Shekhar Saha

The Effect of Including the C2 Insert of Nonmuscle Myosin II-C on Neuritogenesis

Background: The functional importance of the C2 insert-containing isoform of nonmuscle myosin II-C is not known. Results: During the neuritogenesis of Neuro-2a cells, NM II-C1C2 becomes the predominant isoform of NM II-C. Decreasing NM II-C1C2 expression leads to shortening of neurites. Conclusion: NM II-C1C2 plays a role in the later stage of neuritogenesis. Significance: This work contributes to an understanding of the functional importance of NM II-C1C2. 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 β1-integrin in neurites. Altogether, these studies indicate that NM II-C1C2 may be involved in stabilizing neurites by maintaining their structure at adhesion sites.

Dynamics of Gene Silencing in a Live Cell: Stochastic Resonance.

Binding of a specific siRNA to the target mRNA in a live cell (human breast cancer cell, MCF-7) is studied by confocal microscopy. The specific siRNA (labeled with a fluorophore, alexa 488) exhibits much higher intensity of fluorescence in the bound state than in the free (unbound) state. It is observed that repeated unbinding and rebinding of siRNA (to target mRNA) occur before gene silencing. 16 273 on-time periods (residence or dwell time of siRNA in bound form) are detected. They follow a strikingly simple pattern. All of the on-time periods are odd-integral multiples of 5.5 ± 0.05 ms. This is ascribed to stochastic resonance.

Increased expression of nonmuscle myosin IIs is associated with 3MC-induced mouse tumor.

Administration of the chemical carcinogen, 3‐methylcholanthrene (3MC), in the hind leg induces the progressive formation of tumors in mice within 110 days. Previous reports suggest that transformation of muscle cells to atypical cells is one of the causes of tumor formation. Molecular events that lead to transformation of normal cells to atypical cells are not well understood. Here, we investigate the effect of 3MC on the expression of nonmuscle myosin IIs (NM IIs) which are known to be involved in cell migration, division and adhesion. Mass spectroscopy analysis reveals that tumor tissue contains 64.5% NM II‐A, 34% II‐B and only 1.5% II‐C of total NM IIs, whereas these three isoforms of NM IIs are undetectable by mass spectroscopy in normal tissue associated with the tumor (NTAT) from the hind leg. Quantification of heavy chain mRNAs of NM II suggests that tumor tissue contains 25.7‐fold and 19.03‐fold more of NM II‐A and II‐B, respectively, compared with NTAT. Unlike NM II‐B, which is detected only after tumor formation, II‐A is detectable as early as day 7 after a second dose of 3MC. Immunofluorescence confocal microscopy reveals that fibroblast cells which are sparsely distributed in normal tissue are densely populated but of atypical shape in the tumor. These findings suggest that transformation of fibroblasts or non‐fibroblast cells to atypical, cancerous cells is associated with increased levels of NM II‐A and NM II‐B expression in the 3MC‐induced tumor mouse model. 3MC‐induced transformation is further demonstrated in C2C12 myotubes.

A cationic morpholino antisense oligomer conjugate: synthesis, cellular uptake and inhibition of Gli1 in the hedgehog signalling pathway

A guanidinium-linked morpholino pentamer is synthesized and evaluated for cellular penetration properties. A regular morpholino and the pentamer conjugate can exhibit antisense effects when designed against Gli1-transcription factors of the Hh signalling pathway, which is a potential target for cancer therapy. Evidence was found from luciferase assays and western blot analysis.

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

One molecular cue that regulates cellular protrusions such as blebbing and lamellipodia in tumor cells has been less explored than other environmental factors. NM II-A induces blebbing and NM II-C1 induces lamellipodia in tumor cells. NM-II isoforms can change the protrusive activity of a tumor cell.

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.

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.

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.

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.

Amphiphilic random copolymer vesicle induces differentiation of mouse C2C12 myoblasts

Herein we report that vesicular assembly from a simple non-ionic amphiphilic random copolymer initiates extremely efficient myotube formation from C2C12 myoblast cells in standard growth media lacking horse serum. Control experiments with structurally related polymers and a small molecule suggest the absolute necessity of the vesicular assembly as well as the particular hydrophilic functionality in mediating such high yielding muscle cell generation. The LDH assay indicates that the membrane integrity is retained during cell-cell fusion. Expression of various myogenic factors such as MyoD, myogenin and P-21 has been examined in the presence of the polymersome and control molecules to rationalize this serendipitous discovery.