Biology subjects

SHARMA, R.

Publications and source records attributed to SHARMA, R..

2 recordsLinked to original sources

bioRxiv2024

Engineering mtDNA Deletions by Reconstituting End-Joining in Human Mitochondria

Recent breakthroughs in the genetic manipulation of mitochondrial DNA (mtDNA) have enabled the precise introduction of base substitutions and the effective removal of genomes carrying harmful mutations. However, the reconstitution of mtDNA deletions responsible for severe mitochondrial myopathies and age-related diseases has not yet been achieved in human cells. Here, we developed a method to engineer specific mtDNA deletions in human cells by co-expressing end-joining (EJ) machinery and targeted endonucleases. As a proof-of-concept, we used mito-EJ and mito-ScaI to generate a panel of clonal cell lines harboring a [~]3.5 kb mtDNA deletion with the full spectrum of heteroplasmy. Investigating these isogenic cells revealed a critical threshold of [~]75% deleted genomes, beyond which cells exhibited depletion of OXPHOS proteins, severe metabolic disruption, and impaired growth in galactose-containing media. Single-cell multiomic analysis revealed two distinct patterns of nuclear gene deregulation in response to mtDNA deletion accumulation; one triggered at the deletion threshold and another progressively responding to increasing heteroplasmy. In summary, the co-expression of mito-EJ and programable nucleases provides a powerful tool to model disease-associated mtDNA deletions in different cell types. Establishing a panel of cell lines with a large-scale deletion at varying levels of heteroplasmy is a valuable resource for understanding the impact of mtDNA deletions on diseases and guiding the development of potential therapeutic strategies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=143 HEIGHT=200 SRC="FIGDIR/small/618543v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@ab900corg.highwire.dtl.DTLVardef@17e094dorg.highwire.dtl.DTLVardef@194a10corg.highwire.dtl.DTLVardef@d925e5_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICombining prokaryotic end-joining with targeted endonucleases generates specific mtDNA deletions in human cells C_LIO_LIEngineering a panel of cell lines with a large-scale deletion that spans the full spectrum of heteroplasmy C_LIO_LI75% heteroplasmy is the threshold that triggers mitochondrial and cellular dysfunction C_LIO_LITwo distinct nuclear transcriptional programs in response to mtDNA deletions: threshold-triggered and heteroplasmy-sensing C_LI

molecular biology↗

bioRxiv2022

CARP2 regulates the Golgi dynamics upon EGF stimulation

Golgi apparatus regulate diverse cellular functions like protein sorting, vesicular trafficking, secretion, protein modifications like glycosylation etc. In mammalian cells though, Golgi apparatus appear as ribbon architecture, individual stacks laterally linked to each other by tubular structure, its architecture changes dynamically to cater to the needs of the cell under physiological and stress conditions. Loss of Golgi integrity is reported to be associated with pathological conditions like cancer and neurodegeneration. Very little is known of molecular regulators of Golgi dynamics. Here, we demonstrate that CARP2 (Caspase -8 and -10 associated RING containing protein 2), an endosomal ubiquitin ligase and a known regulator of cell migration, modulates the Golgi structure. Stimulation with EGF (Epidermal growth factor) modestly increased CARP2 protein levels. CARP2 exogenous expression or EGF treatment resulted in dispersal of the Golgi apparatus. Conversely, CARP2 deletion suppressed EGF induced Golgi dispersal. CARP2 variants that are defective in their endosomal-association or E3 ligase activity were unable to exhibit Golgi dispersal, indicating importance of both the endosomal localization and the E3 activity for this function. Importantly, we provide evidence that in EGF stimulated cells CARP2 mechanistically functions by targeting one of the Golgi structural proteins, Golgin45 for ubiquitination and degradation. Taken together, our findings unravel the existence of crosstalk between endosomal ubiquitin signaling and the Golgi dynamics. SignificanceThe Golgi is an organelle that exists in mammals in ribbon form - individual stacks laterally linked with each other - is central to protein and lipid modifications, trafficking and secretion. The Golgi architecture is changed dynamically to cater to the physiological needs of the cells (eg: cell division, migration). Dysfunctional or altered Golgi is reported under pathological conditions like cancer, neurodegenerative diseases etc. This study unravels a complex signaling between endosomal ubiquitin ligase, CARP2 and one of the Golgi structural proteins, Golgin45. Here, we delineate CARP2-Golgin45 signaling as a fundamental mechanism that regulates Golgi dynamics underlying in EGF-stimulated cell migration.

cell biology↗