Regulation of functional nuclear pore size in fibroblasts

2001 ◽  
Vol 114 (24) ◽  
pp. 4621-4627
Author(s):  
Carl M. Feldherr ◽  
Debra Akin ◽  
Robert J. Cohen
Keyword(s):  
Pore Size ◽  
Transcriptional Activation ◽  
Pore Diameter ◽  
Nuclear Transport ◽  
Control Level ◽  
Proteasome Inhibitors ◽  
Nuclear Pore ◽  
Transport Capacity ◽  
3T3 Cells ◽  
Gold Particles

Protein-NLS-coated gold particles up to approximately 250 Å in diameter are transported through the nuclear pores in normal, proliferating BALB/c 3T3 cells. This size can increase or decrease, depending on cellular activity. It has been suggested that increases in functional pore size are related to a reduction in the amount of available p53. To further test this hypothesis, we investigated the effects of cycloheximide and pifithrin-α, which inhibits p53-dependent transcriptional activation, on nuclear transport. After 3 hours in cycloheximide, there was a significant increase in the size of the gold particles that entered the nucleoplasm. When the incubation period was extended to 6 hours or longer, transport capacity returned to the control level. By using proteasome inhibitors, it was shown that the cycloheximide-dependent increase in functional pore size was due to the inhibition of protein synthesis, consistent with the fact that p53 is a short-lived protein, and requires the activity of at least two different factors. Although cycloheximide increases the functional diameter of the channel available for signal-mediated transport by approximately 60 Å, it had no significant effect on either the import rate of small NLS-containing substrates (FITC-BSA-NLS), or passive diffusion of fluorescent-labeled proteins across the envelope. This suggests that changes in transport capacity were not caused by an increase in overall pore diameter but instead are due to a transient increase in pore size that accompanies signal-mediated transport. Pifithrin-α also caused an increase in functional pore diameter without altering the import rate of FITC-BSA-NLS, providing further support for the view that p53 can initiate changes in nuclear transport capacity.

Variations in signal-mediated transport through the nuclear pores as a function of cellular activity

1992 ◽  
Vol 50 (1) ◽  
pp. 494-495
Author(s):  
C.M. Feldherr ◽  
D. Akin
Keyword(s):  
Nuclear Localization ◽  
Pore Diameter ◽  
Cellular Activity ◽  
3T3 Cells ◽  
Nuclear Pores ◽  
Gold Particles ◽  
Nuclear Localization Signals ◽  
Nuclear Uptake ◽  
Quiescent Cells ◽  
Transport Channels

Previously, we utilized colloidal gold particles coated with nucleoplasmin, which contains well characterized nuclear localization signals (NLS), to study signal-mediated nuclear transport in proliferating and quiescent BALB/c 3T3 cells. The particles were injected into the cytoplasm, and nuclear uptake (N/C gold ratios) was later determined by TEM. The functional size of the transport channels was also analyzed by measuring the particles that entered the nucleoplasm. When large gold particles (110 to 270 Åin diameter) were injected, nuclear uptake was significantly greater in proliferating as compared to quiescent cells. The average fonctional pore diameter was approximately 230 Å during proliferation, and 140 Åor less in quiescent populations.

scholarly journals Stimulation of nuclear import by simian virus 40-transformed cell extracts is dependent on protein kinase activity.

1995 ◽  
Vol 15 (12) ◽  
pp. 7043-7049 ◽  
Author(s):  
C Feldherr ◽  
D Akin
Keyword(s):  
Protein Kinase ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Kinase Activity ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Protein Kinase Activity ◽  
Transport Capacity ◽  
3T3 Cells ◽  
Gold Particles

We previously reported that both the nuclear import rate of large karyophilic gold particles and the functional size of the pores are significantly greater in simian virus 40-transformed fibroblasts (the SV-T2 cell line) than in nontransformed BALB/c 3T3 cells. In this study, we found that cytosolic fractions obtained from SV-T2 cultures can increase nuclear transport capacity (both import rate and pore size) when microinjected into BALB/c 3T3 cells. The transport-enhancing function of the extracts can be abolished by the protein kinase inhibitors staurosporine and K252a as well as 5'-p-fluorosulfonylbenzoyladenosine and protein phosphatase 2A, which, although less specific, also interfere with kinase activity. Increases in transport capacity of the same magnitude as that produced by the SV-T2 extracts were obtained by microinjecting protein kinase A or C or recombinant mitogen-activated protein kinase. These data provide further support for the interpretation that the enhancer is a protein kinase. From experiments performed with specific kinase inhibitor peptides, it appears likely that protein kinase C is the active factor in the SV-T2 cytosolic fractions; however, this will require further verification. It was also determined, by using gold particles coated with bovine serum albumin conjugated to synthetic nuclear localization signal peptides that lacked phosphorylation sites, that the enhancer affects the transport machinery rather than the activity of the nuclear localization signals.

The Effects of SV40 Large T Antigen and p53 on Nuclear Transport Capacity in BALB/c 3T3 Cells

1994 ◽  
Vol 213 (1) ◽  
pp. 164-171 ◽  
Author(s):  
Carl Feldherr ◽  
Charles Cole ◽  
Robert E. Lanford ◽  
Debra Akin
Keyword(s):  
Nuclear Transport ◽  
T Antigen ◽  
Large T Antigen ◽  
Transport Capacity ◽  
3T3 Cells ◽  
Sv40 Large T Antigen

scholarly journals Signal-mediated nuclear transport in proliferating and growth-arrested BALB/c 3T3 cells.

1991 ◽  
Vol 115 (4) ◽  
pp. 933-939 ◽  
Author(s):  
C M Feldherr ◽  
D Akin
Keyword(s):  
High Cell Density ◽  
Unit Length ◽  
3T3 Cells ◽  
Proliferating Cells ◽  
Gold Particles ◽  
Nuclear Uptake ◽  
Large Particles ◽  
The Mean ◽  
Transport Channels ◽  
Relative Uptake

Mediated transport across the nuclear envelope was investigated in proliferating and growth-arrested (confluent or serum starved) BALB/c 3T3 cells by analyzing the nuclear uptake of nucleoplasmin-coated colloidal gold after injection into the cytoplasm. Compared with proliferating cells the nuclear uptake of large gold particles (110-270 A in diameter, including the protein coat) decreased 5.5-, 33-, and 78-fold, respectively, in 10-, 14-17-, and 21-d-old confluent cultures; however, the relative uptake of small particles (total diameter 50-80 A) did not decrease with increasing age of the cells. This finding suggests that essentially all pores remain functional in confluent populations, but that most pores lose their capacity to transport large particles. By injecting intermediate-sized gold particles, the functional diameters of the transport channels in the downgraded pores were estimated to be approximately to 130 and 110 A, in 14-17- and 21-d-old cultures, respectively. In proliferating cells, the transport channels have a functional diameter of approximately 230 A. The mean diameters of the pores (membrane-to-membrane distance) in proliferating and confluent cells (728 and 712 A, respectively) were significantly different at the 10%, but not the 5%, level. No differences in pore density (pore per unit length of membrane) were detected. Serum-deprived cells (7-8 d in 1% serum or 4 d in 0.5% serum) also showed a significant decrease in the nuclear uptake of large, but not small, gold particles. Thus, the permeability effects are not simply a function of high cell density but appear to be growth related. The possible functional significance of these findings is discussed.

Diffusion of Heavy Oil in SiO2 Model Catalyst and FCC Catalyst

2012 ◽  
Vol 550-553 ◽  
pp. 158-163 ◽  
Author(s):  
Zi Yuan Liu ◽  
Sheng Li Chen ◽  
Peng Dong ◽  
Xiu Jun Ge
Keyword(s):  
Diffusion Coefficient ◽  
Pore Size ◽  
Effective Diffusion Coefficient ◽  
Pore Diameter ◽  
Effective Diffusion ◽  
Model Catalyst ◽  
Average Pore ◽  
Diffusion Factor ◽  
Fcc Catalyst ◽  
Fcc Catalysts

Through the measured effective diffusion coefficients of Dagang vacuum residue supercritical fluid extraction and fractionation (SFEF) fractions in FCC catalysts and SiO2model catalysts, the relation between pore size of catalyst and effective diffusion coefficient was researched and the restricted diffusion factor was calculated. The restricted diffusion factor in FCC catalysts is less than 1 and it is 1~2 times larger in catalyst with polystyrene (PS) template than in conventional FCC catalyst without template, indicating that the diffusion of SFEF fractions in the two FCC catalysts is restricted by the pore. When the average molecular diameter is less than 1.8 nm, the diffusion of SFEF fractions in SiO2model catalyst which average pore diameter larger than 5.6 nm is unrestricted. The diffusion is restricted in the catalyst pores of less than 8 nm for SFEF fractions which diameter more than 1.8 nm. The tortuosity factor of SiO2model catalyst is obtained to be 2.87, within the range of empirical value. The effective diffusion coefficient of the SFEF fractions in SiO2model catalyst is two orders of magnitude larger than that in FCC catalyst with the same average pore diameter. This indicate that besides the ratio of molecular diameter to the pore diameter λ, the effective diffusion coefficient is also closely related to the pore structure of catalyst. Because SiO2model catalyst has uniform pore size, the diffusion coefficient can be precisely correlated with pore size of catalyst, so it is a good model material for catalyst internal diffusion investigation.

scholarly journals Modulation of the transport of a lysosomal enzyme by PDGF.

1990 ◽  
Vol 110 (2) ◽  
pp. 319-326 ◽  
Author(s):  
E M Prence ◽  
J M Dong ◽  
G G Sahagian
Keyword(s):  
Secretory Pathway ◽  
Cathepsin L ◽  
Control Level ◽  
3T3 Cells ◽  
Lysosomal Protease ◽  
Affinity Ligand ◽  
The Media ◽  
Mouse Cells ◽  
Nih 3T3 ◽  
The Relationship

The major excreted protein (MEP) of transformed mouse fibroblasts is the lysosomal protease, cathepsin L. MEP is also secreted by untransformed mouse cells in response to growth factors and tumor promoters, and is thought to play a role in cell growth and transformation. To determine the relationship between MEP synthesis and MEP secretion, we have examined these events in PDGF-treated NIH 3T3 cells. PDGF enhanced MEP synthesis and caused the diversion of MEP from the lysosomal delivery pathway to a secretory pathway. These two effects were found to be regulated independently at various times after growth factor addition. Short PDGF treatments (0.5 or 1 h) resulted in quantitative secretion of MEP although synthesis was near the control level. High levels of both synthesis and secretion occurred between 2 and 14 h of PDGF treatment. Between 18 and 30 h, the amount of secreted MEP returned to the low control level even though synthesis remained elevated. The secretion was specific for MEP; other lysosomal enzymes were not found in the media from PDGF-treated cells. PDGF-induced secretion of MEP was inhibited 84% by cycloheximide, suggesting that protein synthesis is required to elicit this effect. PDGF also caused a time-dependent increase in mannose 6-phosphate (Man-6-P) receptor-mediated endocytosis. These data support a model in which PDGF alters the distribution of Man-6-P receptors such that the Golgi concentration of receptors becomes limiting, thereby causing the selective secretion of the low affinity ligand, MEP.

Prediction of Fluid Flow in Artificial Cancellous Bone

2014 ◽  
Vol 695 ◽  
pp. 393-397
Author(s):  
Elsa Syuhada Abdull Yamin ◽  
Nor Azwadi Che Sidik
Keyword(s):  
Fluid Flow ◽  
Pore Size ◽  
Surface Area ◽  
Pore Diameter ◽  
Bone Surface ◽  
Flow Conditions ◽  
Morphological Aspects ◽  
Flow Through ◽  
Structure Result ◽  
Dynamics Method

The permeability of the blood in the artificial cancellous are affected by certain morphological aspects that include pore diameter, pore size, porosity and the bone surface area. In this study, computational fluid dynamics method is used to study the fluid flow through the cancellous structure. Result of the present work show that geometries with the same porosity and overall volume can have different permeability due to the differences in bone surface area. The hexahedron geometry has the highest permeability under stimulated blood flow conditions, where the cylindrical geometry has the lowest. Linear relationship is found between permeability and the two physical properties, bone surface area and the pore size.

scholarly journals The single transmembrane segment of gp210 is sufficient for sorting to the pore membrane domain of the nuclear envelope.

1992 ◽  
Vol 119 (6) ◽  
pp. 1441-1449 ◽  
Author(s):  
R W Wozniak ◽  
G Blobel
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Immunofluorescence Microscopy ◽  
Nuclear Pore ◽  
Transmembrane Helices ◽  
Wild Type ◽  
Transmembrane Segment ◽  
Membrane Domain ◽  
3T3 Cells ◽  
Type Mutant

The glycoprotein gp210 is located in the "pore membrane," a specialized domain of the nuclear envelope to which the nuclear pore complex (NPC) is anchored. gp210 contains a large cisternal domain, a single transmembrane segment (TM), and a COOH-terminal, 58-amino acid residue cytoplasmic tail (CT) (Wozniak, R. W., E. Bartnik, and G. Blobel. 1989. J. Cell Biol. 108:2083-2092; Greber, U. F., A. Senior, and L. Gerace. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:1495-1502). To locate determinants for sorting of gp210 to the pore membrane, we constructed various cDNAs coding for wild-type, mutant, and chimeric gp210, and monitored localization of the expressed protein in 3T3 cells by immunofluorescence microscopy using appropriate antibodies. The large cisternal domain of gp210 (95% of its mass) did not reveal any sorting determinants. Surprisingly, the TM of gp210 is sufficient for sorting to the pore membrane. The CT also contains a sorting determinant, but it is weaker than that of the TM. We propose specific lateral association of the transmembrane helices of two proteins to yield either a gp210 homodimer or a heterodimer of gp210 and another protein. The cytoplasmically oriented tails of these dimers may bind cooperatively to the adjacent NPCs. In addition, we demonstrate that gp210 co-localizes with cytoplasmically dispersed nucleoporins, suggesting a cytoplasmic association of these components.

scholarly journals SBA-15 with Crystalline Walls Produced via Thermal Treatment with the Alkali and Alkali Earth Metal Ions

Materials ◽  
2021 ◽  
Vol 14 (18) ◽  
pp. 5270
Author(s):  
Sung Soo Park ◽  
Sang-Wook Chu ◽  
Liyi Shi ◽  
Shuai Yuan ◽  
Chang-Sik Ha
Keyword(s):  
Metal Ions ◽  
Pore Size ◽  
Metal Ion ◽  
Pore Diameter ◽  
Earth Metal ◽  
Silica Matrix ◽  
Alkali Earth Metal ◽  
Alkali Earth ◽  
Large Pore ◽  
Large Pore Size

Crystalline walled SBA-15 with large pore size were prepared using alkali and alkali earth metal ions (Na+, Li+, K+ and Ca2+). For this work, the ratios of alkali metal ions (Si/metal ion) ranged from 2.1 to 80, while the temperatures tested ranged from 500 to 700 °C. The SBA-15 prepared with Si/Na+ ratios ranging from 2.1 to 40 at 700 °C exhibited both cristobalite and quartz SiO2 structures in pore walls. When the Na+ amount increased (i.e., Si/Na increased from 80 to 40), the pore size was increased remarkably but the surface area and pore volume of the metal ion-based SBA-15 were decreased. When the SBA-15 prepared with Li+, K+ and Ca2+ ions (Si/metal ion = 40) was thermally treated at 700 °C, the crystalline SiO2 of quartz structure with large pore diameter (i.e., 802.5 Å) was observed for Ca+2 ion-based SBA-15, while no crystalline SiO2 structures were observed in pore walls for both the K+ and Li+ ions treated SBA-15. The crystalline SiO2 structures may be formed by the rearrangement of silica matrix when alkali or alkali earth metal ions are inserted into silica matrix at elevated temperature.

scholarly journals O-GlcNAc modification of nuclear pore complexes accelerates bi-directional transport

2020 ◽  
Author(s):  
Tae Yeon Yoo ◽  
Timothy J Mitchison
Keyword(s):  
Nuclear Import ◽  
Nuclear Transport ◽  
Super Resolution ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Permeability Barrier ◽  
Nuclear Pore Complexes ◽  
Facilitated Diffusion ◽  
Import And Export ◽  
Pore Complexes

AbstractMacromolecular transport across the nuclear envelope depends on facilitated diffusion through nuclear pore complexes (NPCs). The interior of NPCs contains a permeability barrier made of phenylalanine-glycine (FG) repeat domains that selectively facilitates the permeation of cargoes bound to nuclear transport receptors (NTRs). FG repeats in NPC are a major site of O-linked N-acetylglucosamine (O-GlcNAc) modification, but the functional role of this modification in nucleocytoplasmic transport is unclear. We developed high-throughput assays based on optogenetic probes to quantify the kinetics of nuclear import and export in living human cells. We found that increasing O-GlcNAc modification of the NPC accelerated NTR-facilitated nucleocytoplasmic transport of proteins in both directions, and decreasing modification slowed transport. Super-resolution imaging revealed strong enrichment of O-GlcNAc at the FG-repeat barrier. O-GlcNAc modification also accelerated passive permeation of a small, inert protein through NPCs. We conclude that O-GlcNAc modification accelerates nucleocytoplasmic transport by enhancing the non-specific permeability the FG-repeat barrier, perhaps by steric inhibition of interactions between FG repeats.SummaryNuclear pore complexes mediate nuclear transport and are highly modified with O-linked N-acetylglucosamine (O-GlcNAc) on FG repeat domains. Using a new quantitative live-cell imaging assay, Yoo and Mitchison demonstrate acceleration of nuclear import and export by O-GlcNAc modification.

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