Biology subjects

Needs, H. I.

Publications and source records attributed to Needs, H. I..

3 recordsLinked to original sources

bioRxiv2022

Intercellular Mitochondrial Transfer as a Rescue Mechanism in Response to Protein Import Failure

Mitochondria are the powerhouses of eukaryotic cells, composed mostly of nuclear-encoded proteins imported from the cytosol. Thus, problems with the import machinery will disrupt their regenerative capacity and the cells energy (ATP) supplies-particularly troublesome for energy demanding cells like neurons and myocytes. Unsurprisingly then, dysfunctional import is implicated in disease. This study explores the consequences of import failure in mammalian cells; wherein, blocking the import machinery has profound effects on mitochondrial ultra-structure and dynamics, but, surprisingly, does not impact import. The explanation is an astonishing response involving intercellular mitochondrial transfer via tunnelling nanotubes: for the import of healthy mitochondria and jettisoning of those with jammed import sites. These observations support the existence of a widespread mechanism for the rescue of mitochondrial protein import failure. One-Sentence SummaryA mitochondrial import rescue mechanism involving intercellular mitochondrial transport through tunneling nanotubes (TNTs).

cell biology↗

bioRxiv2022

Perturbation of the mitochondrial import machinery by disease prone Tau affects organelle morphology and reduces neuronal complexity

Protein import into mitochondria is an intricate and highly conserved process essential for organellar biogenesis, and maintenance of its structure and function. Defects in the import apparatus impact the assembly of the respiratory chain and ATP synthase complexes required for oxidative phosphorylation, compromising the supply of ATP to the cytosol. The consequences of reduced bioenergetic function are particularly severe for cells with high energetic demands, such as neurons. However, relatively little is known about how defective import contributes to neurodegeneration, or how aggregation prone toxic proteins, characteristic of neurodegenerative disease, impact mitochondrial import efficiency. Here, we used HeLa cells to investigate how expressing Tau, or a disease-causing variant, affects mitochondrial import activity, morphology, and function. We found that a variant associated with frontotemporal dementia (TauP301L), but not the native version, colocalises with mitochondria, associating with TOM40-the protein-channel component of the outer membrane import complex. Interestingly, TauP301L production had no discernible effect on overall mitochondrial import function, despite associating with TOM40 and altering mitochondrial morphology. This raised suspicions of a rescue mechanism manifested by the appearance of microtubule and actin containing tunnelling nanotubes (TNTs), used to recruit healthy mitochondria from neighbouring cells and/ or dispose of mitochondria containing aggregated Tau. Furthermore, in primary neuronal cultures TauP301L induces morphological changes that resemble a neurodegeneration-like phenotype-also mirrored in cells where the import sites are blocked artificially. These results reveal an intriguing link between the production of aggregation prone protein variants, such as TauP301L and others, with the mitochondrial protein import machinery relevant to neurodegenerative disease.

cell biology↗

bioRxiv2022

The NanoLuc Assay System for Accurate Real-Time Monitoring of Mitochondrial Protein Import within Intact Mammalian Cells

Only a few proteins (13 in humans) are encoded by the mammalian mitochondrial genome. Therefore, the other mitochondrial resident proteins (>1000) must be recruited via specialised import pathways. Protein import is critical for mitochondrial biogenesis and bioenergetic function and health; loss of function has been implicated with a wide range of pathologies. Despite this, our understanding of the kinetic and dynamics of import is somewhat limited, particularly within mammalian cells. Here, we report an adaptation of an assay system, established previously to monitor mitochondrial import into isolated yeast mitochondria, to quantitatively monitor mitochondrial import inside mammalian cells. The reporting is based on a split luciferase, whereby the large fragment is segregated in the mitochondrial matrix and the small complementary fragment is fused to the C-terminus of a recombinant precursor protein destined for import. Following import successively through the TOM complex of the outer membrane and the TIM23 complex of the inner membrane, the complementary fragments combine to form an active luciferase. The resultant luminescent signal provides a sensitive, accurate, free of noise and continuous measure of protein import, enabling mathematical model fitting to identify and understand the steps that make up import. This advance allows detailed mechanistic examination of the transport process in live cells. In addition, the assay will enable characterisation of the protein import when the machinery is challenged; for example, in situations associated with disease. Moreover, the assay is compatible with high throughput for large data set collection and kinetic modelling, as well as for drug screening and characterisation. Our set-up also has the potential to be adapted for the analysis of alternative transport systems and different cell types, and even for multicellular model organisms.

biochemistry↗