scholarly journals Recovering mixtures of fast diffusing states from short single particle trajectories

2021 ◽  
Author(s):  
Alec Basil Heckert ◽  
Liza Dahal ◽  
Robert Tjian ◽  
Xavier Darzacq
Keyword(s):  
Single Particle ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Single Particle Tracking ◽  
Depth Of Field ◽  
Cellular Regulation ◽  
Localization Accuracy ◽  
Advantages And Disadvantages ◽  
Trajectory Length ◽  
Cell To Cell Variability

Single particle tracking (SPT) directly measures the dynamics of proteins in living cells and is a powerful tool to dissect molecular mechanisms of cellular regulation. Interpretation of SPT with fast-diffusing proteins in mammalian cells, however, is complicated by technical limitations imposed by fast image acquisition. These limitations include short trajectory length due to photobleaching and shallow depth of field, high localization error due to the low photon budget imposed by short integration times, and cell-to-cell variability. To address these issues, we developed methods to infer distributions of diffusion coefficients from SPT data with short trajectories, variable localization accuracy, and absence of prior knowledge about the number of underlying states. We discuss advantages and disadvantages of these approaches relative to other frameworks for SPT analysis.

scholarly journals Single particle tracking-based reaction progress kinetic analysis reveals a series of molecular mechanisms of cetuximab-induced EGFR processes in a single living cell

2017 ◽  
Vol 8 (7) ◽  
pp. 4823-4832 ◽  
Author(s):  
Do-Hyeon Kim ◽  
Dong-Kyun Kim ◽  
Kai Zhou ◽  
Soyeon Park ◽  
Yonghoon Kwon ◽  
...  
Keyword(s):  
Kinetic Analysis ◽  
Single Molecule ◽  
Particle Tracking ◽  
Single Particle ◽  
Living Cell ◽  
Molecular Mechanisms ◽  
Single Particle Tracking ◽  
Reaction Progress ◽  
Single Living ◽  
Reaction Progress Kinetic Analysis

Reaction progress kinetic analysis utilizing single molecule trajectories revealed the comprehensive molecular mechanisms of cetuximab induced EGFR endocytosis.

Dissection of the molecular mechanisms of protein targeting to the peroxisome using immunofluorescence and immunocryoelectron microscopies

1990 ◽  
Vol 48 (3) ◽  
pp. 44-45
Author(s):  
G-A. Keller ◽  
S. J. Gould ◽  
S. Subramani ◽  
S. Krisans
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Protein Targeting ◽  
Firefly Luciferase ◽  
Peroxisomal Enzyme ◽  
Transfected Cells ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Series Of Experiments ◽  
Peroxisomal Targeting Signal

Subcellular compartments within eukaryotic cells must each be supplied with unique sets of proteins that must be directed to, and translocated across one or more membranes of the target organelles. This transport is mediated by cis- acting targeting signals present within the imported proteins. The following is a chronological account of a series of experiments designed and carried out in an effort to understand how proteins are targeted to the peroxisomal compartment.-We demonstrated by immunocryoelectron microscopy that the enzyme luciferase is a peroxisomal enzyme in the firefly lantern. -We expressed the cDNA encoding firefly luciferase in mammalian cells and demonstrated by immunofluorescence that the enzyme was transported into the peroxisomes of the transfected cells. -Using deletions, linker insertions, and gene fusion to identify regions of luciferase involved in its transport to the peroxisomes, we demonstrated that luciferase contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acid.

A Possible Modulation Mechanism of Intramolecular and Intermolecular Interactions for NCAM Polysialylation and Cell Migration

2019 ◽  
Vol 19 (25) ◽  
pp. 2271-2282 ◽  
Author(s):  
Bo Lu ◽  
Xue-Hui Liu ◽  
Si-Ming Liao ◽  
Zhi-Long Lu ◽  
Dong Chen ◽  
...  
Keyword(s):  
Cell Migration ◽  
Intermolecular Interactions ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Intramolecular Interaction ◽  
Polysialic Acid ◽  
Tumor Cell Migration ◽  
Neural Cell Adhesion ◽  
Polybasic Domains ◽  
Novel Model

Polysialic acid (polySia) is a novel glycan that posttranslationally modifies neural cell adhesion molecules (NCAMs) in mammalian cells. Up-regulation of polySia-NCAM expression or NCAM polysialylation is associated with tumor cell migration and progression in many metastatic cancers and neurocognition. It has been known that two highly homologous mammalian polysialyltransferases (polySTs), ST8Sia II (STX) and ST8Sia IV (PST), can catalyze polysialylation of NCAM, and two polybasic domains, polybasic region (PBR) and polysialyltransferase domain (PSTD) in polySTs play key roles in affecting polyST activity or NCAM polysialylation. However, the molecular mechanisms of NCAM polysialylation and cell migration are still not entirely clear. In this minireview, the recent research results about the intermolecular interactions between the PBR and NCAM, the PSTD and cytidine monophosphate-sialic acid (CMP-Sia), the PSTD and polySia, and as well as the intramolecular interaction between the PBR and the PSTD within the polyST, are summarized. Based on these cooperative interactions, we have built a novel model of NCAM polysialylation and cell migration mechanisms, which may be helpful to design and develop new polysialyltransferase inhibitors.

scholarly journals Information-Efficient, Off-Center Sampling Results in Improved Precision in 3D Single-Particle Tracking Microscopy

Entropy ◽  
2021 ◽  
Vol 23 (5) ◽  
pp. 498
Author(s):  
Chen Zhang ◽  
Kevin Welsher
Keyword(s):  
Fisher Information ◽  
Particle Tracking ◽  
Single Particle ◽  
Biological Samples ◽  
Tracking System ◽  
Single Particle Tracking ◽  
Uniform Sampling ◽  
Single Dimension ◽  
3D Localization ◽  
Particle Localization

In this work, we present a 3D single-particle tracking system that can apply tailored sampling patterns to selectively extract photons that yield the most information for particle localization. We demonstrate that off-center sampling at locations predicted by Fisher information utilizes photons most efficiently. When performing localization in a single dimension, optimized off-center sampling patterns gave doubled precision compared to uniform sampling. A ~20% increase in precision compared to uniform sampling can be achieved when a similar off-center pattern is used in 3D localization. Here, we systematically investigated the photon efficiency of different emission patterns in a diffraction-limited system and achieved higher precision than uniform sampling. The ability to maximize information from the limited number of photons demonstrated here is critical for particle tracking applications in biological samples, where photons may be limited.

scholarly journals The Saccharomyces cerevisiae RAD6 Group Is Composed of an Error-Prone and Two Error-Free Postreplication Repair Pathways

Genetics ◽  
2000 ◽  
Vol 155 (4) ◽  
pp. 1633-1641 ◽  
Author(s):  
Wei Xiao ◽  
Barbara L Chow ◽  
Stacey Broomfield ◽  
Michelle Hanna
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Signal Transducer ◽  
Repair Pathway ◽  
Polyubiquitin Chain ◽  
Postreplication Repair ◽  
Translesion Dna Synthesis ◽  
Radiation Repair ◽  
Repair Pathways ◽  
High Degree

Abstract The RAD6 postreplication repair and mutagenesis pathway is the only major radiation repair pathway yet to be extensively characterized. It has been previously speculated that the RAD6 pathway consists of two parallel subpathways, one error free and another error prone (mutagenic). Here we show that the RAD6 group genes can be exclusively divided into three rather than two independent subpathways represented by the RAD5, POL30, and REV3 genes; the REV3 pathway is largely mutagenic, whereas the RAD5 and the POL30 pathways are deemed error free. Mutants carrying characteristic mutations in each of the three subpathways are phenotypically indistinguishable from a single mutant such as rad18, which is defective in the entire RAD6 postreplication repair/tolerance pathway. Furthermore, the rad18 mutation is epistatic to all single or combined mutations in any of the above three subpathways. Our data also suggest that MMS2 and UBC13 play a key role in coordinating the response of the error-free subpathways; Mms2 and Ubc13 form a complex required for a novel polyubiquitin chain assembly, which probably serves as a signal transducer to promote both RAD5 and POL30 error-free postreplication repair pathways. The model established by this study will facilitate further research into the molecular mechanisms of postreplication repair and translesion DNA synthesis. In view of the high degree of sequence conservation of the RAD6 pathway genes among all eukaryotes, the model presented in this study may also apply to mammalian cells and predicts links to human diseases.

scholarly journals Control of PKR Protein Kinase by Hepatitis C Virus Nonstructural 5A Protein: Molecular Mechanisms of Kinase Regulation

1998 ◽  
Vol 18 (9) ◽  
pp. 5208-5218 ◽  
Author(s):  
Michael Gale ◽  
Collin M. Blakely ◽  
Bart Kwieciszewski ◽  
Seng-Lai Tan ◽  
Michelle Dossett ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Kinase ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Functional Assay ◽  
Binding Region ◽  
Dimerization Domain ◽  
Antiviral Effects

ABSTRACT The PKR protein kinase is a critical component of the cellular antiviral and antiproliferative responses induced by interferons. Recent evidence indicates that the nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) can repress PKR function in vivo, possibly allowing HCV to escape the antiviral effects of interferon. NS5A presents a unique tool by which to study the molecular mechanisms of PKR regulation in that mutations within a region of NS5A, termed the interferon sensitivity-determining region (ISDR), are associated with sensitivity of HCV to the antiviral effects of interferon. In this study, we investigated the mechanisms of NS5A-mediated PKR regulation and the effect of ISDR mutations on this regulatory process. We observed that the NS5A ISDR, though necessary, was not sufficient for PKR interactions; we found that an additional 26 amino acids (aa) carboxyl to the ISDR were required for NS5A-PKR complex formation. Conversely, we localized NS5A binding to within PKR aa 244 to 296, recently recognized as a PKR dimerization domain. Consistent with this observation, we found that NS5A from interferon-resistant HCV genotype 1b disrupted kinase dimerization in vivo. NS5A-mediated disruption of PKR dimerization resulted in repression of PKR function and inhibition of PKR-mediated eIF-2α phosphorylation. Introduction of multiple ISDR mutations abrogated the ability of NS5A to bind to PKR in mammalian cells and to inhibit PKR in a yeast functional assay. These results indicate that mutations within the PKR-binding region of NS5A, including those within the ISDR, can disrupt the NS5A-PKR interaction, possibly rendering HCV sensitive to the antiviral effects of interferon. We propose a model of PKR regulation by NS5A which may have implications for therapeutic strategies against HCV.

scholarly journals Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

1988 ◽  
Vol 8 (10) ◽  
pp. 4185-4189 ◽  
Author(s):  
J A Greenspan ◽  
F M Xu ◽  
R L Davidson
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Shuttle Vector ◽  
Ethyl Methanesulfonate ◽  
Wild Type ◽  
Single Base ◽  
Type Sequence

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

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