Mitochondrial protein import as a quality control sensor.

Mitochondria are organelles involved in various functions related to cellular metabolism and homoeostasis. Though mitochondria contain own genome, their nuclear counterparts encode most of the different mitochondrial proteins. These are synthesised as precursors in the cytosol and have to be delivered into the mitochondria. These organelles hence have elaborate machineries for the import of precursor proteins from cytosol. The protein import machineries present in both mitochondrial membrane and aqueous compartments show great variability in pre-protein recognition, translocation and sorting across or into it. Mitochondrial protein import machineries also interact transiently with other protein complexes of the respiratory chain or those involved in the maintenance of membrane architecture. Hence mitochondrial protein translocation is an indispensable part of the regulatory network that maintains protein biogenesis, bioenergetics, membrane dynamics and quality control of the organelle. Various stress conditions and diseases that are associated with mitochondrial import defects lead to changes in cellular transcriptomic and proteomic profiles. Dysfunction in mitochondrial protein import also causes over-accumulation of precursor proteins and their aggregation in the cytosol. Multiple pathways may be activated for buffering these harmful consequences. Here, we present a comprehensive picture of import machinery and its role in cellular quality control in response to defective mitochondrial import. We also discuss the pathological consequences of dysfunctional mitochondrial protein import in neurodegeneration and cancer.

Protocol for analyzing energy metabolic pathway dependency in human liver cancer cell lines.

Cellular energy metabolism analysis is complex, expensive, and indirect. We present a protocol to analyze relative contribution of metabolic pathways to ATP production by directly measuring ATP levels. We describe steps for cell counting and seeding in 96-well plate, treating with metformin, and systematic inhibition with metabolic inhibitors. We then detail procedures for a viability and ATP assay and calculating energy metabolism dependency. This high-throughput and accessible protocol works with any cell line and allows for flexible perturbation studies.

CMT2A-linked MFN2 mutation, T206I promotes mitochondrial hyperfusion and predisposes cells towards mitophagy.

Mutations in Mitofusin2 (MFN2) associated with the pathology of the debilitating neuropathy Charcot-Marie-Tooth type 2A (CMT2A) are known to alter mitochondrial morphology. Previously, such mutations have been shown to elicit two diametrically opposite phenotypes - while some mutations have been causally linked to enhanced mitochondrial fragmentation, others have been shown to induce hyperfusion. Our study identifies one such MFN2 mutant, T206I that causes mitochondrial hyperfusion. Cells expressing this MFN2 mutant have elongated and interconnected mitochondria. T206I-MFN2 mutation in the GTPase domain increases MFN2 stability and renders cells susceptible to stress. We show that cells expressing T206I-MFN2 have a higher predisposition towards mitophagy under conditions of serum starvation. We also detect increased DRP1 recruitment onto the outer mitochondrial membrane, though the total DRP1 protein level remains unchanged. Here we have characterized a lesser studied CMT2A-linked MFN2 mutant to show that its presence affects mitochondrial morphology and homeostasis.

Role of systems biology and multi-omics analyses in delineating spatial interconnectivity and temporal dynamicity of ER stress mediated cellular responses.

The endoplasmic reticulum (ER) is a membranous organelle involved in calcium storage, lipid biosynthesis, protein folding and processing. Many patho-physiological conditions and pharmacological agents are known to perturb normal ER function and can lead to ER stress, which severely compromise protein folding mechanism and hence poses high risk of proteotoxicity. Upon sensing ER stress, the different stress signaling pathways interconnect with each other and work together to preserve cellular homeostasis. ER stress response is a part of the integrative stress response (ISR) and might play an important role in the pathogenesis of chronic neurodegenerative diseases, where misfolded protein accumulation and cell death are common. The initiation, manifestation and progression of ER stress mediated unfolded protein response (UPR) is a complex procedure involving multiple proteins, pathways and cellular organelles. To understand the cause and consequences of such complex processes, implementation of an integrative holistic approach is required to identify novel players and regulators of ER stress. As multi-omics data-based systems analyses have shown potential to unravel the underneath molecular mechanism of complex biological systems, it is important to emphasize the utility of this approach in understanding the ER stress biology. In this review we first discuss the ER stress signaling pathways and regulatory players, along with their inter-connectivity. We next highlight the importance of systems and network biology approaches using multi-omics data in understanding ER stress mediated cellular responses. This report would help advance our current understanding of the multivariate spatial interconnectivity and temporal dynamicity of ER stress.