scholarly journals Proteomic mapping in live Drosophila tissues using an engineered ascorbate peroxidase

2015 ◽  
Vol 112 (39) ◽  
pp. 12093-12098 ◽  
Author(s):  
Chiao-Lin Chen ◽  
Yanhui Hu ◽  
Namrata D. Udeshi ◽  
Thomas Y. Lau ◽  
Frederik Wirtz-Peitz ◽  
...  
Keyword(s):  
Ascorbate Peroxidase ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Cell Types ◽  
Live Cells ◽  
Specific Cell ◽  
Physiological Conditions ◽  
Subcellular Domains ◽  
Endogenous Proteins

Characterization of the proteome of organelles and subcellular domains is essential for understanding cellular organization and identifying protein complexes as well as networks of protein interactions. We established a proteomic mapping platform in live Drosophila tissues using an engineered ascorbate peroxidase (APEX). Upon activation, the APEX enzyme catalyzes the biotinylation of neighboring endogenous proteins that can then be isolated and identified by mass spectrometry. We demonstrate that APEX labeling functions effectively in multiple fly tissues for different subcellular compartments and maps the mitochondrial matrix proteome of Drosophila muscle to demonstrate the power of APEX for characterizing subcellular proteomes in live cells. Further, we generate “MitoMax,” a database that provides an inventory of Drosophila mitochondrial proteins with subcompartmental annotation. Altogether, APEX labeling in live Drosophila tissues provides an opportunity to characterize the organelle proteome of specific cell types in different physiological conditions.

scholarly journals Automated analysis of invadopodia dynamics in live cells

2014 ◽  
Author(s):  
Matthew E Berginski ◽  
Sarah J Creed ◽  
Shelly Cochran ◽  
David W Roadcap ◽  
James E Bear ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Metastatic Cancer ◽  
Automated Analysis ◽  
Time Lapse ◽  
Cell Types ◽  
Live Cells ◽  
Imaging Data ◽  
The Matrix ◽  
Invadopodia Formation

Multiple cell types form specialized protein complexes, podosomes or invadopodia and collectively referred to as invadosomes, which are used by the cell to actively degrade the surrounding extracellular matrix. Due to their potential importance in both healthy physiology as well as in pathological conditions such as cancer, the characterization of these structures has been of increasing interest. Following early descriptions of invadopodia, assays were developed which labelled the matrix underneath metastatic cancer cells allowing for the assessment of invadopodia activity in motile cells. However, characterization of invadopodia using these methods has traditionally been done manually with time-consuming and potentially biased quantification methods, limiting the number of experiments and the quantity of data that can be analysed. We have developed a system to automate the segmentation, tracking and quantification of invadopodia in time-lapse fluorescence image sets at both the single invadopodia level and whole cell level. We rigorously tested the ability of the method to detect changes in invadopodia formation and dynamics through the use of well-characterized small molecule inhibitors, with known effects on invadopodia. Our results demonstrate the ability of this analysis method to quantify changes in invadopodia formation from live cell imaging data in a high throughput, automated manner.

scholarly journals Quantification of proteins, protein complexes and mRNA in single cells by proximity-sequencing

2020 ◽  
Author(s):  
Luke Vistain ◽  
Hoang Van Phan ◽  
Christian Jordi ◽  
Mengjie Chen ◽  
Sai T. Reddy ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Complexes ◽  
Single Cells ◽  
Cell Types ◽  
Cellular Behaviors ◽  
Functional States ◽  
Drug Studies ◽  
New Protein

Multiplexed analysis of single-cells enables accurate modeling of cellular behaviors, classification of new cell types, and characterization of their functional states. Here we present proximity-sequencing (Prox-seq), a method for simultaneous measurement of an individual cell’s proteins, protein complexes and mRNA. Prox-seq utilizes deep sequencing and barcoded proximity assays to measure proteins and their complexes from all pairwise combinations of targeted proteins, in thousands of single-cells. The number of measured protein complexes scales quadratically with the number of targeted proteins, providing unparalleled multiplexing capacity. We developed a high-throughput experimental and computational pipeline and demonstrated the potential of Prox-Seq for multi-omic analysis with a panel of 13 barcoded proximity probes, enabling the measurement of 91 protein complexes, along with thousands of mRNA molecules in single T-cells and B-cells. Prox-seq provides access to an untapped yet powerful measurement modality for single-cell phenotyping and can discover new protein interactions in signaling and drug studies.

scholarly journals Automated analysis of invadopodia dynamics in live cells

2014 ◽  
Author(s):  
Matthew E Berginski ◽  
Sarah J Creed ◽  
Shelly Cochran ◽  
David W Roadcap ◽  
James E Bear ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Metastatic Cancer ◽  
Automated Analysis ◽  
Time Lapse ◽  
Cell Types ◽  
Live Cells ◽  
Imaging Data ◽  
The Matrix ◽  
Invadopodia Formation

Multiple cell types form specialized protein complexes, podosomes or invadopodia and collectively referred to as invadosomes, which are used by the cell to actively degrade the surrounding extracellular matrix. Due to their potential importance in both healthy physiology as well as in pathological conditions such as cancer, the characterization of these structures has been of increasing interest. Following early descriptions of invadopodia, assays were developed which labelled the matrix underneath metastatic cancer cells allowing for the assessment of invadopodia activity in motile cells. However, characterization of invadopodia using these methods has traditionally been done manually with time-consuming and potentially biased quantification methods, limiting the number of experiments and the quantity of data that can be analysed. We have developed a system to automate the segmentation, tracking and quantification of invadopodia in time-lapse fluorescence image sets at both the single invadopodia level and whole cell level. We rigorously tested the ability of the method to detect changes in invadopodia formation and dynamics through the use of well-characterized small molecule inhibitors, with known effects on invadopodia. Our results demonstrate the ability of this analysis method to quantify changes in invadopodia formation from live cell imaging data in a high throughput, automated manner.

scholarly journals Characterization of major chromatin assembly proteins in tetrahymena thermophile using proeomic and molecular evolutionary analyses

2021 ◽  
Author(s):  
Syed N Shah
Keyword(s):  
Protein Interactions ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Regulation Of Gene Expression ◽  
Chromatin Assembly ◽  
Histone Chaperones ◽  
Protein Protein Interactions ◽  
Selective Forces ◽  
Assembly Proteins

Histones H3/H4 are deposited onto DNA in a replication-dependent or independent fashion by the CAF1 and HIRA protein complexes. Despite the identification of these protein complexes, mechanistic details remain unclear. Recently, we showed that in T. thermophila histone chaperones Nrp1, Asf1 and the Impβ6 importin function together to transport newly synthesized H3/H4 from the cytoplasm to the nucleus. To characterize chromatin assembly proteins in T.thermophila, I used affinity purification combined with mass spectrometry to identify protein-protein interactions of Nrp1, Cac2 subunit of CAF1, HIRA and histone modifying Hat1-complex in T. thermophila. I found that the three-subunit T.thermophila CAF1 complex interacts with Casein Kinase 2 (CKII), possibly accounting for previously reported human CAF1phosphorylation. I also found that Hat2 subunit of HAT1 complex is also shared by CAF1 complex as its Cac3 subunit. This suggests that Hat2/Cac3 might exist in two separate pools of protein complexes. Remarkably, proteomic analysis of Hat2/Cac3 in turn revealed that it forms several complexes with other proteins including SIN3, RXT3, LIN9 and TESMIN, all of which have known roles in the regulation of gene expression. Finally, I asked how selective forces might have impacted on the function of proteins involved in H3/H4 transport. Focusing on NASP which possesses several TPR motifs, I showed that its protein-protein interactions are conserved in T. thermophila. Using molecular evolutionary methods I show that different TPRs in NASP evolve at different rates possibly accounting for the functional diversity observed among different family members.

scholarly journals NMR Characterization of Surface Receptor Protein Interactions in Live Cells Using Methylcellulose Hydrogels

2020 ◽  
Vol 132 (10) ◽  
pp. 3914-3918 ◽  
Author(s):  
Borja Mateos ◽  
Marco Sealey‐Cardona ◽  
Katja Balazs ◽  
Judith Konrat ◽  
Guenther Staffler ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Receptor Protein ◽  
Live Cells ◽  
Nmr Characterization ◽  
Surface Receptor

"U" Protein and Migration Ratios in Paper Electrophoresis

1957 ◽  
Vol 3 (5) ◽  
pp. 599-608 ◽  
Author(s):  
Joel R Stern ◽  
Roland F Mais ◽  
Joseph D Boggs
Keyword(s):  
Protein Complexes ◽  
Periodic Acid ◽  
Paper Electrophoresis ◽  
Evidence Based ◽  
Physiological Conditions ◽  
Sudan Black B ◽  
Carbohydrate Complex ◽  
Liver And Kidney ◽  
And Migration

Abstract Paper electrophoresis of serum from children with liver and kidney disease has led to recognition of a protein which migrates between the 2 and globulins. This protein has been observed by other workers, but because of confusion in nomenclature, a new designation, "U" protein, has been employed. The use of migration ratios in locating peaks of the 2 "U" protein, and globulin when these components resolve poorly has been discussed. Characterization of "U" protein by employing the periodic acid-reduced fuchsin test for carbohydrate-protein complexes and Sudan black B for lipoprotein showed: (1)"U" protein varies in its content of protein-carbohydrate complex, and (2)"U" protein may be lipo-protein. Evidence based on benzidine tests and intentional hemolysis indicated that "U" protein was not an artifact resulting from accidental hemolysis, but a protein which is found in serum under certain physiological conditions.

scholarly journals ETO-2 Associates with SCL in Erythroid Cells and Megakaryocytes and Provides Repressor Functions in Erythropoiesis

2005 ◽  
Vol 25 (23) ◽  
pp. 10235-10250 ◽  
Author(s):  
Anna H. Schuh ◽  
Alex J. Tipping ◽  
Allison J. Clark ◽  
Isla Hamlett ◽  
Boris Guyot ◽  
...  
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
Protein Complexes ◽  
Fetal Liver ◽  
Regulation Of Gene Expression ◽  
Erythroid Differentiation ◽  
Cell Types ◽  
Erythroid Cells ◽  
Proteomic Approach ◽  
Endogenous Proteins

ABSTRACT Lineage specification and cellular maturation require coordinated regulation of gene expression programs. In large part, this is dependent on the activator and repressor functions of protein complexes associated with tissue-specific transcriptional regulators. In this study, we have used a proteomic approach to characterize multiprotein complexes containing the key hematopoietic regulator SCL in erythroid and megakaryocytic cell lines. One of the novel SCL-interacting proteins identified in both cell types is the transcriptional corepressor ETO-2. Interaction between endogenous proteins was confirmed in primary cells. We then showed that SCL complexes are shared but also significantly differ in the two cell types. Importantly, SCL/ETO-2 interacts with another corepressor, Gfi-1b, in red cells but not megakaryocytes. The SCL/ETO-2/Gfi-1b association is lost during erythroid differentiation of primary fetal liver cells. Genetic studies of erythroid cells show that ETO-2 exerts a repressor effect on SCL target genes. We suggest that, through its association with SCL, ETO-2 represses gene expression in the early stages of erythroid differentiation and that alleviation/modulation of the repressive state is then required for expression of genes necessary for terminal erythroid maturation to proceed.

scholarly journals Proximity labeling of protein complexes and cell-type-specific organellar proteomes in Arabidopsis enabled by TurboID

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Andrea Mair ◽  
Shou-Ling Xu ◽  
Tess C Branon ◽  
Alice Y Ting ◽  
Dominique C Bergmann
Keyword(s):  
Transcription Factor ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Cell Types ◽  
Specific Protein ◽  
Cell Type ◽  
Subcellular Compartment ◽  
Cellular Processes ◽  
Highly Active

Defining specific protein interactions and spatially or temporally restricted local proteomes improves our understanding of all cellular processes, but obtaining such data is challenging, especially for rare proteins, cell types, or events. Proximity labeling enables discovery of protein neighborhoods defining functional complexes and/or organellar protein compositions. Recent technological improvements, namely two highly active biotin ligase variants (TurboID and miniTurbo), allowed us to address two challenging questions in plants: (1) what are in vivo partners of a low abundant key developmental transcription factor and (2) what is the nuclear proteome of a rare cell type? Proteins identified with FAMA-TurboID include known interactors of this stomatal transcription factor and novel proteins that could facilitate its activator and repressor functions. Directing TurboID to stomatal nuclei enabled purification of cell type- and subcellular compartment-specific proteins. Broad tests of TurboID and miniTurbo in Arabidopsis and Nicotiana benthamiana and versatile vectors enable customization by plant researchers.

Binding of peanut lectin to specific epithelial cell types in kidney

1982 ◽  
Vol 242 (1) ◽  
pp. C117-C120 ◽  
Author(s):  
M. LeHir ◽  
B. Kaissling ◽  
B. M. Koeppen ◽  
J. B. Wade
Keyword(s):  
Cell Types ◽  
Rabbit Kidney ◽  
Specific Cell ◽  
Luminal Membrane ◽  
Isolation And Characterization ◽  
Collecting Ducts ◽  
Epithelial Membranes ◽  
Outer Stripe ◽  
Specific Affinity

The binding of peanut agglutinin (PNA) to epithelial membranes of the rabbit kidney was evaluated at the light- and electron-microscope level using PNA conjugated to horseradish peroxidase. In the renal cortex and outer stripe of the medulla PNA appears to bind exclusively to the luminal membrane of intercalated cells in connecting tubules and collecting ducts. PNA also binds to the thin descending limb of the loop of Henle in the inner stripe and inner zone of the medulla. This very specific affinity of PNA should be useful in the isolation and characterization of specific cell types in cytologically heterogeneous epithelia.

scholarly journals Harnessing molecular motors for nanoscale pulldown in live cells

2017 ◽  
Vol 28 (3) ◽  
pp. 463-475 ◽  
Author(s):  
Jonathan E. Bird ◽  
Melanie Barzik ◽  
Meghan C. Drummond ◽  
Daniel C. Sutton ◽  
Spencer M. Goodman ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Molecular Motors ◽  
Protein Complexes ◽  
Software Tool ◽  
Epifluorescence Microscopy ◽  
Live Cells ◽  
Data Sets ◽  
Domain Mapping ◽  
Myosin Motors

Protein–protein interactions (PPIs) regulate assembly of macromolecular complexes, yet remain challenging to study within the native cytoplasm where they normally exert their biological effect. Here we miniaturize the concept of affinity pulldown, a gold-standard in vitro PPI interrogation technique, to perform nanoscale pulldowns (NanoSPDs) within living cells. NanoSPD hijacks the normal process of intracellular trafficking by myosin motors to forcibly pull fluorescently tagged protein complexes along filopodial actin filaments. Using dual-color total internal reflection fluorescence microscopy, we demonstrate complex formation by showing that bait and prey molecules are simultaneously trafficked and actively concentrated into a nanoscopic volume at the tips of filopodia. The resulting molecular traffic jams at filopodial tips amplify fluorescence intensities and allow PPIs to be interrogated using standard epifluorescence microscopy. A rigorous quantification framework and software tool are provided to statistically evaluate NanoSPD data sets. We demonstrate the capabilities of NanoSPD for a range of nuclear and cytoplasmic PPIs implicated in human deafness, in addition to dissecting these interactions using domain mapping and mutagenesis experiments. The NanoSPD methodology is extensible for use with other fluorescent molecules, in addition to proteins, and the platform can be easily scaled for high-throughput applications.

Sign in / Sign up

Export Citation Format

Share Document