scholarly journals Selective transport of fluorescent proteins into the phage nucleus

2020 ◽  
Author(s):  
Katrina T. Nguyen ◽  
Joseph Sugie ◽  
Kanika Khanna ◽  
MacKennon E. Egan ◽  
Erica A. Birkholz ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Messenger Rna ◽  
Rna Synthesis ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Amino Acid Residues ◽  
Nuclear Targeting ◽  
Selective Transport ◽  
Insight Into

AbstractUpon infection of Pseudomonas cells, jumbo phages 201Φ2-1, ΦPA3, and ΦKZ assemble a phage nucleus. Viral DNA is enclosed within the phage-encoded proteinaceous shell along with proteins associated with DNA replication, recombination and transcription. Ribosomes and proteins involved in metabolic processes are excluded from the nucleus. RNA synthesis occurs inside the phage nucleus and messenger RNA is presumably transported into the cytoplasm to be translated. Newly synthesized proteins either remain in the cytoplasm or specifically translocate into the nucleus. The molecular mechanisms governing selective protein sorting and nuclear import in these phage infection systems are currently unclear. To gain insight into this process, we studied the localization of five reporter fluorescent proteins (GFP+, sfGFP, GFPmut1, mCherry, CFP). During infection with ΦPA3 or 201Φ2-1, all five fluorescent proteins were excluded from the nucleus as expected; however, we have discovered an anomaly with the ΦKZ nuclear transport system. The fluorescent protein GFPmut1, expressed by itself, was transported into the ΦKZ phage nucleus. We identified the amino acid residues on the surface of GFPmut1 required for nuclear targeting. Fusing GFPmut1 to any protein, including proteins that normally reside in the cytoplasm, resulted in transport of the fusion into the nucleus. Although the mechanism of transport is still unknown, we demonstrate that GFPmut1 is a useful tool that can be used for fluorescent labelling and targeting of proteins into the ΦKZ phage nucleus.

scholarly journals Selective transport of fluorescent proteins into the phage nucleus

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0251429
Author(s):  
Katrina T. Nguyen ◽  
Joseph Sugie ◽  
Kanika Khanna ◽  
MacKennon E. Egan ◽  
Erica A. Birkholz ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Messenger Rna ◽  
Rna Synthesis ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Amino Acid Residues ◽  
Nuclear Targeting ◽  
Selective Transport ◽  
Insight Into

Upon infection of Pseudomonas cells, jumbo phages 201Φ2–1, ΦPA3, and ΦKZ assemble a phage nucleus. Viral DNA is enclosed within the phage-encoded proteinaceous shell along with proteins associated with DNA replication, recombination and transcription. Ribosomes and proteins involved in metabolic processes are excluded from the nucleus. RNA synthesis occurs inside the phage nucleus and messenger RNA is presumably transported into the cytoplasm to be translated. Newly synthesized proteins either remain in the cytoplasm or specifically translocate into the nucleus. The molecular mechanisms governing selective protein sorting and nuclear import in these phage infection systems are currently unclear. To gain insight into this process, we studied the localization of five reporter fluorescent proteins (GFP+, sfGFP, GFPmut1, mCherry, CFP). During infection with ΦPA3 or 201Φ2–1, all five fluorescent proteins were excluded from the nucleus as expected; however, we have discovered an anomaly with the ΦKZ nuclear transport system. The fluorescent protein GFPmut1, expressed by itself, was transported into the ΦKZ phage nucleus. We identified the amino acid residues on the surface of GFPmut1 required for nuclear targeting. Fusing GFPmut1 to any protein, including proteins that normally reside in the cytoplasm, resulted in transport of the fusion into the nucleus. Although the mechanism of transport is still unknown, we demonstrate that GFPmut1 is a useful tool that can be used for fluorescent labelling and targeting of proteins into the ΦKZ phage nucleus.

Fluorescent protein expression in temperature tolerant and susceptible reef-building corals

2021 ◽  
pp. 1-10
Author(s):  
Exequiel Gabriel S. Dizon ◽  
Jeric P. Da-Anoy ◽  
Melissa S. Roth ◽  
Cecilia Conaco
Keyword(s):  
Spectral Properties ◽  
Coral Species ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Antioxidant Properties ◽  
Variable Expression ◽  
Acropora Digitifera ◽  
Insight Into

Abstract Fluorescent proteins (FPs) are reported to play an important role as photoprotectants and antioxidants in corals subjected to stressful conditions. Identifying the various FP genes expressed and FP gene expression patterns under stress in diverse coral species can provide insight into FP function. In this study, we identified 16 putative FP homologues from the transcriptomes of corals with varying susceptibility to elevated temperature, including Acropora digitifera, Favites colemani, Montipora digitata and Seriatopora caliendrum. Each coral expressed a different complement of FP transcripts, which were predicted to have distinct spectral properties. The most diverse and abundant repertoire of FP transcripts, including at least 6 green FPs, were expressed in the temperature-tolerant coral, F. colemani. In comparison, the other corals expressed fewer FP types. Specific FP transcripts exhibited variable expression profiles in coral fragments subjected to 32 ± 1 °C (treatment) or 28 ± 1 °C (control) for up to 72 h, suggesting that distinct FPs may have different roles. Further studies on the expression of the proteins encoded by these FP transcripts, their fluorescence activity, tissue localization, and possible antioxidant properties, are needed to reveal their contribution to thermal stress tolerance in certain species of corals.

scholarly journals The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation

2016 ◽  
Vol 90 (13) ◽  
pp. 5860-5875 ◽  
Author(s):  
Eva Maria Borst ◽  
Rudolf Bauerfeind ◽  
Anne Binz ◽  
Thomas Min Stephan ◽  
Sebastian Neuber ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Viral Genome ◽  
Fluorescent Protein ◽  
Unit Length ◽  
Start Codon ◽  
Nuclear Targeting ◽  
Reading Frame ◽  
A Genome ◽  
Genome Encapsidation ◽  
Insight Into

ABSTRACTSeveral essential viral proteins are proposed to participate in genome encapsidation of human cytomegalovirus (HCMV), among them pUL77 and pUL93, which remain largely uncharacterized. To gain insight into their properties, we generated an HCMV mutant expressing a pUL77-monomeric enhanced green fluorescent protein (mGFP) fusion protein and a pUL93-specific antibody. Immunoblotting demonstrated that both proteins are incorporated into capsids and virions. Conversely to data suggesting internal translation initiation sites within the UL93 open reading frame (ORF), we provide evidence that pUL93 synthesis commences at the first start codon. In infected cells, pUL77-mGFP was found in nuclear replication compartments and dot-like structures, colocalizing with capsid proteins. Immunogold labeling of nuclear capsids revealed that pUL77 is present on A, B, and C capsids. Pulldown of pUL77-mGFP revealed copurification of pUL93, indicating interaction between these proteins, which still occurred when capsid formation was prevented. Correct subnuclear distribution of pUL77-mGFP required pUL93 as well as the major capsid protein (and thus probably the presence of capsids), but not the tegument protein pp150 or the encapsidation protein pUL52, demonstrating that pUL77 nuclear targeting occurs independently of the formation of DNA-filled capsids. When pUL77 or pUL93 was missing, generation of unit-length genomes was not observed, and only empty B capsids were produced. Taken together, these results show that pUL77 and pUL93 are capsid constituents needed for HCMV genome encapsidation. Therefore, the task of pUL77 seems to differ from that of its alphaherpesvirus orthologue pUL25, which exerts its function subsequent to genome cleavage-packaging.IMPORTANCEThe essential HCMV proteins pUL77 and pUL93 were suggested to be involved in viral genome cleavage-packaging but are poorly characterized both biochemically and functionally. By producing a monoclonal antibody against pUL93 and generating an HCMV mutant in which pUL77 is fused to a fluorescent protein, we show that pUL77 and pUL93 are capsid constituents, with pUL77 being similarly abundant on all capsid types. Each protein is required for genome encapsidation, as the absence of either pUL77 or pUL93 results in a genome packaging defect with the formation of empty capsids only. This distinguishes pUL77 from its alphaherpesvirus orthologue pUL25, which is enriched on DNA-filled capsids and exerts its function after the viral DNA is packaged. Our data for the first time describe an HCMV mutant with a fluorescent capsid and provide insight into the roles of pUL77 and pUL93, thus contributing to a better understanding of the HCMV encapsidation network.

scholarly journals Subunits of the Heterotrimeric Transcription Factor NF-Y Are Imported into the Nucleus by Distinct Pathways Involving Importin β and Importin 13

2005 ◽  
Vol 25 (13) ◽  
pp. 5339-5354 ◽  
Author(s):  
Joerg Kahle ◽  
Matthias Baake ◽  
Detlef Doenecke ◽  
Werner Albig
Keyword(s):  
Nuclear Import ◽  
Mutational Analysis ◽  
Dependent Manner ◽  
Amino Acid Residues ◽  
Nuclear Targeting ◽  
Binding Studies ◽  
Histone Fold ◽  
Localization Signal ◽  
Conserved Core

ABSTRACT The transcriptional activator NF-Y is a heterotrimeric complex composed of NF-YA, NF-YB, and NF-YC, which specifically binds the CCAAT consensus present in about 30% of eukaryotic promoters. All three subunits contain evolutionarily conserved core regions, which comprise a histone fold motif (HFM) in the case of NF-YB and NF-YC. Our results of in vitro binding studies and nuclear import assays reveal two different transport mechanisms for NF-Y subunits. While NF-YA is imported by an importin β-mediated pathway, the NF-YB/NF-YC heterodimer is translocated into the nucleus in an importin 13-dependent manner. We define a nonclassical nuclear localization signal (ncNLS) in NF-YA, and mutational analysis indicates that positively charged amino acid residues in the ncNLS are required for nuclear targeting of NF-YA. Importin β binding is restricted to the monomeric, uncomplexed NF-YA subunit. In contrast, the nuclear import of NF-YB and NF-YC requires dimer formation. Only the NF-YB/NF-YC dimer, but not the monomeric components, are recognized by importin 13 and are imported into the nucleus. Importin 13 competes with NF-YA for binding to the NF-YB/NF-YC dimer. Our data suggest that a distinct binding platform derived from the HFM of both subunits, NF-YB/NF-YC, mediates those interactions.

scholarly journals Nuclear Import of Cdk/Cyclin Complexes: Identification of Distinct Mechanisms for Import of Cdk2/Cyclin E and Cdc2/Cyclin B1

1999 ◽  
Vol 144 (2) ◽  
pp. 213-224 ◽  
Author(s):  
Jonathan D. Moore ◽  
Jing Yang ◽  
Ray Truant ◽  
Sally Kornbluth
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Cyclin E ◽  
Cyclin B1 ◽  
Transport Processes ◽  
Nuclear Localization Sequence ◽  
Nuclear Targeting ◽  
Importin Α

Reversible phosphorylation of nuclear proteins is required for both DNA replication and entry into mitosis. Consequently, most cyclin-dependent kinase (Cdk)/cyclin complexes are localized to the nucleus when active. Although our understanding of nuclear transport processes has been greatly enhanced by the recent identification of nuclear targeting sequences and soluble nuclear import factors with which they interact, the mechanisms used to target Cdk/cyclin complexes to the nucleus remain obscure; this is in part because these proteins lack obvious nuclear localization sequences. To elucidate the molecular mechanisms responsible for Cdk/cyclin transport, we examined nuclear import of fluorescent Cdk2/cyclin E and Cdc2/cyclin B1 complexes in digitonin-permeabilized mammalian cells and also examined potential physical interactions between these Cdks, cyclins, and soluble import factors. We found that the nuclear import machinery recognizes these Cdk/cyclin complexes through direct interactions with the cyclin component. Surprisingly, cyclins E and B1 are imported into nuclei via distinct mechanisms. Cyclin E behaves like a classical basic nuclear localization sequence–containing protein, binding to the α adaptor subunit of the importin-α/β heterodimer. In contrast, cyclin B1 is imported via a direct interaction with a site in the NH2 terminus of importin-β that is distinct from that used to bind importin-α.

scholarly journals Orange Fluorescent Proteins: Filling the Spectral Gap

2014 ◽  
Vol 70 (a1) ◽  
pp. C1670-C1670
Author(s):  
Sergei Pletnev ◽  
Daria Shcherbakova ◽  
Oksana Subach ◽  
Vladimir Malashkevich ◽  
Steven Almo ◽  
...  
Keyword(s):  
In Vivo Imaging ◽  
Spectral Properties ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Stokes Shift ◽  
Emission Wavelength ◽  
Microscopy Imaging ◽  
Large Stokes Shift

Fluorescent proteins (FPs) have become valuable tools for molecular biology, biochemistry, medicine, and cancer research. Starting from parent green fluorescent protein (GFP), most challenging task of the FPs studies was the development of FPs with longer excitation/emission wavelength. This pursuit was motivated by advantages of so-called red-shifted FPs, namely, lower background of cellular autofluorescence in microscopy, lower light scattering and reduced tissue absorbance of longer wavelengths for in vivo imaging. In addition to FPs with regular spectral properties, there are proteins of other types available, including FPs with a large Stokes shift and photoconvertible FPs. These special kinds of FPs have become useful in super-resolution microscopy, imaging of enzyme activities, protein-protein interactions, photolabeling, and in vivo imaging. According to their emission wavelength, red-shifted FPs could be divided in the following groups: 520-540 nm yellow FPs (YFPs), 540-570 nm orange FPs (OFPs), 570-620 nm red FPs (RFPs), and > 620 nm far-RFPs. Red shift of the excitation/emission bands of these FPs is predominantly achieved by extension of the conjugated system of the chromophore and its protonation/deprotonation. The variety of spectral properties of FPs (excitation and emission wavelength, quantum yield, brightness, photo- and pH- stability, photoconversion, large Stokes shift, etc) results from the different chromophore structures and its interactions with surrounding amino acid residues. In this work we focus on structural studies and molecular mechanisms of FPs with orange emission.

scholarly journals 275.Calmodulin-dependent nuclear import pathway of the testis-determining factor SRY

2004 ◽  
Vol 16 (9) ◽  
pp. 275
Author(s):  
G. Kaur ◽  
A. Delluc-Clavieres ◽  
I. Poon ◽  
D. A. Jans
Keyword(s):  
Nuclear Import ◽  
Protein Import ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Gonadal Development ◽  
Nuclear Accumulation ◽  
Related Factor ◽  
Testis Development ◽  
New Findings

Modulation of the nuclear entry of transcription factors (TFs) and chromatin components is a means by which eukaryotic cells can regulate gene expression in response to extracellular signals and the cell cycle during differentiation and development. TFs and chromatin components access the nucleus through nuclear localisation sequences (NLSs), which mediate interaction with components of the cellular nuclear import machinery, such as members of the importin superfamily. The Ca2+-binding protein calmodulin (CaM ) has previously been shown to bind at or near NLSs in several nuclear-localising proteins that have important roles in testis development including the Y chromosome-encoded HMG-domain-carrying chromatin remodelling factor SRY, and related factor SOX9, both of which are key regulators of gonadal development. SRY function in the nucleus of somatic cells of the fetal gonad, in particular, is essential for development of a testis in males. Here we present new findings implicating a role for CaM in modulating SRY nuclear accumulation, whereby treatment of transfected cells with CaM antagonists significantly reduces nuclear accumulation of green fluorescent protein (GFP)-fusion proteins encoding either full length SRY or the SRY HMG domain alone. An in vitro nuclear transport assay using bacterially expressed fluorescent proteins showed similar results, with native gel electrophoresis/fluorimaging and fluorescence polarisation assays, indicating direct binding of CaM to the SRY HMG domain in Ca2+-dependent fashion. Since clinical mutations resulting in sex reversal occur within SRY's CaM-binding NLS, these results may shed new insight into CaM-dependent pathways of nuclear protein import, and how this may relate to testis development.

scholarly journals Mapping of functional region conferring nuclear localization and karyopherin α-binding activity of the C2 protein of bhendi yellow vein mosaic virus

2012 ◽  
Vol 93 (6) ◽  
pp. 1367-1374 ◽  
Author(s):  
Sam A. Chandran ◽  
Yael Levy ◽  
Anahit Mett ◽  
Eduard Belausov ◽  
Usha Ramakrishnan ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Molecular Mechanisms ◽  
Fluorescent Proteins ◽  
Mosaic Disease ◽  
Yellow Vein ◽  
Transcriptional Gene Silencing ◽  
Functional Region ◽  
Yellow Vein Mosaic Virus

Bhendi yellow vein mosaic disease is caused by a complex consisting of a monopartite begomovirus associated with a β-satellite. The C2 protein of bhendi yellow vein mosaic virus (BYVMV) is a suppressor of post-transcriptional gene silencing and also functions as a transcriptional activator. To explore the molecular mechanisms of its nuclear trafficking and self-interaction, fusion proteins of fluorescent proteins with wild-type or mutated constructs of BYVMV C2 were expressed in tobacco protoplasts. Analyses revealed that the BYVMV C2 nuclear localization signal (NLS) was located in the N terminus of the protein, comprising aa 17–31 of C2. NLSs are recognized by a class of soluble transport receptors termed karyopherins α and β. The BYVMV C2 NLS was found to be necessary for this protein’s interaction with its nuclear import mediator, karyopherin α, ensuring its nuclear localization. Nevertheless, when deleted, C2 was found in both the cytoplasm and the nucleus, suggesting NLS-independent nuclear import of this protein. Homotypic interaction of BYVMV C2 was also found, which correlates with the nuclear localization needed for efficient activation of transcription.

Fluorescent proteins for in vivo imaging, where's the biliverdin?

2020 ◽  
Vol 48 (6) ◽  
pp. 2657-2667
Author(s):  
Felipe Montecinos-Franjola ◽  
John Y. Lin ◽  
Erik A. Rodriguez
Keyword(s):  
Hydrogen Peroxide ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Imaging ◽  
Infrared Light ◽  
Red Fluorescent Protein ◽  
Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

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