scholarly journals Laser switching contrast microscopy to monitor free and restricted diffusion inside the cell nucleus

2016 ◽  
Vol 3 (1) ◽  
Author(s):  
Franz-Josef Schmitt ◽  
Cornelia Junghans ◽  
Matthias Sturm ◽  
Csongor Keuer ◽  
Hans Joachim Eichler ◽  
...  
Keyword(s):  
Cell Nucleus ◽  
Fluorescent Protein ◽  
Mean Square Displacement ◽  
Chinese Hamster ◽  
Microscopic Technique ◽  
Cell Interior ◽  
Cell Compartments ◽  
Diffusion Constants ◽  
Laser Focus ◽  
Contrast Microscopy

AbstractA novel microscopic technique termed laser switching contrast microscopy (LSCM) allows for the imaging of the dynamics of optically switchable proteins in single cell compartments. We present an application for the monitoring of diffusive properties of single molecules of the photo-switchable fluorescent protein Dreiklang (DRK). LSCM in the cell nucleus of Chinese hamster ovary (CHO) cells cytoplasmically expressing DRK unravels quick diffusive equilibration of the DRK molecules inside the whole cytoplasm and inside the cell nucleus within seconds. The nuclear membrane is also highly permeable for DRK. Inside the nucleus entirely distinct regions are found that only partially enable diffusive protein redistribution with mean square displacement proportional to time while in other regions the mobility of the proteins seems to be restricted. After photo-switching string like patterns of light DRK molecules are observed in the cell nucleus. In addition a fraction of these DRK molecules appears immobile. The findings support recent theories of the cell interior described as a random obstacle model with an additional immobile fraction of DRK. Numerical simulations show that at different illumination intensity and different distance from the laser focus similar patterns for fluorescence recovery might be obtained in spite of strongly varying diffusion constants.

scholarly journals Evidence for a Menkes-like protein with a nuclear targeting sequence

2000 ◽  
Vol 350 (3) ◽  
pp. 855-863 ◽  
Author(s):  
Manchi C. M. REDDY ◽  
Sudeep MAJUMDAR ◽  
Edward D. HARRIS
Keyword(s):  
Cell Nucleus ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Menkes Disease ◽  
Transcript Variant ◽  
Nuclear Localization Sequence ◽  
Nuclear Targeting ◽  
Metal Binding Domain

Extracts from three human cell lines were found to contain abridged Menkes disease gene transcripts with novel insertion sequences. The transcript variant that is the focus of the present study codes for a 103-residue protein containing the first heavy-metal-binding domain (Hmb1) of ATP7A, the Cu-ATPase associated with Menkes disease. This transcript variant has a 45-bp nucleotide insert interposed between exons 1 and 2 of ATP7A that starts with a 5´ ATG that is in-frame with the downstream ATG translation start site of ATP7A. We report here that the 66-bp nucleotides positioned between the upstream and downstream ATG sites encode 22 amino acid residues whose primary structure in part meets the criteria for a nuclear-localization sequence (NLS). We have referred to the transcript as nuclear Menkes-like (NML) 45. A green fluorescent protein (GFP) construct with NML45 when transfected in Chinese hamster ovary cells localized to the cell nucleus. A similar construct without the 66-bp segment exhibited a random dispersed fluorescent pattern in the cytosol. GFP constructs encoding ATP7A exons likewise failed to direct GFP into the cell nucleus, suggesting the nuclear determinant is not in an internal domain of the protein. The data suggest that the 22-residue segment contains an NLS for an 11.2-kDa protein with one Cu-binding site that may function as a chaperone to transport Cu into the nucleus of mammalian cells.

scholarly journals Tyrosine Phosphorylation of Selected Secretory Carrier Membrane Proteins, SCAMP1 and SCAMP3, and Association with the EGF Receptor

1998 ◽  
Vol 9 (7) ◽  
pp. 1661-1674 ◽  
Author(s):  
Theodore T. Wu ◽  
J. David Castle
Keyword(s):  
Membrane Proteins ◽  
Egf Receptor ◽  
Tyrosine Phosphatase ◽  
Chinese Hamster ◽  
Tyrosine Residue ◽  
Cell Interior ◽  
Murine Fibroblasts ◽  
Secretory Carrier Membrane Proteins

Secretory carrier membrane proteins (SCAMPs) are ubiquitously expressed proteins of post-Golgi vesicles. In the presence of the tyrosine phosphatase inhibitor vanadate, or after overexpression in Chinese hamster ovary (CHO) cells, SCAMP1 and SCAMP3 are phosphorylated selectively on tyrosine residue(s). Phosphorylation is reversible after vanadate washout in situ or when isolated SCAMP3 is incubated with the recombinant tyrosine phosphatase PTP1B. Vanadate also causes the partial accumulation of SCAMP3, but not SCAMP1, in “patches” at or near the cell surface. A search for SCAMP kinase activities has shown that SCAMPs 1 and 3, but not SCAMP2, are tyrosine phosphorylated in EGF-stimulated murine fibroblasts overexpressing the EGF receptor (EGFR). EGF catalyzes the progressive phosphorylation of the SCAMPs up to 1 h poststimulation and may enhance colocalization of the EGFR and SCAMP3 within the cell interior. EGF also induces SCAMP–EGFR association, as detected by coimmunoprecipitation, and phosphorylation of SCAMP3 is stimulated by the EGFR in vitro. These results suggest that phosphorylation of SCAMPs, either directly or indirectly, may be functionally linked to the internalization/down-regulation of the EGFR.

scholarly journals Hydrophobic Regions Adjacent to Transmembrane Domains 1 and 5 Are Important for the Targeting of the 70-kDa Peroxisomal Membrane Protein

2007 ◽  
Vol 282 (46) ◽  
pp. 33831-33844 ◽  
Author(s):  
Yoshinori Kashiwayama ◽  
Kota Asahina ◽  
Masashi Morita ◽  
Tsuneo Imanaka
Keyword(s):  
Amino Acid ◽  
Membrane Protein ◽  
Protein Targeting ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Intracellular Localization ◽  
Chinese Hamster ◽  
Transmembrane Domains ◽  
Peroxisomal Membrane ◽  
Peroxisomal Membrane Protein

The 70-kDa peroxisomal membrane protein (PMP70) is a major component of peroxisomal membranes. Human PMP70 consists of 659 amino acid residues and has six putative transmembrane domains (TMDs). PMP70 is synthesized on cytoplasmic ribosomes and targeted posttranslationally to peroxisomes by an unidentified peroxisomal membrane protein targeting signal (mPTS). In this study, to examine the mPTS within PMP70 precisely, we expressed various COOH-terminally or NH2-terminally deleted constructs of PMP70 fused with green fluorescent protein (GFP) in Chinese hamster ovary cells and determined their intracellular localization by immunofluorescence. In the COOH-terminally truncated PMP70, PMP70(AA.1-144)-GFP, including TMD1 and TMD2 of PMP70, was still localized to peroxisomes. However, by further removal of TMD2, PMP70(AA.1-124)-GFP lost the targeting ability, and PMP70(TMD2)-GFP did not target to peroxisomes by itself. The substitution of TMD2 in PMP70(AA.1-144)-GFP for TMD4 or TMD6 did not affect the peroxisomal localization, suggesting that PMP70(AA.1-124) contains the mPTS and an additional TMD is required for the insertion into the peroxisomal membrane. In the NH2-terminal 124-amino acid region, PMP70 possesses hydrophobic segments in the region adjacent to TMD1. By the disruption of these hydrophobic motifs by the mutation of L21Q/L22Q/L23Q or I70N/L71Q, PMP70(AA.1-144)-GFP lost targeting efficiency. The NH2-terminally truncated PMP70, GFP-PMP70(AA.263-375), including TMD5 and TMD6, exhibited the peroxisomal localization. PMP70(AA.263-375) also possesses hydrophobic residues (Ile307/Leu308) in the region adjacent to TMD5, which were important for targeting. These results suggest that PMP70 possesses two distinct targeting signals, and hydrophobic regions adjacent to the first TMD of each region are important for targeting.

scholarly journals mTORC1 controls phase-separation and the biophysical properties of the cytoplasm by tuning crowding

2017 ◽  
Author(s):  
M. Delarue ◽  
G.P. Brittingham ◽  
S. Pfeffer ◽  
I.V. Surovtsev ◽  
S. Ping-lay ◽  
...  
Keyword(s):  
Phase Separation ◽  
Fluorescent Protein ◽  
Effective Diffusion ◽  
Reaction Rates ◽  
Biophysical Properties ◽  
Cell Interior ◽  
Profound Impact ◽  
Mtorc1 Pathway

Summary (Abstract)Macromolecular crowding has a profound impact on reaction rates and the physical properties of the cell interior, but the mechanisms that regulate crowding are poorly understood. We developed Genetically Encoded Multimeric nanoparticles (GEMs) to dissect these mechanisms. GEMs are homomultimeric scaffolds fused to a fluorescent protein. GEMs self-assemble into bright, stable fluorescent particles of defined size and shape. By combining tracking of GEMs with genetic and pharmacological approaches, we discovered that the mTORC1 pathway can tune the effective diffusion coefficient of macromolecules ≥15 nm in diameter more than 2-fold without any discernable effect on the motion of molecules ≥5 nm. These mTORCI-dependent changes in crowding and rheology affect phase-separation both in vitro and in vivo. Together, these results establish a role for mTORCI in controlling both the biophysical properties of the cytoplasm and the phase-separation of biopolymers.

scholarly journals Fluorescent protein-tagged Vpr dissociates from HIV-1 core after viral fusion and rapidly enters the cell nucleus

Retrovirology ◽  
2015 ◽  
Vol 12 (1) ◽  
Author(s):  
Tanay M. Desai ◽  
Mariana Marin ◽  
Chetan Sood ◽  
Jiong Shi ◽  
Fatima Nawaz ◽  
...  
Keyword(s):  
Cell Nucleus ◽  
Fluorescent Protein ◽  
Viral Fusion ◽  
Hiv 1

scholarly journals Characterization of the transcriptional repressor RBP in Epstein–Barr virus-transformed B cells

1999 ◽  
Vol 80 (12) ◽  
pp. 3217-3226 ◽  
Author(s):  
Kenia G. Krauer ◽  
Marion Buck ◽  
Tom Sculley
Keyword(s):  
B Cells ◽  
Cell Nucleus ◽  
Epstein Barr Virus ◽  
Fluorescent Protein ◽  
Transcriptional Repressor ◽  
Free Form ◽  
Nuclear Fraction ◽  
Nuclear Staining ◽  
Barr Virus ◽  
Epstein Barr

RBP, a transcriptional repressor, is intricately involved in Epstein–Barr virus (EBV) transformation of human B cells. The EBV nuclear proteins EBNA-2, -3, -4 and -6 all utilize RBP to regulate the transcription of both cellular and viral genes. This study investigates the isoforms of the RBP protein in Burkitt’s lymphoma (BL) cells and in EBV-transformed lymphoblastoid cell lines (LCLs). Two-dimensional gel electrophoresis showed the presence of two different cellular isoforms of RBP; the molecular masses and isoelectric points of these two isoforms corresponded to RBP-Jκ and RBP-2N. Fractionation studies and green fluorescent protein (GFP)-tagged expression studies demonstrated that both RBP isoforms were located predominantly in the cell nucleus. Interestingly, GFP-tagged RBP-Jκ showed diffuse, uniform nuclear staining, whereas GFP-tagged RBP-2N showed a discrete nuclear pattern, demonstrating differences between the two isoforms. Within the nuclear fraction of EBV-negative BL cells, RBP existed both in a free form and bound to chromatin, whereas in LCLs the intranuclear RBP was predominantly chromatin-bound. Expression of the EBV latent proteins was found to lead to the sequestering of RBP from the cytoplasm into the cell nucleus and to an increase in the chromatin-bound forms of RBP.

scholarly journals Peptidoglycan Synthesis Machinery in Agrobacterium tumefaciens During Unipolar Growth and Cell Division

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Todd A. Cameron ◽  
James Anderson-Furgeson ◽  
John R. Zupan ◽  
Justin J. Zik ◽  
Patricia C. Zambryski
Keyword(s):  
Cell Division ◽  
Agrobacterium Tumefaciens ◽  
Fluorescent Protein ◽  
Polar Growth ◽  
Growth Strategies ◽  
Cell Width ◽  
Content Type ◽  
E Coli ◽  
Cell Compartments ◽  
New Material

ABSTRACT The synthesis of peptidoglycan (PG) in bacteria is a crucial process controlling cell shape and vitality. In contrast to bacteria such as Escherichia coli that grow by dispersed lateral insertion of PG, little is known of the processes that direct polar PG synthesis in other bacteria such as the Rhizobiales. To better understand polar growth in the Rhizobiales Agrobacterium tumefaciens, we first surveyed its genome to identify homologs of (~70) well-known PG synthesis components. Since most of the canonical cell elongation components are absent from A. tumefaciens, we made fluorescent protein fusions to other putative PG synthesis components to assay their subcellular localization patterns. The cell division scaffolds FtsZ and FtsA, PBP1a, and a Rhizobiales- and Rhodobacterales-specific l,d-transpeptidase (LDT) all associate with the elongating cell pole. All four proteins also localize to the septum during cell division. Examination of the dimensions of growing cells revealed that new cell compartments gradually increase in width as they grow in length. This increase in cell width is coincident with an expanded region of LDT-mediated PG synthesis activity, as measured directly through incorporation of exogenous d-amino acids. Thus, unipolar growth in the Rhizobiales is surprisingly dynamic and represents a significant departure from the canonical growth mechanism of E. coli and other well-studied bacilli. IMPORTANCE Many rod-shaped bacteria, including pathogens such as Brucella and Mycobacteriu, grow by adding new material to their cell poles, and yet the proteins and mechanisms contributing to this process are not yet well defined. The polarly growing plant pathogen Agrobacterium tumefaciens was used as a model bacterium to explore these polar growth mechanisms. The results obtained indicate that polar growth in this organism is facilitated by repurposed cell division components and an otherwise obscure class of alternative peptidoglycan transpeptidases (l,d-transpeptidases). This growth results in dynamically changing cell widths as the poles expand to maturity and contrasts with the tightly regulated cell widths characteristic of canonical rod-shaped growth. Furthermore, the abundance and/or activity of l,d-transpeptidases appears to associate with polar growth strategies, suggesting that these enzymes may serve as attractive targets for specifically inhibiting growth of Rhizobiales, Actinomycetales, and other polarly growing bacterial pathogens.

scholarly journals Generation of Transduction-Competent Retroviral Vectors by Infection with a Single Hybrid Vaccinia Virus

2003 ◽  
Vol 77 (12) ◽  
pp. 7017-7025 ◽  
Author(s):  
Christian Konetschny ◽  
Georg W. Holzer ◽  
Carsten Urban ◽  
Thomas Hämmerle ◽  
Josef Mayrhofer ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Host Range ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Retroviral Vectors ◽  
Host Cells ◽  
Rabbit Kidney ◽  
Single Infection ◽  
Broad Host Range

ABSTRACT Recombinant vaccinia viruses that express defective retroviral vectors upon a single infection event in normal host cells were constructed. The gag-pol and envelope genes and a retroviral vector unit were inserted as vaccinia virus promoter-controlled transcription units at three separate loci. The triple recombinant virus was used to infect such diverse cell types as monkey and rabbit kidney, human lung, and primary chicken cells, resulting in the production of transduction-competent defective retroviral vectors. Infection of Chinese hamster ovary cells, which are nonpermissive for vaccinia virus replication, also resulted in production of retroviral vectors and secondary permanent transduction of the host cells. Since vaccinia virus supports the expression of cytotoxic proteins, the vesicular stomatitis virus G glycoprotein could be chosen as the envelope allowing a broad host range of transduction. Functionality of particles was monitored by expression of the green fluorescent protein in transduced 3T3 cell clones. This is the first description of a single chimeric virus encoding and releasing functional retroviral vectors, providing proof of principle of the new concept. No replication-competent retrovirus was detectable by sensitive reverse transcriptase assays. Since vaccinia virus has a broad host range, is extremely robust, and can be obtained at high titers and safe nonreplicating vaccinia virus strains are available, the hybrid system may open new perspectives for gene delivery.

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