Jaime Santo-Domingo

Calcium uptake mechanisms of mitochondria

The ability of mitochondria to capture Ca2+ ions has important functional implications for cells, because mitochondria shape cellular Ca2+ signals by acting as a Ca2+ buffer and respond to Ca2+ elevations either by increasing the cell energy supply or by triggering the cell death program of apoptosis. A mitochondrial Ca2+ channel known as the uniporter drives the rapid and massive entry of Ca2+ ions into mitochondria. The uniporter operates at high, micromolar cytosolic Ca2+ concentrations that are only reached transiently in cells, near Ca2+ release channels. Mitochondria can also take up Ca2+ at low, nanomolar concentrations, but this high affinity mode of Ca2+ uptake is not well characterized. Recently, leucine-zipper-EF hand-containing transmembrane region (Letm1) was proposed to be an electrogenic 1:1 mitochondrial Ca2+/H+ antiporter that drives the uptake of Ca2+ into mitochondria at nanomolar cytosolic Ca2+ concentrations. In this article, we will review the properties of the Ca2+ import systems of mitochondria and discuss how Ca2+ uptake via an electrogenic 1:1 Ca2+/H+ antiport challenges our current thinking of the mitochondrial Ca2+ uptake mechanism.

Dynamic Regulation of the Mitochondrial Proton Gradient during Cytosolic Calcium Elevations

Mitochondria extrude protons across their inner membrane to generate the mitochondrial membrane potential (ΔΨm) and pH gradient (ΔpHm) that both power ATP synthesis. Mitochondrial uptake and efflux of many ions and metabolites are driven exclusively by ΔpHm, whose in situ regulation is poorly characterized. Here, we report the first dynamic measurements of ΔpHm in living cells, using a mitochondrially targeted, pH-sensitive YFP (SypHer) combined with a cytosolic pH indicator (5-(and 6)-carboxy-SNARF-1). The resting matrix pH (∼7.6) and ΔpHm (∼0.45) of HeLa cells at 37 °C were lower than previously reported. Unexpectedly, mitochondrial pH and ΔpHm decreased during cytosolic Ca2+ elevations. The drop in matrix pH was due to cytosolic acid generated by plasma membrane Ca2+-ATPases and transmitted to mitochondria by Pi/H+ symport and K+/H+ exchange, whereas the decrease in ΔpHm reflected the low H+-buffering power of mitochondria (∼5 mm, pH 7.8) compared with the cytosol (∼20 mm, pH 7.4). Upon agonist washout and restoration of cytosolic Ca2+ and pH, mitochondria alkalinized and ΔpHm increased. In permeabilized cells, a decrease in bath pH from 7.4 to 7.2 rapidly decreased mitochondrial pH, whereas the addition of 10 μm Ca2+ caused a delayed and smaller alkalinization. These findings indicate that the mitochondrial matrix pH and ΔpHm are regulated by opposing Ca2+-dependent processes of stimulated mitochondrial respiration and cytosolic acidification.

Perspectives on: SGP Symposium on Mitochondrial Physiology and Medicine: The renaissance of mitochondrial pH.

The generation of a proton gradient across the inner mitochondrial membrane (IMM) is an essential energy conservation event that couples the oxidation of carbohydrates and fat to the synthesis of ATP. Studies in isolated mitochondria have established that the chemical gradient for protons (ΔpH m )

OPA1 promotes pH flashes that spread between contiguous mitochondria without matrix protein exchange

The chemical nature and functional significance of mitochondrial flashes associated with fluctuations in mitochondrial membrane potential is unclear. Using a ratiometric pH probe insensitive to superoxide, we show that flashes reflect matrix alkalinization transients of ∼0.4 pH units that persist in cells permeabilized in ion‐free solutions and can be evoked by imposed mitochondrial depolarization. Ablation of the pro‐fusion protein Optic atrophy 1 specifically abrogated pH flashes and reduced the propagation of matrix photoactivated GFP (paGFP). Ablation or invalidation of the pro‐fission Dynamin‐related protein 1 greatly enhanced flash propagation between contiguous mitochondria but marginally increased paGFP matrix diffusion, indicating that flashes propagate without matrix content exchange. The pH flashes were associated with synchronous depolarization and hyperpolarization events that promoted the membrane potential equilibration of juxtaposed mitochondria. We propose that flashes are energy conservation events triggered by the opening of a fusion pore between two contiguous mitochondria of different membrane potentials, propagating without matrix fusion to equilibrate the energetic state of connected mitochondria.

Securin and Separase Modulate Membrane Traffic by Affecting Endosomal Acidification

Securin and separase play a key role in sister chromatid separation during anaphase. However, a growing body of evidence suggests that in addition to regulating chromosome segregation, securin and separase display functions implicated in membrane traffic in Caenorhabditis elegans and Drosophila. Here we show that in mammalian cells both securin and separase associate with membranes and that depletion of either protein causes robust swelling of the trans‐Golgi network (TGN) along with the appearance of large endocytic vesicles in the perinuclear region. These changes are accompanied by diminished constitutive protein secretion as well as impaired receptor recycling and degradation. Unexpectedly, cells depleted of securin or separase display defective acidification of early endosomes and increased membrane recruitment of vacuolar (V‐) ATPase complexes, mimicking the effect of the specific V‐ATPase inhibitor Bafilomycin A1. Taken together, our findings identify a new functional role of securin and separase in the modulation of membrane traffic and protein secretion that implicates regulation of V‐ATPase assembly and function.

Modulation of the matrix redox signaling by mitochondrial Ca2

Mitochondria sense, shape and integrate signals, and thus function as central players in cellular signal transduction. Ca(2+) waves and redox reactions are two such intracellular signals modulated by mitochondria. Mitochondrial Ca(2+) transport is of utmost physio-pathological relevance with a strong impact on metabolism and cell fate. Despite its importance, the molecular nature of the proteins involved in mitochondrial Ca(2+) transport has been revealed only recently. Mitochondrial Ca(2+) promotes energy metabolism through the activation of matrix dehydrogenases and down-stream stimulation of the respiratory chain. These changes also alter the mitochondrial NAD(P)H/NAD(P)(+) ratio, but at the same time will increase reactive oxygen species (ROS) production. Reducing equivalents and ROS are having opposite effects on the mitochondrial redox state, which are hard to dissect. With the recent development of genetically encoded mitochondrial-targeted redox-sensitive sensors, real-time monitoring of matrix thiol redox dynamics has become possible. The discoveries of the molecular nature of mitochondrial transporters of Ca(2+) combined with the utilization of the novel redox sensors is shedding light on the complex relation between mitochondrial Ca(2+) and redox signals and their impact on cell function. In this review, we describe mitochondrial Ca(2+) handling, focusing on a number of newly identified proteins involved in mitochondrial Ca(2+) uptake and release. We further discuss our recent findings, revealing how mitochondrial Ca(2+) influences the matrix redox state. As a result, mitochondrial Ca(2+) is able to modulate the many mitochondrial redox-regulated processes linked to normal physiology and disease.

Advances in pancreatic islet monolayer culture on glass surfaces enable super-resolution microscopy and insights into beta cell ciliogenesis and proliferation

A robust and reproducible method for culturing monolayers of adherent and well-spread primary islet cells on glass coverslips is required for detailed imaging studies by super-resolution and live-cell microscopy. Guided by an observation that dispersed islet cells spread and adhere well on glass surfaces in neuronal co-culture and form a monolayer of connected cells, we demonstrate that in the absence of neurons, well-defined surface coatings combined with components of neuronal culture media collectively support robust attachment and growth of primary human or rat islet cells as monolayers on glass surfaces. The islet cell monolayer cultures on glass stably maintain distinct mono-hormonal insulin+, glucagon+, somatostatin+ and PP+ cells and glucose-responsive synchronized calcium signaling as well as expression of the transcription factors Pdx-1 and NKX-6.1 in beta cells. This technical advance enabled detailed observation of sub-cellular processes in primary human and rat beta cells by super-resolution microscopy. The protocol is envisaged to have broad applicability to sophisticated analyses of pancreatic islet cells that reveal new biological insights, as demonstrated by the identification of an in vitro protocol that markedly increases proliferation of primary beta cells and is associated with a reduction in ciliated, ostensibly proliferation-suppressed beta cells.