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 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.

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.

scholarly journals Vpx proteins of SIVmac239 and HIV-2ROD interact with the cytoskeletal protein α-actinin 1

2004 ◽  
Vol 85 (11) ◽  
pp. 3291-3303 ◽  
Author(s):  
Sandra M. Mueller ◽  
Ronny Jung ◽  
Sigrid Weiler ◽  
Sabine M. Lang
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Cytoskeletal Protein ◽  
Yeast Two Hybrid ◽  
Nuclear Localization Signals ◽  
Quiescent Cells ◽  
Immunodeficiency Virus ◽  
Sequence Elements ◽  
Two Hybrid

vpx genes of human immunodeficiency virus type 2 (HIV-2) and immunodeficiency viruses from macaques (SIVmac), sooty mangabeys (SIVsm) and red-capped mangabeys (SIVrcm) encode a 112 aa protein that is packed into virion particles via interaction with the p6 domain of p55gag. Vpx localizes to the nucleus when expressed in the absence of other viral proteins. Moreover, Vpx is necessary for efficient nuclear import of the pre-integration complex (PIC) and critical for virus replication in quiescent cells, such as terminally differentiated macrophages and memory T cells. Vpx does not contain sequence elements that are homologous to previously characterized nuclear localization signals (NLSs). Therefore, it is likely that Vpx-dependent import of the PIC is mediated by interaction of Vpx with cellular proteins that do not belong to the classical import pathways. By using a yeast two-hybrid screen, α-actinin 1, a cytoskeletal protein, was identified to interact with SIVmac239 Vpx. Interestingly, deletion of the proline-rich C-terminal domain (aa 101–112) of Vpx, which is important for nuclear localization, resulted in loss of interaction with α-actinin 1. These findings suggest that the interaction with α-actinin 1 may play an important role in the transport of Vpx to the nucleus and in Vpx-mediated nuclear import of the PIC.

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.

Energy filtering TEM of transfected DNA

1996 ◽  
Vol 54 ◽  
pp. 924-925
Author(s):  
Marek Maleckl
Keyword(s):  
Nuclear Localization ◽  
Plasmid Dna ◽  
Intracellular Trafficking ◽  
Spatial Separation ◽  
Nuclear Pores ◽  
Transfected Cells ◽  
Nuclear Localization Signals ◽  
Organometallic Clusters ◽  
Energy Filtering ◽  
Transmission Electron

Energy filtering transmission electron microscopy (EFTEM) relies upon spatial separation of imaging electrons based upon their energy within an energy loss spectrum (Ottensmeyer 1986). In particular, EFTEM allows contrast enhancement in the zero loss mode and element mapping with electron spectroscopic imaging.These capabilities find a new application in studies of transgenesis in which constructs, probes, and antibodies are marked with organometallic clusters. Since the basic routes of intracellular trafficking of the transfected DNA have become recognized along with the crucial role played by nuclear pores as the selection gates (Malecki et al., 1995, Malecki and Skowron 1995),the current research is pursued by means of ultramicroscopy.Two strategies were developed for ultrastructural imaging of the transfected plasmid DNA with EFTEM. In both strategies, the transfected cells were cryo-immobilized, embedded in Lowlcryl K4M, and sectioned;plasmid constructs, transfection complexes containing nuclear localization signals, and transfection procedures were described previously (Malecki 1995).In the first strategy, the plasmid DNA was covalently conjugated to Nanogold (Nanoprobes) prior to transfections.

scholarly journals Nuclear translocation of adeno-associated virus type 2 capsid proteins for virion assembly

2012 ◽  
Vol 93 (9) ◽  
pp. 1887-1898 ◽  
Author(s):  
Ruth Popa-Wagner ◽  
Florian Sonntag ◽  
Kristin Schmidt ◽  
Jason King ◽  
Jürgen A. Kleinschmidt
Keyword(s):  
Nuclear Localization ◽  
Nuclear Translocation ◽  
Nuclear Transport ◽  
Capsid Proteins ◽  
Capsid Assembly ◽  
Protein Distribution ◽  
Adeno Associated Virus ◽  
Nuclear Localization Signals ◽  
Nuclear Uptake

Adeno-associated virus (AAV) capsid assembly occurs in the nucleus. Newly synthesized capsid proteins VP1, VP2 and VP3 contain several basic regions (BRs), which may act as nuclear localization signals (NLSs). Mutation of BR2 and BR3, located at the VP1 and VP2 N termini, marginally reduced nuclear uptake of VP1 or VP2, but not of VP3, when expressed in the context of the whole AAV type 2 (AAV2) genome. Combined mutation of BR1, BR2 and BR3 resulted in capsids with slightly reduced amounts of VP1. Expression of isolated VP1/2 N termini revealed an influence of BR3 on nuclear transport, whilst BR1 or BR2 had no effect. However, deletion of an N-terminal fragment in front of the BR elements strongly reduced nuclear uptake of VP1/2 N termini. Mutation of BR4, present in all three capsid proteins, led to their retention in the cytoplasm and to the formation of speckles, resulting in a lack of capsid formation and a significant reduction in VP levels. In a VP fragment comprising BR2, BR3 and BR4, the BR4 element was not necessary for nuclear localization. Mutation of BR5 in the C-terminal part of the VPs resulted in a speckled protein distribution in the nucleus, strongly reduced capsid assembly, and low VP1 and VP2 levels. Taken together, these results showed that BR2 and BR3 have a weak influence on nuclear transport of VP1 and VP2, whilst combined mutation of BR1, BR2 and BR3 influences the stoichiometry of VPs in assembled capsids. BR4 and BR5 play a crucial role in capsid assembly but have no NLS activity.

A nuclear localization signal can enhance both the nuclear transport and expression of 1 kb DNA

1999 ◽  
Vol 112 (12) ◽  
pp. 2033-2041
Author(s):  
J.J. Ludtke ◽  
G. Zhang ◽  
M.G. Sebestyen ◽  
J.A. Wolff
Keyword(s):  
Active Transport ◽  
Nuclear Localization ◽  
Transport Process ◽  
Nuclear Transport ◽  
Passive Diffusion ◽  
Viral Gene ◽  
Nuclear Localization Signals ◽  
Size Limit ◽  
Cytosolic Extract ◽  
Active Transport Process

Although the entry of DNA into the nucleus is a crucial step of non-viral gene delivery, fundamental features of this transport process have remained unexplored. This study analyzed the effect of linear double stranded DNA size on its passive diffusion, its active transport and its NLS-assisted transport. The size limit for passive diffusion was found to be between 200 and 310 bp. DNA of 310–1500 bp entered the nuclei of digitonin treated cells in the absence of cytosolic extract by an active transport process. Both the size limit and the intensity of DNA nuclear transport could be increased by the attachment of strong nuclear localization signals. Conjugation of a 900 bp expression cassette to nuclear localization signals increased both its nuclear entry and expression in microinjected, living cells.

Modulation of alpha-actinin levels affects cell motility and confers tumorigenicity on 3T3 cells

1994 ◽  
Vol 107 (7) ◽  
pp. 1773-1782 ◽  
Author(s):  
U. Gluck ◽  
A. Ben-Ze'ev
Keyword(s):  
Cell Motility ◽  
Colloidal Gold ◽  
Actin Filaments ◽  
Cell Types ◽  
Cell Behavior ◽  
Regulatory Pathway ◽  
3T3 Cells ◽  
Quiescent Cells ◽  
Beta 1 Integrin ◽  
Cdna Construct

alpha-Actinin is an abundant actin crosslinking protein, also localized at adherens type junctions. In adhesion plaques, alpha-actinin can link the actin filaments to integrin via vinculin and talin, or directly by binding to the cytoplasmic domain of beta 1-integrin. The expression of alpha-actinin is rapidly elevated in growth-activated quiescent cells, and is reduced in SV40-transformed 3T3 cells and various differentiating cell types (reviewed by Gluck, U., Kwiatkowski, D. J. and Ben-Ze'ev, A. Proc. Nat. Acad. Sci. USA 90, 383–387, 1993). To study the effect of changes in alpha-actinin levels on cell behavior, alpha-actinin expression was elevated in 3T3 cells by transfection with a full-length human nonmuscle alpha-actinin cDNA. To suppress alpha-actinin levels, 3T3 cells were transfected with an antisense alpha-actinin cDNA construct. Cells overexpressing alpha-actinin by 40–60% displayed a significant reduction in cell motility, as demonstrated by their slower locomotion into an artificial wound, and by forming shorter phagokinetic tracks on colloidal gold-coated substrata. 3T3 cells in which the expression of alpha-actinin was reduced to 25–60% of control levels, after antisense alpha-actinin transfection, had an increased cell motility. Moreover, such alpha-actinin-deficient 3T3 cells formed tumors upon injection into nude mice. The results demonstrate that modulations in alpha-actinin expression can affect, in a major way, the motile and tumorigenic properties of cells, and support the view that decreased alpha-actinin expression could be a common regulatory pathway to malignant transformation of 3T3 cells.

scholarly journals OsWRKY62 and OsWRKY76 interact with Importin α1s for Negative Regulation of Defensive Responses in Nucleus

2021 ◽  
Author(s):  
Xiaohui Xu ◽  
Han Wang ◽  
Jiqin Liu ◽  
Shuying Han ◽  
Miaomiao Lin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nuclear Localization ◽  
Nuclear Export ◽  
Nuclear Translocation ◽  
Nuclear Export Signal ◽  
Defense Responses ◽  
Nuclear Localization Signals ◽  
Defensive Responses ◽  
Export Signal ◽  
Positively Charged

Abstract Background: OsWRKY62 and OsWRKY76, two close members of WRKY transcription factors, function together as transcriptional repressors. OsWRKY62 is predominantly localized in the cytosol. What are the regulatory factors for OsWRKY62 nuclear translocation?Results: In this study, we characterized they interacted with rice importin, OsIMα1a and OsIMα1b, for nuclear translocation. Chimeric OsWRKY62.1-GFP, which is predominantly localized in the cytoplasm, was translocated to the nucleus of Nicotiana benthamiana leaf cells in the presence of OsIMα1a or OsIMαDIBB1a lacking the auto-inhibitory importin β-binding domain. OsIMαDIBB1a interacted with the WRKY domain of OsWRKY62.1, which has specific bipartite positively charged concatenated amino acids functioning as a nuclear localization signal. Similarly, we found that OsIMαDIBB1a interacted with the AvrPib effector of rice blast fungus Magnaporthe oryzae, which contains a scattered distribution of positively charged amino acids. Furthermore, we identified a nuclear export signal in OsWRKY62.1 that inhibited nuclear transportation. Overexpression of OsIMα1a or OsIMα1b enhanced resistance to M. oryzae, whereas knockout mutants decreased resistance to the pathogen. However, overexpressing both OsIMα1a and OsWRKY62.1 were slightly more susceptible to M. oryzae than OsWRKY62.1 alone. Ectopic overexpression of OsWRKY62.1 with an extra nuclear export signal compromised the enhanced susceptibility of OsWRKY62.1 to M. oryzae.Conclusion: These results indicated that OsWRKY62 localization is a consequence of competition binding between rice importins and exportins. OsWRKY62, OsWRKY76, and AvrPib effector translocate to nucleus in association with importin α1s through new types of nuclear localization signals for negatively regulating defense responses.

scholarly journals Nuclear localization of the Epstein–Barr virus EBNA3B protein

2006 ◽  
Vol 87 (4) ◽  
pp. 789-793 ◽  
Author(s):  
Anita Burgess ◽  
Marion Buck ◽  
Kenia Krauer ◽  
Tom Sculley
Keyword(s):  
Nuclear Localization ◽  
Epstein Barr Virus ◽  
Fluorescent Protein ◽  
Site Directed Mutagenesis ◽  
Nuclear Antigen ◽  
Nuclear Localization Signals ◽  
Barr Virus ◽  
Computer Analyses ◽  
Epstein Barr ◽  
Green Fluorescent

The Epstein–Barr virus nuclear antigen (EBNA) 3B is a hydrophilic, proline-rich, charged protein that is thought to be involved in transcriptional regulation and is targeted exclusively to the cell nucleus, where it localizes to discrete subnuclear granules. Co-localization studies utilizing a fusion protein between enhanced green fluorescent protein (EGFP) and EBNA3B with FLAG-tagged EBNA3A and EBNA3C proteins demonstrated that EBNA3B co-localized with both EBNA3A and EBNA3C in the nuclei of cells when overexpressed. Computer analyses identified four potential nuclear-localization signals (NLSs) in the EBNA3B amino acid sequence. By utilizing fusion proteins with EGFP, deletion constructs of EBNA3B and site-directed mutagenesis, three of the four NLSs (aa 160–166, 430–434 and 867–873) were shown to be functional in truncated forms of EBNA3B, whilst an additional NLS (aa 243–246) was identified within the N-terminal region of EBNA3B. Only two of the NLSs were found to be functional in the context of the full-length EBNA3B protein.

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