Generation of fluorescent HCV pseudoparticles to study early viral entry events-involvement of Rab1a in HCV entry

Understanding the early events in viral biology holds the key to the development of preventives. In this study fluorescent Hepatitis C Virus pseudoparticles have been generated where the envelope glycoprotein has a GFP tag. Using these pseudoparticles entry assays were conducted where the entry of the pseudoparticles was tracked via confocal microscopy. Using this system, fusion of host and viral membranes is predicted to occur within 15 minutes of entry in HCV. Using cells with a knockdown for Rab1a, HCV trafficking was observed to be altered, indicating a role of Rab1a in HCV trafficking. In conclusion, this study reports the generation and use of fluorescent pseudoparticles which may be used to understand the early events of viral entry. This system may be adapted for the study of other enveloped viruses as well.HighlightsFluorescent HCV pseudoparticles have been created to study early entry events.HCV entry tracking via confocal microscopy reveals fusion within 15 minutes.Rab1a is important for HCV trafficking within the cell.

Gold labelling of Green Fluorescent Protein (GFP)-tag inside cells using recombinant nanobodies conjugated to 2.4 nm thiolate-coated gold nanoparticles

The advances in the microscopy technology have prompted efforts to improve the reagents required to recognize specific molecules within the intracellular environment. For high-resolution electron microscopy, conjugation of selective binders...

Light-Regulated Transcription of a Mitochondrial-Targeted K+ Channel

The inner membranes of mitochondria contain several types of K+ channels, which modulate the membrane potential of the organelle and contribute in this way to cytoprotection and the regulation of cell death. To better study the causal relationship between K+ channel activity and physiological changes, we developed an optogenetic platform for a light-triggered modulation of K+ conductance in mitochondria. By using the light-sensitive interaction between cryptochrome 2 and the regulatory protein CIB1, we can trigger the transcription of a small and highly selective K+ channel, which is in mammalian cells targeted into the inner membrane of mitochondria. After exposing cells to very low intensities (≤0.16 mW/mm2) of blue light, the channel protein is detectable as an accumulation of its green fluorescent protein (GFP) tag in the mitochondria less than 1 h after stimulation. This system allows for an in vivo monitoring of crucial physiological parameters of mitochondria, showing that the presence of an active K+ channel causes a substantial depolarization compatible with the effect of an uncoupler. Elevated K+ conductance also results in a decrease in the Ca2+ concentration in the mitochondria but has no impact on apoptosis.

Tagging the proteasome active site β5 causes tag specific phenotypes in yeast

The efficient and timely degradation of proteins is crucial for many cellular processes and to maintain general proteostasis. The proteasome, a complex multisubunit protease, plays a critical role in protein degradation. Therefore, it is important to understand the assembly, regulation, and localization of proteasome complexes in the cell under different conditions. Fluorescent tags are often utilized to study proteasomes. A GFP-tag on the β5 subunit, one of the core particle (CP) subunits with catalytic activity, has been shown to be incorporated into proteasomes and commonly used by the field. We report here that a tag on this subunit results in aberrant phenotypes that are not observed when several other CP subunits are tagged. These phenotypes appear in combination with other proteasome mutations and include poor growth, and, more significantly, altered 26S proteasome localization. In strains defective for autophagy, β5-GFP tagged proteasomes, unlike other CP tags, localize to granules upon nitrogen starvation. These granules are reflective of previously described proteasome storage granules but display unique properties. This suggests proteasomes with a β5-GFP tag are specifically recognized and sequestered depending on physiological conditions. In all, our data indicate the intricacy of tagging proteasomes, and possibly, large complexes in general.

TLR5: A prognostic and monitoring indicator for triple-negative breast cancer

A novel, highly selective biomarker is urgently needed to predict and monitor triple-negative breast cancer (TNBC) because targeting molecules are not currently available. Although associated with various malignant tumors, the role of toll-like receptor 5 (TLR5) in TNBC remains uncertain. We aimed to define the effects of TLR5 in TNBC to determine whether it could serve as a prognostic and monitoring indicator for TNBC. We established TNBC cell line 4T1 with low TLR5 expression (GFP tag; TLR5− 4T1) and with normal TLR5 expression (GFP tag; TLR5+ 4T1) using lentivirus-shRNA-TLR5 knockdown transfection and negative lentivirus transfection, respectively. Detected by western blot and qPCR, we found knockdown of TLR5 resulted in decreased expression of TLR5 and E-cadherin and increased expression of N-cadherin, vimentin, fibronectin, TRAF6, SOX2, and Twist1, which were related to EMT (epithelial–mesenchymal transition). In addition, downregulation of TLR5 increased the invasion and migration of 4T1 cells in vitro, which were investigated by CCK-8 and wound healing, as well as transwell assay and colony formation. Furthermore, the metastatic ability of TLR5− 4T1 cells to the lungs was also increased compared to TLR5+ 4T1 cells in vivo. To verify the effect of TLR5 as a monitor indicator, mice bearing TLR5+ and TLR5− 4T1 tumors injected with 125I-anti-TLR5 mAb or isotype 125I-IgG were assessed by whole body phosphor-autoradiography and fluorescence imaging in vivo. Phosphor-autoradiography of model mice revealed early tumors at 6 days after inoculation with TLR5+ 4T1, but not TLR5− 4T1 cells. Intratumoral accumulation of radioactivity positively correlated with TLR5 expression, and fluorescence imaging in vivo revealed both TLR5+ and TLR5− 4T1 tumors. Our results suggested that downregulation of TLR5 in TNBC increased tumor invasiveness and EMT expression via TRAF6 and SOX2 pathway and TLR5 could serve as a prognostic and monitoring indicator for TLR5-positive tumors.

Unintended inhibition of protein function using GFP nanobodies in human cells

Tagging a protein-of-interest with GFP using genome editing is a popular approach to study protein function in cell and developmental biology. To avoid re-engineering cell lines or organisms in order to introduce additional tags, functionalized nanobodies that bind GFP can be used to extend the functionality of the GFP tag. We developed functionalized nanobodies, which we termed “dongles”, that could add, for example, an FKBP tag to a GFP-tagged protein-of-interest; enabling knocksideways experiments in GFP knock-in cell lines. The power of knocksideways is that it allows investigators to rapidly switch the protein from an active to an inactive state. We show that dongles allow for effective knocksideways of GFP-tagged proteins in genome-edited human cells. However, we discovered that nanobody binding to dynamin-2-GFP caused inhibition of dynamin function prior to knocksideways. While this limitation might be specific to the protein studied, it was significant enough to convince us not to pursue development of dongle technology further.

Influence of the combination of last sense codon and stop codon on expression efficiency of green fluorescent protein gene in Escherichia coli when the expression vector pKK223-3

In this paper, the influence of the combination of last sense codon and stop codon on expression efficiency was studied. By our study, for the last sense codon CCG, the RFI of GFP(CCG) was 2.1 fold when the stop codon was UAA, but in comparison, the RFI was 1.1 fold when the stop codon was changed from UAA to UAG. For last sense codon TAG, the RFI of GFP(TAG) with the stop codon UAG was stronger than that with the stop codons UAA and UGA.