Biology subjects

Sivaguru, M.

Publications and source records attributed to Sivaguru, M..

3 recordsLinked to original sources

bioRxiv2022

CHCHD2 mediates glioblastoma cell proliferation, mitochondrial metabolism, hypoxia-induced invasion, and therapeutic resistance

Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults and remains incurable. The mitochondrial coiled-coil-helix-coiled-coil-helix domain-containing protein 2 (CHCHD2) is demonstrated to mediate mitochondrial respiration, nuclear gene expression, and cell migration, but evidence of this in GBM is lacking. We hypothesized that CHCHD2 would serve a functional role in U87 GBM cells expressing the constitutively active epidermal growth factor receptor variant III (EGFRvIII). Amplification of the CHCHD2 gene was found to be associated with decreased patient overall survival and progression-free survival. CHCHD2 mRNA levels were increased in high-versus low-grade glioma, IDH-wt GBMs, and in tumor versus non-tumor tissue. Additionally, CHCHD2 protein expression was greatest in invasive, EGFRvIII-expressing patient-derived samples. CRISPR-Cas9-mediated knockout of CHCHD2 in EGFRvIII-expressing U87 cells resulted in altered mitochondrial respiration and glutathione status, decreased cell growth and invasion in both normoxia and hypoxia, and increased sensitivity to cytotoxic agents. CHCHD2 was distributed in both mitochondria and nuclei of U87 and U87vIII cells, and U87vIII displayed greater nuclear CHCHD2 compared to isogenic U87 cells. Incubation in hypoxia, serum starvation, and reductive unfolding of CHCHD2 induced nuclear accumulation of CHCHD2 in both cell lines. Collectively, these data indicate that CHCHD2 mediates a variety of GBM cell hallmark characteristics and highlights mitonuclear retrograde signaling as a pathway of interest in GBM cell biology. ImplicationsThese data demonstrate CHCHD2 as a mediator of a number of GBM cell functions representing disease hallmarks, as well as highlight its subcellular distribution in response to metabolic stressors. These results may inspire therapeutic strategies undermining mitochondrial biology to potentially improve GBM tumor management.

cancer biology↗

bioRxiv2020

Threshold illumination for non-invasive imaging of cells and tissues

Fluorescent microscopy employs monochromatic light which can affect the cells being observed. We reported earlier that fibroblasts relax their contractile force in response to green light of typical intensity. Here we show that such effects are independent of extracellular matrix and type of cell. In addition, we establish a threshold light that invokes minimal effect on cells. We cultured fibroblasts on soft 2D elastic hydrogels embedded with fluorescent beads to trace substrate deformation. The beads move towards cell center when cells contract, but they move away when cells relax. We use relaxation/contraction ratio,{lambda} r, as a measure of cell response to light. The cells were exposed to green (wavelength,{lambda} = 545-580 nm) and red ({lambda} = 635-650 nm) light with a range of intensities. We find red light with intensity less than ~ 57 W/m2 results in{lambda} r = 1, i.e., cells maintain force homeostasis. Higher intensities and smaller wavelengths result in widespread force-relaxation in cells with{lambda} r > 1. We suggest the use of{lambda} > 650 nm light with low intensity (I [≤] 57 W/m2) for time-lapse imaging of cells and tissues in order to avoid light-induced artifacts in experimental observations.

biophysics↗

bioRxiv2017

CRISPR/Cas9-mediated Knock-in of an Optimized TetO Repeat for Live Cell Imaging of Endogenous Loci

Nuclear organization has an important role in determining genome function; however, it is not clear how spatiotemporal organization of the genome relates to functionality. To elucidate this relationship, a high-throughput method for tracking any locus of interest is desirable. Here, we report an efficient and scalable method named SHACKTeR (Short Homology and CRISPR/Cas9-mediated Knock-in of a TetO Repeat) for live cell imaging of specific chromosomal regions. Compared to alternatives, our method does not require a nearby repetitive sequence and it requires only two modifications to the genome: CRISPR/Cas9-mediated knock-in of an optimized TetO repeat and its visualization by TetR-EGFP expression. Our simplified knock-in protocol, utilizing short homology arms integrated by PCR, was successful at labeling 9 different loci in HCT116 cells with up to 20% efficiency. These loci included both nuclear speckle-associated, euchromatin regions and nuclear lamina-associated, heterochromatin regions. We anticipate the general applicability and scalability of our method will enhance causative analyses between gene function and compartmentalization in a high-throughput manner.

cell biology↗