New insights into the role of mitochondrial calcium homeostasis in cell migration

Mitochondria are dynamic organelles involved in numerous physiological functions. Beyond their function in ATP production, mitochondria regulate cell death, reactive oxygen species (ROS) generation, immunity and metabolism. Mitochondria also play a key role in the buffering of cytosolic calcium, and calcium transported into the matrix regulates mitochondrial metabolism. Recently, the identification of the mitochondrial calcium uniporter (MCU) and associated regulators has allowed the characterization of new physiological roles for calcium in both mitochondrial and cellular homeostasis. Indeed, recent work has highlighted the importance of mitochondrial calcium homeostasis in regulating cell migration. Cell migration is a property common to all metazoans and is critical to embryogenesis, cancer progression, wound-healing and immune surveillance. Previous work has established that cytoplasmic calcium is a key regulator of cell migration, as oscillations in cytosolic calcium activate cytoskeletal remodelling, actin contraction and focal adhesion (FA) turnover necessary for cell movement. Recent work using animal models and $\textit{in cellulo}$ experiments to genetically modulate MCU and partners have shed new light on the role of mitochondrial calcium dynamics in cytoskeletal remodelling through the modulation of ATP and ROS production, as well as intracellular calcium signalling. This review focuses on MCU and its regulators in cell migration during physiological and pathophysiological processes including development and cancer. We also present hypotheses to explain the molecular mechanisms by which MCU may regulate mitochondrial dynamics and motility to drive cell migration.This work was supported by the Medical Research Council, UK (MC_UP_1601/1). V.P. is supported by a Medical Research Council postdoctoral fellowship

Observations of Colloidal Gold Labelled Platelet Microtubules: High Voltage Electron Microscopy and Low Voltage-High Resolution Scanning Electron Microscopy

18 nm colloidal gold-antitubulin and 4 nm colloidal gold-antitubulin were used to label microtubules in adherent, fully spread platelets. Both sizes of marker effectively labelled microtubules in the partially extracted platelets. However only the 4 nm gold penetrated the dense microfilament matrix of the inner filamentous zone so that portions of microtubules within this cytoskeletal zone could be tracked. The gold marker could be visualized well with 1 MeV high voltage transmission EM and with 5 kV or greater secondary imaging or 20 kV backscattered imaging of carbon only coated samples. 1 kV secondary imaging permitted high resolution imaging of the surface of tubules and the microfilaments with their respective associated material. Individual gold-antibody complexes were difficult to identify by shape alone due to the tendency of the antibody coats to blend together when in very close approximation and due to the presence of other molecules or molecular aggregates similar in size to the gold-antibody labels. Microtubules were seen to wind in and out of the inner and outer filamentous zones as they encircled the granulomere. Some tubules were seen to dead end at the peripheral web. Numerous smaller microtubule loops were present principally in the outer filamentous zone and tubules could be followed as they went from the outer filamentous zone through the inner filamentous zone and into the granulomere

The mitochondria–endoplasmic reticulum contact sites: a signalling platform for cell death

Mitochondria evolved as an endosymbiont providing the cell with a dizzying array of catabolic and anabolic processes essential for life. However, mitochondria have retained the ability to kill from within, and are widely considered the final executioners of programmed cell death. The groundbreaking discovery over 25 years ago that mitochondrial cytochrome c is released into the cytosol shone new and unexpected light onto this old organelle, revitalizing the field. The Bcl-2 family of proteins plays a central role in the maintenance of mitochondrial membrane integrity, but other factors are also involved in the cell death program. Indeed, contacts with the endoplasmic reticulum (ER), mitochondrial division and inner membrane cristae remodeling have emerged as key regulators of cytochrome c release. This review will focus on recent progress to define the functional contribution of the apoptotic ER/mitochondrial interface, which couples mitochondrial fission and cristae remodeling to calcium and lipid fluxes.Studies in this area were funded by Canadian Institutes of Health Research (CIHR) (PT-71405) and Canadian Cancer Society Research Institute (CCSRI) (#704826) to HMM, and Medical Research Council, UK (MC_UP_1601/1) to JP. HMM was supported by a Canada Research Chair Tier 1

Interplay between Mitochondrial Protein Import and Respiratory Complexes Assembly in Neuronal Health and Degeneration

The fact that >99% of mitochondrial proteins are encoded by the nuclear genome and synthesised in the cytosol renders the process of mitochondrial protein import fundamental for normal organelle physiology. In addition to this, the nuclear genome comprises most of the proteins required for respiratory complex assembly and function. This means that without fully functional protein import, mitochondrial respiration will be defective, and the major cellular ATP source depleted. When mitochondrial protein import is impaired, a number of stress response pathways are activated in order to overcome the dysfunction and restore mitochondrial and cellular proteostasis. However, prolonged impaired mitochondrial protein import and subsequent defective respiratory chain function contributes to a number of diseases including primary mitochondrial diseases and neurodegeneration. This review focuses on how the processes of mitochondrial protein translocation and respiratory complex assembly and function are interlinked, how they are regulated, and their importance in health and disease

Mejora del comportamiento en la extrusión de pastas cerámicas de baja plasticidad.

Shaping by extrusion may be used for the manufacture of tiles, bricks, and also refractory products. The three fundamental aspects that must be considered to achieve ideal extrusion conditions are the nature of the material, particle size and Plastic behaviour. Of these, obtaining a mass of adequate plasticity is essential during the processing in order to prevent defects such as cracks, laminations or migrations of the liquid phase [1]. Plastic behaviour depends on numerous factors [2]: size and shape of the particle, mineralogical composition, presence of electrolytes, organic matter, etc. With regard to traditional ceramics, as is the case of the manufacture of ceramic tiles, clay materials are those with the necessary plasticity to carry out the extrusion process. However, in the absence of clays with sufficient plasticity or when the composition incorporates a high proportion of other non-plastic ingredients, the extrusion operation may be seriously compromised, particularly for the manufacture of large or complex tiles. In these cases it is necessary to add plasticizers or binders to the pastes to achieve rheological behaviour and plasticity enabling correct processing of the composition [3]. In this report the influence of different additives in different proportions on the plasticity index of a ceramic composition was evaluated and the variation of the plasticity index was related to the behaviour of the paste during extrusion.El conformado por extrusión se puede utilizar para la fabricación de baldosas, ladrillos, tejas o también productos refractarios. Los tres aspectos fundamentales que se deben considerar para lograr unas condiciones de extrusión idóneas son la naturaleza del material, el tamaño de partícula y el comportamiento plástico. De éstas, obtener una masa de plasticidad adecuada es fundamental durante el procesado, para evitar la aparición de defectos tales como grietas, laminaciones o migraciones de la fase líquida [1]. El comportamiento plástico depende de numerosos factores [2]: tamaño y forma de la partícula, composición mineralógica, presencia de electrolitos, materia orgánica, etc. En lo que concierne a las cerámicas tradicionales, como es el caso de la fabricación de baldosas cerámicas, los materiales arcillosos son los que confieren la plasticidad necesaria para llevar a cabo el proceso de extrusión. Sin embargo, cuando no se dispone de arcillas con la plasticidad suficiente o cuando la composición incorpora otros ingredientes no plásticos en elevada proporción, la operación de extrusión se puede ver seriamente comprometida, sobre todo en la fabricación de baldosas de gran formato o de geometría compleja. En estos casos es necesario añadir a las pastas aditivos plastificantes o ligantes para conseguir un comportamiento reológico y plasticidad que permitan un correcto procesado de la composición [3]. En este trabajo se evaluó la influencia de la incorporación de diferentes aditivos en distintas proporciones sobre el índice de plasticidad de una composición cerámica y se relacionó la variación del índice de plasticidad con el comportamiento de la pasta durante la extrusión

Mass spectrometry of B. subtilis CopZ: Cu(I)-binding and interactions with bacillithiol

CopZ from Bacillus subtilis is a well-studied member of the highly conserved family of Atx1-like copper chaperones. It was previously shown via solution and crystallographic studies to undergo Cu(I)-mediated dimerisation, where the CopZ dimer can bind between one and four Cu(I) ions. However, these studies could not provide information about the changing distribution of species at increasing Cu(I) levels. To address this, electrospray ionisation mass spectrometry using soft ionisation was applied to CopZ under native conditions. Data revealed folded, monomeric CopZ in apo- and Cu(I)-bound forms, along with Cu(I)-bound dimeric forms of CopZ at higher Cu(I) loading. Cu4(CopZ)2 was the major dimeric species at loadings >1 Cu(I)/CopZ, indicating the cooperative formation of the tetranuclear Cu(I)-bound species. As the principal low molecular weight thiol in B. subtilis, bacillithiol (BSH) may play a role in copper homeostasis. Mass spectrometry showed that increasing BSH led to a reduction in Cu(I)-bound dimeric forms, and the formation of S-bacillithiolated apo-CopZ and BSH adducts of Cu(I)-bound forms of CopZ, where BSH likely acts as a Cu(I) ligand. These data, along with the high affinity of BSH for Cu(I), determined here to be β2(BSH) = ∼4 × 1017 M−2, are consistent with a role for BSH alongside CopZ in buffering cellular Cu(I) levels. Here, mass spectrometry provides a high resolution overview of CopZ–Cu(I) speciation that cannot be obtained from less discriminating solution-phase methods, thus illustrating the potential for the wider application of this technique to studies of metal–protein interactions