Transcriptomic Analysis of Alzheimer's Disease Pathways in a Pakistani Population.

Background: Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder that is most prevalent in elderly individuals, especially in developed countries, and its prevalence is now increasing in developing countries like Pakistan. Objective: Our goal was to characterize key genes and their levels of expression and related molecular transcriptome networks associated with AD pathogenesis in a pilot case-control study in a Pakistani population. Methods: To obtain the spectrum of molecular networks associated with pathogenesis in AD patients in Pakistan (comparing cases and controls), we used high-throughput qRT-PCR (TaqMan Low-Density Array; n = 33 subjects) coupled with Affymetrix Arrays (n = 8) and Ingenuity Pathway Analysis (IPA) to identify signature genes associated with Amyloid processing and disease pathways. Results: We confirmed 16 differentially expressed AD-related genes, including maximum fold changes observed in CAPNS2 and CAPN1. The global gene expression study observed that 61% and 39% of genes were significantly (p-value 0.05) up- and downregulated, respectively, in AD patients compared to healthy controls. The key pathways include, e.g., Amyloid Processing, Neuroinflammation Signaling, and ErbB4 Signaling. The top-scoring networks in Diseases and Disorders Development were Neurological Disease, Organismal Injury and Abnormalities, and Psychological Disorders. Conclusions: Our pilot study offers a non-invasive and efficient way of investigating gene expression patterns by combining TLDA and global gene expression method in AD patients by utilizing whole blood. This provides valuable insights into the expression status of genes related to Amyloid Processing, which could play potential role in future studies to identify sensitive, early biomarkers of AD in general.

Expression of nonmuscle myosin IIC is regulated by non-canonical binding activity of miRNAs.

The expression of mechanoresponsive nonmuscle myosin II (NMII)C is found to be inducible during tumor progression, but its mechanism is yet to be explored. Here, we report a group of microRNAs (mmu-miR-200a-5p, mmu-miR-532-3p, mmu-miR-680, and mmu-miR-1901) can significantly repress the expression of nonmuscle myosin IIC (NMIIC). Interestingly, these microRNAs have both canonical and non-canonical binding sites at 3/UTR and coding sequence (CDS) of NMIIC's heavy chain (HC) mRNA. Each of the miRNA downregulates NMHC-IIC to a different degree as assessed by dual-luciferase and immunoblot analyses. When we abolish the complementary base pairing at canonical binding site, mmu-miR-532-3p can still bind at non-canonical binding site and form Argonaute2 (AGO2)-miRNA complex to downregulate the expression of NMIIC. Modulating the expression of NMIIC by miR-532-3p in mouse mammary tumor cells, 4T1, increases its tumorigenic potential both in vitro and in vivo. Together, these studies provide the functional role of miRNA's non-canonical binding mediated NMIIC regulation in tumor cells.

A brain specific alternatively spliced isoform of nonmuscle myosin IIA lacks its mechanoenzymatic activities.

Recent genomic studies reported that 90 to 95% of human genes can undergo alternative splicing, by which multiple isoforms of proteins are synthesized. However, the functional consequences of most of the isoforms are largely unknown. Here, we report a novel alternatively spliced isoform of nonmuscle myosin IIA (NM IIA), called NM IIA2, which is generated by the inclusion of 21 amino acids near the actin-binding region (loop 2) of the head domain of heavy chains. Expression of NM IIA2 is found exclusively in the brain tissue, where it reaches a maximum level at 24 h during the circadian rhythm. The actin-dependent Mg2+-ATPase activity and in vitro motility assays reveal that NM IIA2 lacks its motor activities but localizes with actin filaments in cells. Interestingly, NM IIA2 can also make heterofilaments with NM IIA0 (noninserted isoform of NM IIA) and can retard the in vitro motility of NM IIA, when the two are mixed. Altogether, our findings provide the functional importance of a previously unknown alternatively spliced isoform, NM IIA2, and its potential physiological role in regulating NM IIA activity in the brain.

Impact of HMGB1 binding on the structural alterations of platinum drug-treated single dsDNA molecule.

High mobility group B1 (HMGB1) is an architectural protein that recognizes the DNA damage sites formed by the platinum anticancer drugs. However, the impact of HMGB1 binding on the structural alterations of the platinum drug-treated single dsDNA molecules has remained largely unknown. Herein, the structural alterations induced by the platinum drugs, the mononuclear cisplatin and it's analog the trinuclear BBR3464, have been probed in presence of HMGB1, by atomic force microscopy (AFM) and AFM-based force spectroscopy. It is observed that the drug-induced DNA loop formation enhanced upon HMGB1 binding, most likely as a result of HMGB1-induced increase in DNA conformational flexibility that allowed the drug-binding sites to come close and form double adducts, thereby resulting in enhanced loop formation via inter-helix cross-linking. Since HMGB1 enhances DNA flexibility, the near-reversible structural transitions as observed in the force-extension curves (for 1 h drug treatment), generally occurred at lower forces in presence of HMGB1. The DNA structural integrity was largely lost after 24 h drug treatment as no reversible transition could be observed. The Young's modulus of the dsDNA molecules, as estimated from the force-extension analysis, increased upon drug treatment, due to formation of the drug-induced covalent cross-links and consequent reduction in DNA flexibility. The Young's modulus increased further in presence of HMGB1 due to HMGB1-induced enhancement in DNA flexibility that could ease formation of the drug-induced covalent cross-links. To our knowledge, this is the first report that shows an increase in the stiffness of the platinum drug-treated DNA molecules in presence of HMGB1.

Assembly and disassembly dynamics of nonmuscle myosin II control endosomal fission.

Endocytic vesicular trafficking requires merging of two lipid bilayers, but how the two lipid bilayers can come close together during fusion and fission in endocytic trafficking is not well explored. Here, we establish that knocking down nonmuscle myosin IIs (NM IIs) by small interfering RNA (siRNA) or inhibition of their activities by (-) blebbistatin causes the formation of a ring-like assembly of early endosomes (raEE). Inhibition of NM II assembly by an inhibitor of regulatory light-chain (RLC) kinase results in the formation of raEE, whereas inhibition of NM II disassembly by inhibitors of heavy chain kinases, protein kinase C (PKC) and casein kinase 2 (CK2), causes the dispersion of early endosomes. The raEEs retain EEA1, Rab7, and LAMP2 markers. Overexpression of an assembly incompetent form, RLC-AA, and disassembly incompetent form, NMHCIIB-S6A or NMHCIIA-1916A, induces such defects, respectively. Altogether, these data support that NM II assembly and disassembly dynamics participate in endocytic trafficking by regulating fission to maintain the size of early endosomes.

D155Y substitution of SARS-CoV-2 ORF3a weakens binding with Caveolin-1.

The clinical manifestation of the recent pandemic COVID-19, caused by the novel SARS-CoV-2 virus, varies from mild to severe respiratory illness. Although environmental, demographic and co-morbidity factors have an impact on the severity of the disease, contribution of the mutations in each of the viral genes towards the degree of severity needs a deeper understanding for designing a better therapeutic approach against COVID-19. Open Reading Frame-3a (ORF3a) protein has been found to be mutated at several positions. In this work, we have studied the effect of one of the most frequently occurring mutants, D155Y of ORF3a protein, found in Indian COVID-19 patients. Using computational simulations we demonstrated that the substitution at 155th changed the amino acids involved in salt bridge formation, hydrogen-bond occupancy, interactome clusters, and the stability of the protein compared with the other substitutions found in Indian patients. Protein-protein docking using HADDOCK analysis revealed that substitution D155Y weakened the binding affinity of ORF3a with caveolin-1 compared with the other substitutions, suggesting its importance in the overall stability of ORF3a-caveolin-1 complex, which may modulate the virulence property of SARS-CoV-2.

Nonmuscle Myosin II in cancer cell migration and mechanotransduction.

Cell migration is a key step of cancer metastasis, immune-cell navigation, homing of stem cells and development. What adds complexity to it is the heterogeneity of the tissue environment that gives rise to a vast diversity of migratory mechanisms utilized by cells. A majority of cell motility mechanisms reported elsewhere largely converge in depicting the importance of the activity and complexity of actomyosin networks in the cell. In this review, we highlight the less discussed functional diversity of these actomyosin complexes and describe in detail how the major cellular actin-binding molecular motor proteins, nonmuscle myosin IIs are regulated and how they participate and mechanically reciprocate to changes in the microenvironment during cancer cell migration and tumor progression. Understanding the role of nonmuscle myosin IIs in the cancer cell is important for designing efficient therapeutic strategies to prevent cancer metastasis.

MRX8, the conserved mitochondrial YihA GTPase family member, is required for de novo Cox1 synthesis at suboptimal temperatures in Saccharomyces cerevisiae.

The synthesis of Cox1, the conserved catalytic-core subunit of Complex IV, a multisubunit machinery of the mitochondrial oxidative phosphorylation (OXPHOS) system under environmental stress, has not been sufficiently addressed. In this study, we show that the putative YihA superfamily GTPase, Mrx8, is a bona fide mitochondrial protein required for Cox1 translation initiation and elongation during suboptimal growth condition at 16°C. Mrx8 was found in a complex with mitochondrial ribosomes, consistent with a role in protein synthesis. Cells expressing mutant Mrx8 predicted to be defective in guanine nucleotide binding and hydrolysis were compromised for robust cellular respiration. We show that the requirement of Pet309 and Mss51 for cellular respiration is not bypassed by overexpression of Mrx8 and vice versa. Consistently the ribosomal association of Mss51 is independent of Mrx8. Significantly, we find that GTPBP8, the human orthologue, complements the loss of cellular respiration in Δmrx8 cells and GTPBP8 localizes to the mitochondria in mammalian cells. This strongly suggests a universal role of the MRX8 family of proteins in regulating mitochondrial function.

Switching between blebbing and lamellipodia depends on the degree of non-muscle myosin II activity.

Cells can adopt both mesenchymal and amoeboid modes of migration through membrane protrusive activities, namely formation of lamellipodia and blebbing. How the molecular players control the transition between lamellipodia and blebs is yet to be explored. Here, we show that addition of the ROCK inhibitor Y27632 or low doses of blebbistatin, an inhibitor of non-muscle myosin II (NMII) ATPase activity and filament partitioning, induces blebbing to lamellipodia conversion (BLC), whereas addition of low doses of ML7, an inhibitor of myosin light chain kinase (MLCK), induces lamellipodia to blebbing conversion (LBC) in human MDA-MB-231 cells. Similarly, siRNA-mediated knockdown of ROCK and MLCK induces BLC and LBC, respectively. Interestingly, both blebs and lamellipodia membrane protrusions are able to maintain the ratio of phosphorylated to unphosphorylated regulatory light chain at cortices when MLCK and ROCK, respectively, are inhibited either pharmacologically or genetically, suggesting that MLCK and ROCK activities are interlinked in BLC and LBC. Such BLCs and LBCs are also inducible in other cell lines, including MCF7 and MCF10A. These studies reveal that the relative activity of ROCK and MLCK, which controls both the ATPase activity and filament-forming property of NMII, is a determining factor in whether a cell exhibits blebbing or lamellipodia.

Nonmuscle myosin IIA and IIB differentially modulate migration and alter gene expression in primary mouse tumorigenic cells.

Though many cancers are known to show up-regulation of nonmuscle myosin (NM) IIA and IIB, the mechanism by which NMIIs aid in cancer development remains unexplored. Here we demonstrate that tumor-generating, fibroblast-like cells isolated from 3-methylcholanthrene (3MC)-induced murine tumor exhibit distinct phospho-dependent localization of NMIIA and NMIIB at the perinuclear area and tip of the filopodia and affect cell migration differentially. While NMIIA-KD affects protrusion dynamics and increases cell directionality, NMIIB-KD lowers migration speed and increases filopodial branching. Strategically located NMIIs at the perinuclear area colocalize with the linker of nucleoskeleton and cytoskeleton (LINC) protein Nesprin2 and maintain the integrity of the nuclear-actin cap. Interestingly, knockdown of NMIIs results in altered expression of genes involved in epithelial-to-mesenchymal transition, angiogenesis, and cellular senescence. NMIIB-KD cells display down-regulation of Gsc and Serpinb2, which is strikingly similar to Nesprin2-KD cells as assessed by quantitative PCR analysis. Further gene network analysis predicts that NMIIA and NMIIB may act on similar pathways but through different regulators. Concomitantly, knockdown of NMIIA or NMIIB lowers the growth rate and tumor volume of 3MC-induced tumor in vivo. Altogether, these results open a new window to further investigate the effect of LINC-associated perinuclear actomyosin complex on mechanoresponsive gene expression in the growing tumor.

Differential role of nonmuscle myosin II isoforms during blebbing of MCF-7 cells.

Bleb formation has been correlated with nonmuscle myosin II (NM-II) activity. Whether three isoforms of NM-II (NM-IIA, -IIB and -IIC) have the same or differential roles in bleb formation is not well understood. Here we report that ectopically expressed, GFP-tagged NM-II isoforms exhibit different types of membrane protrusions, such as multiple blebs, lamellipodia, combinations of both, or absence of any such protrusions in MCF-7 cells. Quantification suggests that 50% of NM-IIA-GFP-, 29% of NM-IIB-GFP-, and 19% of NM-IIC1-GFP-expressing MCF-7 cells show multiple bleb formation, compared with 36% of cells expressing GFP alone. Of interest, NM-IIB has an almost 50% lower rate of dissociation from actin filament than NM-IIA and -IIC1 as determined by FRET analysis both at cell and bleb cortices. We induced bleb formation by disruption of the cortex and found that all three NM-II-GFP isoforms can reappear and form filaments but to different degrees in the growing bleb. NM-IIB-GFP can form filaments in blebs in 41% of NM-IIB-GFP-expressing cells, whereas filaments form in only 12 and 3% of cells expressing NM-IIA-GFP and NM-IIC1-GFP, respectively. These studies suggest that NM-II isoforms have differential roles in the bleb life cycle.

N-terminal polar amino acids of the C2 insert of nonmuscle myosin II-C2 regulate its functional properties.

In this study, we investigated the regions in the alternatively spliced C2 insert of nonmuscle myosin (NM) II-C conferring unique functional properties to the protein. We used constructs carrying deletions within different regions of C2 in neuronal cells; namely, the polar N terminus, the proline/serine-rich middle, and the nonpolar C terminus. We compared the wild-type NM II-C2 and deletion mutants with respect to ATPase activity, coassembly with NM II-B, regulation by myosin light-chain kinase (MLCK), and solubility, to determine the C2 region(s) involved in these processes. In addition, we examined the ability of the mutants to rescue the neurite-shortening phenotype upon NM II-C2 knockdown in Neuro-2a cells. Our data highlight the importance of the polar N terminus in NM II-C2 function.

miRepress: modelling gene expression regulation by microRNA with non-conventional binding sites.

Some earlier studies have reported an alternative mode of microRNA-target interaction. We detected target regions within mRNA transcripts from AGO PAR-CLIP that did not contain any conventional microRNA seed pairing but only had non-conventional binding sites with microRNA 3' end. Our study from 7 set of data that measured global protein fold change after microRNA transfection pointed towards the association of target protein fold change with 6-mer and 7-mer target sites involving microRNA 3' end. We developed a model to predict the degree of microRNA target regulation in terms of protein fold changes from the number of different conventional and non-conventional target sites present in the target, and found significant correlation of its output with protein expression changes. We validated the effect of non-conventional interactions with target by modulating the abundance of microRNA in a human breast cancer cell line MCF-7. The validation was done using luciferase assay and immunoblot analysis for our predicted non-conventional microRNA-target pair WNT1 (3' UTR) and miR-367-5p and immunoblot analysis for another predicted non-conventional microRNA-target pair MYH10 (coding region) and miR-181a-5p. Both experiments showed inhibition of targets by transfection of microRNA mimics that were predicted to have only non-conventional sites.

Molecular association of glucose-6-phosphate isomerase and pyruvate kinase M2 with glyceraldehyde-3-phosphate dehydrogenase in cancer cells.

BACKGROUND: For a long time cancer cells are known for increased uptake of glucose and its metabolization through glycolysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key regulatory enzyme of this pathway and can produce ATP through oxidative level of phosphorylation. Previously, we reported that GAPDH purified from a variety of malignant tissues, but not from normal tissues, was strongly inactivated by a normal metabolite, methylglyoxal (MG). Molecular mechanism behind MG mediated GAPDH inhibition in cancer cells is not well understood. METHODS: GAPDH was purified from Ehrlich ascites carcinoma (EAC) cells based on its enzymatic activity. GAPDH associated proteins in EAC cells and 3-methylcholanthrene (3MC) induced mouse tumor tissue were detected by mass spectrometry analysis and immunoprecipitation (IP) experiment, respectively. Interacting domains of GAPDH and its associated proteins were assessed by in silico molecular docking analysis. Mechanism of MG mediated GAPDH inactivation in cancer cells was evaluated by measuring enzyme activity, Circular dichroism (CD) spectroscopy, IP and mass spectrometry analyses. RESULT: Here, we report that GAPDH is associated with glucose-6-phosphate isomerase (GPI) and pyruvate kinase M2 (PKM2) in Ehrlich ascites carcinoma (EAC) cells and also in 3-methylcholanthrene (3MC) induced mouse tumor tissue. Molecular docking analyses suggest C-terminal domain preference for the interaction between GAPDH and GPI. However, both C and N termini of PKM2 might be interacting with the C terminal domain of GAPDH. Expression of both PKM2 and GPI is increased in 3MC induced tumor compared with the normal tissue. In presence of 1 mM MG, association of GAPDH with PKM2 or GPI is not perturbed, but the enzymatic activity of GAPDH is reduced to 26.8 ± 5 % in 3MC induced tumor and 57.8 ± 2.3 % in EAC cells. Treatment of MG to purified GAPDH complex leads to glycation at R399 residue of PKM2 only, and changes the secondary structure of the protein complex. CONCLUSION: PKM2 may regulate the enzymatic activity of GAPDH. Increased enzymatic activity of GAPDH in tumor cells may be attributed to its association with PKM2 and GPI. Association of GAPDH with PKM2 and GPI could be a signature for cancer cells. Glycation at R399 of PKM2 and changes in the secondary structure of GAPDH complex could be one of the mechanisms by which GAPDH activity is inhibited in tumor cells by MG.

Phosphorylation of Nonmuscle myosin II-A regulatory light chain resists Sendai virus fusion with host cells.

Enveloped viruses enter host cells through membrane fusion and the cells in turn alter their shape to accommodate components of the virus. However, the role of nonmuscle myosin II of the actomyosin complex of host cells in membrane fusion is yet to be understood. Herein, we show that both (-) blebbistatin, a specific inhibitor of nonmuscle myosin II (NMII) and small interfering RNA markedly augment fusion of Sendai virus (SeV), with chinese hamster ovary cells and human hepatocarcinoma cells. Inhibition of RLC phosphorylation using inhibitors against ROCK, but not PKC and MRCK, or overexpression of phospho-dead mutant of RLC enhances membrane fusion. SeV infection increases cellular stiffness and myosin light chain phosphorylation at two hour post infection. Taken together, the present investigation strongly indicates that Rho-ROCK-NMII contractility signaling pathway may provide a physical barrier to host cells against viral fusion.

Role of Nonmuscle Myosin II in Migration of Wharton's Jelly-Derived Mesenchymal Stem Cells.

It is the promise of regeneration and therapeutic applications that has sparked an interest in mesenchymal stem cells (MSCs). Following infusion, MSCs migrate to sites of injury or inflammation by virtue of their homing property. To exert optimal clinical benefits, systemically delivered MSCs need to migrate efficiently and in adequate numbers to pathological areas in vivo. However, underlying molecular mechanisms responsible for MSC migration are still not well understood. The Wharton's jelly (WJ) of the umbilical cord is an attractive source of MSCs for stem cell therapy because of its abundant availability and painless collection. In this study, we attempted to identify the role of nonmuscle myosin II (NMII), if any, in the migration of WJ-derived MSCs (WJ-MSCs). Expression of NMII isoforms, NMIIA, and NMIIB was observed both at RNA and protein levels in WJ-MSCs. Inhibition of NMII or its regulator ROCK, by pharmacological inhibitors, resulted in significant reduction in the migration of WJ-MSCs as confirmed by the scratch migration assay and time-lapse microscopy. Next, trying to dissect the role of each NMII isoform in migration of WJ-MSCs, we found that siRNA-mediated downregulation of NMIIA, but not NMIIB expression, led to cells failing to retract their trailing edge and losing cell-cell cohesiveness, while exhibiting a nondirectional migratory pathway. Migration, moreover, is also dependent on optimal affinity adhesion, which would allow rapid attachment and release of cells and, hence, can be influenced by extracellular matrix (ECM) and adhesion molecules. We demonstrated that inhibition of NMII and more specifically NMIIA resulted in increased gene expression of ECM and adhesion molecules, which possibly led to stronger adhesions and, hence, decreased migration. Therefore, these data suggest that NMII acts as a regulator of cell migration and adhesion in WJ-MSCs.

Regulation of nonmuscle myosin II during 3-methylcholanthrene induced dedifferentiation of C2C12 myotubes.

3-Methylcholanthrene (3MC) induces tumor formation at the site of injection in the hind leg of mice within 110 days. Recent reports reveal that the transformation of normal muscle cells to atypical cells is one of the causes for tumor formation, however the molecular mechanism behind this process is not well understood. Here, we show in an in vitro study that 3MC induces fragmentation of multinucleate myotubes into viable mononucleates. These mononucleates form colonies when they are seeded into soft agar, indicative of cellular transformation. Immunoblot analysis reveals that phosphorylation of myosin regulatory light chain (RLC20) is 5.6±0.5 fold reduced in 3MC treated myotubes in comparison to vehicle treated myotubes during the fragmentation of myotubes. In contrast, levels of myogenic factors such as MyoD, Myogenin and cell cycle regulators such as Cyclin D, Cyclin E1 remain unchanged as assessed by real-time PCR array and reverse transcriptase PCR analysis, respectively. Interestingly, addition of the myosin light chain kinase inhibitor, ML-7, enhances the fragmentation, whereas phosphatase inhibitor perturbs the 3MC induced fragmentation of myotubes. These results suggest that decrease in RLC20 phosphorylation may be associated with the fragmentation step of dedifferentiation.

Inhibition of non-muscle myosin II leads to G0/G1 arrest of Wharton's jelly-derived mesenchymal stromal cells.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) have remarkable clinical potential for cell-based therapy. Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs) from umbilical cord share unique properties with both embryonic and adult stem cells. MSCs are found at low frequency in vivo, and their successful therapeutic application depends on rapid and efficient large-scale expansion in vitro. Non-muscle myosin II (NMII) has pivotal roles in different cellular activities, such as cell division, migration and differentiation. We performed this study to understand the role of NMII in proliferation and cell cycle progression in WJ-MSCs. METHODS: WJ-MSCs were cultured in the presence of blebbistatin, and cell cycle analysis was performed using flow cytometry, proliferation kinetics, senescence assay and gene expression profile using polymerase chain reaction array. RESULTS: When cultured in the presence of blebbistatin, an inhibitor of NMII adenosine triphosphatase activity, WJ-MSCs exhibited dose-dependent reduction in proliferative potential along with increase in cell size and induction of early senescence. Inhibition of NMII activity also affected cell cycle progression in WJ-MSCs and led to an increase in the percentage of cells in G0/G1 phase with a corresponding reduction in the percentage of cells in G2/M phase. Blebbistatin-induced G0/G1 arrest of WJ-MSCs was further associated with up-regulation of cell cycle inhibitory genes CDKN1A, CDKN2A and CDKN2B and down-regulation of numerous genes related to progression through S and M phases of the cell cycle. CONCLUSIONS: Our study demonstrates that inhibition of NMII activity in WJ-MSCs leads to G0/G1 arrest and alteration in the expression levels of certain key cell cycle-related genes.

Hydrogen-bonding-induced chain folding and vesicular assembly of an amphiphilic polyurethane.

We have reported synthesis and vesicular assembly of a novel amphiphilic polyurethane with hydrophobic backbone and hydrophilic pendant carboxylic acid groups which were periodically grafted to the backbone via a tertiary amine group. In aqueous medium the polymer chain adopted a folded conformation which was stabilized by intrachain H-bonding among the urethane groups. Such a model was supported by concentration and solvent-dependent FT-IR, powder XRD, and urea-mediated "denaturation" experiments. Folded polymer chains further formed vesicular assembly which was probed by dynamic light scattering, TEM, AFM, SEM, and fluorescence microscopic studies, and dye encapsulation experiments. pH-dependent DLS and fluorescence microscopic studies revealed stable polymersome in entire tested pH window of 3.5-11.0. Zeta potential measurements showed a negatively charged surface in basic pH while a charge-neutral surface in neutral and acidic pH. MTT assay with CHO cell line indicated good cell viability.

The effect of including the C2 insert of nonmuscle myosin II-C on neuritogenesis.

The functional role of the C2 insert of nonmuscle myosin II-C (NM II-C) is poorly understood. Here, we report for the first time that the expression of the C2 insert-containing isoform, NM II-C1C2, is inducible in Neuro-2a cells during differentiation both at mRNA and protein levels. Immunoblot and RT-PCR analysis reveal that expression of NM II-C1C2 peaks between days 3 and 6 of differentiation. Localization of NM II-C1C2 in Neuro-2a cells suggests that the C2 insert-containing isoform is localized in the cytosol and along the neurites, specifically at the adherence point to substratum. Inhibition of endogenous NM II-C1C2 using siRNA decreases the neurite length by 43% compared with control cells treated with nonspecific siRNA. Time lapse image analysis reveals that neurites of C2-siRNA-treated cells have a net negative change in neurite length per minute, leading to a reduction of overall neurite length. During neuritogenesis, NM II-C1C2 can interact and colocalize with ß1-integrin in neurites. Altogether, these studies indicate that NM II-C1C2 may be involved in stabilizing neurites by maintaining their structure at adhesion sites.