Statistical modeling of mRNP transport in dendrites: A comparative analysis of ß-actin and Arc mRNP dynamics.

Localization of messenger RNA (mRNA) in dendrites is crucial for regulating gene expression during long-term memory formation. mRNA binds to RNA-binding proteins (RBPs) to form messenger ribonucleoprotein (mRNP) complexes that are transported by motor proteins along microtubules to their target synapses. However, the dynamics by which mRNPs find their target locations in the dendrite have not been well understood. Here, we investigated the motion of endogenous ß-actin and Arc mRNPs in dissociated mouse hippocampal neurons using the MS2 and PP7 stem-loop systems, respectively. By evaluating the statistical properties of mRNP movement, we found that the aging Lévy walk model effectively describes both ß-actin and Arc mRNP transport in proximal dendrites. A critical difference between ß-actin and Arc mRNPs was the aging time, the time lag between transport initiation and measurement initiation. The longer mean aging time of ß-actin mRNP (~100 s) compared with that of Arc mRNP (~30 s) reflects the longer half-life of constitutively expressed ß-actin mRNP. Furthermore, our model also permitted us to estimate the ratio of newly generated and pre-existing ß-actin mRNPs in the dendrites. This study offers a robust theoretical framework for mRNP transport, which provides insight into how mRNPs locate their targets in neurons.

Purification of human ß- and γ-actin from budding yeast.

Biochemical studies of human actin and its binding partners rely heavily on abundant and easily purified α-actin from skeletal muscle. Therefore, muscle actin has been used to evaluate and determine the activities of most actin regulatory proteins but there is an underlying concern that these proteins perform differently from actin present in non-muscle cells. To provide easily accessible and relatively abundant sources of human ß- or γ-actin (i.e. cytoplasmic actins), we developed Saccharomyces cerevisiae strains that express each as their sole source of actin. Both ß- or γ-actin purified in this system polymerize and interact with various binding partners, including profilin, mDia1 (formin), fascin and thymosin-ß4 (Tß4). Notably, Tß4 and profilin bind to ß- or γ-actin with higher affinity than to α-actin, emphasizing the value of testing actin ligands with specific actin isoforms. These reagents will make specific isoforms of actin more accessible for future studies on actin regulation.

The importance of m6A topology in chicken embryo mRNA: a precise mapping of m6A at the conserved chicken ß-actin zipcode.

N6-methyladenosine (m6A) in mRNA regulates almost every stage in the mRNA life cycle, and the development of methodologies for the high-throughput detection of methylated sites in mRNA using m6A-specific methylated RNA immunoprecipitation with next-generation sequencing (MeRIPSeq) or m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) have revolutionized the m6A research field. Both of these methods are based on immunoprecipitation of fragmented mRNA. However, it is well documented that antibodies often have nonspecific activities, thus verification of identified m6A sites using an antibody-independent method would be highly desirable. We mapped and quantified the m6A site in the chicken ß-actin zipcode based on the data from chicken embryo MeRIPSeq results and our RNA-Epimodification Detection and Base-Recognition (RedBaron) antibody-independent assay. We also demonstrated that methylation of this site in the ß-actin zipcode enhances ZBP1 binding in vitro, while methylation of a nearby adenosine abolishes binding. This suggests that m6A may play a role in regulating localized translation of ß-actin mRNA, and the ability of m6A to enhance or inhibit a reader protein's RNA binding highlights the importance of m6A detection at nucleotide resolution.

Dynamics of axonal ß-actin mRNA in live hippocampal neurons.

Localization of mRNA facilitates spatiotemporally controlled protein expression in neurons. In axons, mRNA transport followed by local protein synthesis plays a critical role in axonal growth and guidance. However, it is not yet clearly understood how mRNA is transported to axonal subcellular sites and what regulates axonal mRNA localization. Using a transgenic mouse model in which endogenous ß-actin mRNA is fluorescently labeled, we investigated ß-actin mRNA movement in axons of hippocampal neurons. We cultured neurons in microfluidic devices to separate axons from dendrites and performed single-particle tracking of axonal ß-actin mRNA. Compared with dendritic ß-actin mRNA, axonal ß-actin mRNA showed less directed motion and exhibited mostly subdiffusive motion, especially near filopodia and boutons in mature dissociated hippocampal neurons. We found that axonal ß-actin mRNA was likely to colocalize with actin patches (APs), regions that have a high density of filamentous actin (F-actin) and are known to have a role in branch initiation. Moreover, simultaneous imaging of F-actin and axonal ß-actin mRNA in live neurons revealed that moving ß-actin mRNA tended to be docked in the APs. Our findings reveal that axonal ß-actin mRNA localization is facilitated by actin networks and suggest that localized ß-actin mRNA plays a potential role in axon branch formation.

Quantitative and structural changes of blood platelet cytoskeleton proteins in multiple sclerosis (MS).

Epidemiological studies show that cardiovascular events related to platelet hyperactivity remain the leading causes of death among multiple sclerosis (MS) patients. Quantitative or structural changes of platelet cytoskeleton alter their morphology and function. Here, we demonstrated, for the first time, the structural changes in MS platelets that may be related to their hyperactivity. MS platelets were found to form large aggregates compared to control platelets. In contrast to the control, the images of overactivated, irregularly shaped MS platelets show changes in the cytoskeleton architecture, fragmented microtubule rings. Furthermore, MS platelets have long and numerous pseudopodia rich in actin filaments. We showed that MS platelets and megakaryocytes, overexpress ß1-tubulin and ß-actin mRNAs and proteins and have altered post-translational modification patterns. Moreover, we identified two previously undisclosed mutations in the gene encoding ß1-tubulin in MS. We propose that the demonstrated structural changes of platelet cytoskeleton enhance their ability to adhere, aggregate, and degranulate fueling the risk of adverse cardiovascular events in MS.

GnRH Induces Citrullination of the Cytoskeleton in Murine Gonadotrope Cells.

Peptidylarginine deiminases (PADs or PADIs) catalyze the conversion of positively charged arginine to neutral citrulline, which alters target protein structure and function. Our previous work established that gonadotropin-releasing hormone agonist (GnRHa) stimulates PAD2-catalyzed histone citrullination to epigenetically regulate gonadotropin gene expression in the gonadotrope-derived LßT2 cell line. However, PADs are also found in the cytoplasm. Given this, we used mass spectrometry (MS) to identify additional non-histone proteins that are citrullinated following GnRHa stimulation and characterized the temporal dynamics of this modification. Our results show that actin and tubulin are citrullinated, which led us to hypothesize that GnRHa might induce their citrullination to modulate cytoskeletal dynamics and architecture. The data show that 10 nM GnRHa induces the citrullination of ß-actin, with elevated levels occurring at 10 min. The level of ß-actin citrullination is reduced in the presence of the pan-PAD inhibitor biphenyl-benzimidazole-Cl-amidine (BB-ClA), which also prevents GnRHa-induced actin reorganization in dispersed murine gonadotrope cells. GnRHa induces the citrullination of ß-tubulin, with elevated levels occurring at 30 min, and this response is attenuated in the presence of PAD inhibition. To examine the functional consequence of ß-tubulin citrullination, we utilized fluorescently tagged end binding protein 1 (EB1-GFP) to track the growing plus end of microtubules (MT) in real time in transfected LßT2 cells. Time-lapse confocal microscopy of EB1-GFP reveals that the MT average lifetime increases following 30 min of GnRHa treatment, but this increase is attenuated by PAD inhibition. Taken together, our data suggest that GnRHa-induced citrullination alters actin reorganization and MT lifetime in gonadotrope cells.

Salvianolic Acid B Significantly Suppresses the Migration of Melanoma Cells via Direct Interaction with ß-Actin.

Melanoma is the most aggressive and difficult to treat of all skin cancers. Despite advances in the treatment of melanoma, the prognosis for melanoma patients remains poor, and the recurrence rate remains high. There is substantial evidence that Chinese herbals effectively prevent and treat melanoma. The bioactive ingredient Salvianolic acid B (SAB) found in Salvia miltiorrhiza, a well-known Chinese herbal with various biological functions, exhibits inhibitory activity against various cancers. A375 and mouse B16 cell lines were used to evaluate the main targets and mechanisms of SAB in inhibiting melanoma migration. Online bioinformatics analysis, Western blotting, immunofluorescence, molecular fishing, dot blot, and molecular docking assays were carried out to clarify the potential molecular mechanism. We found that SAB prevents the migration and invasion of melanoma cells by inhibiting the epithelial-mesenchymal transition (EMT) process of melanoma cells. As well as interacting directly with the N-terminal domain of ß-actin, SAB enhanced its compactness and stability, thereby inhibiting the migration of cells. Taken together, SAB could significantly suppress the migration of melanoma cells via direct binding with ß-actin, suggesting that SAB could be a helpful supplement that may enhance chemotherapeutic outcomes and benefit melanoma patients.

ß-Actin: An Emerging Biomarker in Ischemic Stroke.

At present, the diagnosis of ischemic stroke mainly depends on neuroimaging technology, but it still has many limitations. Therefore, it is very important to find new biomarkers of ischemic stroke. Recently, ß-actin has attracted extensive attention as a biomarker of a variety of cancers. Although several recent studies have been investigating its role in ischemic stroke and other cerebrovascular diseases, the understanding of this emerging biomarker in neurology is still limited. We examined human and preclinical studies to gain a comprehensive understanding of the literature on the subject. Most relevant literatures focus on preclinical research, and pay more attention to the role of ß-actin in the process of cerebral ischemia, but some recent literatures reported that in human studies, serum ß-actin increased significantly in the early stage of acute cerebral ischemia. This review will investigate the basic biology of ß-actin, pay attention to the potential role of serum ß-actin as an early diagnostic blood biomarker of ischemic stroke, and explore its potential mechanism in ischemic stroke and new strategies for stroke treatment in the future.

Optimization of mRNA extraction from human nasal mucosa biopsies for gene expression profile analysis by qRT-PCR.

Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is the gold-standard method for analyzing modifications in gene expression in cells and tissues. However, large quantities of high-quality RNA samples are needed for analyzing the expression of multiple genes from one human tissue sample. Here, we provide an optimized protocol for extracting large amounts of RNA from human nasal mucosal biopsies. The quality and quantity of samples were sufficient for qRT-PCR analyses of the expressions of various genes, in duplicate. In contrast to other protocols, we optimized RNA isolation to increase the amount from nasal biopsy samples for analyses of multiple genes. In most previous publications, expressions of only one or a few genes, including housekeeping genes, were analyzed because the amount of biological material was small. We were able to improve our protocol with respect to the yield and quality of RNA. This is likely to produce better results from molecular analyses of very small biopsy samples of human nasal mucosa.

No-stain protein labeling as a potential normalization marker for small extracellular vesicle proteins.

Western blot analysis of relative protein expression relies on appropriate reference proteins for data normalization. Small extracellular vesicles (sEVs), or exosomes, are increasingly recognized as potential indicators of the physiological state of cells due to their protein composition. Therefore, accurate relative sEVs protein quantification is crucial for disease detection and prognosis applications. Currently, no documented ubiquitous reference proteins are identified for precise normalization of a protein of interest in sEVs. Here we showed the use of total protein staining method for sEVs protein normalization in western blots of samples where conventional housekeeping proteins like ß-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are not always detected in the sEVs western blots. The No-Stain™ Protein Labeling (NSPL) method showed high sensitivity in sEVs-protein labeling and facilitated quantitative evaluation of changes in the expression pattern of the protein of interest. Further, to show the robustness of NSPL for expression analysis, the results were compared with quantitative mass spectroscopy analysis results. Here, we outline a comprehensive method for protein normalization in sEVs that will increase the value of protein expression study of therapeutically significant sEVs.

Low anti-CFL1 antibody with high anti-ACTB antibody is a poor prognostic factor in esophageal squamous cell carcinoma.

BACKGROUND: Cofilin (CFL1, actin-binding protein) and ß-actin (ACTB) are key molecules in the polymerization and depolymerization of actin microfilaments. The levels of these antibodies were analyzed, and the clinicopathological significance in patients with esophageal carcinoma were evaluated. METHODS: The levels of anti-CFL1 and anti-ACTB antibodies were analyzed in serum samples of patients with esophageal carcinoma and of healthy donors. Eighty-seven cases underwent radical surgery and the clinicopathological characteristics and prognosis was examined. RESULTS: Serum anti-CFL1 antibody (s-CFL1-Ab) levels and anti-ACTB antibody (s-ACTB-Ab) levels were significantly higher in patients with esophageal carcinoma than in healthy donors. Following the receiver operating characteristic curve analysis between healthy donors and esophageal carcinoma, the sensitivity and specificity for serum anti-CFL1 antibody (s-CFL1-Ab) were 53.3% and 68.8%. The sensitivity and specificity for serum anti-ACTB antibody (s-ACTB-Ab) were 54.9% and 67.7%, respectively. Univariate and multivariate analysis showed that s-CFL1-Ab and s-ACTB-Ab levels were not associated with sex, age, tumor depth, lymph node metastasis, or anti-p53-antibody levels. s-ACTB-Ab levels but not s-CFL1-Ab levels significantly correlated with squamous cell carcinoma antigen. Neither s-CFL1-Ab nor s-ACTB-Ab levels alone were obviously related to overall survival. However, patients with low s-CFL1-Ab levels and high s-ACTB-Ab levels exhibited significantly more unfavorable prognoses than those with high s-CFL1-Ab and low s-ACTB-Ab levels. CONCLUSIONS: Serum levels of anti-CFL1 and anti-ACTB antibodies were significantly higher in patients with esophageal carcinoma than in healthy donors. A combination of low anti-CFL1 and high anti-ACTB antibodies is a poor prognostic factor in esophageal carcinoma.

Cloning, characterization, and transcriptional activity of ß-actin promoter of African catfish (Clarias gariepinus).

Selection of suitable promoters is crucial for the efficient expression of exogenous genes in transgenic animals. Although one of the most effective promoters, the ß-actin promoter, has been widely studied in fish species, it still remains unknown in the economical important African catfish (Clarias gariepinus). In this study, the ß-actin promoter of African catfish (cgß-actinP) was cloned and characterized. In addition, recombinant plasmid pcgß-actinP-EGFP with enhanced green fluorescent protein (GFP) gene as the reporter gene was constructed to verify the transcriptional activity. We obtained a cgß-actinP fragment length of 1405 bp, consisting 104 bp of the 5' proximal promoter, 96 bp of the first exon, and 1205 bp of the first intron. Similar to those of other fish species, cgß-actinP contains three key transcription regulatory elements (CAAT box, CArG motif, and TATA box). GFP-specific fluorescent signals were detected in chicken embryonic fibroblasts cells (DF-1 cells) transfected with pcgß-actinP-EGFP, which was approximately 1.11 times of the positive control. In addition, GFP was effectively expressed in zebrafish larvae microinjected with linearized cgß-actinP-EGFP, with expression rate reaching approximately 49.84%. Our data indicate that cgß-actinP could be a potential candidate promoter in the practice of constructing "all fish" transgenic fish.

Essential nucleotide- and protein-dependent functions of Actb/ß-actin.

The highly similar cytoplasmic ß- and γ-actins differ by only four functionally similar amino acids, yet previous in vitro and in vivo data suggest that they support unique functions due to striking phenotypic differences between Actb and Actg1 null mouse and cell models. To determine whether the four amino acid variances were responsible for the functional differences between cytoplasmic actins, we gene edited the endogenous mouse Actb locus to translate γ-actin protein. The resulting mice and primary embryonic fibroblasts completely lacked ß-actin protein, but were viable and did not present with the most overt and severe cell and organismal phenotypes observed with gene knockout. Nonetheless, the edited mice exhibited progressive high-frequency hearing loss and degeneration of actin-based stereocilia as previously reported for hair cell-specific Actb knockout mice. Thus, ß-actin protein is not required for general cellular functions, but is necessary to maintain auditory stereocilia.

Single-molecule analysis of endogenous ß-actin mRNA trafficking reveals a mechanism for compartmentalized mRNA localization in axons.

During embryonic nervous system assembly, mRNA localization is precisely regulated in growing axons, affording subcellular autonomy by allowing controlled protein expression in space and time. Different sets of mRNAs exhibit different localization patterns across the axon. However, little is known about how mRNAs move in axons or how these patterns are generated. Here, we couple molecular beacon technology with highly inclined and laminated optical sheet microscopy to image single molecules of identified endogenous mRNA in growing axons. By combining quantitative single-molecule imaging with biophysical motion models, we show that ß-actin mRNA travels mainly as single copies and exhibits different motion-type frequencies in different axonal subcompartments. We find that ß-actin mRNA density is fourfold enriched in the growth cone central domain compared with the axon shaft and that a modicum of directed transport is vital for delivery of mRNA to the axon tip. Through mathematical modeling we further demonstrate that directional differences in motor-driven mRNA transport speeds are sufficient to generate ß-actin mRNA enrichment at the growth cone. Our results provide insight into how mRNAs are trafficked in axons and a mechanism for generating different mRNA densities across axonal subcompartments.

The Cytoplasmic Actins in the Regulation of Endothelial Cell Function.

The primary function of the endothelial cells (EC) lining the inner surface of all vessels is to regulate permeability of vascular walls and to control exchange between circulating blood and tissue fluids of organs. The EC actin cytoskeleton plays a crucial role in maintaining endothelial barrier function. Actin cytoskeleton reorganization result in EC contraction and provides a structural basis for the increase in vascular permeability, which is typical for many diseases. Actin cytoskeleton in non-muscle cells presented two actin isoforms: non-muscle ß-cytoplasmic and γ-cytoplasmic actins (ß-actins and γ-actins), which are encoded by ACTB and ACTG1 genes, respectively. They are ubiquitously expressed in the different cells in vivo and in vitro and the ß/γ-actin ratio depends on the cell type. Both cytoplasmic actins are essential for cell survival, but they perform various functions in the interphase and cell division and play different roles in neoplastic transformation. In this review, we briefly summarize the research results of recent years and consider the features of the cytoplasmic actins: The spatial organization in close connection with their functional activity in different cell types by focusing on endothelial cells.

[Hypoxia Accelerate ß-Actin Expression through Transcriptional Activation of ACTB by Nuclear Respiratory Factor-1].

Cytoskeletal protein ß-actin is abundant both in the cytoplasm and the nucleus, its mRNA is commonly utilized an internal control for gene expression analysis. Recent reports demostrated that hypoxia influences the levels of ß-actin in a variety of cells. The mechanism underlying this change are not yet elucidated. In this work, we show that the changes in the levels of hypoxia-induced Nuclear respiratory factor-1 (NRF-1) lead to the change in expression of ß-actin. We compared the protein levels of NRF-1 and ß-actin in gastric cancer and adjacent tissues and found their significantly upregulation in cancer (33% patitents). When gastric cancer cells and normal gastric cells were treated with 1% O2 for 48 h, the trends in expression levels of NRF-1 and ß-actin were similar. When NRF-1 expression was modified by its overexpressing or silencing, the levels of ß-actin changed accordingly. In ß-actin gene (ACTB), three binding sites for NRF-1 were found. These sites are conserved in human, mouse and rat genomes. In ChIP experiments, we showed that NRF-1 directly binds to human ACTB and mouse Actb coding regions. Its seems that the transcription of ß-actin encoding gene is NRF-1 dependent.

ACTB Loss-of-Function Mutations Result in a Pleiotropic Developmental Disorder.

ACTB encodes ß-actin, an abundant cytoskeletal housekeeping protein. In humans, postulated gain-of-function missense mutations cause Baraitser-Winter syndrome (BRWS), characterized by intellectual disability, cortical malformations, coloboma, sensorineural deafness, and typical facial features. To date, the consequences of loss-of-function ACTB mutations have not been proven conclusively. We describe heterozygous ACTB deletions and nonsense and frameshift mutations in 33 individuals with developmental delay, apparent intellectual disability, increased frequency of internal organ malformations (including those of the heart and the renal tract), growth retardation, and a recognizable facial gestalt (interrupted wavy eyebrows, dense eyelashes, wide nose, wide mouth, and a prominent chin) that is distinct from characteristics of individuals with BRWS. Strikingly, this spectrum overlaps with that of several chromatin-remodeling developmental disorders. In wild-type mouse embryos, ß-actin expression was prominent in the kidney, heart, and brain. ACTB mRNA expression levels in lymphoblastic lines and fibroblasts derived from affected individuals were decreased in comparison to those in control cells. Fibroblasts derived from an affected individual and ACTB siRNA knockdown in wild-type fibroblasts showed altered cell shape and migration, consistent with known roles of cytoplasmic ß-actin. We also demonstrate that ACTB haploinsufficiency leads to reduced cell proliferation, altered expression of cell-cycle genes, and decreased amounts of nuclear, but not cytoplasmic, ß-actin. In conclusion, we show that heterozygous loss-of-function ACTB mutations cause a distinct pleiotropic malformation syndrome with intellectual disability. Our biological studies suggest that a critically reduced amount of this protein alters cell shape, migration, proliferation, and gene expression to the detriment of brain, heart, and kidney development.

Reconsidering an active role for G-actin in cytoskeletal regulation.

Globular (G)-actin, the actin monomer, assembles into polarized filaments that form networks that can provide structural support, generate force and organize the cell. Many of these structures are highly dynamic and to maintain them, the cell relies on a large reserve of monomers. Classically, the G-actin pool has been thought of as homogenous. However, recent work has shown that actin monomers can exist in distinct groups that can be targeted to specific networks, where they drive and modify filament assembly in ways that can have profound effects on cellular behavior. This Review focuses on the potential factors that could create functionally distinct pools of actin monomers in the cell, including differences between the actin isoforms and the regulation of G-actin by monomer binding proteins, such as profilin and thymosin ß4. Owing to difficulties in studying and visualizing G-actin, our knowledge over the precise role that specific actin monomer pools play in regulating cellular actin dynamics remains incomplete. Here, we discuss some of these unanswered questions and also provide a summary of the methodologies currently available for the imaging of G-actin.

DNA repair protein RAD51 enhances the CRISPR/Cas9-mediated knock-in efficiency in brain neurons.

Gene knock-in using the CRISPR/Cas9 system can be achieved in a specific population of neurons in the mouse brain, by using in utero electroporation to introduce DNA fragments into neural progenitor cells. Using this strategy, we previously knocked-in the EGFP coding sequence into the N-terminal region of the ß-actin gene specifically in the pyramidal neurons in layer 2/3 of the somatosensory cortex. However, the knock-in efficiency was less than 2% of the transfected neurons. In this study, we sought to improve the knock-in efficiency using this system. First, we varied the length of the homology arms of the ß-actin donor template DNA, and found that the knock-in efficiency was increased to ∼14% by extending the length of the 5' and 3' homology arms to 1.6 kb and 2.0 kb, respectively. We then tested the effect of the DNA repair protein RAD51 and the knock-in efficiency was increased up to 2.5-fold when co-transfecting with two different ß-actin and a camk2a targeting EGFP knock-in modules. The RAD51 overexpression did not alter the migration of developing neurons, density or morphology of the dendritic spines compared to those in neurons not transfected with RAD51. RAD51 expression will be useful for increasing the knock-in efficiency in neurons in vivo by CRISPR/Cas9-mediated homology directed repair (HDR).

Profilin 1 knockdown prevents ischemic brain damage by promoting M2 microglial polarization associated with the RhoA/ROCK pathway.

Microglial polarization to the anti-inflammatory M2 phenotype is essential in resolving neuroinflammation, making it a promising therapeutic strategy for stroke intervention. The actin cytoskeleton is known to be important for the physiological functions of microglia, including migration and phagocytosis. Profilin 1 (PFN1), an actin-binding protein, is involved in the dynamic transformation and reorganization of actin. However, the role of PFN1 in microglial polarization and ischemia/reperfusion injury is unclear. The role of PFN1 on microglial polarization was examined in vitro in BV2 microglial cells subjected to oxygen-glucose deprivation/reoxygenation (OGDR) and in vivo in male mice after transient middle cerebral artery occlusion (MCAO). Knockdown of PFN1 inhibited M1 microglial polarization and promoted M2 microglia polarization 48 hr after OGDR stimulation in BV2 cells and 7 days after MCAO-induced injury in male mice. RhoA/ROCK pathway was involved in the regulation of PFN1 during microglial polarization. Knockdown of PFN1 also significantly attenuated brain infarcts and edema, improved cerebral blood flow and neurological deficits in MCAO-injured mice. Inhibition of PFN1 effectively protected the brain against ischemia/reperfusion injuries by promoting M2 microglial polarization in vitro and in vivo.