Recently, highly sensitive nanotubular structures mediating membrane continuity between mammalian cells have been discovered. With respect to their peculiar architecture, these membrane channels were ...termed tunneling nanotubes (TNTs). TNTs could form de novo between animal cells leading to the generation of complex cellular networks. They have been shown to facilitate the intercellular transfer of organelles as well as, on a limited scale, of membrane components and cytoplasmic molecules. It has been proposed that TNTs represent a novel and general biological principle of cell-to-cell communication and it becomes increasingly apparent that they fulfill important functions in the physiological processes of multicellular organisms.
Virus‐like particles‐based vaccines have been gaining interest in recent years. The manufacturing of these particles includes their production by cell culture followed by their purification to meet ...the requirements of its final use. The presence of host cell extracellular vesicles represents a challenge for better virus‐like particles purification, because both share similar characteristics which hinders their separation. The present study aims to compare some of the most used downstream processing technologies for capture and purification of virus‐like particles. Four steps of the purification process were studied, including a clarification step by depth filtration and filtration, an intermediate step by tangential flow filtration or multimodal chromatography, a capture step by ion exchange, heparin affinity and hydrophobic interaction chromatography and finally, a polishing step by size exclusion chromatography. In each step, the yields were evaluated by percentage of recovery of the particles of interest, purity, and elimination of main contaminants. Finally, a complete purification train was implemented using the best results obtained in each step. A final concentration of 1.40 × 1010 virus‐like particles (VLPs)/mL with a purity of 64% after the polishing step was achieved, with host cell DNA and protein levels complaining with regulatory standards, and an overall recovery of 38%. This work has resulted in the development of a purification process for HIV‐1 Gag‐eGFP virus‐like particles suitable for scale‐up.
Oxidases are of interest to chemical and pharmaceutical industries because they catalyze highly selective oxidations. However, oxidases found in nature often need to be re‐engineered for synthetic ...applications. Herein, we developed a versatile and robust flow cytometry‐based screening platform “FlOxi” for directed oxidase evolution. FlOxi utilizes hydrogen peroxide produced by oxidases expressed in E. coli to oxidize Fe2+ to Fe3+ (Fenton reaction). Fe3+ mediates the immobilization of a His6‐tagged eGFP (eGFPHis) on the E. coli cell surface, ensuring the identification of beneficial oxidase variants by flow cytometry. FlOxi was validated with two oxidases—a galactose oxidase (GalOx) and a D‐amino acid oxidase (D‐AAO)—yielding a GalOx variant (T521A) with a 4.4‐fold lower Km value and a D‐AAO variant (L86M/G14/A48/T205) with a 4.2‐fold higher kcat than their wildtypes. Thus, FlOxi can be used for the evolution of hydrogen peroxide‐producing oxidases and applied for non‐fluorescent substrates.
An ultrahigh‐throughput screening platform “FlOxi” was developed by integrating oxidation/Fenton reaction cascade, Fe3+ mediated eGFPHis decoration, and fluorescence‐activated‐cell‐sorting into a general strategy. FlOxi was validated in two evolution campaigns targeting two oxidases, exemplifying the suitability of the platform for screening oxidase variants in an ultrahigh‐throughput manner.
Aggregated Aβ peptides which cause amyloid deposits, a characteristic of Alzheimer's disease (AD), activate a stress response in the endoplasmic reticulum (ER), known as the unfolded protein ...response, UPRER. Nascent UPRER induction helps in reducing ER stress by eliminating accumulated misfolded/aggregated secretory proteins. However, prolonged UPRER induction may trigger apoptosis. Here we show that, when expressed in yeast with an NH2-terminal secretory signal sequence (ss), the 42-amino acid human Aβ42 (h_Aβ42), but not the mouse/ratAβ42 (m_Aβ42) which reportedly does not misfold/aggregate, induces UPRER as monitored via an eGFP reporter. We also show that expression of ss-h_Aβ42, not ss-m_Aβ42, blocks yeast cell growth, with cells expressing ss-h_Aβ42 manifesting distinctive features of apoptosis such as loss of mitochondrial membrane potential, increase in ROS levels and DNA fragmentation. Screening for suppressors of ss-h_Aβ42-activated UPRER-eGFP induction, in a computationally-designed 29-compound methoxy-stilbene library, revealed three compounds that reduce >95% of UPRER-eGFP induction at 5 μM concentration, with EC50 values of 40–50 nM. Surprisingly, the compounds also rescue yeast cells from ss-h_Aβ42-mediated apoptosis, with EC50-s of 50–60 nM. These results provide direct evidence, probably for the first time, that there is a direct correlation between deactivation of UPRER and attenuation of apoptosis.
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Acrosomal exocytosis (AR) is a critical process that sperm need to undergo to fertilize an egg. The evaluation of the presence or absence of the acrosome is usually performed by using lectins or dyes ...in fixed cells. With this approach, it is neither possible to monitor the dynamic process of exocytosis and related molecular events while discriminating between live and dead cells, nor to evaluate the acrosomal status while sperm reside in the female reproductive tract. However, over the last two decades, several new methodologies have been used to assess the occurrence of AR in living cells allowing different groups to obtain information that was not possible in the past. These techniques have revolutionized the whole study of this process. This review summarizes current methods available to analyze AR in living cells as well as the important information that emerged from studies using these approaches.
This review summarizes current methods available to analyze the acrosomal exocytosis in living sperm as well as the important information that emerged from studies using these approaches.
We describe the utility of a tandem-tagged autophagy reporter mouse model (CAG-RFP-EGFP-MAP1LC3B) in investigating basal macroautophagic/autophagic flux in the neural retina. Western blot, in situ ...hybridization, immunohistochemistry, and confocal microscopy showed that CAG promoter-driven expression of RFP-EGFP-MAP1LC3B increased "cytosolic" RFP-EGFP-LC3B-I levels, whereas RFP-EGFP-LC3B-II decorates true phagosomes. We verified that the electroretinographic (ERG) responses of tandem-tagged LC3B mice were comparable to those of age-matched controls. Optimized microscope settings detected lipofuscin autofluorescence in retinas of abca4
−/-
mice. The majority of retinal phagosomes in the reporter mice exhibited only RFP (not EGFP) fluorescence, suggesting rapid maturation of phagosomes. Only ~1.5% of the total phagosome population was EGFP-labeled; RFP-labeled (mature) phagosomes colocalized with lysosomal markers LAMP2 and CTSD. In the outer retina, phagosome sizes were as follows (in µm
2
, ave ± SEM): RPE, 0.309 ± 0.015; photoreceptor inner segment-myoid, 0.544 ± 0.031; and outer nuclear layer, 0.429 ± 0.011. Detection of RPE phagosomes by fluorescence microscopy is challenging, due to the presence of melanin. Increased lipofuscin autofluorescence, such as observed in the abca4
−/-
mouse model of Stargardt disease, is a strong confounding factor when attempting to study autophagy in the RPE. In addition to RPE and photoreceptor cells, phagosomes were detected in inner retinal cell types, microglia, astrocytes, and endothelial cells. We conclude that the tandem-tagged LC3B mouse model serves as a useful system for studying autophagy in the retina. This utility, however, is dependent upon several technical and biological factors, including microscope settings, transgene expression, choice of fluorophores, and lipofuscin autofluorescence.
Abbreviations: ACTB: actin, beta; AIF1: allograft inflammatory factor 1; ATG: autophagy related; CTSD: cathepsin D; DAPI: (4',6-diamido-2-phenylindole); DIC: differential interference contrast; EGFP: enhanced green fluorescent protein; ELM: external limiting membrane; ERG: electroretinography; GCL: ganglion cell layer; GLUL: glutamine-ammonia ligase (glutamine synthetase); INL: inner nuclear layer; IS-E/M: inner segment - ellipsoid/myoid; ISH: in situ hybridization; LAMP2: lysosomal-associated membrane protein 2; L.I.: laser Intensity; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; O.C.T.: optimal cutting temperature; OS: outer segment; ONL: outer nuclear layer; PE: phosphatidylethanolamine; RFP: red fluorescent protein; R.O.I.: region of interest; RPE: retinal pigment epithelium
The HIV-1 capsid protein performs multiple roles in virus replication both during assembly and particle release and during virus trafficking into the nucleus. In order to decipher the roles of capsid ...protein during early replication, a reliable method to follow its intracellular distribution is required. To complement existing approaches to track HIV-1 capsid during early infection, we developed an HIV-1 imaging strategy, relying on viruses incorporating enhanced green fluorescent protein (eGFP)-tagged capsid (CA-eGFP) protein and mCherry-tagged integrase (IN-mCherry). Wild-type infectivity and sensitivity to inhibition by PF74 point to the functionality of CA-eGFP-containing complexes. Low numbers of CA-eGFP molecules were located inside the viral core and imported into the nucleus without significant loss in intensity. Less than 5% of particles carrying both CA-eGFP and IN-mCherry retained both labelled proteins after nuclear entry, implying a major uncoating event at the nuclear envelope dissociating IN and CA. Still, 20% of all CA-eGFP-containing complexes were detected in the nucleus. Unlike for IN-mCherry complexes, addition of the integrase inhibitor raltegravir had no effect on CA-eGFP-containing complexes, suggesting that these may be not (yet) competent for integration. Our imaging strategy offers alternative visualization of viral capsid trafficking and helps clarify its potential role during integration.
HIV-1 capsid protein (CA) builds a conical shell protecting viral genomic RNA inside the virus particles. Upon entry into host cells, this shell disassembles in a process of uncoating, which is coordinated with reverse transcription of viral RNA into DNA. After uncoating, a portion of CA remains associated with the viral DNA and mediates its nuclear import and, potentially, integration into host DNA. In this study, we tagged CA with eGFP to follow its trafficking in host cells and address potential CA roles in the nucleus. We found that while functional viruses import the tagged CA into the nucleus, this capsid protein is not part of integration-competent complexes. The roles of nuclear CA thus remain to be established.
Abstract Transgenic mice that express enhanced green fluorescent protein (EGFP) under the control of a cell-specific promoter have been used with great success to identify and label specific cell ...types of the retina. We studied the expression of EGFP in the retina of mice making use of four transgenic mouse lines. Expression of EGFP driven by the calretinin promoter was found in amacrine, displaced amacrine and ganglion cells. Comparison of the EGFP expression and calretinin immunolabeling showed that many but not all cells appear to be double labeled. Expression of EGFP under the control of the choline acetyltransferase promoter was found in amacrine cells; however, the cells did not correspond to the well known cholinergic (starburst) cells of the mouse retina. The expression of EGFP under the control of the parvalbumin promoter was restricted to amacrine cells of the inner nuclear layer and to cells of the ganglion cell layer (displaced amacrine cells and ganglion cells). Most of the cells were also immunoreactive for parvalbumin, however, differences in labeling intensity were observed. The expression of EGFP driven by the promoter for the 5-HT3 A receptor (5-HTR3A) was restricted to type 5 bipolar cells. In contrast, immunostaining for 5-HTR3A was found in synaptic hot spots in sublamina 1 of the inner plexiform layer and was not related to type 5 bipolar cells. The results show that these transgenic mice are very useful for future electrophysiological studies of specific types of amacrine and bipolar cells that express EGFP and thus permit directed microelectrode targeting under microscopic control.
Our understanding of how otoferlin, the major calcium sensor in inner hair cells (IHCs) synaptic transmission, contributes to the overall dynamics of synaptic vesicle (SV) trafficking remains ...limited. To address this question, we generated a knock-in mouse model expressing an otoferlin-GFP protein, where GFP was fused to its C-terminal transmembrane domain. Similar to the wild type protein, the GFP-tagged otoferlin showed normal expression and was associated with IHC SV. Surprisingly, while the heterozygote Otof+/GFP mice exhibited a normal hearing function, homozygote OtofGFP/GFP mice were profoundly deaf attributed to severe reduction in SV exocytosis. Fluorescence recovery after photobleaching revealed a markedly increased mobile fraction of the otof-GFP-associated SV in Otof GFP/GFP IHCs. Correspondingly, 3D-electron tomographic of the ribbon synapses indicated a reduced density of SV attached to the ribbon active zone. Collectively, these results indicate that otoferlin requires a free intravesicular C-terminal end for normal SV docking and fusion.
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•We engineered a knock-in mouse model expressing an Otof-GFP fusion gene (with GFP fused to the Otof intravesicular C-ter).•This model allowed us to monitor synaptic vesicle mobility in live inner hair cells.•Homozygote Otof GFP/GFP mice were profoundly deaf while heterozygote Otof +/GFP had normal hearing function.•The hearing deficit in Otof GFP/GFP mice is caused by a severe reduction in synaptic vesicle exocytosis.•We propose that the proper docking and fusion of the synaptic vesicles depend on the self-dimerization of Otof via its TMD.
A novel bacterial display vector based on Escherichia coli has been engineered for recombinant protein production and purification. Accordingly, a construct harboring the enhanced green fluorescent ...protein (EGFP) and the ice nucleation protein (INP) was designed to produce EGFP via the surface display in E. coli cells. The fusion EGFP-expressed cells were then investigated using fluorescence measurement, SDS- and native-PAGE before and after TEV protease digestion. The displayed EGFP was obtained with a recovery of 57.7 % as a single band on SDS-PAGE. Next, the efficiency of the cell surface display for mutant EGFP (EGFP S202H/Q204H) was examined in sensing copper ions. Under optimal conditions, a satisfactorily linear range for copper ions concentrations up to 10 nM with a detection limit of 0.073 nM was obtained for cell-displayed mutant EGFP (mEGFP). In the presence of bacterial cell lysates and purified mEGFP, response to copper was linear in the 2–10 nM and 0.1–2 μM concentration range, respectively, with a 1.3 nM and 0.14 μM limit of detection. The sensitivity of bacterial cell lysates and surface-displayed mEGFP in the detection of copper ions is higher than the purified mEGFP.
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•Engineering a novel bacterial display system based on NTD of InaK as the efficient carrier.•The display system provides an efficient approach to purifying the recombinant proteins.•Mutant EGFP displayed on the surface of bacteria cells provides a highly sensitive fluorescence sensor for Cu2+.
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