Using this new methodology, we have also examined the role that the mitochondrial calcium uniporter (MCU), an inner pore-forming unit of the mitochondrial membrane, plays in mitochondrial movement. Biochemical analysis reveals that this is due to an interaction with Miro1, the master regulator of mitochondrial transport. While Miro1 and MCU are outer and inner mitochondrial membrane proteins, respectively, MCU is able to bind to Miro1 through its previously defined mitochondrial targeting sequence (MTS).
We found that the MTS of MCU is localized to the mitochondrial outer membrane and is dispensable for the localization of MCU to the mitochondria. Taken together, this body of work explores multiple aspects of mitochondrial transport in axons, revealing levels of complexity not previously described. 96 Figure 3.7: The putative amino-terminal mitochondria targeting sequence of MCU is neither necessary nor sufficient for directing the mitochondrial localization of MCU.
102 Figure 3.10: Domain mapping of MCU and Miro1 interaction shows a new secondary target for the putative MCU MTS. TIM – Translocase of the inner mitochondrial membrane TOMM20 – Translocase of the outer mitochondrial membrane TRAK -Trafficking Kinesin Protein.
Introduction
Retrograde movement, however, returns components back to the cell body, usually for processing by aspects of the cell that do not exist in the axon. For example, Parkinson's disease can occur when components of the mitochondrial maintenance pathway are genetically defective (28, 29), preventing the removal of dysfunctional mitochondria from the general population of organelles and increasing levels of reactive oxygen species (ROS) in the cell. Small sections of the axon are observed and the results extrapolated to the entire axon.
The discovery of this endogenous property of cells renewed interest in mitochondrial calcium influx, leading to the discovery of the proteins responsible for this function. In this study, MCUR1 was found to be associated with cytochrome oxidase (COX) complex assembly, which is part of the electron transport chain, and not the mitochondrial calcium uniplex. Knockout of this protein does not affect mitochondrial calcium influx, showing that it is not part of the overall calcium influx mechanism(108).
Furthermore, overexpression of the same calcium-null Miro1 reduced the amount of calcium entering mitochondria in both cell bodies and axons, demonstrating that Miro1 plays a role in calcium influx. Our next goal is to investigate the molecular nature of mitochondrial movement, and how the components of the mitochondrial matrix may play in the overall regulation of mitochondrial movement of axons.
Large scale analysis of mitochondrial movement in axons
Using this dispersion curve, we derived a formulation of the Fokker-Planck equation that fits our velocity dispersion curves to create an accurate method to compare the effect that different conditions have on mitochondrial movement. Anterograde mitochondria, or those leaving the soma, can reach the ends of the axons without difficulty. Indeed, the axon possesses many of the normal cytoplasmic components, such as ER(122), Golgi apparatus(122), cytoskeletal components(14), and organelles such as mitochondria(12).
In our initial examination of mitochondria throughout the axon over two hours (Figure 2.1), we found that different parts of the axon were shown very differently. Thus, the mitochondria of the soma, or those located in the most distal part of the axon, can be photoconverted and tracked on a large scale. This approach allows us to study mitochondria in the context of the whole axon and to eliminate confounding variables caused by observing only a small part of the axon.
Half of the maintenance medium is replaced every 3-4 days to compensate for evaporation over time. The ImageJ straighten function was used on the traced axon lines to produce a straightened time course of the axon for analysis. To further characterize our formula for the study of mitochondrial motion, we used the Kolmogorov–Smirnov test to determine the significance of the overall derivative curve.
We found that, as the measured time increases, the variability of the raw data decreases. Regardless of this variability, however, none of the data resembled those obtained from the whole axon (Figure 2.8B). Photoconversion of the soma will convert hundreds of mitochondria for analysis, whereas photoconversion of the distal tip converts a much smaller population of mitochondria, leading to the numbers obtained.
Mitochondria moving in the anterograde direction were found to be able to travel the entire length of the axon (Fig. 2.5A + 2.12C). The dynamics of mitochondria moving in the anterograde direction were normalized to the length of the axon. By forming a velocity distribution and calculating a derivative of the Fokker-Planck equation for analysis, we have cemented several key features regarding mitochondrial velocity.
Anterograde movement is significantly faster compared to retrograde movement, and is capable of transporting mitochondria from the proximal to the distal end of the axon. Anterograde velocity is dependent on the location of transport, as mitochondria closer to the soma are significantly slower compared to those further away.
MCU modulates mitochondria movement in axons through interaction with Miro1
Analysis of isolated mitochondria reveals that this interaction occurs within the mitochondrial outer membrane and is regulated via cleavage of the MCU N-terminal domain, a process stimulated by calcium influx. Analysis of HEK293 cells transfected with MCU-Flag and Myc-Miro1 confirmed the presence of a biochemical relationship between these two proteins (Fig. 3.5A, B). Intriguingly, Miro1 showed a bias towards co-immunoprecipitation with the crude 40 kDa band of MCU (Fig. 3.5B).
Flag-MCU and MCU-Flag both colocalized with Myc-Miro1 (Fig. 3.6), although Flag-MCU did show a less intense signal. However, transfection of this vector into HEK cells revealed that MCU's MTS linked to sfGFP does not direct localization to mitochondria, appearing instead in the cytoplasm (Fig. 3.7D), suggesting that the N-terminal domain of MCU is not sufficient to direct. MCU localization in mitochondria. While the N-terminus of MCU appears to colocalize with the outer mitochondrial membrane, it is unclear whether it occurs on the inside or outside of the membrane.
Protease K, confirming that the N-terminus of MCU is located on the cytoplasmic side of the outer mitochondrial membrane. To next investigate the role of the N-terminal domain of MCU (Fig. 3.10A) in the interaction with Miro1 (Fig. 3.10B), we performed immunoprecipitation analysis using cell extracts containing MCU-Flag or MCU(Δ2-57 ) contain - Flag and Myc-Miro1. Removing the MTS from MCU (Fig. 3.10C) eliminates the interaction between MCU and Miro1, demonstrating that this domain is essential for interaction.
The DIME motif of MCU is predicted to project into the mitochondrial intermembrane space and is important for Ca2+ pore formation (90) (Fig. 3.10A). This interaction is specific for Miro1 and MCU, as TOMM20-Myc could not be immunoprecipitated with MCU-Flag (Fig. 3.10F). To further verify interaction between MCU and Miro1 in mitochondria, we performed FRET experiment using acceptor photobleaching with isolated mitochondria (Fig. 3.10G).
In addition to the FRET analysis, we performed dSTORM imaging with isolated mitochondria (Fig. 3.11), which allowed us to demonstrate co-localization between Flag-MCU and Myc-Miro1 at the nanoscale. MCU is present in both unprocessed and processed form, and Miro1 binds preferentially to unprocessed from MCU in mitochondria (Fig. 3.3B). Combined with our data showing that Miro1 preferentially binds to full-length MCU containing the N-terminal domain (amino acids 2-57) of MCU in the outer mitochondrial membrane (Fig. 3.5B), these results suggest that Ca2+.
Conversely, MCUΔ2-57-Flag was able to rescue calcium influx to the same level of MCU-Flag (Fig. 3.14A-C). While both MCU and Miro1 knockdown negatively affect calcium influx (Fig. 3.4), eliminating the interaction between them does not affect calcium influx.
Discussion and Future efforts
Furthermore, our new technique allowed us to establish that MCU, the primary component of the mitochondrial calcium uniplex (90, 91), also plays a crucial but previously unknown role in the regulation of mitochondrial movement in axons. Although the majority of functional domains in MCU have been identified and categorized ( 94 , 143 ), the putative mitochondrial targeting sequence has been largely ignored. While this approach is not necessarily wrong and answers many questions about the nature and function of mitochondria in this area, it does leave gaps in knowledge about the general purpose of this transport in the larger context of the axon.
Our work in identifying MCU as an accessory component of the mitochondrial movement regulation complex has confirmed our previous results showing that internal components of mitochondria play an important role in mitochondrial mobility ( 48 ). This component has been largely ignored in calcium uniporter research, as most studies have been conducted in immortal cell lines ( 90 , 91 ) or cardiac tissue ( 111 ), as mitochondrial calcium fluctuations have been shown to play an important role. in the event of a heart attack there. However, the fact that the uniplex has a neuron-exclusive component suggests that there is a neuron-exclusive functional aspect of the uniporter.
An important concern in this study is the native expression level of the larger interactive form of MCU, which we propose is responsible for the regulation of mitochondrial transport versus the mitochondrial transport machinery. As the neuron matures, the total number of mobile mitochondria also decreases (52, 127), which would result in an even lower presence of the larger form of MCU. If the total number of motors available on a given mitochondrion were disproportionate to the amount of calcium uniplex, it would be difficult to understand the role MCU plays in mitochondrial transport.
Lewit-Bentley A & Rety S (2000) EF-hand calcium-binding proteins. 2013) Structural coupling of the EF-hand and C-terminal GTPase domains in the mitochondrial protein Miro. Montero M, Lobaton CD, Moreno A, & Alvarez J (2002) A novel regulatory mechanism of the mitochondrial Ca2+ uniporter revealed by the p38 mitogen-activated protein kinase inhibitor SB202190. The physiological role of mitochondrial calcium revealed by mice lacking the mitochondrial calcium uniporter. 2013) MICU1 controls both the threshold and cooperative activation of the mitochondrial Ca(2)(+) uniporter.
Kamer KJ & Mootha VK (2014) MICU1 and MICU2 play non-redundant roles in the regulation of the mitochondrial calcium uniporter. Paupe V, Prudent J, Dassa EP, Rendon OZ, & Shoubridge EA (2015) CCDC90A (MCUR1) is a cytochrome c oxidase assembly factor and not a regulator of the mitochondrial calcium uniporter. 2015 ) MCUR1, CCDC90A, is a regulator of the Mitochondrial Calcium Uniporter. De La Fuente S, et al. 2016) Strategic Positioning and Biased Activity of the Mitochondrial Calcium Uniporter in Cardiac Muscle.
