Dual labeling of the fibronectin matrix and actin cytoskeleton with green fluorescent protein variants

We have prepared 3T3 cells doubly labeled to visualize simultaneously the extracellular fibronectin (FN) matrix and intracellular actin cytoskeleton in living cell cultures. We used FN-yellow fluorescent protein (FN-yfp) for the FN matrix, and the actin-binding domain of moesin fused to cyan fluorescent protein (cfp-Moe) to stain actin. Actin filament bundles were clearly seen in the protruding lamellae of the cells. FN matrix assembly appeared to be initiated as small spots of FN at the ends of actin filament bundles. The spots then elongated along the actin filament bundle toward the cell center to form FN fibrils. The end of the fibril towards the cell edge appeared immobile, and probably attached to the substrate, whereas the end toward the cell center frequently showed movements, suggesting attachment to the cell. Combining our data with the observations of Pankov et al. we suggest that fibrils grow by stretching this mobile end toward the cell center while adding new FN molecules at the end and along the entire lenght. When the cell culture was treated with cytochalasin to disrupt the actin cytoskeleton, some fibrils contracted substantially, suggesting that the segment attached primarily to the cell surface is stretched.

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A Green Fluorescent Protein Fusion to Actin-Binding Domain 2 of Arabidopsis Fimbrin Highlights New Features of a Dynamic Actin Cytoskeleton in Live Plant Cells

PLANT PHYSIOLOGY ◽

10.1104/pp.104.049411 ◽

2004 ◽

Vol 136 (4) ◽

pp. 3968-3978 ◽

Cited By ~ 161

Author(s):

Michael B. Sheahan ◽

Chris J. Staiger ◽

Ray J. Rose ◽

David W. McCurdy

Keyword(s):

Green Fluorescent Protein ◽

Actin Cytoskeleton ◽

Fluorescent Protein ◽

Plant Cells ◽

Binding Domain ◽

Actin Binding ◽

Green Fluorescent Protein Fusion ◽

Protein Fusion ◽

Actin Binding Domain ◽

Green Fluorescent

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Regulation of the Cortical Actin Cytoskeleton in Budding Yeast by Twinfilin, a Ubiquitous Actin Monomer-sequestering Protein

The Journal of Cell Biology ◽

10.1083/jcb.142.3.723 ◽

1998 ◽

Vol 142 (3) ◽

pp. 723-733 ◽

Cited By ~ 85

Author(s):

Bruce L. Goode ◽

David G. Drubin ◽

Pekka Lappalainen

Keyword(s):

Actin Cytoskeleton ◽

Actin Filament ◽

Fluorescent Protein ◽

Budding Yeast ◽

Yeast Cells ◽

Actin Binding ◽

Actin Monomer ◽

Nucleotide Exchange ◽

Cortical Actin ◽

Green Fluorescent

Here we describe the identification of a novel 37-kD actin monomer binding protein in budding yeast. This protein, which we named twinfilin, is composed of two cofilin-like regions. In our sequence database searches we also identified human, mouse, and Caenorhabditis elegans homologues of yeast twinfilin, suggesting that twinfilins form an evolutionarily conserved family of actin-binding proteins. Purified recombinant twinfilin prevents actin filament assembly by forming a 1:1 complex with actin monomers, and inhibits the nucleotide exchange reaction of actin monomers. Despite the sequence homology with the actin filament depolymerizing cofilin/actin-depolymerizing factor (ADF) proteins, our data suggests that twinfilin does not induce actin filament depolymerization. In yeast cells, a green fluorescent protein (GFP)–twinfilin fusion protein localizes primarily to cytoplasm, but also to cortical actin patches. Overexpression of the twinfilin gene (TWF1) results in depolarization of the cortical actin patches. A twf1 null mutation appears to result in increased assembly of cortical actin structures and is synthetically lethal with the yeast cofilin mutant cof1-22, shown previously to cause pronounced reduction in turnover of cortical actin filaments. Taken together, these results demonstrate that twinfilin is a novel, highly conserved actin monomer-sequestering protein involved in regulation of the cortical actin cytoskeleton.

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αE-catenin actin-binding domain alters actin filament conformation and regulates binding of nucleation and disassembly factors

Molecular Biology of the Cell ◽

10.1091/mbc.e13-07-0388 ◽

2013 ◽

Vol 24 (23) ◽

pp. 3710-3720 ◽

Cited By ~ 62

Author(s):

Scott D. Hansen ◽

Adam V. Kwiatkowski ◽

Chung-Yueh Ouyang ◽

HongJun Liu ◽

Sabine Pokutta ◽

...

Keyword(s):

Actin Filament ◽

Actin Filaments ◽

Binding Domain ◽

Actin Binding ◽

Filamentous Actin ◽

Filament Structure ◽

Actin Binding Domain ◽

Filament Bundles ◽

Underlying Mechanisms ◽

Cell Cell

The actin-binding protein αE-catenin may contribute to transitions between cell migration and cell–cell adhesion that depend on remodeling the actin cytoskeleton, but the underlying mechanisms are unknown. We show that the αE-catenin actin-binding domain (ABD) binds cooperatively to individual actin filaments and that binding is accompanied by a conformational change in the actin protomer that affects filament structure. αE-catenin ABD binding limits barbed-end growth, especially in actin filament bundles. αE-catenin ABD inhibits actin filament branching by the Arp2/3 complex and severing by cofilin, both of which contact regions of the actin protomer that are structurally altered by αE-catenin ABD binding. In epithelial cells, there is little correlation between the distribution of αE-catenin and the Arp2/3 complex at developing cell–cell contacts. Our results indicate that αE-catenin binding to filamentous actin favors assembly of unbranched filament bundles that are protected from severing over more dynamic, branched filament arrays.

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Fluorescent protein-based reporters of the actin cytoskeleton in living plant cells: Fluorophore variant, actin binding domain, and promoter considerations

Cytoskeleton ◽

10.1002/cm.21174 ◽

2014 ◽

Vol 71 (5) ◽

pp. 311-327 ◽

Cited By ~ 24

Author(s):

Julia Dyachok ◽

J. Alan Sparks ◽

Fuqi Liao ◽

Yuh-Shuh Wang ◽

Elison B. Blancaflor

Keyword(s):

Actin Cytoskeleton ◽

Fluorescent Protein ◽

Plant Cells ◽

Binding Domain ◽

Actin Binding ◽

Living Plant ◽

Actin Binding Domain

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Improved imaging of actin filaments in transgenic Arabidopsis plants expressing a green fluorescent protein fusion to the C- and N-termini of the fimbrin actin-binding domain 2

New Phytologist ◽

10.1111/j.1469-8137.2007.02261.x ◽

2007 ◽

Vol 0 (0) ◽

pp. 071120093824004-??? ◽

Cited By ~ 12

Author(s):

Yuh-Shuh Wang ◽

Cheol-Min Yoo ◽

Elison B. Blancaflor

Keyword(s):

Green Fluorescent Protein ◽

Actin Filaments ◽

Fluorescent Protein ◽

Transgenic Arabidopsis ◽

Actin Binding ◽

Green Fluorescent Protein Fusion ◽

Protein Fusion ◽

Actin Binding Domain ◽

C And N ◽

Green Fluorescent

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The Actin-Driven Movement and Formation of Acetylcholine Receptor Clusters

The Journal of Cell Biology ◽

10.1083/jcb.150.6.1321 ◽

2000 ◽

Vol 150 (6) ◽

pp. 1321-1334 ◽

Cited By ~ 83

Author(s):

Zhengshan Dai ◽

Xiaoyan Luo ◽

Hongbo Xie ◽

H. Benjamin Peng

Keyword(s):

Actin Cytoskeleton ◽

Acetylcholine Receptor ◽

Actin Polymerization ◽

Fluorescent Protein ◽

Actin Binding ◽

Heparin Binding ◽

Actin Assembly ◽

Achr Cluster ◽

Green Fluorescent ◽

Receptor Clusters

A new method was devised to visualize actin polymerization induced by postsynaptic differentiation signals in cultured muscle cells. This entails masking myofibrillar filamentous (F)-actin with jasplakinolide, a cell-permeant F-actin–binding toxin, before synaptogenic stimulation, and then probing new actin assembly with fluorescent phalloidin. With this procedure, actin polymerization associated with newly induced acetylcholine receptor (AChR) clustering by heparin-binding growth-associated molecule–coated beads and by agrin was observed. The beads induced local F-actin assembly that colocalized with AChR clusters at bead–muscle contacts, whereas both the actin cytoskeleton and AChR clusters induced by bath agrin application were diffuse. By expressing a green fluorescent protein–coupled version of cortactin, a protein that binds to active F-actin, the dynamic nature of the actin cytoskeleton associated with new AChR clusters was revealed. In fact, the motive force generated by actin polymerization propelled the entire bead-induced AChR cluster with its attached bead to move in the plane of the membrane. In addition, actin polymerization is also necessary for the formation of both bead and agrin-induced AChR clusters as well as phosphotyrosine accumulation, as shown by their blockage by latrunculin A, a toxin that sequesters globular (G)-actin and prevents F-actin assembly. These results show that actin polymerization induced by synaptogenic signals is necessary for the movement and formation of AChR clusters and implicate a role of F-actin as a postsynaptic scaffold for the assembly of structural and signaling molecules in neuromuscular junction formation.

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Stable transformation and actin visualization in callus cultures of dodder (Cuscuta europaea)

Biologia ◽

10.2478/s11756-013-0188-0 ◽

2013 ◽

Vol 68 (4) ◽

Cited By ~ 2

Author(s):

Renáta Švubová ◽

Alžbeta Blehová

Keyword(s):

Actin Cytoskeleton ◽

Fluorescent Protein ◽

Callus Cultures ◽

Visual Selection ◽

Actin Binding ◽

Selection Marker ◽

Nptii Gene ◽

Cortical Actin ◽

Cuscuta Europaea ◽

Green Fluorescent

AbstractAgrobacterium tumefaciens-mediated transformation of callus culture, combined with a visual selection of GFP-tagged fimbrin actin binding domain (FABD2) expression is described for parasitic species (Cuscuta europaea). The conditions for callus induction from 1 cm-long explants from the basal part of 7-day-old dodder seedlings were defined. We obtained light-green calli, which were transformed with A. tumefaciens bacterial strain GV3101 carrying plasmid pCB302 (35S::ABD2:gfp) with neomycin phosphotransferase (nptII) gene. The limitations of selection procedures based on antibiotics were avoided using green fluorescent protein (GFP) detection, as a visual selection marker subcellularly targeted to the actin cytoskeleton. Fluorescence microscopy analyses demonstrated a network of nucleus-associated actin arrays and dense cortical actin arrangements in stably transformed Cuscuta callus cells. RT-PCR analyses confirmed gfp expression in transformed calli 7, 14 and 21 days after transformation. Although the GFP fluorescence associated with the actin cytoskeleton has retained for at least six months without silencing, no shoot regeneration was observed. It can be concluded that, C. europaea callus cells are competent for transformation, but under given conditions, these cells failed to realize their morphogenic and regeneration potentials.

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Dynamin Is Required for GnRH Signaling to L-Type Calcium Channels and Activation of ERK

Endocrinology ◽

10.1210/en.2015-1575 ◽

2015 ◽

Vol 157 (2) ◽

pp. 831-843 ◽

Cited By ~ 9

Author(s):

Brian S. Edwards ◽

An K. Dang ◽

Dilyara A. Murtazina ◽

Melissa G. Dozier ◽

Jennifer D. Whitesell ◽

...

Keyword(s):

Plasma Membrane ◽

Green Fluorescent Protein ◽

Actin Cytoskeleton ◽

Fluorescent Protein ◽

Leading Edge ◽

Dominant Negative ◽

Actin Binding ◽

Erk Phosphorylation ◽

Green Fluorescent ◽

Ca2 Channels

Abstract We have shown that GnRH-mediated engagement of the cytoskeleton induces cell movement and is necessary for ERK activation. It also has previously been established that a dominant negative form of the mechano-GTPase dynamin (K44A) attenuates GnRH activation of ERK. At present, it is not clear at what level these cellular events might be linked. To explore this, we used live cell imaging in the gonadotrope-derived αT3–1 cell line to determine that dynamin-green fluorescent protein accumulated in GnRH-induced lamellipodia and plasma membrane protrusions. Coincident with translocation of dynamin-green fluorescent protein to the plasma membrane, we demonstrated that dynamin colocalizes with the actin cytoskeleton and the actin binding protein, cortactin at the leading edge of the plasma membrane. We next wanted to assess the physiological significance of these findings by inhibiting dynamin GTPase activity using dynasore. We find that dynasore suppresses activation of ERK, but not c-Jun N-terminal kinase, after exposure to GnRH agonist. Furthermore, exposure of αT3–1 cells to dynasore inhibited GnRH-induced cyto-architectural rearrangements. Recently it has been discovered that GnRH induced Ca2+ influx via the L-type Ca2+ channels requires an intact cytoskeleton to mediate ERK phosphorylation. Interestingly, not only does dynasore attenuate GnRH-mediated actin reorganization, it also suppresses Ca2+ influx through L-type Ca2+ channels visualized in living cells using total internal reflection fluorescence microscopy. Collectively, our data suggest that GnRH-induced membrane remodeling events are mediated in part by the association of dynamin and cortactin engaging the actin cytoskeleton, which then regulates Ca2+ influx via L-type channels to facilitate ERK phosphorylation.

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A machine-learning-guided mutagenesis platform for accelerated discovery of novel functional proteins

10.1101/282996 ◽

2018 ◽

Author(s):

Yutaka Saito ◽

Misaki Oikawa ◽

Hikaru Nakazawa ◽

Teppei Niide ◽

Tomoshi Kameda ◽

...

Keyword(s):

Machine Learning ◽

Molecular Evolution ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Screening Experiments ◽

Novel Approach ◽

Machine Learning Model ◽

High Enrichment ◽

Green Fluorescent ◽

Protein Variants

AbstractMolecular evolution based on mutagenesis is widely used in protein engineering. However, optimal proteins are often difficult to obtain due to a large sequence space that requires high costs for screening experiments. Here, we propose a novel approach that combines molecular evolution with machine learning. In this approach, we conduct two rounds of mutagenesis where an initial library of protein variants is used to train a machine-learning model to guide mutagenesis for the second-round library. This enables to prepare a small library suited for screening experiments with high enrichment of functional proteins. We demonstrated a proof-of-concept of our approach by altering the reference green fluorescent protein (GFP) so that its fluorescence is changed to yellow while improving its fluorescence intensity. Using 155 and 78 variants for the initial and the second-round libraries, respectively, we successfully obtained a number of proteins showing yellow fluorescence, 12 of which had better fluorescence performance than the reference yellow fluorescent protein (YFP). These results show the potential of our approach as a powerful platform for accelerated discovery of functional proteins.

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