoskeleton to the cytoskeleton have been implicated in maintaining NE spacing. Surprisingly, the NE morphology of most Caenorhabditis elegans nuclei was normal in the absence of functional SUN proteins. Distortions of the perinuclear space observed in unc-84 mutant muscle nuclei resembled those previously observed in HeLa cells, suggesting that SUN proteins are required to maintain NE architecture in cells under high mechanical strain. The UNC-84 protein with large deletions in its luminal domain was able to form functional NE bridges but had no observable effect on NE architecture. Therefore, SUN-KASH bridges are only required to maintain NE spacing in cells subjected to increased mechanical forces. Furthermore, SUN proteins do not dictate the width of the NE.

scholarly journals Endoplasmic reticulum-to-Golgi transitions upon herpes virus infection

F1000Research ◽  
2018 ◽  
Vol 6 ◽  
pp. 1804 ◽  
Author(s):  
Peter Wild ◽  
Andres Kaech ◽  
Elisabeth M. Schraner ◽  
Ladina Walser ◽  
Mathias Ackermann
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Golgi Complex ◽  
Nuclear Membrane ◽  
Progeny Virus ◽  
Cytoplasmic Matrix ◽  
Outer Nuclear Membrane ◽  
Nuclear Membranes ◽  
Perinuclear Space ◽  
Hsv 1

Background: Herpesvirus capsids are assembled in the nucleus, translocated to the perinuclear space by budding, acquiring tegument and envelope, or released to the cytoplasm via impaired nuclear envelope. One model proposes that envelopment, “de-envelopment” and “re-envelopment” is essential for production of infectious virus. Glycoproteins gB/gH were reported to be essential for de-envelopment, by fusion of the “primary” envelope with the outer nuclear membrane. Yet, a high proportion of enveloped virions generated from genomes with deleted gB/gH were found in the cytoplasm and extracellular space, suggesting the existence of alternative exit routes.Methods: We investigated the relatedness between the nuclear envelope and membranes of the endoplasmic reticulum and Golgi complex, in cells infected with either herpes simplex virus 1 (HSV-1) or a Us3 deletion mutant thereof, or with bovine herpesvirus 1 (BoHV-1) by transmission and scanning electron microscopy, employing freezing technique protocols.Results:  The Golgi complex is a compact entity in a juxtanuclear position covered by a membrane on thecisface. Golgi membranes merge with membranes of the endoplasmic reticulum forming an entity with the perinuclear space. All compartments contained enveloped virions. After treatment with brefeldin A, HSV-1 virions aggregated in the perinuclear space and endoplasmic reticulum, while infectious progeny virus was still produced.Conclusions: The data suggest that virions derived by budding at nuclear membranes are intraluminally transported from the perinuclear space via Golgi -endoplasmic reticulum transitions into Golgi cisternae for packaging. Virions derived by budding at nuclear membranes are infective like Us3 deletion mutants, which  accumulate in the perinuclear space. Therefore, i) de-envelopment followed by re-envelopment is not essential for production of infective progeny virus, ii) the process taking place at the outer nuclear membrane is budding not fusion, and iii) naked capsids gain access to the cytoplasmic matrix via impaired nuclear envelope as reported earlier.

scholarly journals The Caenorhabditis elegans SUN protein UNC-84 interacts with lamin to transfer forces from the cytoplasm to the nucleoskeleton during nuclear migration

2014 ◽  
Vol 25 (18) ◽  
pp. 2853-2865 ◽  
Author(s):  
Courtney R. Bone ◽  
Erin C. Tapley ◽  
Mátyás Gorjánácz ◽  
Daniel A. Starr
Keyword(s):  
Caenorhabditis Elegans ◽  
Nuclear Envelope ◽  
Missense Mutation ◽  
Nuclear Membrane ◽  
Nuclear Migration ◽  
Live Imaging ◽  
Mechanical Forces ◽  
The Sun ◽  
Critical Component ◽  
Linc Complex

Nuclear migration is a critical component of many cellular and developmental processes. The nuclear envelope forms a barrier between the cytoplasm, where mechanical forces are generated, and the nucleoskeleton. The LINC complex consists of KASH proteins in the outer nuclear membrane and SUN proteins in the inner nuclear membrane that bridge the nuclear envelope. How forces are transferred from the LINC complex to the nucleoskeleton is poorly understood. The Caenorhabditis elegans lamin, LMN-1, is required for nuclear migration and interacts with the nucleoplasmic domain of the SUN protein UNC-84. This interaction is weakened by the unc-84(P91S) missense mutation. These mutant nuclei have an intermediate nuclear migration defect—live imaging of nuclei or LMN-1::GFP shows that many nuclei migrate normally, others initiate migration before subsequently failing, and others fail to begin migration. At least one other component of the nucleoskeleton, the NET5/Samp1/Ima1 homologue SAMP-1, plays a role in nuclear migration. We propose a nut-and-bolt model to explain how forces are dissipated across the nuclear envelope during nuclear migration. In this model, SUN/KASH bridges serve as bolts through the nuclear envelope, and nucleoskeleton components LMN-1 and SAMP-1 act as both nuts and washers on the inside of the nucleus.

Temporal and spatial interrelationships of confronting cisternae to nuclear envelope

1992 ◽  
Vol 50 (1) ◽  
pp. 930-931
Author(s):  
John R. Palisano
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Tumor Cells ◽  
Hela Cells ◽  
Microscopic Investigation ◽  
Electron Microscopic Investigation ◽  
Serial Sections ◽  
Electron Microscopic ◽  
Fetal Rat ◽  
Temporal And Spatial

Although confronting cistemae (CC) have been observed in a variety of tumor cells and normal fetal rat, mouse, and human epithelial tissues, little is known about their origin or role in mitotic cells. While several investigators have suggested that CC arise from nuclear envelope (NE) folding back on itself during prophase, others have suggested that CC arise when fragments of NE pair with endoplasmic reticulum. An electron microscopic investigation of 0.25 um thick serial sections was undertaken to examine the origin of CC in HeLa cells.

Annona muricata silver nanoparticles exhibit strong anticancer activities against cervical and prostate adenocarcinomas through regulation of CASP9 and the CXCL1/CXCR2 genes axis

Tumor Biology ◽  
2021 ◽  
Vol 43 (1) ◽  
pp. 37-55
Author(s):  
Yahaya Gavamukulya ◽  
Esther N. Maina ◽  
Hany A. El-Shemy ◽  
Amos M. Meroka ◽  
Geoffrey K. Kangogo ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
Anticancer Drugs ◽  
Hela Cells ◽  
Selectivity Index ◽  
Annona Muricata ◽  
Anticancer Activities ◽  
Apoptosis Pathway ◽  
Migration Assay ◽  
Intrinsic Apoptosis Pathway ◽  
In Cells

BACKGROUND: Green synthesized nanoparticles have been earmarked for use in nanomedicine including for the development of better anticancer drugs. OBJECTIVE: The aim of this study was to undertake biochemical evaluation of anticancer activities of green synthesized silver nanoparticles (AgNPs) from ethanolic extracts of fruits (AgNPs-F) and leaves (AgNPs-L) of Annona muricata. METHODS: Previously synthesized silver nanoparticles were used for the study. The effects of the AgNPs and 5-Fluorouracil were studied on PC3, HeLa and PNT1A cells. The resazurin, migration and colonogenic assays as well as qRT-PCR were employed. RESULTS: The AgNPs-F displayed significant antiproliferative effects against HeLa cells with an IC50 of 38.58μg/ml and PC3 cells with an IC50 of 48.17μg/ml but selectively spared normal PNT1A cells (selectivity index of 7.8), in comparison with first line drug 5FU and AgNPs-L whose selectivity index were 3.56 and 2.26 respectively. The migration assay revealed potential inhibition of the metastatic activity of the cells by the AgNPs-F while the colonogenic assay indicated the permanent effect of the AgNPs-F on the cancer cells yet being reversible on the normal cells in contrast with 5FU and AgNPs-L. CASP9 was significantly over expressed in all HeLa cells treated with the AgNPs-F (1.53-fold), AgNPs-L (1.52-fold) and 5FU (4.30-fold). CXCL1 was under expressed in HeLa cells treated with AgNPs-F (0.69-fold) and AgNPs-L (0.58-fold) and over expressed in cells treated with 5FU (4.95-fold), but the difference was not statistically significant. CXCR2 was significantly over expressed in HeLa cells treated with 5FU (8.66-fold) and AgNPs-F (1.12-fold) but under expressed in cells treated with AgNPs-L (0.76-fold). CONCLUSIONS: Here we show that biosynthesized AgNPs especially AgNPs-F can be used in the development of novel and better anticancer drugs. The mechanism of action of the AgNPs involves activation of the intrinsic apoptosis pathway through upregulation of CASP9 and concerted down regulation of the CXCL1/ CXCR2 gene axis.

BAF is required for emerin assembly into the reforming nuclear envelope

2001 ◽  
Vol 114 (24) ◽  
pp. 4575-4585 ◽  
Author(s):  
Tokuko Haraguchi ◽  
Takako Koujin ◽  
Miriam Segura-Totten ◽  
Kenneth K. Lee ◽  
Yosuke Matsuoka ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Hela Cells ◽  
Core Region ◽  
Lamin B ◽  
The Core ◽  
Double Stranded Dna ◽  
Recessive Form ◽  
Nuclear Envelope Assembly

Mutations in emerin cause the X-linked recessive form of Emery-Dreifuss muscular dystrophy (EDMD). Emerin localizes at the inner membrane of the nuclear envelope (NE) during interphase, and diffuses into the ER when the NE disassembles during mitosis. We analyzed the recruitment of wildtype and mutant GFP-tagged emerin proteins during nuclear envelope assembly in living HeLa cells. During telophase, emerin accumulates briefly at the ‘core’ region of telophase chromosomes, and later distributes over the entire nuclear rim. Barrier-to-autointegration factor (BAF), a protein that binds nonspecifically to double-stranded DNA in vitro, co-localized with emerin at the ‘core’ region of chromosomes during telophase. An emerin mutant defective for binding to BAF in vitro failed to localize at the ‘core’ in vivo, and subsequently failed to localize at the reformed NE. In HeLa cells that expressed BAF mutant G25E, which did not show ‘core’ localization, the endogenous emerin proteins failed to localize at the ‘core’ region during telophase, and did not assemble into the NE during the subsequent interphase. BAF mutant G25E also dominantly dislocalized LAP2β and lamin A from the NE, but had no effect on the localization of lamin B. We conclude that BAF is required for the assembly of emerin and A-type lamins at the reforming NE during telophase, and may mediate their stability in the subsequent interphase.

scholarly journals In Vivo Dynamics of Nuclear Pore Complexes in Yeast

1997 ◽  
Vol 136 (6) ◽  
pp. 1185-1199 ◽  
Author(s):  
Mirella Bucci ◽  
Susan R. Wente
Keyword(s):  
Nuclear Envelope ◽  
Recipient Cell ◽  
Nucleocytoplasmic Transport ◽  
Yeast Cells ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Central Location ◽  
Nuclear Membranes ◽  
Mutant Strains ◽  
Pore Complexes

While much is known about the role of nuclear pore complexes (NPCs) in nucleocytoplasmic transport, the mechanism of NPC assembly into pores formed through the double lipid bilayer of the nuclear envelope is not well defined. To investigate the dynamics of NPCs, we developed a live-cell assay in the yeast Saccharomyces cerevisiae. The nucleoporin Nup49p was fused to the green fluorescent protein (GFP) of Aequorea victoria and expressed in nup49 null haploid yeast cells. When the GFP–Nup49p donor cell was mated with a recipient cell harboring only unlabeled Nup49p, the nuclei fused as a consequence of the normal mating process. By monitoring the distribution of the GFP–Nup49p, we could assess whether NPCs were able to move from the donor section of the nuclear envelope to that of the recipient nucleus. We observed that fluorescent NPCs moved and encircled the entire nucleus within 25 min after fusion. When assays were done in mutant kar1-1 strains, where nuclear fusion does not occur, GFP–Nup49p appearance in the recipient nucleus occurred at a very slow rate, presumably due to new NPC biogenesis or to exchange of GFP– Nup49p into existing recipient NPCs. Interestingly, in a number of existing mutant strains, NPCs are clustered together at permissive growth temperatures. This has been explained with two different hypotheses: by movement of NPCs through the double nuclear membranes with subsequent clustering at a central location; or, alternatively, by assembly of all NPCs at a central location (such as the spindle pole body) with NPCs in mutant cells unable to move away from this point. Using the GFP–Nup49p system with a mutant in the NPCassociated factor Gle2p that exhibits formation of NPC clusters only at 37°C, it was possible to distinguish between these two models for NPC dynamics. GFP– Nup49p-labeled NPCs, assembled at 23°C, moved into clusters when the cells were shifted to growth at 37°C. These results indicate that NPCs can move through the double nuclear membranes and, moreover, can do so to form NPC clusters in mutant strains. Such clusters may result by releasing NPCs from a nuclear tether, or by disappearance of a protein that normally prevents pore aggregation. This system represents a novel approach for identifying regulators of NPC assembly and movement in the future.

scholarly journals Nuclear envelope rupture is induced by actin-based nucleus confinement

2016 ◽  
Vol 215 (1) ◽  
pp. 27-36 ◽  
Author(s):  
Emily M. Hatch ◽  
Martin W. Hetzer
Keyword(s):  
Nuclear Envelope ◽  
Cancer Cells ◽  
Nuclear Membrane ◽  
Nuclear Lamina ◽  
Membrane Stability ◽  
Linc Complex ◽  
Actin Bundles ◽  
In Cells

Repeated rounds of nuclear envelope (NE) rupture and repair have been observed in laminopathy and cancer cells and result in intermittent loss of nucleus compartmentalization. Currently, the causes of NE rupture are unclear. Here, we show that NE rupture in cancer cells relies on the assembly of contractile actin bundles that interact with the nucleus via the linker of nucleoskeleton and cytoskeleton (LINC) complex. We found that the loss of actin bundles or the LINC complex did not rescue nuclear lamina defects, a previously identified determinant of nuclear membrane stability, but did decrease the number and size of chromatin hernias. Finally, NE rupture inhibition could be rescued in cells treated with actin-depolymerizing drugs by mechanically constraining nucleus height. These data suggest a model of NE rupture where weak membrane areas, caused by defects in lamina organization, rupture because of an increase in intranuclear pressure from actin-based nucleus confinement.

Novel biochemical and functional insights into nuclear Ca2+ transport through IP3Rs and RyRs in osteoblasts

2000 ◽  
Vol 278 (5) ◽  
pp. F784-F791 ◽  
Author(s):  
Olugbenga A. Adebanjo ◽  
Gopa Biswas ◽  
Baljit S. Moonga ◽  
Hindupur K. Anandatheerthavarada ◽  
Li Sun ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Ryanodine Receptors ◽  
Functional Characterization ◽  
Inositol Trisphosphate Receptors ◽  
Nuclear Membranes ◽  
Sds Page ◽  
Direct Regulation

We report the first biochemical and functional characterization of inositol trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) in the nuclear membrane of bone-forming (MC3T3-E1) osteoblasts. Intact nuclei fluoresced intensely with anti-RyR (Ab34) and anti-IP3R (Ab40) antisera in a typically peripheral nuclear membrane pattern. Isolated nuclear membranes were next subjected to SDS-PAGE and blotted with isoform-specific anti-receptor antisera, notably Ab40, anti-RyR-1, anti-RyR-2 (Ab129), and anti-RyR-3 (Ab180). Only anti-RyR-1 and Ab40 showed bands corresponding, respectively, to full-length RyR-1 (∼500 kDa) and IP3R-1 (∼250 kDa). Band intensity was reduced by just ∼20% after brief tryptic proteolysis of intact nuclei; this confirmed that isolated nuclear membranes were mostly free of endoplasmic reticular contaminants. Finally, the nucleoplasmic Ca2+ concentration ([Ca2+]np) was measured in single nuclei by using fura-dextran. The nuclear envelope was initially loaded with Ca2+ via Ca2+-ATPase activation (1 mM ATP and ∼100 nM Ca2+). Adequate Ca2+ loading was next confirmed by imaging the nuclear envelope (and nucleoplasm). Exposure of Ca2+-loaded nuclei to IP3 or cADP ribose resulted in a rapid and sustained [Ca2+]np elevation. Taken together, the results provide complementary evidence for nucleoplasmic Ca2+ influx in osteoblasts through nuclear membrane-resident IP3Rs and RyRs. Our findings may conceivably explain the direct regulation of osteoblastic gene expression by hormones that use the IP3-Ca2+pathway.

scholarly journals Differentiating the Roles of UL16, UL21, and Us3 in the Nuclear Egress of Herpes Simplex Virus Capsids

2020 ◽  
Vol 94 (13) ◽  
Author(s):  
Jie Gao ◽  
Renée L. Finnen ◽  
Maxwell R. Sherry ◽  
Valerie Le Sage ◽  
Bruce W. Banfield
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Nuclear Envelope ◽  
Viral Proteins ◽  
Virus Infections ◽  
Antiviral Therapies ◽  
Nuclear Egress ◽  
Simplex Virus ◽  
Herpes Simplex Virus 2 ◽  
In Cells

ABSTRACT Viral proteins pUL16 and pUL21 are required for efficient nuclear egress of herpes simplex virus 2 capsids. To better understand the role of these proteins in nuclear egress, we established whether nuclear egress complex (NEC) distribution and/or function was altered in the absence of either pUL16 or pUL21. NEC distribution in cells infected with pUL16-deficient viruses was indistinguishable from that observed in cells infected with wild-type viruses. In contrast, NEC distribution was aberrant in cells infected with pUL21-deficient virus and, instead, showed some similarity to the aberrant NEC distribution pattern observed in cells infected with pUs3-deficient virus. These results indicated that pUL16 plays a role in nuclear egress that is distinct from that of pUL21 and pUs3. Higher-resolution examination of nuclear envelope ultrastructure in cells infected with pUL21-deficient viruses by transmission electron microscopy showed different types of nuclear envelope perturbations, including some that were not observed in cells infected with pUs3 deficient virus. The formation of the nuclear envelope perturbations observed in pUL21-deficient virus infections was dependent on a functional NEC, revealing a novel role for pUL21 in regulating NEC activity. The results of comparisons of nuclear envelope ultrastructure in cells infected with viruses lacking pUs3, pUL16, or both pUs3 and pUL16 were consistent with a role for pUL16 in advance of primary capsid envelopment and shed new light on how pUs3 functions in nuclear egress. IMPORTANCE The membrane deformation activity of the herpesvirus nuclear egress complex (NEC) allows capsids to transit through both nuclear membranes into the cytoplasm. NEC activity must be precisely controlled during viral infection, and yet our knowledge of how NEC activity is controlled is incomplete. To determine how pUL16 and pUL21, two viral proteins required for nuclear egress of herpes simplex virus 2, function in nuclear egress, we examined how the lack of each protein impacted NEC distribution. These analyses revealed a function of pUL16 in nuclear egress distinct from that of pUL21, uncovered a novel role for pUL21 in regulating NEC activity, and shed new light on how a viral kinase, pUs3, regulates nuclear egress. Nuclear egress of capsids is required for all herpesviruses. A complete understanding of all aspects of nuclear egress, including how viral NEC activity is controlled, may yield strategies to disrupt this process and aid the development of herpes-specific antiviral therapies.

scholarly journals Regulation of transferrin-induced endocytosis by wild-type and C282Y-mutant HFE in transfected HeLa cells

2002 ◽  
Vol 282 (5) ◽  
pp. C973-C979 ◽  
Author(s):  
Lukas Schwake ◽  
Andreas W. Henkel ◽  
Hans D. Riedel ◽  
Thorsten Schlenker ◽  
Matthias Both ◽  
...  
Keyword(s):  
Hela Cells ◽  
Single Cells ◽  
Hereditary Hemochromatosis ◽  
Inhibitory Effect ◽  
Membrane Capacitance ◽  
Wild Type ◽  
Time Resolved ◽  
Cell Membrane Capacitance ◽  
Endocytic Vesicles ◽  
In Cells

The hereditary hemochromatosis protein HFE is known to complex with the transferrin receptor; however, its function regarding endocytosis of transferrin is unclear. We performed patch-clamp capacitance measurements in transfected HeLa cells carrying wild-type or C282Y-mutant HFE cDNA under the control of a tetracycline-sensitive promoter. Whole cell experiments in cells with suppressed expression of wild-type HFE revealed a decrease in membrane capacitance, reflecting predominance of endocytosis in the presence of transferrin. Cells overexpressing C282Y-mutant HFE displayed less intense capacitance decreases, whereas no significant decrease was observed in cells overexpressing wild-type HFE. The formation of single endocytic vesicles in cells with suppressed expression of wild-type HFE was greatly increased in the presence of transferrin as revealed by cell-attached recordings. According to their calculated diameters, many of these vesicles corresponded to clathrin-coated vesicles. These results suggest that wild-type HFE negatively modulates the endocytic uptake of transferrin. This inhibitory effect is attenuated in cells expressing C282Y-mutant HFE. Time-resolved measurements of cell membrane capacitance provide a powerful tool to study transferrin-induced endocytosis in single cells.

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