Biology subjects

Shutt, T. E.

Publications and source records attributed to Shutt, T. E..

5 recordsLinked to original sources

bioRxiv2025

A viral transduction approach for iPSC differentiation into cortisol-producing steroidogenic cells

Steroid hormones are important signaling molecules that are primarily produced by specialized cells. The ability to culture steroidogenic cells is critical to study this important process. While a few steroidogenic cell lines are available for study, they are typically derived from cancer patients and do not reflect genetic alterations in steroidogenic genes that cause human disease. In this regard, iPSCs are a powerful tool for modeling human disease, as patient-derived iPSCs can be differentiated into a variety of different cell types. While other approaches exist to differentiate iPSCs into steroidogenic cells, they are typically complex and time consuming. In contrast, a simple approach based on lentivirus transduction of SF1, the master regulator of steroidogenesis, can differentiate multiple types of cultured cells into a steroidogenic state. However, we show here that this approach does not work for iPSCs. To circumvent this limitation, we report a simple adaptation that first differentiates iPSCs to embryoid bodies prior of SF1 transduction. With this modified approach, we provide a straightforward cost-effective approach to differentiate iPSCs into actively steroidogenic cells.

developmental biology↗

bioRxiv2023

A novel genetic fluorescent reporter to visualize mitochondrial nucleoids

Mitochondria contain their own genome (mtDNA), which is present in hundreds of copies per cell and organized into nucleoid structures that are distributed throughout the dynamic mitochondrial network. Beyond encoding essential protein subunits for oxidative phosphorylation, mtDNA can also serve as a signalling molecule when it is present into the cytosol. Despite the importance of this genome, there are still many unknowns with respect to its regulation. To study mtDNA dynamics in living cells, we have developed a genetic fluorescent reporter, mt-HI-NESS, which is based on the HI-NESS reporter that uses the bacterial H-NS DNA binding domain. Here, we describe how this reporter can be used to image mtDNA nucleoids for live cell imaging without affecting the replication or expression of the mtDNA. In addition to demonstrating the adaptability of the mt-HI-NESS reporter for multiple fluorescent proteins, we also emphasize important factors to consider during the optimization and application of this reporter.

cell biology↗

bioRxiv2021

Functional characterization of two variants in the mitochondrial topoisomerase gene TOP1MT that impact regulation of the mitochondrial genome

TOP1MT encodes a mitochondrial topoisomerase that is important for mtDNA regulation, and that is involved in mitochondrial replication, transcription and translation. Two variants predicted to affect TOP1MT function (V1 - R198C and V2 - V338L) were identified by exome sequencing of a newborn with hypertrophic cardiomyopathy. As no pathogenic TOP1MT variants had been confirmed previously, we characterized these variants for their ability to rescue several TOP1MT functions in knockout cells. Consistent with these TOP1MT variants contributing to the patient phenotype, comprehensive characterization suggests that both variants had impaired activity. Critically, neither variant was able to restore steady state levels of mitochondrial-encoded proteins, nor reduced oxidative phosphorylation when re-expressed in TOP1MT knockout cells. However, the two variants behaved differently in some respects. While the V1 variant was better at restoring transcript levels, the V2 variant was able to restore mtDNA copy number and replication. These findings suggest that the different TOP1MT variants affect distinct TOP1MT functions. Altogether, these findings begin to provide insight into the many roles that TOP1MT plays in the maintenance and expression of the mitochondrial genome, and how impairments in this important protein may lead to human pathology.

molecular biology↗

bioRxiv2021

Characterization of a novel variant in the HR1 domain of MFN2 in a patient with ataxia, optic atrophy and sensorineural hearing loss

Pathogenic variants in MFN2 cause Charcot-Marie-Tooth disease (CMT) type 2A (CMT2A) and are the leading cause of the axonal subtypes of CMT. CMT2A is characterized by predominantly distal motor weakness and muscle atrophy, with highly variable severity and onset age. Notably, some MFN2 variants can also lead to other phenotypes such as optic atrophy, hearing loss and lipodystrophy. Despite the clear link between MFN2 and CMT2A, our mechanistic understanding of how dysfunction of the MFN2 protein causes human disease pathologies remains incomplete. This lack of understanding is due in part to the multiple cellular roles of MFN2. Though initially characterized for its role in mediating mitochondrial fusion, MFN2 also plays important roles in mediating interactions between mitochondria and other organelles, such as the endoplasmic reticulum and lipid droplets. Additionally, MFN2 is also important for mitochondrial transport, mitochondrial autophagy, and has even been implicated in lipid transfer. Though over 100 pathogenic MFN2 variants have been described to date, only a few have been characterized functionally, and even then, often only for one or two functions. Here, we describe a novel homozygous MFN2 variant, D414V, in a patient presenting with cerebellar ataxia, deafness, blindness, and diffuse cerebral and cerebellar atrophy. Characterization of patient fibroblasts reveals phenotypes consistent with impaired MFN2 functions and expands the phenotypic presentation of MFN2 variants to include cerebellar ataxia.

genetics↗

bioRxiv2018

PISD is a mitochondrial disease gene causing skeletal dysplasia, cataracts and white matter changes

Exome sequencing of two sisters with congenital cataracts, short stature and white matter changes identified compound heterozygous variants in the PISD gene, encoding the phosphatidylserine decarboxylase enzyme that converts phosphatidylserine (PS) to phosphatidylethanolamine (PE) in the inner mitochondrial membrane (IMM). Decreased conversion of PS to PE, and depletion of total cellular PE levels in patient fibroblasts are consistent with impaired PISD enzyme activity. Meanwhile, as evidence for mitochondrial dysfunction, patient fibroblasts exhibited more fragmented mitochondrial networks, enlarged lysosomes, decreased maximal oxygen consumption rates and increased sensitivity to 2-deoxyglucose. Moreover, treatment with lyso-PE, which can replenish the mitochondrial pool of PE, restored mitochondrial and lysosome morphology in patient fibroblasts. Functional characterization of the PISD mutations demonstrates that the maternal variant causes an alternative splice product. Meanwhile, the paternal variant impairs autocatalytic self-processing of the PISD protein required for its activity. Finally, evidence for impaired activity of mitochondrial IMM proteases explains why the phenotypes of these PISD patients resemble recently described \"mitochondrial chaperonopathies\". Collectively, these findings demonstrate that PISD is a novel mitochondrial disease gene.

molecular biology↗