scholarly journals Imaging analysis to quantitate the Interplay of membrane and cytoplasm protein dynamics

2021 ◽  
Author(s):  
N Kislev ◽  
M Egozi ◽  
D Benayahu
Keyword(s):  
Membrane Proteins ◽  
Protein Expression ◽  
Glucose Transporter ◽  
Imaging Method ◽  
Protein Distribution ◽  
Cellular Functions ◽  
Membrane Expression ◽  
Cellular Processes ◽  
Imaging Tool

AbstractPlasma membrane proteins are extremely important in cell signaling and cellular functions. Protein expression and localization alter in response to various signals in a way that is dependent on cell type and niche. Compartmental quantification of the expression of particular proteins is a very useful means of understanding their role in cellular processes. Immunofluorescence staining is frequently used to investigate the distribution of proteins of interest. Here, we present an imaging method for quantifying the membrane to cytoplasm ratio (MCR) of proteins analyzed at single-cell resolution. This technique provides a robust quantification of membrane proteins and contributes new insights into membrane expression dynamics. We have developed a protocol that uses immunostaining to assess protein expression according to the fluorescent cellular distribution and to compute the MCR. The method was applied to measure the MCR of glucose transporter 4 (GLUT4) in response to insulin in 3T3-L1 cells, an in-vitro model for adipocyte function and adipogenesis. The results revealed informative changes in the subcellular localization of GLUT4 following insulin induction. MCR analysis is a powerful imaging tool that can be generally applied to membrane proteins to provide a rapid and efficient quantitative analysis of protein distribution and sub-cellular processes in cells.

scholarly journals CRISPR-mediated isogenic cell-SELEX approach for generating highly specific aptamers against native membrane proteins

2020 ◽  
Author(s):  
Jonah C. Rosch ◽  
Emma H. Neal ◽  
Daniel A. Balikov ◽  
Mohsin Rahim ◽  
Ethan S. Lippmann
Keyword(s):  
Membrane Proteins ◽  
Glucose Transporter ◽  
High Specificity ◽  
Epithelial Cell Line ◽  
Target Cells ◽  
Wild Type ◽  
Simultaneous Exposure ◽  
Affinity Reagents ◽  
A Cell

AbstractIntroductionThe generation of affinity reagents that bind native membrane proteins with high specificity remains challenging. Most in vitro selection paradigms utilize different cell types for positive and negative rounds of selection (where the positive selection is against a cell that expresses the desired membrane protein and the negative selection is against a cell that lacks the protein). However, this strategy can yield affinity reagents that bind unintended membrane proteins on the target cells. To address this issue, we developed a systematic evolution of ligands by exponential enrichment (SELEX) scheme that utilizes isogenic pairs of cells generated via CRISPR techniques.MethodsUsing a Caco-2 epithelial cell line with constitutive Cas9 expression, we knocked out the SLC2A1 gene (encoding the GLUT1 glucose transporter) via lipofection with synthetic gRNAs. Cell-SELEX rounds were carried out against wild-type and GLUT1-null cells using a single-strand DNA (ssDNA) library. Next-generation sequencing (NGS) was used to quantify enrichment of prospective binders to the wild-type cells.Results10 rounds of cell-SELEX were conducted via simultaneous exposure of ssDNA pools to wild-type and GLUT1-null Caco-2 cells under continuous perfusion. The top binders identified from NGS were validated by flow cytometry and immunostaining for their specificity to the GLUT1 receptor.ConclusionsOur data indicate that highly specific aptamers can be isolated with a SELEX strategy that utilizes isogenic cell lines. This approach should be broadly useful for generating affinity reagents that selectively bind to membrane proteins in their native conformations on the cell surface.

scholarly journals A Human DUB Protein Array for Clarification of Linkage Specificity of Polyubiquitin Chain and Application to Evaluation of Its Inhibitors

Biomedicines ◽  
2020 ◽  
Vol 8 (6) ◽  
pp. 152
Author(s):  
Hirotaka Takahashi ◽  
Satoshi Yamanaka ◽  
Shohei Kuwada ◽  
Kana Higaki ◽  
Kohki Kido ◽  
...  
Keyword(s):  
Drug Targets ◽  
Protein Array ◽  
Polyubiquitin Chain ◽  
Deubiquitinating Enzymes ◽  
Cellular Functions ◽  
Cellular Processes ◽  
Model Study ◽  
Biochemical Analyses ◽  
Almost All

Protein ubiquitinations play pivotal roles in many cellular processes, including homeostasis, responses to various stimulations, and progression of diseases. Deubiquitinating enzymes (DUBs) remove ubiquitin molecules from ubiquitinated proteins and cleave the polyubiquitin chain, thus negatively regulating numerous ubiquitin-dependent processes. Dysfunctions of many DUBs reportedly cause various diseases; therefore, DUBs are considered as important drug targets, although the biochemical characteristics and cellular functions of many DUBs are still unclear. Here, we established a human DUB protein array to detect the activity and linkage specificity of almost all human DUBs. Using a wheat cell-free protein synthesis system, 88 full-length recombinant human DUB proteins were prepared and termed the DUB array. In vitro DUB assays were performed with all of these recombinant DUBs, using eight linkage types of diubiquitins as substrates. As a result, 80 DUBs in the array showed DUB activities, and their linkage specificities were determined. These 80 DUBs included many biochemically uncharacterized DUBs in the past. In addition, taking advantage of these active DUB proteins, we applied the DUB array to evaluate the selectivities of DUB inhibitors. We successfully developed a high-throughput and semi-quantitative DUB assay based on AlphaScreen technology, and a model study using two commercially available DUB inhibitors revealed individual selectivities to 29 DUBs, as previously reported. In conclusion, the DUB array established here is a powerful tool for biochemical analyses and drug discovery for human DUBs.

Dynamic Mechanical Properties Control Adult Stem Cell Fate

2012 ◽  
Author(s):  
Murat Guvendiren ◽  
Jason A. Burdick
Keyword(s):  
Stem Cell ◽  
Cell Fate ◽  
Adult Stem Cell ◽  
Dynamic Mechanical Properties ◽  
Cellular Functions ◽  
Cellular Processes ◽  
Proliferation And Differentiation ◽  
Important Field ◽  
Control Adult

Stem cells respond to many microenvironmental cues towards their decisions to spread, migrate, and differentiate and these cues can be incorporated into materials for regenerative medicine.1 In the last decade, matrix stiffness alone has been implicated in regulating cellular functions such as migration, proliferation and differentiation. With this in mind, a variety of natural and synthetic polymer systems were used in vitro to mimic the elasticity of native tissues. Despite helping to develop this important field and gather valuable information, these substrates are primarily static and lack the dynamic nature that is observed during many cellular processes such as development, fibrosis and cancer. Thus, it is of great interest to temporally manipulate matrix elasticity in vitro to better understand and develop strategies to control these biological processes. In this work, we utilize a sequential crosslinking approach (initial gelation via addition reaction, secondary crosslinking through light-mediated radical polymerization) to fabricate hydrogel substrates that stiffen (e.g., ∼3 to 30 kPa) either immediately or at later times and in the presence of cells. We demonstrate the utility of this technique by investigating the short-term (several minutes to hours) and long-term (several days to weeks) stem cell response to dynamic stiffening

scholarly journals Ecklonia cavaInhibits Glucose Absorption and Stimulates Insulin Secretion in Streptozotocin-Induced Diabetic Mice

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Hye Kyung Kim
Keyword(s):  
Insulin Secretion ◽  
Protein Expression ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Islet Cells ◽  
Glucose Absorption ◽  
Oral Glucose ◽  
Diabetic Mice ◽  
Glucose Transporter 1

Aims of study. Present study investigated the effect ofEcklonia cava(EC) on intestinal glucose uptake and insulin secretion.Materials and methods. Intestinal Na+-dependent glucose uptake (SGU) and Na+-dependent glucose transporter 1 (SGLT1) protein expression was determined using brush border membrane vesicles (BBMVs). Glucose-induced insulin secretion was examined in pancreatic β-islet cells. The antihyperglycemic effects of EC, SGU, and SGLT1 expression were determined in streptozotocin (STZ)-induced diabetic mice.Results. Methanol extract of EC markedly inhibited intestinal SGU of BBMV with the IC50value of 345 μg/mL. SGLT1 protein expression was dose dependently down regulated with EC treatment. Furthermore, insulinotrophic effect of EC extract was observed at high glucose media in isolated pancreatic β-islet cellsin vitro. We next conducted the antihyperglycemic effect of EC in STZ-diabetic mice. EC supplementation markedly suppressed SGU and SGLT1 abundance in BBMV from STZ mice. Furthermore, plasma insulin level was increased by EC treatment in diabetic mice. As a result, EC supplementation improved postprandial glucose regulation, assessed by oral glucose tolerance test, in diabetic mice.Conclusion. These results suggest that EC play a role in controlling dietary glucose absorption at the intestine and insulinotrophic action at the pancreas contributing blood glucose homeostasis in diabetic condition.

scholarly journals Dose-dependent phorbol 12-myristate-13-acetate-mediated monocyte-to-macrophage differentiation induces unique proteomic signatures in THP-1 cells

2020 ◽  
Author(s):  
Sneha M. Pinto ◽  
Hera Kim ◽  
Yashwanth Subbannayya ◽  
Miriam Giambelluca ◽  
Korbinian Bösl ◽  
...  
Keyword(s):  
Immune Responses ◽  
Protein Expression ◽  
Macrophage Differentiation ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Cellular Processes ◽  
Human Monocytic Cell Line ◽  
Human Monocytic Cell ◽  
Altered Gene

AbstractMacrophages are sentinels of the innate immune system, and the human monocytic cell line THP-1 is one of the widely used in vitro models to study immune responses. Several monocyte-to-macrophage differentiation protocols exist, with phorbol 12-myristate-13-acetate (PMA) being the widely used and accepted method. However, the concentrations and duration of PMA treatment vary widely in the published literature and their effect on protein expression is not fully deciphered. In this study, we employed a dimethyl labeling-based quantitative proteomics approach to determine the changes in the protein repertoire of macrophage-like cells differentiated from THP-1 monocytes by three commonly used PMA-based differentiation protocols. Our analysis shows that variations in PMA concentration and duration of rest post-stimulation result in downstream differences in the protein expression and cellular processes. We demonstrate that these differences result in altered gene expression of cytokines upon stimulation with various TLR agonists. Together, these findings provide a valuable resource that significantly expands the knowledge of protein expression dynamics with one of the most common in vitro models for macrophages, which in turn has a profound impact on the immune responses being studied.

scholarly journals Epigenetic upregulation by valproic acid of transferrin receptor 1, and not glucose transporter 1 or CD98hc, at the blood-brain barrier

2022 ◽  
Author(s):  
Steinunn Sara Helgudóttir ◽  
Kasper Bendix Johnsen ◽  
Lisa Juul Routhe ◽  
Charlotte L.M. Rasmussen ◽  
Azra Karamehmedovic ◽  
...  
Keyword(s):  
Valproic Acid ◽  
Endothelial Cells ◽  
Protein Expression ◽  
Transferrin Receptor ◽  
Glucose Transporter ◽  
Brain Capillaries ◽  
Glucose Transporter 1 ◽  
Transferrin Receptor 1

Abstract BackgroundThe objectives of the present study were to investigate whether the expression of transferrin receptor 1 (TfR1), glucose transporter 1 (Glut1), or Cluster of Differentiation 98 Heavy Chain (CD98hc) is epigenetically regulated in brain capillary endothelial cells (BCECs) denoting the blood-brain barrier (BBB).MethodsThe expression of these targets was investigated both in vitro and in vivo following treatment with the histone deacetylase inhibitor (HDACi) valproic acid (VPA). Mice were injected intraperitoneally with VPA followed by analysis of isolated brain capillaries, and the capillary depleted brain samples. Brain tissue, isolated brain capillaries, and cultured primary endothelial cells were analyzed by RT-qPCR, immunolabeling and ELISA for expression of TfR1, Glut1 and CD98hc. We also studied the vascular targeting in VPA-treated mice injected with monoclonal anti-transferrin receptor (Ri7) conjugated with 1.4 nm gold nanoparticles. ResultsValidating the effects of VPA on gene transcription in BCECs, transcriptomic analysis identified 24,371 expressed genes, of which 305 were differentially expressed with 192 upregulated and 113 downregulated genes. In vitro using BCECs co-cultured with glial cells, the mRNA expression of Tfrc was significantly higher after VPA treatment for 6 h with its expression returning to baseline after 24 h. Conversely, the mRNA expression of Glut1 and Cd98hc was unaffected by VPA treatment. In vivo, the TfR1 protein expression in brain capillaries increased significantly after treatment with both 100 mg/kg and 400 mg/kg VPA. Conversely, VPA treatment did not increase GLUT1 or CD98hc. Using ICP-MS-based quantification, the brain uptake of nanogold conjugated anti-TfR1 antibodies was non-significant in spite of increased expression of TfR1. ConclusionsWe report that VPA treatment upregulates TfR1 at the BBB both in vivo and in vitro in isolated primary endothelial cells. In contrast, VPA treatment does not influence the expression of GLUT1 and CD98hc. The increase in the overall TfR1 protein expression however does not increase transport of TfR-targeted monoclonal antibody and indicates that targeted delivery using the transferrin receptor should aim for increased mobilization of already available transferrin receptor molecules to improve trafficking through the BBB.

scholarly journals F-Actin Cytoskeleton Network Self-Organization Through Competition and Cooperation

2020 ◽  
Vol 36 (1) ◽  
pp. 35-60
Author(s):  
Rachel S. Kadzik ◽  
Kaitlin E. Homa ◽  
David R. Kovar
Keyword(s):  
Actin Binding ◽  
Filamentous Actin ◽  
Cellular Functions ◽  
Actin Organization ◽  
Actin Networks ◽  
Cellular Processes ◽  
Competition And Cooperation ◽  
A Cell ◽  
Overlapping Sets

Many fundamental cellular processes such as division, polarization, endocytosis, and motility require the assembly, maintenance, and disassembly of filamentous actin (F-actin) networks at specific locations and times within the cell. The particular function of each network is governed by F-actin organization, size, and density as well as by its dynamics. The distinct characteristics of different F-actin networks are determined through the coordinated actions of specific sets of actin-binding proteins (ABPs). Furthermore, a cell typically assembles and uses multiple F-actin networks simultaneously within a common cytoplasm, so these networks must self-organize from a common pool of shared globular actin (G-actin) monomers and overlapping sets of ABPs. Recent advances in multicolor imaging and analysis of ABPs and their associated F-actin networks in cells, as well as the development of sophisticated in vitro reconstitutions of networks with ensembles of ABPs, have allowed the field to start uncovering the underlying principles by which cells self-organize diverse F-actin networks to execute basic cellular functions.

scholarly journals Experimental platform for the functional investigation of membrane proteins in giant unilamellar vesicles

2021 ◽  
Author(s):  
Nicolas Dolder ◽  
Philipp Mueller ◽  
Christoph von Ballmoos
Keyword(s):  
Membrane Proteins ◽  
Model Membrane ◽  
Giant Unilamellar Vesicles ◽  
Cellular Functions ◽  
Unilamellar Vesicles ◽  
Synthetic Cells ◽  
Buffer Composition ◽  
Protein Incorporation ◽  
Functional Investigation

Giant unilamellar vesicles (GUVs) are micrometer-sized model membrane systems that can be viewed directly under the microscope. They serve as scaffolds for the bottom-up creation of synthetic cells, targeted drug delivery and have been used in many in vitro studies of membrane related phenomena. GUVs are also of interest for the functional investigation of membrane proteins that carry out many key cellular functions. A major hurdle to a wider application of GUVs in this field is the diversity of existing protocols that are optimized for individual proteins. Here, we compare PVA assisted and electroformation techniques for GUV formation under physiologically relevant conditions, and analyze the effect of immobilization on vesicle structure and membrane tightness towards small substrates and protons. There, differences in terms of yield, size, and leakage of GUVs produced by PVA assisted swelling and electroformation were found, dependent on salt and buffer composition. Using fusion of oppositely charged membranes to reconstitute a model membrane protein, we find that empty vesicles and proteoliposomes show similar fusion behavior, which allows for a rapid estimation of protein incorporation using fluorescent lipids.

scholarly journals RAB6 GTPase regulates mammary secretory function by controlling the activation of STAT5

Development ◽  
2020 ◽  
Vol 147 (19) ◽  
pp. dev190744 ◽  
Author(s):  
Surya Cayre ◽  
Marisa M. Faraldo ◽  
Sabine Bardin ◽  
Stéphanie Miserey-Lenkei ◽  
Marie-Ange Deugnier ◽  
...  
Keyword(s):  
Prolactin Receptor ◽  
Retrograde Transport ◽  
Mammary Epithelial ◽  
Epithelial Cell Line ◽  
Rab Gtpases ◽  
Cellular Functions ◽  
Membrane Expression ◽  
Transport Pathways

ABSTRACTThe Golgi-associated RAB GTPases, RAB6A and RAB6A′, regulate anterograde and retrograde transport pathways from and to the Golgi. In vitro, RAB6A/A′ control several cellular functions including cell division, migration, adhesion and polarity. However, their role remains poorly described in vivo. Here, we generated BlgCre; Rab6aF/F mice presenting a specific deletion of Rab6a in the mammary luminal secretory lineage during gestation and lactation. Rab6a loss severely impaired the differentiation, maturation and maintenance of the secretory tissue, compromising lactation. The mutant epithelium displayed a decreased activation of STAT5, a key regulator of the lactogenic process primarily governed by prolactin. Data obtained with a mammary epithelial cell line suggested that defective STAT5 activation might originate from a perturbed transport of the prolactin receptor, altering its membrane expression and signaling cascade. Despite the major functional defects observed upon Rab6a deletion, the polarized organization of the mammary epithelial bilayer was preserved. Altogether, our data reveal a crucial role for RAB6A/A′ in the lactogenic function of the mammary gland and suggest that the trafficking pathways controlled by RAB6A/A′ depend on cell-type specialization and tissue context.

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