Dynamic Changes In Localization Research Articles

Emerging SARS-CoV-2 Diversity Revealed by Rapid Whole-Genome Sequence Typing.

Discrete classification of SARS-CoV-2 viral genotypes can identify emerging strains and detect geographic spread, viral diversity, and transmission events. We developed a tool (GNU-based Virus IDentification [GNUVID]) that integrates whole-genome multilocus sequence typing and a supervised machine learning random forest-based classifier. We used GNUVID to assign sequence type (ST) profiles to all high-quality genomes available from GISAID. STs were clustered into clonal complexes (CCs) and then used to train a machine learning classifier. We used this tool to detect potential introduction and exportation events and to estimate effective viral diversity across locations and over time in 16 US states. GNUVID is a highly scalable tool for viral genotype classification (https://github.com/ahmedmagds/GNUVID) that can quickly classify hundreds of thousands of genomes in a way that is consistent with phylogeny. Our genotyping ST/CC analysis uncovered dynamic local changes in ST/CC prevalence and diversity with multiple replacement events in different states, an average of 20.6 putative introductions and 7.5 exportations for each state over the time period analyzed. We introduce the use of effective diversity metrics (Hill numbers) that can be used to estimate the impact of interventions (e.g., travel restrictions, vaccine uptake, mask mandates) on the variation in circulating viruses. Our classification tool uncovered multiple introduction and exportation events, as well as waves of expansion and replacement of SARS-CoV-2 genotypes in different states. GNUVID classification lends itself to measures of ecological diversity, and, with systematic genomic sampling, it could be used to track circulating viral diversity and identify emerging clones and hotspots.

464. Rapid whole genome sequence typing reveals multiple waves of SARS-CoV-2 spread

BackgroundAs the pandemic SARS-CoV-2 virus has spread globally its genome has diversified and distinct clones can now be recognized, tracked, and traced. Identifying clonal groups allows for assessment of geographic spread, transmission events, and identification of more virulent or transmissible emerging strains.MethodsAll SARS-CoV-2 genomes (n=17,504) that are complete and high coverage were downloaded from GISAID on May 17th 2020. We developed a GNU-based Virus IDentification (GNUVID) tool that implements a whole genome multilocus sequence typing (wgMLST) scheme composed of all ten ORFs in the SARS-CoV-2 genome. The 10,422 genomes that passed our quality check were fed to the GNUVID tool, which assigned a ST profile to each genome. Global optimum eBURST was then used to cluster the STs in clonal complexes (CCs).ResultsOur ST/CC analysis uncovered strong associations of ST/CCs with certain geographical regions but also dynamic local changes in ST/CC prevalence. We also identified several unexpected putative global transmission events (e.g., from the US to the Middle East and reintroduction to China later in the pandemic). We have made our tool (GNUVID) available so that new WG sequences can be rapidly assigned to an ST/CC (https://github.com/ahmedmagds/GNUVID).ConclusionOur sequence typing system uncovered previously unappreciated transmission events and waves of expansion and replacement of SARS-CoV-2 STs and CCs in different geographical locations, suggesting complex dynamics in viral populations that previously seemed monomorphic. Because, our tool can be rapidly updated with new sequencing data it can track emerging clones and identifying new hotspots.Disclosures All Authors: No reported disclosures

Rapid Whole Genome Sequence Typing Reveals Multiple Waves of SARS-CoV-2 Spread

Background: As the pandemic SARS-CoV-2 virus has spread globally its genome has diversified and distinct clones can now be recognized, tracked, and traced. Identifying clonal groups allows for assessment of geographic spread, transmission events, and identification of more virulent or transmissible emerging strains. Methods: All SARS-CoV-2 genomes (n=17,504) that are complete and high coverage were downloaded from GISAID on May 17 th 2020. We developed a GNU-based Virus IDentification (GNUVID) tool that implements a whole genome multilocus sequence typing (wgMLST) scheme composed of all ten ORFs in the SARS-CoV-2 genome. The 10,422 genomes that passed our quality check were fed to the GNUVID tool, which assigned a ST profile to each genome. Global optimum eBURST was then used to cluster the STs in clonal complexes (CCs). Findings: Our ST/CC analysis uncovered strong associations of ST/CCs with certain geographical regions but also dynamic local changes in ST/CC prevalence. We also identified several unexpected putative global transmission events (e.g., from the US to the Middle East and reintroduction to China later in the pandemic). We have made our tool (GNUVID) available so that new WG sequences can be rapidly assigned to an ST/CC (https://github.com/ahmedmagds/GNUVID). Interpretation: Our sequence typing system uncovered previously unappreciated transmission events and waves of expansion and replacement of SARS-CoV-2 STs and CCs in different geographical locations, suggesting complex dynamics in viral populations that previously seemed monomorphic. Because, our tool can be rapidly updated with new sequencing data it can track emerging clones and identifying new hotspots. Funding: NIH1R01AI137526-01, PLANET19G0, 1R21AI144561-01A1 and R01NR015639. Declaration of Interests: The authors declare that they have no competing interests.

Fluorogenic probes reveal a role of GLUT4 N-glycosylation in intracellular trafficking.

Glucose transporter 4 (GLUT4) is an N-glycosylated protein that maintains glucose homeostasis by regulating the protein translocation. To date, it has been unclear whether the N-glycan of GLUT4 contributes to its intracellular trafficking. Here, to clarify the role of the N-glycan, we developed fluorogenic probes that label cytoplasmic and plasma-membrane proteins for multicolor imaging of GLUT4 translocation. One of the probes, which is cell impermeant, selectively detected exocytosed GLUT4. Using this probe, we verified the 'log' of the trafficking, in which N-glycan-deficient GLUT4 was transiently translocated to the cell membrane upon insulin stimulation and was rapidly internalized without retention on the cell membrane. The results strongly suggest that the N-glycan functions in the retention of GLUT4 on the cell membrane. This study showed the utility of the fluorogenic probes and indicated that this imaging tool will be applicable for research on various membrane proteins that show dynamic changes in localization.

B-Adrenergic Stimulation Accelerates Local Recovery of Cardiac Ca 2+ Release

In cardiac myocytes, Ca2+ sparks terminate reliably and exhibit time-dependent refractoriness after termination. Compelling evidence suggests that dynamic local changes in SR [Ca2+] play an important role in the control of these processes. We examined Ca2+ spark refractoriness by exposing fluo-3 loaded quiescent rat ventricular myocytes to 50 nM ryanodine, recording Ca2+ sparks with a confocal microscope, and analyzing the repeated sparks that were produced at a limited number of ryanodine receptor (RyR) clusters. Previous experiments showed that altering RyR sensitivity (caffeine or tetracaine) influenced the time between consecutive sparks but did not affect the recovery of spark amplitude (time constant = ∼100 ms in all cases). Here we examined repeated Ca2+ sparks after application of 100 nM isoproterenol to determine how β-adrenergic stimulation influences spark restitution. Isoproterenol dramatically decreased the median interval between consecutive sparks (192 ms vs. 280 ms in control) and led to faster recovery of Ca2+ spark amplitude (time constant = 58 ms). Mechanisms underlying these results were explored through simulations with an established mathematical model of the Ca2+ spark. Simulations showed that faster SR refilling led to earlier triggering of Ca2+ sparks due to the greater flux of Ca2+ through each open RyR, but this effect was insufficient to explain the experimental data. The results could be reproduced if we assumed that isoproterenol both increased the rate of local SR refilling and increased RyR sensitivity. Together, our results indicate that β-adrenergic stimulation influences both: 1) Ca2+ spark amplitude recovery, through changes in the time course of local SR refilling; and 2) Ca2+ spark triggering, through changes in both refilling and RyR sensitivity.

A Monomeric Photoconvertible Fluorescent Protein for Imaging of Dynamic Protein Localization

The use of green-to-red photoconvertible fluorescent proteins (FPs) enables researchers to highlight a subcellular population of a fusion protein of interest and to image its dynamics in live cells. In an effort to enrich the arsenal of photoconvertible FPs and to overcome the limitations imposed by the oligomeric structure of natural photoconvertible FPs, we designed and optimized a new monomeric photoconvertible FP. Using monomeric versions of Clavularia sp. cyan FP as template, we employed sequence-alignment-guided design to create a chromophore environment analogous to that shared by known photoconvertible FPs. The designed gene was synthesized and, when expressed in Escherichia coli, found to produce green fluorescent colonies that gradually switched to red after exposure to white light. We subjected this first-generation FP [named mClavGR1 (monomeric Clavularia-derived green-to-red photoconvertible 1)] to a combination of random and targeted mutageneses and screened libraries for efficient photoconversion using a custom-built system for illuminating a 10-cm Petri plate with 405-nm light. Following more than 15 rounds of library creation and screening, we settled on an optimized version, known as mClavGR2, that has eight mutations relative to mClavGR1. Key improvements of mClavGR2 relative to mClavGR1 include a 1.4-fold brighter red species, 1.8-fold higher photoconversion contrast, and dramatically improved chromophore maturation in E. coli. The monomeric status of mClavGR2 has been demonstrated by gel-filtration chromatography and the functional expression of a variety of mClavGR2 chimeras in mammalian cells. Furthermore, we have exploited mClavGR2 to determine the diffusion kinetics of the membrane protein intercellular adhesion molecule 1 both when the membrane is in contact with a T-lymphocyte expressing leukocyte-function-associated antigen 1 and when it is not. These experiments clearly establish that mClavGR2 is well suited for rapid photoconversion of protein subpopulations and subsequent tracking of dynamic changes in localization in living cells.

Sensing tension

Measuring the tension forces at specific sites in living cells is technically challenging. Now, a fluorescent biosensor protein can be used to characterize dynamic local changes in tension in migrating cells.

Predicting Local SR Ca2+ Dynamics during Ca2+ Wave Propagation in Ventricular Myocytes

Of the many ongoing controversies regarding the workings of the sarcoplasmic reticulum (SR) in cardiac myocytes, two unresolved and interconnected topics are 1), mechanisms of calcium (Ca2+) wave propagation, and 2), speed of Ca2+ diffusion within the SR. Ca2+ waves are initiated when a spontaneous local SR Ca2+ release event triggers additional release from neighboring clusters of SR release channels (ryanodine receptors (RyRs)). A lack of consensus regarding the effective Ca2+ diffusion constant in the SR (DCa,SR) severely complicates our understanding of whether dynamic local changes in SR [Ca2+] can influence wave propagation. To address this problem, we have implemented a computational model of cytosolic and SR [Ca2+] during Ca2+ waves. Simulations have investigated how dynamic local changes in SR [Ca2+] are influenced by 1), DCa,SR; 2), the distance between RyR clusters; 3), partial inhibition or stimulation of SR Ca2+ pumps; 4), SR Ca2+ pump dependence on cytosolic [Ca2+]; and 5), the rate of transfer between network and junctional SR. Of these factors, DCa,SR is the primary determinant of how release from one RyR cluster alters SR [Ca2+] in nearby regions. Specifically, our results show that local increases in SR [Ca2+] ahead of the wave can potentially facilitate Ca2+ wave propagation, but only if SR diffusion is relatively slow. These simulations help to delineate what changes in [Ca2+] are possible during SR Ca2+release, and they broaden our understanding of the regulatory role played by dynamic changes in [Ca2+]SR.

Engineering a polarity-sensitive biosensor for time-lapse imaging of apoptotic processes and degeneration

Apoptosis is of central importance to many areas of biological research, but there is a lack of methods that permit continuous monitoring of apoptosis or cell viability in a nontoxic and noninvasive manner. Here we report the development of a tool applicable to live-cell imaging that facilitates the visualization of real-time apoptotic changes without perturbing the cellular environment. We designed a polarity-sensitive annexin-based biosensor (pSIVA) with switchable fluorescence states, which allows detection only when bound to apoptotic cells. Using pSIVA with live-cell imaging, we observed dynamic local changes in individual rat neurons during degeneration in vitro and in vivo. Furthermore, we observed that pSIVA binding was reversible and clearly defined the critical period for neurons to be rescued. We anticipate pSIVA can be widely applied to address questions concerning spatiotemporal events in apoptotic processes, its reversibility and the general viability of cells in culture.

The DLK-1 Kinase Promotes mRNA Stability and Local Translation in C. elegans Synapses and Axon Regeneration

Growth cone guidance and synaptic plasticity involve dynamic local changes in proteins at axons and dendrites. The Dual-Leucine zipper Kinase MAPKKK (DLK) has been previously implicated in synaptogenesis and axon outgrowth in C. elegans and other animals. Here we show that in C. elegans DLK-1 regulates not only proper synapse formation and axon morphology but also axon regeneration by influencing mRNA stability. DLK-1 kinase signals via a MAPKAP kinase, MAK-2, to stabilize the mRNA encoding CEBP-1, a bZip protein related to CCAAT/enhancer-binding proteins, via its 3'UTR. Inappropriate upregulation of cebp-1 in adult neurons disrupts synapses and axon morphology. CEBP-1 and the DLK-1 pathway are essential for axon regeneration after laser axotomy in adult neurons, and axotomy induces translation of CEBP-1 in axons. Our findings identify the DLK-1 pathway as a regulator of mRNA stability in synapse formation and maintenance and also in adult axon regeneration.

An Evolutionarily Conserved Nuclear Export Signal Facilitates Cytoplasmic Localization of the Tbx5 Transcription Factor

During cardiac development, the T-box transcription factor Tbx5 displays dynamic changes in localization from strictly nuclear to both nuclear and cytoplasmic to exclusively cytoplasmic along the actin cytoskeleton in cells coexpressing its binding protein LMP4. Although nuclear localization signals (NLSs) have been described, the mechanism by which Tbx5 exits the nucleus remained elusive. Here, we describe for Tbx5 a nuclear export signal (NES) that is recognized by the CRM1 export protein. Site-directed mutagenesis of a critical amino acid(s) within this sequence determined the functionality of this NES. Confocal localization studies and luciferase transcriptional reporter assays with NES mutant Tbx5 forms demonstrated retention in the nucleus, regardless of the presence of LMP4. Coimmunoprecipitation and pharmacological interference studies demonstrated a direct interaction between Tbx5 and CRM1, revealing that Tbx5 is using the CRM1 pathway for nuclear export. In addition to Tbx5, we identified NESs in all T-box proteins and demonstrated interaction of the family members Tbx3 and Brachyury with the CRM1 exporter, suggesting general significance. This first demonstration of evolutionarily conserved NESs in all T-box proteins in conjunction with NLSs indicates a primordial function of T-box proteins to dynamically shuttle between nuclear and cytoplasmic compartments of the cell.

Dynamic changes in localization of chromobox (CBX) family members during the maternal to embryonic transition

The Chromobox domain (Cbx) gene family, consisting of Polycomb and Heterochromatin Protein 1 genes, is involved in transcriptional repression, cell cycle regulation and chromatin remodeling. We report the first study of gene expression and protein localization of the Cbx genes in in vitro produced bovine embryos. All but one gene (Cbx6) were expressed. This was confirmed by immunolocalization for HP1alpha, beta, gamma, and Pc2, 3. HP1beta was found in the nuclei of embryos from the two-cell stage onwards, whereas HP1gamma showed diffuse cytoplasmic/nuclear localization at the two- and eight-cell stages, and predominantly nuclear localization at the four-cell stage and the 16-cell stage onwards. Leptomycin B (LMB), a specific inhibitor of the nuclear export protein CRM-1 (chromosomal regional maintenance-1), was found to increase nuclear localization of HP1gamma at the eight-cell stage, and to prevent progression past this stage of embryogenesis. This indicates that HP1gamma possesses a CRM-1-dependent nuclear export pathway which may represent part of the basis of HP1gamma's ability to shuttle between the nucleus and the cytoplasm in dynamic fashion. HP1alpha was expressed in embryonic nuclei at all stages, but was found to relocalise from euchromatin to heterochromatin during the maternal to embryonic transition (MET). In contrast, Pc2 and Pc3 were evenly distributed between cytoplasm and nucleus until the eight- and sixteen-cell stages or the morula stage, respectively, before relocating preferentially to the cytoplasm. Collectively, the results suggest that dynamic changes of the nuclear-cytoplasmic and subnuclear distribution of members of the Cbx family may be central to the MET.

Threshold Energy Effects in Secondary Electron Emission

The work function ϕ, the bandgap Eg, the threshold energy level Et, for the inelastic scattering of excited electrons and the threshold energy transfer Ed for the onset of structural ionisation damage are clearly of major significance in various actively developing forms of hot carrier imaging. Two exciting examples here are the ability to image small and dynamic local changes in work function by PEEM and as well as mapping the variations in electronic structure between p and n-type regions of a semiconductor by SE imaging in the SEM. More recently still there have been indications that the SE signal in the ESEM might be sensitive to local changes in bandgap and suggestions that it might be even possible to image spatial variations in pH. It is increasingly clear that if these attractive opportunities are to be efficiently explored and developed, systematic and preferably quantitative observations are needed. Such work requires specimens whose atomic and electronic structure is either fully known beforehand or can be deduced from other signals available in a situation where the physical processes in the microscope are well understood.

Localization of the site of Ca2+ release at the level of a single sarcomere in skeletal muscle fibres.

The development of mechanical force in skeletal muscle fibres is brought about by rapid increases in the intracellular calcium concentration (Ca2+ transients) which can be detected by optical methods. Local stimulation experiments and ultrastructural evidence suggest that, at a microscopic level, these Ca2+ transients are generated by the release of Ca2+ ions from the terminal cisternae of the sarcoplasmic reticulum in response to the depolarization of the transverse tubules (t-tubules). Nevertheless, to date, there is no functional information on the exact location at which Ca2+ release takes place. The present experiments were designed to obtain direct evidence about dynamic changes in localization and microscopic distribution of Ca2+ in a single sarcomere using two independent novel methodologies: confocal spot detection of Ca2+ transients and Ca2+ imaging with pulsed laser excitation.