DASC, a sensitive classifier for measuring discrete early stages in clathrin-mediated endocytosis

Clathrin-mediated endocytosis (CME) in mammalian cells is driven by resilient machinery that includes >70 endocytic accessory proteins (EAP). Accordingly, perturbation of individual EAPs often results in minor effects on biochemical measurements of CME, thus providing inconclusive/misleading information regarding EAP function. Live-cell imaging can detect earlier roles of EAPs preceding cargo internalization; however, this approach has been limited because unambiguously distinguishing abortive coats (ACs) from bona fide clathrin-coated pits (CCPs) is required but unaccomplished. Here, we develop a thermodynamics-inspired method, “disassembly asymmetry score classification (DASC)”, that resolves ACs from CCPs based on single channel fluorescent movies. After extensive verification, we use DASC-resolved ACs and CCPs to quantify CME progression in 11 EAP knockdown conditions. We show that DASC is a sensitive detector of phenotypic variation in CCP dynamics that is uncorrelated to the variation in biochemical measurements of CME. Thus, DASC is an essential tool for uncovering EAP function.

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DASC, a sensitive classifier for measuring discrete early stages in clathrin-mediated endocytosis

10.1101/2020.01.28.924019 ◽

2020 ◽

Author(s):

Xinxin Wang ◽

Zhiming Chen ◽

Marcel Mettlen ◽

Jungsik Noh ◽

Sandra L. Schmid ◽

...

Keyword(s):

Phenotypic Variation ◽

Mammalian Cells ◽

Live Cell Imaging ◽

Cell Imaging ◽

Accessory Proteins ◽

Sensitive Detector ◽

Coated Pits ◽

Misleading Information ◽

Bona Fide ◽

Biochemical Measurements

AbstractClathrin-mediated endocytosis (CME) in mammalian cells is driven by resilient machinery that includes >70 endocytic accessory proteins (EAP). Accordingly, perturbation of individual EAPs often results in minor effects on biochemical measurements of CME, thus providing inconclusive/misleading information regarding EAP function. Live-cell imaging can detect earlier roles of EAPs preceding cargo internalization; however, this approach has been limited because unambiguously distinguishing abortive-clathrin coats (ACs) from bona fide clathrin-coated pits (CCPs) is required but unaccomplished. Here, we develop a thermodynamics-inspired method, “disassembly asymmetry score classification (DASC)”, that unambiguously separates ACs from CCPs without an additional marker. After extensive verification, we use DASC-resolved ACs and CCPs to quantify CME progression in 11 EAP knockdown conditions. We show that DASC is a sensitive detector of phenotypic variation in CCP dynamics that is orthogonal to the variation in biochemical measurements of CME. Thus, DASC is an essential tool for uncovering the function of individual EAPs.

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Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells

Molecular Biology of the Cell ◽

10.1091/mbc.e14-06-1091 ◽

2014 ◽

Vol 25 (22) ◽

pp. 3610-3618 ◽

Cited By ~ 34

Author(s):

Robert Mahen ◽

Birgit Koch ◽

Malte Wachsmuth ◽

Antonio Z. Politi ◽

Alexis Perez-Gonzalez ◽

...

Keyword(s):

Single Molecule ◽

Protein Function ◽

Mammalian Cells ◽

Live Cell Imaging ◽

Cell Imaging ◽

Quantitative Imaging ◽

Live Cells ◽

Single Molecule Spectroscopy ◽

Mitotic Kinase ◽

Endogenous Proteins

Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells. However, the extent to which different mammalian transgene methods faithfully report on the properties of endogenous proteins has not been studied comparatively. Here we use quantitative live-cell imaging and single-molecule spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B. We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quantitative imaging studies in mammalian cells.

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Live-cell imaging study of mitochondrial morphology in mammalian cells exposed to X-rays

Radiation Protection Dosimetry ◽

10.1093/rpd/ncv157 ◽

2015 ◽

Vol 166 (1-4) ◽

pp. 101-103 ◽

Cited By ~ 2

Author(s):

M. Noguchi ◽

Y. Kanari ◽

A. Yokoya ◽

A. Narita ◽

K. Fujii

Keyword(s):

Mammalian Cells ◽

Live Cell Imaging ◽

Cell Imaging ◽

Imaging Study ◽

Live Cell ◽

Mitochondrial Morphology ◽

X Rays

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MyTH4-FERM myosins have an ancient and conserved role in filopod formation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1615392113 ◽

2016 ◽

Vol 113 (50) ◽

pp. E8059-E8068 ◽

Cited By ~ 11

Author(s):

Karl J. Petersen ◽

Holly V. Goodson ◽

Ashley L. Arthur ◽

G. W. Gant Luxton ◽

Anne Houdusse ◽

...

Keyword(s):

Mammalian Cells ◽

Live Cell Imaging ◽

Cell Imaging ◽

Null Mutant ◽

Minimal Elements ◽

Social Amoeba ◽

Functional Conservation ◽

Functional Homolog ◽

The Social ◽

Functional Features

The formation of filopodia in Metazoa and Amoebozoa requires the activity of myosin 10 (Myo10) in mammalian cells and of Dictyostelium unconventional myosin 7 (DdMyo7) in the social amoeba Dictyostelium. However, the exact roles of these MyTH4-FERM myosins (myosin tail homology 4-band 4.1, ezrin, radixin, moesin; MF) in the initiation and elongation of filopodia are not well defined and may reflect conserved functions among phylogenetically diverse MF myosins. Phylogenetic analysis of MF myosin domains suggests that a single ancestral MF myosin existed with a structure similar to DdMyo7, which has two MF domains, and that subsequent duplications in the metazoan lineage produced its functional homolog Myo10. The essential functional features of the DdMyo7 myosin were identified using quantitative live-cell imaging to characterize the ability of various mutants to rescue filopod formation in myo7-null cells. The two MF domains were found to function redundantly in filopod formation with the C-terminal FERM domain regulating both the number of filopodia and their elongation velocity. DdMyo7 mutants consisting solely of the motor plus a single MyTH4 domain were found to be capable of rescuing the formation of filopodia, establishing the minimal elements necessary for the function of this myosin. Interestingly, a chimeric myosin with the Myo10 MF domain fused to the DdMyo7 motor also was capable of rescuing filopod formation in the myo7-null mutant, supporting fundamental functional conservation between these two distant myosins. Together, these findings reveal that MF myosins have an ancient and conserved role in filopod formation.

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Live Cell Imaging of Bioorthogonally Labelled Proteins Generated With a Single Pyrrolysine tRNA Gene

10.1101/161984 ◽

2017 ◽

Author(s):

Noa Aloush ◽

Tomer Schvartz ◽

Andres I. König ◽

Sarit Cohen ◽

Eugene Brozgol ◽

...

Keyword(s):

Mammalian Cells ◽

Live Cell Imaging ◽

Cell Imaging ◽

Stop Codon ◽

Signal To Noise Ratio ◽

Trna Synthetase ◽

Live Cell ◽

Live Cells ◽

Copy Numbers ◽

Intracellular Proteins

ABSTRACTGenetic code expansion enables the incorporation of non-canonical amino acids (ncAAs) into expressed proteins. ncAAs are usually encoded by a stop codon that is decoded by an exogenous orthogonal aminoacyl tRNA synthetase and its cognate suppressor tRNA, such as the pyrrolysine synthetase/ pair. In such systems, stop codon suppression is dependent on the intracellular levels of the exogenous tRNA. Therefore, multiple copies of the tRNAPyl gene (PylT) are encoded to improve ncAA incorporation. However, certain applications in mammalian cells, such as live-cell imaging applications, where labelled tRNA contributes to background fluorescence, can benefit from the use of less invasive minimal expression systems. Accordingly, we studied the effect of tRNAPyl on live-cell fluorescence imaging of bioorthogonally-labelled intracellular proteins. We found that in COS7 cells, a decrease in PylT copy numbers had no measurable effect on protein expression levels. Importantly, reducing PylT copy numbers improved the quality of live-cells images by enhancing the signal-to-noise ratio and reducing an immobile tRNAPyl population. This enabled us to improve live cell imaging of bioorthogonally labelled intracellular proteins, and to simultaneously label two different proteins in a cell. Our results indicate that the number of introduced PylT genes can be minimized according to the transfected cell line, incorporated ncAA, and application.

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Sortase-Modified Cholera Toxoids Show Specific Golgi Localization

10.26434/chemrxiv.9125201.v1 ◽

2019 ◽

Cited By ~ 2

Author(s):

Darren Machin ◽

Daniel Williamson ◽

Peter Fisher ◽

victoria miller ◽

Gemma Wildsmith ◽

...

Keyword(s):

Lipid Rafts ◽

Cell Biology ◽

Mammalian Cells ◽

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell ◽

Neuronal Tracing ◽

Future Role ◽

Golgi Localization

Cholera toxoid is an established tool for use in cellular tracing in neuroscience and cell biology. We use a sortase-labelling approach to generate site-specifically N-terminally modified variants of both the A2-B5 heterohexamer and B5 pentamer forms of the toxoid. Both forms of the toxoid are endocytosed by GM1-positive mammalian cells, and while the heterohexameric toxoid was principally localized in the ER, the B5 pentamer showed an unexpected localization in the medial/trans Golgi. This study suggests a future role for specifically-labelled cholera toxoids in live-cell imaging beyond their current applications in neuronal tracing and labelling of lipid-rafts in fixed cells.

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A Protocol to Engineer Bacteriophages for Live-Cell Imaging of Bacterial Prophage Induction Inside Mammalian Cells

STAR Protocols ◽

10.1016/j.xpro.2020.100084 ◽

2020 ◽

Vol 1 (2) ◽

pp. 100084 ◽

Cited By ~ 1

Author(s):

Katie Bodner ◽

Arin L. Melkonian ◽

Markus W. Covert

Keyword(s):

Mammalian Cells ◽

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell ◽

Prophage Induction

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Golgi Inheritance in Mammalian Cells Is Mediated through Endoplasmic Reticulum Export Activities

Molecular Biology of the Cell ◽

10.1091/mbc.e05-02-0155 ◽

2006 ◽

Vol 17 (2) ◽

pp. 990-1005 ◽

Cited By ~ 78

Author(s):

Nihal Altan-Bonnet ◽

Rachid Sougrat ◽

Wei Liu ◽

Erik L. Snapp ◽

Theresa Ward ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Mammalian Cells ◽

Live Cell Imaging ◽

Cell Imaging ◽

Transmembrane Proteins ◽

Temperature Sensitive ◽

Golgi Membranes ◽

Sequential Inactivation ◽

Er Export ◽

Microscopy Techniques

Golgi inheritance during mammalian cell division occurs through the disassembly, partitioning, and reassembly of Golgi membranes. The mechanisms responsible for these processes are poorly understood. To address these mechanisms, we have examined the identity and dynamics of Golgi proteins within mitotic membranes using live cell imaging and electron microscopy techniques. Mitotic Golgi fragments, seen in prometaphase and telophase, were found to localize adjacent to endoplasmic reticulum (ER) export domains, and resident Golgi transmembrane proteins cycled rapidly into and out of these fragments. Golgi proteins within mitotic Golgi haze—seen during metaphase—were found to redistribute with ER markers into fragments when the ER was fragmented by ionomycin treatment. The temperature-sensitive misfolding mutant ts045VSVG protein, when localized to the Golgi at the start of mitosis, became trapped in the ER at the end of mitosis in cells shifted to 40°C. Finally, reporters for Arf1 and Sar1 activity revealed that Arf1 and Sar1 undergo sequential inactivation during mitotic Golgi breakdown and sequential reactivation upon Golgi reassembly at the end of mitosis. Together, these findings support a model of mitotic Golgi inheritance that involves inhibition and subsequent reactivation of cellular activities controlling the cycling of Golgi components into and out of the ER.

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