Soluble expression and characterization of a GFP-fused pea actin isoform (PEAc1)

Liu, Ai Xiao; Zhang, Shao Bin; Xu, Xiao; Ren, Dongtao; Liu, Guo Qin

doi:10.1038/sj.cr.7290241

Cited by 11 publications

(11 citation statements)

References 52 publications

(50 reference statements)

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“…However, there has been few reports of visualizing GFP-actin in plant cells, such as enrichment of actin-GFP around the nuclei and near the tip and cell wall in N . tabacum BY2 suspension cells 23 , and bundle-like patches of actin-GFP on mitochondria in N . tabacum protoplasts 24 .…”

Section: Discussionmentioning

confidence: 99%

“…Many studies successfully visualized actin filaments in other live eukaryotic cells, including Dictyostelium discoideum 20 and mammalian cells 21 , 22 , by expression of GFP-actin. In plant cells, two studies reported expression of actin-GFP in Nicotiana tabacum BY2 cells and protoplasts, respectively, but no long filamentous structures were observed even though localized GFP fluorescence was detected 23 , 24 . For these reasons, GFP fused with the actin-binding domain (ABD) of mouse talin1 (GFP-mTalin1), was the first GFP-based probe to observe actin filaments in living plant cells 25 .…”

Section: Introductionmentioning

confidence: 99%

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Arabidopsis vegetative actin isoforms, AtACT2 and AtACT7, generate distinct filament arrays in living plant cells

Kijima

Staiger²,

Katoh

et al. 2018

Sci Rep

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Flowering plants express multiple actin isoforms. Previous studies suggest that individual actin isoforms have specific functions; however, the subcellular localization of actin isoforms in plant cells remains obscure. Here, we transiently expressed and observed major Arabidopsis vegetative actin isoforms, AtACT2 and AtACT7, as fluorescent-fusion proteins. By optimizing the linker sequence between fluorescent protein and actin, we succeeded in observing filaments that contained these expressed actin isoforms fused with green fluorescent protein (GFP) in Arabidopsis protoplasts. Different colored fluorescent proteins fused with AtACT2 and AtACT7 and co-expressed in Nicotiana benthamiana mesophyll cells co-polymerized in a segregated manner along filaments. In epidermal cells, surprisingly, AtACT2 and AtACT7 tended to polymerize into different types of filaments. AtACT2 was incorporated into thinner filaments, whereas AtACT7 was incorporated into thick bundles. We conclude that different actin isoforms are capable of constructing unique filament arrays, depending on the cell type or tissue. Interestingly, staining patterns induced by two indirect actin filament probes, Lifeact and mTalin1, were different between filaments containing AtACT2 and those containing AtACT7. We suggest that filaments containing different actin isoforms bind specific actin-binding proteins in vivo, since the two probes comprise actin-binding domains from different actin-binding proteins.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Arabidopsis vegetative actin isoforms, AtACT2 and AtACT7, generate distinct filament arrays in living plant cells

Kijima

Staiger²,

Katoh

et al. 2018

Sci Rep

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“…Although its use as an agent to facilitate the soluble expression of proteins that misfold or aggregate when overproduced in Escherichia coli has been approached with caution [85], success has been reported for a plant actin [86]. We reasoned that, given its robust solubility, it might be used to facilitate soluble expression of the intractably insoluble IS 2 TPase under native conditions.…”

Section: Discussionmentioning

confidence: 99%

Soluble expression, purification and characterization of the full length IS2 Transposase

et al. 2011

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BackgroundThe two-step transposition pathway of insertion sequences of the IS3 family, and several other families, involves first the formation of a branched figure-of-eight (F-8) structure by an asymmetric single strand cleavage at one optional donor end and joining to the flanking host DNA near the target end. Its conversion to a double stranded minicircle precedes the second insertional step, where both ends function as donors. In IS2, the left end which lacks donor function in Step I acquires it in Step II. The assembly of two intrinsically different protein-DNA complexes in these F-8 generating elements has been intuitively proposed, but a barrier to testing this hypothesis has been the difficulty of isolating a full length, soluble and active transposase that creates fully formed synaptic complexes in vitro with protein bound to both binding and catalytic domains of the ends. We address here a solution to expressing, purifying and structurally analyzing such a protein.ResultsA soluble and active IS2 transposase derivative with GFP fused to its C-terminus functions as efficiently as the native protein in in vivo transposition assays. In vitro electrophoretic mobility shift assay data show that the partially purified protein prepared under native conditions binds very efficiently to cognate DNA, utilizing both N- and C-terminal residues. As a precursor to biophysical analyses of these complexes, a fluorescence-based random mutagenesis protocol was developed that enabled a structure-function analysis of the protein with good resolution at the secondary structure level. The results extend previous structure-function work on IS3 family transposases, identifying the binding domain as a three helix H + HTH bundle and explaining the function of an atypical leucine zipper-like motif in IS2. In addition gain- and loss-of-function mutations in the catalytic active site define its role in regional and global binding and identify functional signatures that are common to the three dimensional catalytic core motif of the retroviral integrase superfamily.ConclusionsIntractably insoluble transposases, such as the IS2 transposase, prepared by solubilization protocols are often refractory to whole protein structure-function studies. The results described here have validated the use of GFP-tagging and fluorescence-based random mutagenesis in overcoming this limitation at the secondary structure level.

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“…PCR ampliWcations were performed on 1-l cDNA template using the kit PCR Master (Roche). The sequences of the primers used for PCR ampliWcations were the following ones: for PsMPK2 (AF154329), forward 5Ј-GGATCCAT GGCGACTCCGGTCGAGCC-3Јand reverse 5Ј-CAGAAT ATATATCGAGCTCACCGATGACCG-3Ј corresponding with the 3Ј UTR; for the actin PEAct1 (X67666) (Liu et al 2004), forward 5Ј-GGATCCAAAAGATGGCCGATGCT GAGGAT-3Ј and reverse 5Ј-GCAAAATCACTTACCATT ATACGCGC-3Ј.…”

Section: Rt-pcrmentioning

confidence: 99%

Characterization of PsMPK2, the first C1 subgroup MAP kinase from pea (Pisum sativum L.)

Ortiz-Masiá

Pérez‐Amador

Carbonell

et al. 2008

Planta

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Mitogen-activated protein kinase (MAPK) cascades play a key role in plant growth and development as well as in biotic and abiotic stress responses. They are classified according to their sequence homology into four major groups (A-D). A large amount of information about MAPKs in groups A and B is available but few data of the C group have been reported. In this study, a C1 subgroup MAP kinase cDNA, PsMPK2, was isolated from Pisum sativum. PsMPK2 is expressed in vegetative (root and leaf) and reproductive (stamen, pistil and fruit) organs. Expression of PsMPK2 in Arabidopsis thaliana shows that mechanical injury and other stress signals as abscisic acid, jasmonic acid and hydrogen peroxide increase its kinase activity, extending previous results indicating that C1 subgroup MAPKs may be involved in the response to stress.

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Soluble expression and characterization of a GFP-fused pea actin isoform (PEAc1)

Cited by 11 publications

References 52 publications

Arabidopsis vegetative actin isoforms, AtACT2 and AtACT7, generate distinct filament arrays in living plant cells

Arabidopsis vegetative actin isoforms, AtACT2 and AtACT7, generate distinct filament arrays in living plant cells

Soluble expression, purification and characterization of the full length IS2 Transposase

Characterization of PsMPK2, the first C1 subgroup MAP kinase from pea (Pisum sativum L.)

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