scholarly journals Real-Time Imaging of Polioviral RNA Translocation across a Membrane

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Krishanthi S. Karunatilaka ◽  
David J. Filman ◽  
Mike Strauss ◽  
Joseph J. Loparo ◽  
James M. Hogle
Keyword(s):  
Real Time ◽  
Cellular Membrane ◽  
Cell Entry ◽  
Viral Genomes ◽  
Cellular Assays ◽  
Complex Process ◽  
Nonenveloped Virus ◽  
A Cell ◽  
Membrane Spanning

ABSTRACT Genome transfer from a virus into a cell is a critical early step in viral replication. Enveloped viruses achieve the delivery of their genomes into the cytoplasm by merging the viral membrane with the cellular membrane via a conceptually simple mechanism called membrane fusion. In contrast, genome translocation mechanisms in nonenveloped viruses, which lack viral membranes, remain poorly understood. Although cellular assays provide useful information about cell entry and genome release, it is difficult to obtain detailed mechanistic insights due both to the inherent technical difficulties associated with direct visualization of these processes and to the prevalence of nonproductive events in cellular assays performed at a very high multiplicity of infection. To overcome these issues, we developed an in vitro single-particle fluorescence assay to characterize genome release from a nonenveloped virus (poliovirus) in real time using a tethered receptor-decorated liposome system. Our results suggest that poliovirus genome release is a complex process that consists of multiple rate-limiting steps. Interestingly, we found that the addition of exogenous wild-type capsid protein VP4, but not mutant VP4, enhanced the efficiency of genome translocation. These results, together with prior structural analysis, suggest that VP4 interacts with RNA directly and forms a protective, membrane-spanning channel during genome translocation. Furthermore, our data indicate that VP4 dynamically interacts with RNA, rather than forming a static tube for RNA translocation. This study provides new insights into poliovirus genome translocation and offers a cell-free assay that can be utilized broadly to investigate genome release processes in other nonenveloped viruses. IMPORTANCE The initial transfer of genomic material from a virus into a host cell is a key step in any viral infection. Consequently, understanding how viruses deliver their genomes into cells could reveal attractive therapeutic targets. Although conventional biochemical and cellular assays have provided useful information about cell entry, the mechanism used to deliver the viral genomes across the cellular membrane into the cytoplasm is not well characterized for nonenveloped viruses such as poliovirus. In this study, we developed a fluorescence imaging assay to visualize poliovirus genome release using a synthetic vesicle system. Our results not only provide new mechanistic insights into poliovirus genome translocation but also offer a cell-free assay to bridge gaps in understanding of this process in other nonenveloped viruses.

scholarly journals A cell-free platform for rapid synthesis and testing of active oligosaccharyltransferases

2017 ◽  
Author(s):  
Jennifer A. Schoborg ◽  
Jasmine Hershewe ◽  
Jessica C. Stark ◽  
Weston Kightlinger ◽  
James E. Kath ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Cellular Membrane ◽  
Production Method ◽  
Protein Glycosylation ◽  
Campylobacter Coli ◽  
Amino Acid Residues ◽  
Target Proteins ◽  
A Cell ◽  
Campylobacter Lari

AbstractProtein glycosylation, or the attachment of sugar moieties (glycans) to proteins, is important for protein stability, activity, and immunogenicity. However, understanding the roles and regulations of site-specific glycosylation events remains a significant challenge due to several technological limitations. These limitations include a lack of available tools for biochemical characterization of enzymes involved in glycosylation. A particular challenge is the synthesis of oligosaccharyltransferases (OSTs), which catalyze the attachment of glycans to specific amino acid residues in target proteins. The difficulty arises from the fact that canonical OSTs are large (>70 kDa) and possess multiple transmembrane helices, making them difficult to overexpress in living cells. Here, we address this challenge by establishing a bacterial cell-free protein synthesis platform that enables rapid production of a variety of OSTs in their active conformations. Specifically, by using lipid nanodiscs as cellular membrane mimics, we obtained yields of up to 440 µg/mL for the single-subunit OST enzyme, ‘Protein glycosylation B’ (PglB) from Campylobacter jejuni, as well as for three additional PglB homologs from Campylobacter coli, Campylobacter lari, and Desulfovibrio gigas. Importantly, all of these enzymes catalyzed N-glycosylation reactions in vitro with no purification or processing needed. Furthermore, we demonstrate the ability of cell-free synthesized OSTs to glycosylate multiple target proteins with varying N-glycosylation acceptor sequons. We anticipate that this broadly applicable production method will advance glycoengineering efforts by enabling preparative expression of membrane-embedded OSTs from all kingdoms of life.

scholarly journals Cell adhesion molecule IGPR-1 activates AMPK connecting cell adhesion to autophagy

2020 ◽  
Vol 295 (49) ◽  
pp. 16691-16699
Author(s):  
Razie Amraei ◽  
Tooba Alwani ◽  
Rachel Xi-Yeen Ho ◽  
Zahra Aryan ◽  
Shawn Wang ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Cellular Assays ◽  
Iκb Kinase ◽  
Promising Therapeutic Target ◽  
A Cell ◽  
Subsequent Activation

Autophagy plays critical roles in the maintenance of endothelial cells in response to cellular stress caused by blood flow. There is growing evidence that both cell adhesion and cell detachment can modulate autophagy, but the mechanisms responsible for this regulation remain unclear. Immunoglobulin and proline-rich receptor-1 (IGPR-1) is a cell adhesion molecule that regulates angiogenesis and endothelial barrier function. In this study, using various biochemical and cellular assays, we demonstrate that IGPR-1 is activated by autophagy-inducing stimuli, such as amino acid starvation, nutrient deprivation, rapamycin, and lipopolysaccharide. Manipulating the IκB kinase β activity coupled with in vivo and in vitro kinase assays demonstrated that IκB kinase β is a key serine/threonine kinase activated by autophagy stimuli and that it catalyzes phosphorylation of IGPR-1 at Ser220. The subsequent activation of IGPR-1, in turn, stimulates phosphorylation of AMP-activated protein kinase, which leads to phosphorylation of the major pro-autophagy proteins ULK1 and Beclin-1 (BECN1), increased LC3-II levels, and accumulation of LC3 punctum. Thus, our data demonstrate that IGPR-1 is activated by autophagy-inducing stimuli and in response regulates autophagy, connecting cell adhesion to autophagy. These findings may have important significance for autophagy-driven pathologies such cardiovascular diseases and cancer and suggest that IGPR-1 may serve as a promising therapeutic target.

Single Molecule Force Spectroscopy Maps to Study Receptors Clustering

2001 ◽  
Vol 7 (S2) ◽  
pp. 860-861
Author(s):  
R. Bhatia ◽  
N. Almqvist ◽  
S. Banerjee ◽  
G. Primbs ◽  
N. Desai ◽  
...  
Keyword(s):  
Cell Surface ◽  
Real Time ◽  
Single Molecule ◽  
Regional Distribution ◽  
Cellular Membrane ◽  
Angiogenic Factor ◽  
Extracellular Region ◽  
Force Volume ◽  
Endothelial Growth Factor

An atomic force microscope (AFM) allows molecular resolution imaging of hydrated specimens. However, it is often limited in providing identity of the imaged structures, especially in a complex system such as a cellular membrane. Cell surface macromolecules such as ion channels and receptors serve as the interface between the cytoplasm and the extracellular region and toward which many regulatory signals are directed. Their density, distribution and clustering are key spatial features influencing effective and proper physiological responses. We used a method that uses AFM “force-volume maps” to identify and map regional distribution as well as ligand-, or antibody-induced real-time clustering of receptors on the cell surface. This technique also allows simultaneous imaging of the resultant changes in cellular micromechanical properties, such as elasticity and cytoskeletal reorganization of the cell. As an appropriate physiological sample, we have examined spatial distribution and real-time clustering of VEGFR, the receptor for vascular endothelial growth factor which is an important angiogenic factor in human and animal tissues.We have used AFM probes conjugated with anti-VEGFR-antibody (anti-Flk-1 antibody) to examine binding (or unbinding) forces between VEGF-R2 (Flk-1) in both in vitro as well as in live endothelial cells. A quantal set of binding and unbinding forces was measured between the antibody conjugated to the AFM tip and purified VEGFRs adsorbed on to a mica surface (Fig 1). The unbinding force varied between 60 and 240 pN and was a multiple of discrete quantized strength of approximately 60 pN (Figure 1B).

scholarly journals Virus-mediated release of endosomal content in vitro: different behavior of adenovirus and rhinovirus serotype 2.

1995 ◽  
Vol 131 (1) ◽  
pp. 111-123 ◽  
Author(s):  
E Prchla ◽  
C Plank ◽  
E Wagner ◽  
D Blaas ◽  
R Fuchs
Keyword(s):  
Vitro Assay ◽  
Forming Mechanism ◽  
Endosomal Membrane ◽  
Influenza Virus Hemagglutinin ◽  
Late Endosomes ◽  
Nonenveloped Virus ◽  
Capsid Protein Vp1 ◽  
Membrane Spanning

Endosomal penetration by nonenveloped viruses might be accomplished by either local breakdown of the endosomal membrane (e.g., adenovirus) or formation of a membrane-spanning pore by capsid proteins. Uncoating of the nonenveloped virus human rhinovirus serotype 2 (HRV2) has been shown to occur from late endosomes and to be entirely dependent on the acidic pH in this compartment (Prchla, E., E. Kuechler, D. Blaas, and R. Fuchs. 1994. J. Virol. 68: 3713-3723). To investigate further the mechanism of uncoating of HRV2, an in vitro assay was established to test viruses or virus-derived peptides for their capacity to release cointernalized biotin-dextran of different molecular mass (10 and 70 kD) from isolated endosomes. The suitability of the assay was demonstrated by use of a fusogenic peptide derived from influenza virus hemagglutinin (GALA-INF3). Whereas adenovirus induced a low pH-dependent release of up to 46% of the internalized biotin-dextran and did not show any significant size selectivity (as expected for endosome disruption), HRV2 mediated release of 27% of the 10 kD dextran and only traces of the 70-kD dextran. Similarly, GALA-INF3-induced release of biotin-dextran was also size dependent. The potential role of the capsid protein VP1 in HRV2 uncoating in vivo was also substantiated in our in vitro system using an amphipathic, NH2-terminal peptide of VP1. Taken together, these data favor the model of a specific pore-forming mechanism for HRV2 uncoating which is in contrast to the membrane-disrupting mechanism of adenovirus.

scholarly journals Endothelial-specific Gata3 expression is required for haematopoietic stem cell generation

2021 ◽  
Author(s):  
Nada Zaidan ◽  
Evangelia Diamanti ◽  
Leslie Nitsche ◽  
Antonella Fidanza ◽  
Nicola Wilson ◽  
...  
Keyword(s):  
Haematopoietic Stem Cell ◽  
Quiescent State ◽  
The Core ◽  
Complex Process ◽  
Gata3 Expression ◽  
A Cell ◽  
Specific Deletion ◽  
Supportive Function

To generate sufficient numbers of transplantable haematopoietic stem cells (HSCs) in vitro, a detailed understanding of how this process takes place in vivo is essential. The endothelial-to-haematopoietic transition (EHT), which culminates in the production of the first HSCs, is a highly complex process during which key regulators are switched on and off at precise moments and which is embedded into a myriad of microenvironmental signals from surrounding cells and tissues. We have previously demonstrated an HSC-supportive function for Gata3 within the sympathetic nervous system and the sub-aortic mesenchyme, but show here that it also plays a cell-intrinsic role during the EHT. It is expressed in haemogenic endothelial cells and early HSC precursors, where its expression correlates with a more quiescent state. Importantly, endothelial-specific deletion of Gata3 shows that it is functionally required for these cells to mature into HSCs, placing Gata3 at the core of the EHT regulatory network.

scholarly journals Vaccinia virus hijacks ESCRT-mediated multivesicular body formation for virus egress

2021 ◽  
Vol 4 (8) ◽  
pp. e202000910
Author(s):  
Moona Huttunen ◽  
Jerzy Samolej ◽  
Robert J Evans ◽  
Artur Yakimovich ◽  
Ian J White ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Membrane Trafficking ◽  
Multivesicular Body ◽  
Cellular Membrane ◽  
Double Membrane ◽  
Endosomal Sorting ◽  
Complex Process ◽  
A Cell ◽  
Infected Cells ◽  
Virus Egress

Poxvirus egress is a complex process whereby cytoplasmic single membrane–bound virions are wrapped in a cell-derived double membrane. These triple-membrane particles, termed intracellular enveloped virions (IEVs), are released from infected cells by fusion. Whereas the wrapping double membrane is thought to be derived from virus-modified trans-Golgi or early endosomal cisternae, the cellular factors that regulate virus wrapping remain largely undefined. To identify cell factors required for this process the prototypic poxvirus, vaccinia virus (VACV), was subjected to an RNAi screen directed against cellular membrane-trafficking proteins. Focusing on the endosomal sorting complexes required for transport (ESCRT), we demonstrate that ESCRT-III and VPS4 are required for packaging of virus into multivesicular bodies (MVBs). EM-based characterization of MVB-IEVs showed that they account for half of IEV production indicating that MVBs are a second major source of VACV wrapping membrane. These data support a model whereby, in addition to cisternae-based wrapping, VACV hijacks ESCRT-mediated MVB formation to facilitate virus egress and spread.

Cytotoxic Activity of CD48 Monoclonal Antibodies Against Human Lymphoma Cells

2008 ◽  
Vol 2 (1) ◽  
pp. 219-223 ◽  
Author(s):  
Jiewei Wei ◽  
David Y. Chin ◽  
Stephen M. Mahler
Keyword(s):  
Chromosomal Dna ◽  
Cell Shrinkage ◽  
Raji Cells ◽  
Cellular Assays ◽  
Viable Cells ◽  
Human Lymphoma ◽  
A Cell ◽  
Human Igg1 ◽  
Potent Growth

CD48 is a cell surface, glycosylphosphatidylinositol-linked glycoprotein, and a potential target for treatment of leukemia and lymphoma. Two anti-CD48 mAbs, murine HuLy-m3 and human IgG1-N2A, were compared in cellular assays using a human lymphoma cell line (Raji) for their ability to inhibit cell growth and induce apoptosis. In vitro studies revealed both HuLy-m3 and IgG1-N2A mAbs were able to induce potent growth inhibition, reflected by a reduction in viable cells of approximately 70% compared to controls after 90 h. Furthermore, Raji cells treated with IgG1-N2A showed evidence of apoptosis, including increased ethidium bromide uptake, cell shrinkage and chromosomal DNA degradation.

A cell impedance-based real-time in vitro assay to assess the toxicity of amphotericin B formulations

2017 ◽  
Vol 334 ◽  
pp. 18-23 ◽  
Author(s):  
Jean Menotti ◽  
Alexandre Alanio ◽  
Aude Sturny-Leclère ◽  
Sandrine Vitry ◽  
Félix Sauvage ◽  
...  
Keyword(s):  
Real Time ◽  
Amphotericin B ◽  
In Vitro Assay ◽  
Vitro Assay ◽  
Cell Impedance ◽  
A Cell

scholarly journals TAOK2 is an ER-localized Kinase that Catalyzes the Dynamic Tethering of ER to Microtubules

2021 ◽  
Author(s):  
Kimya Nourbakhsh ◽  
Amy A. Ferreccio ◽  
Matthew J. Bernard ◽  
Smita Yadav
Keyword(s):  
Endoplasmic Reticulum ◽  
Catalytic Activity ◽  
Membrane Dynamics ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
High Affinity ◽  
Amphipathic Helices ◽  
Cellular Assays ◽  
Membrane Spanning

SummaryThe endoplasmic reticulum (ER) depends on extensive association with the microtubule cytoskeleton for its structure, function and mitotic inheritance. The identity of molecular tethers that mediate ER-microtubule coupling, and mechanisms through which dynamic tethering is regulated are poorly understood. Here, we identify, Thousand And One amino acid Kinase 2 (TAOK2) as a pleiotropic protein kinase that mediates tethering of ER to microtubules. We show that TAOK2 is a unique multipass membrane spanning serine/threonine kinase localized in distinct ER domains via four transmembrane and amphipathic helices. Using in vitro and cellular assays, we find that TAOK2 directly binds microtubules with high affinity. We define the minimal TAOK2 determinants that induce ER-microtubule tethering, and delineate the mechanism for its autoregulation. While ER membrane dynamics are increased in TAOK2 knockout cells, the movement of ER along growing microtubule plus-ends is disrupted. We show that ER-microtubule tethering is tightly regulated by catalytic activity of TAOK2 in both interphase and mitotic cells, perturbation of which leads to profound defects in ER morphology and cell division. Our study identifies TAOK2 as an ER-microtubule tether, and reveals a kinase-regulated mechanism for control of ER dynamics critical for cell growth and division.

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