CLPB disaggregase dysfunction impacts the functional integrity of the proteolytic SPY complex.

CLPB is a mitochondrial intermembrane space AAA+ domain-containing disaggregase. CLPB mutations are associated with 3-methylglutaconic aciduria and neutropenia; however, the molecular mechanism underscoring disease and the contribution of CLPB substrates to disease pathology remains unknown. Interactions between CLPB and mitochondrial quality control (QC) factors, including PARL and OPA1, have been reported, hinting at dysregulation of organelle QC in disease. Utilizing proteomic and biochemical approaches, we show a stress-specific aggregation phenotype in a CLPB-null environment and define the CLPB substrate profile. We illustrate an interplay between intermembrane space proteins including CLPB, HAX1, HTRA2, and the inner membrane quality control proteins (STOML2, PARL, YME1L1; SPY complex), with CLPB deficiency impeding SPY complex function by virtue of protein aggregation in the intermembrane space. We conclude that there is an interdependency of mitochondrial QC components at the intermembrane space/inner membrane interface, and perturbations to this network may underscore CLPB disease pathology.

Human Tim8a, Tim8b and Tim13 are auxiliary assembly factors of mature Complex IV.

Human Tim8a and Tim8b are paralogous intermembrane space proteins of the small TIM chaperone family. Yeast small TIMs function in the trafficking of proteins to the outer and inner mitochondrial membranes. This putative import function for hTim8a and hTim8b has been challenged in human models, but their precise molecular function(s) remains undefined. Likewise, the necessity for human cells to encode two Tim8 proteins and whether any potential redundancy exists is unclear. We demonstrate that hTim8a and hTim8b function in the assembly of cytochrome c oxidase (Complex IV). Using affinity enrichment mass spectrometry, we define the interaction network of hTim8a, hTim8b and hTim13, identifying subunits and assembly factors of the Complex IV COX2 module. hTim8-deficient cells have a COX2 and COX3 module defect and exhibit an accumulation of the Complex IV S2 subcomplex. These data suggest that hTim8a and hTim8b function in assembly of Complex IV via interactions with intermediate-assembly subcomplexes. We propose that hTim8-hTim13 complexes are auxiliary assembly factors involved in the formation of the Complex IV S3 subcomplex during assembly of mature Complex IV.

Super-resolution microscopy reveals the arrangement of inner membrane protein complexes in mammalian mitochondria.

The mitochondrial inner membrane is a protein-rich environment containing large multimeric complexes, including complexes of the mitochondrial electron transport chain, mitochondrial translocases and quality control machineries. Although the inner membrane is highly proteinaceous, with 40-60% of all mitochondrial proteins localised to this compartment, little is known about the spatial distribution and organisation of complexes in this environment. We set out to survey the arrangement of inner membrane complexes using stochastic optical reconstruction microscopy (STORM). We reveal that subunits of the TIM23 complex, TIM23 and TIM44 (also known as TIMM23 and TIMM44, respectively), and the complex IV subunit COXIV, form organised clusters and show properties distinct from the outer membrane protein TOM20 (also known as TOMM20). Density based cluster analysis indicated a bimodal distribution of TIM44 that is distinct from TIM23, suggesting distinct TIM23 subcomplexes. COXIV is arranged in larger clusters that are disrupted upon disruption of complex IV assembly. Thus, STORM super-resolution microscopy is a powerful tool for examining the nanoscale distribution of mitochondrial inner membrane complexes, providing a 'visual' approach for obtaining pivotal information on how mitochondrial complexes exist in a cellular context.

Mitochondrial protein import dysfunction: mitochondrial disease, neurodegenerative disease and cancer.

The majority of proteins localised to mitochondria are encoded by the nuclear genome, with approximately 1500 proteins imported into mammalian mitochondria. Dysfunction in this fundamental cellular process is linked to a variety of pathologies including neuropathies, cardiovascular disorders, myopathies, neurodegenerative diseases and cancer, demonstrating the importance of mitochondrial protein import machinery for cellular function. Correct import of proteins into mitochondria requires the co-ordinated activity of multimeric protein translocation and sorting machineries located in both the outer and inner mitochondrial membranes, directing the imported proteins to the destined mitochondrial compartment. This dynamic process maintains cellular homeostasis, and its dysregulation significantly affects cellular signalling pathways and metabolism. This review summarises current knowledge of the mammalian mitochondrial import machinery and the pathological consequences of mutation of its components. In addition, we will discuss the role of mitochondrial import in cancer, and our current understanding of the role of mitochondrial import in neurodegenerative diseases including Alzheimer's disease, Huntington's disease and Parkinson's disease.

Function of hTim8a in complex IV assembly in neuronal cells provides insight into pathomechanism underlying Mohr-Tranebjærg syndrome.

Human Tim8a and Tim8b are members of an intermembrane space chaperone network, known as the small TIM family. Mutations in TIMM8A cause a neurodegenerative disease, Mohr-Tranebjærg syndrome (MTS), which is characterised by sensorineural hearing loss, dystonia and blindness. Nothing is known about the function of hTim8a in neuronal cells or how mutation of this protein leads to a neurodegenerative disease. We show that hTim8a is required for the assembly of Complex IV in neurons, which is mediated through a transient interaction with Complex IV assembly factors, in particular the copper chaperone COX17. Complex IV assembly defects resulting from loss of hTim8a leads to oxidative stress and changes to key apoptotic regulators, including cytochrome c, which primes cells for death. Alleviation of oxidative stress with Vitamin E treatment rescues cells from apoptotic vulnerability. We hypothesise that enhanced sensitivity of neuronal cells to apoptosis is the underlying mechanism of MTS.

Mitochondria-hubs for regulating cellular biochemistry: emerging concepts and networks.

Mitochondria are iconic structures in biochemistry and cell biology, traditionally referred to as the powerhouse of the cell due to a central role in energy production. However, modern-day mitochondria are recognized as key players in eukaryotic cell biology and are known to regulate crucial cellular processes, including calcium signalling, cell metabolism and cell death, to name a few. In this review, we will discuss foundational knowledge in mitochondrial biology and provide snapshots of recent advances that showcase how mitochondrial function regulates other cellular responses.

Four-Dimensional Imaging of Cardiac Trabeculae Contracting In Vitro Using Gated OCT.

Cardiac trabeculae are widely used as experimental muscle preparations for studying heart muscle. However, their geometry (diameter, length, and shape) can vary not only among samples, but also within a sample, leading to inaccuracies in estimating their stress production, volumetric energy output, and/or oxygen consumption. Hence, it is desirable to have a system that can accurately image each trabecula in vitro during an experiment. To this end, we constructed an optical coherence tomography system and implemented a gated imaging procedure to image actively contracting trabeculae and reconstruct their time-varying geometry. By imaging a single cross section while monitoring the developed force, we found that gated stimulation of the muscle was sufficiently repeatable to allow us to reconstruct multiple contractions to form a four-dimensional representation of a single muscle contraction cycle. The complete muscle was imaged at various lengths and the cross-sectional area along the muscle was quantified during the contraction cycle. The variation of cross-sectional area along the length during a contraction tended to increase as the muscle was contracting, and this increase was greater at longer muscle lengths. To our knowledge, this is the first system that is able to measure the geometric change of cardiac trabeculae in vitro during a contraction, allowing cross-sectional stress and other volume-dependent parameters to be estimated with greater accuracy.

Characterization of a Novel Collagen Scaffold for Corneal Tissue Engineering.

Globally millions are blind due to corneal disease, yet tissue for transplantation is a limited resource. This study characterizes the physical and biological properties of a novel collagen-based scaffold. Transparency, optical coherence tomography (OCT), and scanning electron microscopy (SEM) were used to analyze the structure of the scaffold, synthesized using rat tail collagen I. Water content was determined. The tensile strength was assessed using a micro-mechanical analyzer. In vitro biocompatibility was assessed by culturing the scaffold with epithelial or keratocyte spheres. The mean scaffold transmittance was 0.72 at 358 nm, 0.88 at 570 nm, and 0.92 at 900 nm. OCT imaging confirmed that the scaffold maintained a corneal shape, with a central thickness of 502 µm and a reflectivity profile comparable to that of a normal human cornea. SEM of the scaffold revealed multiple lamellae on cross section. The mean water content was 88.7% ± 0.7%. Ultimate tensile strength for the noncross-linked scaffold was 1.23 ± 0.27 MPa compared with 2.21 ± 0.70 MPa for the cross-linked scaffold (human corneal anterior stroma 1.53 ± 0.86 MPa) at a strain rate of 0.5%/s. Epithelial cells migrated over the scaffold to confluence. Keratocytes populated the scaffold and maintained a lamellar arrangement. The properties of this novel scaffold suggest that it has potential to be developed into a corneal tissue substitute for human transplantation.

An investigation into the viability of image processing for the measurement of sarcomere length in isolated cardiac trabeculae.

A preliminary investigation was performed into the viability of using frequency domain image processing techniques to determine sarcomere length from bright-field images of isolated cardiac trabecula in real-time. Hardware based data processing was used to compute the average sarcomere length in a cardiac trabecula undergoing stretch. Our technique estimated the increase in mean sarcomere length with increasing muscle length as the trabecula was stretched to and beyond the normal physiological limit of 2.4 µm. The standard error in the mean sarcomere length extracted from each image was typically10 nm.