Dual role of Miro protein clusters in mitochondrial cristae organisation and ER-Mitochondria Contact Sites

Mitochondrial Rho (Miro) GTPases localize to the outer mitochondrial membrane and are essential machinery for the regulated trafficking of mitochondria to defined subcellular locations. However, their sub-mitochondrial localization and relationship with other critical mitochondrial complexes remains poorly understood. Here, using super-resolution fluorescence microscopy, we report that Miro proteins form nanometer-sized clusters along the mitochondrial outer membrane in association with the Mitochondrial Contact Site and Cristae Organizing System (MICOS). Using knockout mouse embryonic fibroblasts (MEF) we show that Miro1 and Miro2 are required for normal mitochondrial cristae architecture and endoplasmic reticulum-mitochondria contacts sites (ERMCS). Further, we show that Miro couples MICOS to TRAK motor protein adaptors to ensure the concerted transport of the two mitochondrial membranes and the correct distribution of cristae on the mitochondrial membrane. The Miro nanoscale organization, association with MICOS complex and regulation of ERMCS reveal new levels of control of the Miro GTPases on mitochondrial functionality

The Loop2 Insertion of Type IX Myosin Acts as an Electrostatic Actin Tether that Permits Processive Movement

<div><p>Although class IX myosins are single-headed, they demonstrate characteristics of processive movement along actin filaments. Double-headed myosins that move processively along actin filaments achieve this by successive binding of the two heads in a hand-over-hand mechanism. This mechanism, obviously, cannot operate in single-headed myosins. However, it has been proposed that a long class IX specific insertion in the myosin head domain at loop2 acts as an F-actin tether, allowing for single-headed processive movement. Here, we tested this proposal directly by analysing the movement of deletion constructs of the class IX myosin from <i>Caenorhabditis elegans</i> (Myo IX). Deletion of the large basic loop2 insertion led to a loss of processive behaviour, while deletion of the N-terminal head extension, a second unique domain of class IX myosins, did not influence the motility of Myo IX. The processive behaviour of Myo IX is also abolished with increasing salt concentrations. These observations directly demonstrate that the insertion located in loop2 acts as an electrostatic actin tether during movement of Myo IX along the actin track.</p></div

Size distribution of purified myosn IX head.

<p>A volume of 200 µl purified Myo IX head was loaded on a size exclusion column (Superdex 200; 10/300 GL) in assay buffer including 130 mM KCl. <b>A:</b> Optical density was monitored at 280 nm and plotted against the elution volume. Myo IX head could be detected by SDS-PAGE in peak 1 and peak 2 as shown. <b>B:</b> The column was calibrated by the use of standard proteins (closed triangles) and K_av was calculated (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084874#pone.0084874.e001" target="_blank">equation 1</a>). The arrow marks K_av determined for peak 2 (open circle) that corresponds to a molecular weight of 192 kDa.</p

Movement of Qdot-labelled myosin IX head^(a) and myosin IX headΔext.

<p>Purified Myo IX head and Myo IX headΔext as indicated were labelled with streptavidin coated Qdots in a mixing ratio of 2 Qdots/Myo IX and introduced into a flow cell with immobilized actin filaments. Histograms of the velocity of moving Qdots fit a Gaussian distribution. Average run length of moving Qdots was determined by fitting the distribution to a function of an exponential decay as described in experimental procedures. The mean velocities (v) and run lengths (λ) are indicated. Exemplary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084874#pone.0084874.s006" target="_blank">movies (S6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084874#pone.0084874.s007" target="_blank">S7</a>) can be found in supporting information. ^(a) published by Liao <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084874#pone.0084874-Liao1" target="_blank">[12]</a>, shown for comparative reasons.</p

Influence of ionic strength on myosin IX head processivity.

<p>Filament gliding assays were performed as described in experimental procedures. <b>A:</b> Myo IX head (black), Myo IX headΔins (green) and Myo IX headΔext (blue) were immobilized specifically by biotin-streptavidin interaction on the surface of a flow cell. Velocity of F-actin gliding was determined at 30 mM, 80 mM, 130 mM and 180 mM KCl. <b>B:</b> Varying amounts of purified Myo IX head were immobilized on the surface of a flow cell. Velocity of F-actin gliding was determined at 30 mM KCl (closed circles) and 180 mM KCl (open circles), respectively. Errors presented are standard errors of varying numbers of tracked filaments.</p

F-actin gliding velocity driven by myosin IX head constructs.

<p>Filament gliding assays were performed as described in experimental procedures. Varying amounts of purified Myo IX head (black), Myo IX headΔins (green) or Myo IX headΔext (blue) were immobilized specifically by biotin-streptavidin interaction on the surface of a flow cell. Velocity of F-actin gliding was determined at different motor densities on the surface. The velocity as a function of motor density was fitted using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084874#pone.0084874.e002" target="_blank">equation 2</a>. Errors presented are standard deviations of varying numbers of tracked filaments. Exemplary movies for each construct can be found under supporting information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084874#pone.0084874.s003" target="_blank">movies S3</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084874#pone.0084874.s005" target="_blank">S5</a>).</p

Purification of myosin IX head constructs.

<p>Different Myo IX head constructs as shown in panel <b>A</b> were purified by α-flag affinity chromatography from Sf9-cells and separated by 10–15% gradient SDS-PAGE. Proteins were either stained by coomassie blue (<b>B</b>) or transferred to a PVDF membrane followed by specific detection of biotinated proteins by HRP-coupled streptavidin (<b>C</b>). CaM  =  recombinant calmodulin.</p

Actin filament landing rates as a function of myosin IX head construct density.

<p>Successful filament landing and gliding events were determined at varying motor densities on the surface for Myo IX head (black) Myo IX headΔins (green) and Myo IX headΔext (blue). Error bars represent the S.E.M. calculated from counting statistics (S.E.M.  =  landing rate/√n; n  =  number of landing events). For each data set, two theoretical curves were generated by non-linear least-squares fitting of parameters l_max and A of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084874#pone.0084874.e003" target="_blank">equation 3</a> with fixed n = 1 (solid lines) or n = 2 (dashed lines). Correlation coefficients indicate that n = 2 yields the best fit for headΔins, whereas head and headΔext are best fitted with n = 1 (head: R²_(n = 1) = 0.9845; R²_(n = 2) = 0.9370; headΔins: R²_(n = 1) = 0.9663; R²_(n = 2) = 0.9805; headΔext: R²_(n = 1) = 0.9729; R²_(n = 2) = 0.9268). The horizontal dotted grey line represents the experimental limit of detection given by the time and area of observation.</p

Parameters of <i>in vitro</i> motility of different myosin IX head constructs.

<p>F-actin gliding velocity v driven by different Myo IX head constructs is given (gliding assay). Mean velocity v and run length λ of Qdot-labelled Myo IX head and Myo IX headΔext moving along immobilized actin filaments (stepping assay) were determined from histograms shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084874#pone-0084874-g006" target="_blank">figure 6</a>. (n.d.  =  not detectable).</p