scholarly journals Identification of Six Autographa californica Multicapsid Nucleopolyhedrovirus Early Genes That Mediate Nuclear Localization of G-Actin

2002 ◽  
Vol 76 (23) ◽  
pp. 12281-12289 ◽  
Author(s):  
Taro Ohkawa ◽  
Annette R. Rowe ◽  
Loy E. Volkman
Keyword(s):  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Early Stage ◽  
Autographa Californica ◽  
Host Cells ◽  
Filamentous Actin ◽  
Progeny Production ◽  
Cellular Mechanisms ◽  
Early Genes ◽  
Green Fluorescent

ABSTRACT Nuclear filamentous actin (F-actin) is required for nucleopolyhedrovirus (NPV) progeny production in NPV-infected, cultured lepidopteran cells. We have determined that monomeric G-actin is localized within the nuclei of host cells during the early stage of infection by Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). With a library of cloned AcMNPV genomic fragments, along with a plasmid engineered to express enhanced green fluorescent protein-Bombyx mori G-actin in transient transfection experiments, we identified six AcMNPV early genes that mediate nuclear localization of G-actin in TN-368 cells: ie-1, pe38, he65, Ac004, Ac102, and Ac152. Within this subset, ie-1 and pe38 encode immediate-early transcriptional transactivators, he65 encodes a delayed-early product, and the products encoded by Ac004, Ac102, and Ac152 have not been characterized. We found that when driven by foreign promoters, ie-1, pe38, and Ac004 had to be expressed prior to Ac102 or he65 for nuclear G-actin to accumulate and that expression of Ac152 was no longer required. These results and others suggested that the product of Ac152 was a transactivator (directly or indirectly) of both Ac102 and he65 and that recruitment of G-actin to the nucleus was a temporally regulated process. Determining the functions of each of the six AcMNPV gene products with respect to our assay should provide valuable clues to basic cellular mechanisms of actin regulation and how AcMNPV infection affects them.

scholarly journals Optical reconstruction of murine colorectal mucosa at cellular resolution

2015 ◽  
Vol 308 (9) ◽  
pp. G721-G735 ◽  
Author(s):  
Cambrian Y. Liu ◽  
Philip E. Dubé ◽  
Nandini Girish ◽  
Ajay T. Reddy ◽  
D. Brent Polk
Keyword(s):  
Cell Function ◽  
Fluorescent Protein ◽  
Confocal Imaging ◽  
Host Cells ◽  
Cellular Mechanisms ◽  
Colorectal Mucosa ◽  
Cellular Resolution ◽  
Volume Imaging ◽  
Mucosal Layer ◽  
Optical Reconstruction

The mucosal layer of the colon is a unique and dynamic site where host cells interface with one another and the microbiome, with major implications for physiology and disease. However, the cellular mechanisms mediating colonic regeneration, inflammation, dysplasia, and dysbiosis remain undercharacterized, partly because the use of thin tissue sections in many studies removes important volumetric context. To address these challenges in visualization, we have developed the deep mucosal imaging (DMI) method to reconstruct continuous extended volumes of mouse colorectal mucosa at cellular resolution. Use of ScaleA2 and SeeDB clearing agents enabled full visualization of the colonic crypt, the fundamental unit of adult colon. Confocal imaging of large colorectal expanses revealed epithelial structures involved in repair, inflammation, tumorigenesis, and stem cell function, in fluorescent protein-labeled, immunostained, paraffin-embedded, or human biopsy samples. We provide freely available software to reconstruct and explore on computers with standard memory allocations the large DMI datasets containing in toto representations of distal colonic mucosal volume. Extended-volume imaging of colonic mucosa through the novel, extensible, and readily adopted DMI approach will expedite mechanistic investigations of intestinal physiology and pathophysiology at intracrypt to multicrypt length scales.

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.

scholarly journals Live-Cell Fluorescence Imaging Reveals the Dynamics of Protein Kinase CK2 Individual Subunits

2003 ◽  
Vol 23 (3) ◽  
pp. 975-987 ◽  
Author(s):  
Odile Filhol ◽  
Arsenio Nueda ◽  
Véronique Martel ◽  
Delphine Gerber-Scokaert ◽  
Maria José Benitez ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nuclear Localization ◽  
Protein Kinase Ck2 ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Nuclear Accumulation ◽  
Nucleocytoplasmic Trafficking ◽  
Green Fluorescent ◽  
Nucleocytoplasmic Distribution ◽  
Kinase Ck2

ABSTRACT Protein kinase CK2 is a multifunctional enzyme which has long been described as a stable heterotetrameric complex resulting from the association of two catalytic (α or α′) and two regulatory (β) subunits. To track the spatiotemporal dynamics of CK2 in living cells, we fused its catalytic α and regulatory β subunits with green fluorescent protein (GFP). Both CK2 subunits contain nuclear localization domains that target them independently to the nucleus. Imaging of stable cell lines expressing low levels of GFP-CK2α or GFP-CK2β revealed the existence of CK2 subunit subpopulations exhibiting differential dynamics. Once in the nucleus, they diffuse randomly at different rates. Unlike CK2β, CK2α can shuttle, showing the dynamic nature of the nucleocytoplasmic trafficking of the kinase. When microinjected in the cytoplasm, the isolated CK2 subunits are rapidly translocated into the nucleus, whereas the holoenzyme complex remains in this cell compartment, suggesting an intramolecular masking of the nuclear localization sequences that suppresses nuclear accumulation. However, binding of FGF-2 to the holoenzyme triggers its nuclear translocation. Since the substrate specificity of CK2α is dramatically changed by its association with CK2β, the control of the nucleocytoplasmic distribution of each subunit may represent a unique potential regulatory mechanism for CK2 activity.

scholarly journals Detection of Oil Palm Root Penetration by Agrobacterium-Mediated Transformed Ganoderma boninense, Expressing Green Fluorescent Protein

2017 ◽  
Vol 107 (4) ◽  
pp. 483-490 ◽  
Author(s):  
Nisha Govender ◽  
Mui-Yun Wong
Keyword(s):  
Oil Palm ◽  
Fluorescent Protein ◽  
Fusion Gene ◽  
Early Stage ◽  
Marker Gene ◽  
Induction Medium ◽  
Root Penetration ◽  
Ganoderma Boninense ◽  
Agrobacterium Strain ◽  
Green Fluorescent

A highly efficient and reproducible Agrobacterium-mediated transformation protocol for Ganoderma boninense was developed to facilitate observation of the early stage infection of basal stem rot (BSR). The method was proven amenable to different explants (basidiospore, protoplast, and mycelium) of G. boninense. The transformation efficiency was highest (62%) under a treatment combination of protoplast explant and Agrobacterium strain LBA4404, with successful expression of an hyg marker gene and gus-gfp fusion gene under the control of heterologous p416 glyceraldehyde 3-phosphate dehydrogenase promoter. Optimal transformation conditions included a 1:100 Agrobacterium/explant ratio, induction of Agrobacterium virulence genes in the presence of 250 μm acetosyringone, co-cultivation at 22°C for 2 days on nitrocellulose membrane overlaid on an induction medium, and regeneration of transformants on potato glucose agar prepared with 0.6 M sucrose and 20 mM phosphate buffer. Evaluated transformants were able to infect root tissues of oil palm plantlets with needle-like microhyphae during the penetration event. The availability of this model pathogen system for BSR may lead to a better understanding of the pathogenicity factors associated with G. boninense penetration into oil palm roots.

scholarly journals Nature and consequences of interactions between Salmonella enterica serovar Dublin and host cells in cattle

2019 ◽  
Vol 50 (1) ◽  
Author(s):  
Prerna Vohra ◽  
Christina Vrettou ◽  
Jayne C. Hope ◽  
John Hopkins ◽  
Mark P. Stevens
Keyword(s):  
Lymph Nodes ◽  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Host Cells ◽  
Large Animal ◽  
Ileal Mucosa ◽  
Zoonotic Infections ◽  
Infected Cells ◽  
Green Fluorescent ◽  
Salmonella Enterica Serovar Dublin

AbstractSalmonella enterica is a veterinary and zoonotic pathogen of global importance. While murine and cell-based models of infection have provided considerable knowledge about the molecular basis of virulence of Salmonella, relatively little is known about salmonellosis in naturally-affected large animal hosts such as cattle, which are a reservoir of human salmonellosis. As in humans, Salmonella causes bovine disease ranging from self-limiting enteritis to systemic typhoid-like disease and exerts significant economic and welfare costs. Understanding the nature and consequences of Salmonella interactions with bovine cells will inform the design of effective vaccines and interventions to control animal and zoonotic infections. In calves challenged orally with S. Dublin expressing green fluorescent protein (GFP) we observed that the bacteria were predominantly extracellular in the distal ileal mucosa and within gut-associated lymph nodes 48 h post-infection. Intracellular bacteria, identified by flow cytometry using the GFP signal, were predominantly within MHCII+ macrophage-like cells. In contrast to observations from murine models, these S. Dublin-infected cells had elevated levels of MHCII and CD40 compared to both uninfected cells from the same tissue and cells from the cognate tissue of uninfected animals. Moreover, no gross changes of the architecture of infected lymph nodes were observed as was described previously in a mouse model. In order to further investigate Salmonella-macrophage interactions, net replication of S. enterica serovars that differ in virulence in cattle was measured in bovine blood-derived macrophages by enumeration of gentamicin-protected bacteria and fluorescence dilution, but did not correlate with host-specificity.

scholarly journals Nuclear targeting of the β isoform of Type II phosphatidylinositol phosphate kinase (phosphatidylinositol 5-phosphate 4-kinase) by its α-helix 7

2000 ◽  
Vol 346 (3) ◽  
pp. 587-591 ◽  
Author(s):  
Antonio CIRUELA ◽  
Katherine A. HINCHLIFFE ◽  
Nullin DIVECHA ◽  
Robin F. IRVINE
Keyword(s):  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Physiological Role ◽  
Nuclear Localization Sequence ◽  
Type Ii ◽  
Nuclear Targeting ◽  
Phosphatidylinositol Phosphate ◽  
Localization Sequence ◽  
Α Helix ◽  
Green Fluorescent

Type II phosphatidylinositol phosphate kinases (PIPkins) have recently been found to be primarily phosphatidylinositol 5-phosphate 4-kinases, and their physiological role remains unclear. We have previously shown that a Type II PIPkin [isoform(s) unknown], is localized partly in the nucleus [Divecha, Rhee, Letcher and Irvine (1993) Biochem. J. 289, 617-620], and here we show, by transfection of HeLa cells with green-fluorescent-protein-tagged Type II PIPkins, that this is likely to be the Type IIβ isoform. Type IIβ PIPkin has no obvious nuclear localization sequence, and a detailed analysis of the localization of chimaeras and mutants of the α (cytosolic) and β PIPkins shows that the nuclear localization requires the presence of a 17-amino-acid length of α-helix (α-helix 7) that is specific to the β isoform, and that this helix must be present in its entirety, with a precise orientation. This resembles the nuclear targeting of the HIV protein Vpr, and Type IIβ PIPkin is apparently therefore the first example of a eukaryotic protein that uses the same mechanism.

scholarly journals Plant Hormones Differentially Control the Sub-Cellular Localization of Plasma Membrane Microdomains during the Early Stage of Soybean Nodulation

Genes ◽  
2019 ◽  
Vol 10 (12) ◽  
pp. 1012 ◽  
Author(s):  
Zhenzhen Qiao ◽  
Prince Zogli ◽  
Marc Libault
Keyword(s):  
Plasma Membrane ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Early Stage ◽  
Cellular Level ◽  
Infection Thread ◽  
Membrane Microdomains ◽  
Symbiotic Interaction ◽  
Green Fluorescent ◽  
Plasma Membrane Microdomains

Phytohormones regulate the mutualistic symbiotic interaction between legumes and rhizobia, nitrogen-fixing soil bacteria, notably by controlling the formation of the infection thread in the root hair (RH). At the cellular level, the formation of the infection thread is promoted by the translocation of plasma membrane microdomains at the tip of the RH. We hypothesize that phytohormones regulate the translocation of plasma membrane microdomains to regulate infection thread formation. Accordingly, we treated with hormone and hormone inhibitors transgenic soybean roots expressing fusions between the Green Fluorescent Protein (GFP) and GmFWL1 or GmFLOT2/4, two microdomain-associated proteins translocated at the tip of the soybean RH in response to rhizobia. Auxin and cytokinin treatments are sufficient to trigger or inhibit the translocation of GmFWL1 and GmFLOT2/4 to the RH tip independently of the presence of rhizobia, respectively. Unexpectedly, the application of salicylic acid, a phytohormone regulating the plant defense system, also promotes the translocation of GmFWL1 and GmFLOT2/4 to the RH tip regardless of the presence of rhizobia. These results suggest that phytohormones are playing a central role in controlling the early stages of rhizobia infection by regulating the translocation of plasma membrane microdomains. They also support the concept of crosstalk of phytohormones to control nodulation.

scholarly journals Pak1 Protein Kinase Regulates Activation and Nuclear Localization of Snf1-Gal83 Protein Kinase

2004 ◽  
Vol 24 (18) ◽  
pp. 8255-8263 ◽  
Author(s):  
Kristina Hedbacker ◽  
Seung-Pyo Hong ◽  
Marian Carlson
Keyword(s):  
Protein Kinase ◽  
Nuclear Localization ◽  
Kinase Activity ◽  
Fluorescent Protein ◽  
Protein Kinase Activity ◽  
Activation Loop ◽  
Glucose Limitation ◽  
Subunit Isoforms ◽  
Green Fluorescent ◽  
Amp Activated Protein Kinase

ABSTRACT Three kinases, Pak1, Tos3, and Elm1, activate Snf1 protein kinase in Saccharomyces cerevisiae. This cascade is conserved in mammals, where LKB1 activates AMP-activated protein kinase. We address the specificity of the activating kinases for the three forms of Snf1 protein kinase containing the β-subunit isoforms Gal83, Sip1, and Sip2. Pak1 is the most important kinase for activating Snf1-Gal83 in response to glucose limitation, but Elm1 also has a significant role; moreover, both Pak1 and Elm1 affect Snf1-Sip2. These findings exclude the possibility of a one-to-one correspondence between the activating kinases and the Snf1 complexes. We further identify a second, unexpected role for Pak1 in regulating Snf1-Gal83: the catalytic activity of Pak1 is required for the nuclear enrichment of Snf1-Gal83 in response to carbon stress. The nuclear enrichment of Snf1 fused to green fluorescent protein (GFP) depends on both Gal83 and Pak1 and is abolished by a mutation of the activation loop threonine; in contrast, the nuclear enrichment of Gal83-GFP occurs in a snf1Δ mutant and depends on Pak1 only when Snf1 is present. Snf1-Gal83 is the only form of the kinase that localizes to the nucleus. These findings, that Pak1 both activates Snf1-Gal83 and controls its nuclear localization, implicate Pak1 in regulating nuclear Snf1 protein kinase activity.

Nuclear localization of angiotensinogen in astrocytes

2005 ◽  
Vol 288 (2) ◽  
pp. R539-R546 ◽  
Author(s):  
Mikhiela Sherrod ◽  
Xuebo Liu ◽  
Xiaoji Zhang ◽  
Curt D. Sigmund
Keyword(s):  
Fusion Protein ◽  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Primary Cultures ◽  
Endocrine Function ◽  
Astrocytoma Cell ◽  
Protein Encoding ◽  
Localization Signal ◽  
Green Fluorescent ◽  
The Brain

In the brain, angiotensinogen (AGT) is primarily expressed in astrocytes; brain ANG II derived from locally produced AGT has been shown to influence blood pressure. To better understand the molecular basis of AGT expression in the brain, we identified a human astrocytoma cell line, CCF-STTG1, that expresses endogenous AGT mRNA and produces AGT protein. Studies examining CCF-STTG1 cell AGT after N- and O-glycosidase suggest that AGT may not be posttranslationally modified by glycosylation in these cells as it is in plasma. Small amounts of AGT (5% of HepG2) were detected in the culture medium, suggesting a low rate of AGT secretion. Immunocytochemical examination of AGT in CCF-STTG1 cells revealed mainly nuclear localization. Although this has not been previously reported, it is consistent with nuclear localization of other serpin family members. To examine this further, we generated a fusion protein consisting of green fluorescent protein (GFP) and human AGT and examined subcellular localization by confocal microscopy after confirming expression of the fusion protein by Western blot. In CCF-STTG1 cells, a control GFP construct lacking AGT was mainly localized in the cytoplasm, whereas the GFP-AGT fusion protein was primarily localized in the nucleus. To map the location of a potential nuclear localization signal, overlapping 500-bp fragments of human AGT cDNA were fused in frame downstream of GFP. Although four of the fusion proteins exhibited either perinuclear or cytoplasmic localization, one fusion protein encoding the COOH terminus of AGT was localized in the nucleus. Importantly, nuclear localization of human AGT was confirmed in primary cultures of glial cells isolated from transgenic mice expressing the human AGT under the control of its own endogenous promoter. Our results suggest that AGT may have a novel intracellular role in the brain apart from its predicted endocrine function.

Sign in / Sign up

Export Citation Format

Share Document