TOWARD UNDERSTANDING THE CELLULAR CONTROL OF VERTEBRATE MINERALIZATION: THE POTENTIAL ROLE OF MITOCHONDRIA

Abstract

This review examines the possible role of mitochondria in maintaining calcium and phosphate ion homeostasis and participating in the mineralization of bone, cartilage and other vertebrate hard tissues. The paper builds on the known structural features of mitochondria and the documented observations in these tissues that the organelles contain calcium phosphate granules. Such deposits in mitochondria putatively form to buffer excessively high cytosolic calcium ion concentrations and prevent metabolic deficits and even cell death. While mitochondria protect cytosolic enzyme systems through this buffering capacity, the accumulation of calcium ions by mitochondria promotes the activity of enzymes of the tricarboxylic acid (TCA/Krebs) cycle, increases oxidative phosphorylation and ATP synthesis, and leads to changes in intramitochondrial pH. These pH alterations influence ion solubility and possibly the transitions and composition in the mineral phase structure of the granules. Based on these considerations, mitochondria are proposed to support the mineralization process by providing a mobile store of calcium and phosphate ions, in smaller cluster or larger granule form, while maintaining critical cellular activities. The rise in the mitochondrial calcium level also increases the generation of citrate and other TCA cycle intermediates that contribute to cell function and the development of extracellular mineral. This paper suggests that another key role of the mitochondrion, along with the effects just noted, is to supply phosphate ions, derived from the breakdown of ATP, to endolysosomes and autophagic vesicles originating in the endoplasmic reticulum and Golgi and at the plasma membrane. These many separate but interdependent mitochondrial functions emphasize the critical importance of this organelle in the cellular control of vertebrate mineralization.

Introduction

On May 31, 1678, Antoni van Leeuwenhoek, later acknowledged as the first microscopist and the father of microbiology, wrote to the plant botanist, Nehemia Grew, that he had observed “a fragment of bone which even through a good microscope will appear to be composed of globules.” [1] van Leeuwenhoek also commented that bone contained “pipes,” which today are assumed to represent the osteonal structures of lamellar bone. [1] Almost three centuries later, electron microscopy revealed globules termed matrix vesicles, suggested as forming initial sites for mineral deposition in the tissue. [2,3] Since the time of matrix vesicle discovery, an explosion of information has occurred concerning their biogenesis and function. However, questions still remain about the means by which matrix vesicles and other organelles in bone-forming cells (osteoblasts), as well as the cells of several additional mineralizing vertebrate tissues such as those in cartilage (chondrocytes), dentin (odontoblasts), and tendon (tenocytes), may sequester, transit and export calcium and phosphate ions to mediate extracellular mineral deposition.

In this context, numerous studies have confirmed that a general property of mitochondria in a wide variety of cells is to accumulate calcium and phosphate ions. [4–8] These ion stores can be observed microscopically as dense mineral granules in the mitochondrial matrix. [6,9–17] In terms of understanding a possible role of such granules in the mineralization process, most experimental investigations have focused on the importance of mitochondria in calcium ion homeostasis, [8,18–20] and few reports have considered the idea that the organelle may mediate the extracellular deposition of mineral. [4,21] Herein, this concept is addressed through a discussion of mechanisms governing calcium and phosphate ion accumulation by mitochondria, the relationship of these ions to mineral granule formation and mitochondrial function, the interaction of mitochondria with other cellular organelles, and finally the initiation of extracellular mineralization by mitochondria in vivo.

The earliest clues to the source of ions for the formation of mineral were provided by the pioneering work of Albert Lehninger, who established the importance of mitochondria as the major organelle regulating cell bioenergetics. [5–7] He observed that mitochondria functioned to accumulate high levels of the calcium and phosphate ions that comprised hydroxyapatite [7], and he wrote that “Mitochondria may also function as packaging plants in which are manufactured the bricks and mortar of hard tissues.” [5]

General structure and occurrence of mitochondria

Mitochondria are intracellular organelles appearing by electron microscopy as rounded, oval, cylindrical or tubular in a variety of cells. [22,23] The average diameter of tubular mitochondria is reported between 0.5 and 1.0 μm [24] with cross-sectional areas of different shaped organelles ranging up to 3 μm². [25] The number of mitochondria in a cell depends on tissue type. For example, muscle cells contain about 5000 mitochondria; human liver cells are estimated to have about 1000-2000 mitochondria per cell, comprising up to 1/5 of the cell volume, and a mature erythrocyte contains no mitochondria. [26–28] The number or volume of mitochondria in osteoblasts or chondrocytes is currently unknown, although microscopic images show that these organelles are morphologically similar to those in many other soft tissues. [29,30] In contrast to osteoblasts, osteoclasts, the cells that principally resorb bone, exhibit a far greater number of mitochondria. [30] The structure and certain additional characteristics of mitochondria distinguish them from other intracellular organelles in that they are bounded by two distinct enveloping membranes, each a phospholipid bilayer; they contain a unique DNA; and they divide by fission to reproduce their genome. [31–32]

As just noted, there are two mitochondrial membranes. They consist of discrete permeable outer and relatively impermeable inner components, separated by an intermembranous space [Figure 1]. The outer membrane is smooth while the inner membrane is invaginated, forming interconnected lamellar and tubular structures, the mitochondrial cristae, which demarcate an intramitochondrial matrix [Figure 1]. [31] The smooth outer membrane consists in part of protein porins (voltage-dependent anion channels; VDACs) that freely allow the passage of molecules up to 5 kD [Figure 2]. [33–35] Porins facilitate outer membrane entry of ions and metabolites whose concentration in the intermembranous space is similar to that of the cell cytosol. [33–35] Compared to the outer mitochondrial membrane, the highly folded cristae are far more selective in their passage of molecules to the intramitochondrial matrix, the site maintaining mitochondrial DNA and the Krebs/tricarboxylic acid (TCA) cycle as described below. The membranous infolded cristae are permeable to oxygen, carbon dioxide, and water and also permit controlled transport of ADP, ATP, pyruvate, and calcium and phosphate ions through specific protein systems. [31] These transporters include the adenine nucleotide translocase (ANT), which shuttles ADP and ATP into and out of the intramitochondrial matrix, respectively; a lithium-dependent sodium/calcium exchanger (NCLX), moving sodium ions into the intramitochondrial matrix and calcium ions from the matrix to the intermembranous space; a sodium/hydrogen exchanger (NHE), transporting sodium ions to the intermembranous space and hydrogen ions to the intramitochondrial matrix; and leucine zipper-EF-hand-containing transmembrane protein 1 (Letm 1), which, in exchange for calcium ions, also provides hydrogen ions to the intramitochondrial matrix. [36,37] A highly selective calcium channel protein complex, the mitochondrial calcium uniporter (MCU), transports calcium ions across the cristae into the intramitochondrial matrix, [38,39] and a proton/phosphate symporter (PiC) permits phosphate ion entry into the same space [Figure 2]. [40] The folded cristae additionally contain the five complexes (I-V) and associated factors (electron carriers) comprising the electron transport chain (ETC) [Figure 1]. The last of these complexes (V) is an enzyme system, F₁F₀ATP synthase, responsible for the generation of ATP as a fundamental energy source for the cell. [41] Other details of the MCU and synthase system will be described subsequently.

Figure 1.

Schematic representation of energy generation in mitochondria. The inset (top left) shows a cross section of a mitochondrion with a segment of the organelle enlarged to illustrate its characteristic membrane structure as well as components of the Krebs/tricarboxylic acid (TCA) cycle and electron transport chain (ETC). Catalyzed by enzymes of the Krebs cycle, mitochondria generate NADH and FADH₂, which interact with specific molecules of the ETC. The ETC is comprised of the multienzyme complexes (complexes I-IV) together with cytochrome C (Cyto C) and ubiquinone (Q). Complex I (NADH-ubiquinone oxidoreductase) catalyzes the transfer of electrons (e-) from NADH to ubiquinone. Following acceptance of a pair of high energy electrons from NADH, Complex I undergoes a redox-related conformational change that promotes the pumping of 4 protons (H⁺) across the inner mitochondrial membrane (IMM) and its infoldings (cristae). Complex II (succinate-ubiquinone oxidoreductase) receives reducing equivalents (FADH₂) from succinate dehydrogenase. Although this conformational change generates less energy than that of Complex I, it is sufficient to reduce ubiquinone, which can then interact with Complex III. Complex III (ubiquinone-cytochrome C reductase) allows the stepwise flow of two electrons from Complex I and II and shuttles other electrons to Cyto C, a protein located partly in the mitochondrial intermembranous space. Complex IV (cytochrome oxidase) is the final electron acceptor molecule in the ETC. Complex IV has two functions. First, it transfers electrons to oxygen, which react with protons to form water. Second, undergoing a conformational change, Complex IV causes the movement of two protons from the mitochondrial matrix to the intermembranous space. Pumping of these and the other protons from Complexes I and III across the inner membrane of the mitochondrion creates a proton motive force (ΔΨm, 180 – 200 mV; dashed arrows) that drives F₁F₀ATP synthase to phosphorylate ADP to form ATP. Additional protons resulting from ETC reactions involving Complexes I, III and IV are utilized with oxygen to change Cyto C from a reduced state (Cyto C_r) to an oxidized state (Cyto C_o) with the generation of water. Citrate, an important molecule in mineralization and other cellular processes, is a product of the Krebs cycle. ADP: adenosine diphosphate; ATP: adenosine triphosphate; P_i: inorganic phosphate; GDP: guanosine diphosphate; GTP: guanosine triphosphate; NAD⁺/NADH: reduced and oxidized forms of nicotinamide adenine dinucleotide, respectively; FAD/FADH₂: reduced and oxidized forms of flavin adenine dinucleotide, respectively; OMM: outer mitochondrial membrane.

Figure 2.

Schematic of calcium and phosphate ion uptake and granule formation in mitochondria of cells of vertebrate mineralized tissues. A segment of a mitochondrion, a neighboring region of endoplasmic reticulum (ER), and aspects of the intracellular and intramitochondrial matrices are depicted in the diagram. Following depletion of calcium ions from the ER, STIM1 and associated proteins at the plasma membrane (not shown) promote the entry of extracellular calcium ions (Ca²⁺, red dots) into the cytosol (intracellular matrix) as well as the saccules, tubules and lamellae of the ER. Calcium ions enter mitochondria from the intracellular matrix and endoplasmic reticulum-mitochondria attachment complexes (mitochondria associated membranes, MAM). Calcium ion transfer from the ER to mitochondria is mediated by activation of inositol 1,4,5-trisphosphate (IP3R) and ryanodine receptors (RyR) in the ER membrane and voltage-dependent anion channel (VDAC) activity in the outer membrane of the mitochondria (OMM). The inner mitochondrial membrane (IMM) contains a mitochondrial calcium uniporter (MCU), which transfers calcium ions into the mitochondrial matrix. GRP75 enhances the stability of the MAM complex and increases the efficiency of ion transfer. Phosphate ion (Pi, blue dots) entry into mitochondria is mediated by a solute carrier protein, SLC253A3, and a mitochondrial proton/phosphate symporter (PiC). Other calcium ion mitochondrial transporters include voltage-operated calcium channels (VOC), receptor-operated calcium channels (ROC), and members of the transient receptor potential family of channels (TRP). Within the mitochondrial matrix, calcium and phosphate ions may cluster and form calcium phosphate mitochondrial granules (red/blue). A rise in intramitochondrial levels of calcium ions increases TCA cycle enzyme activity, whose initial metabolic reaction is conversion of pyruvate into acetyl CoA [Figure 1]. The subsequent synthesis of citrate both promotes TCA cycle activity and provides a source of this anion for apatite crystallite interactions [Figure 1]. The increase in mitochondrial metabolic activity influences oxidative phosphorylation and ATP synthase function [Figure 1]. With the upregulation in oxidative phosphorylation activity and the release of protons, there is an increase in the mitochondrial pH, enhancing granule formation.

Responding to the energy and metabolic needs of the cell, mitochondria exhibit changes that result in their fusion and fission, as briefly mentioned above. Both fission and mitochondrial biogenesis are stress responses regulated by the peroxisome proliferator-activated receptor-γ (PPARγ) family of proteins interacting with the mitochondrial transcription factor A (TFAM). [42] TFAM, together with other transcription factors different from PPARγ, modulates the activity of the mitochondrial genome by controlling the replacement of mitochondria throughout the life cycle of the cell. [43] Thus, as mitochondria and their cargo are removed through mitophagy (See below), they are rapidly replaced, thereby maintaining the energetic and mineralization-related needs of the cell.

Mitochondrial function: Calcium ion uptake and transport

Ion transport into and out of mitochondria is part of a more generalized homeostatic system, one purpose of which is to maintain the cytosolic calcium ion concentration at a very low (nanomolar) level. In cells of both hard and soft tissues, a 10,000-fold difference exists between the high calcium ion levels in the extracellular fluid and the very low ion content of the cytosol. Although the plasma membrane of a cell exerts a powerful barrier function to ion traffic, rapid changes in cytoplasmic calcium ion concentration are often observed and occur as sudden spikes, bursts, or oscillations that return to baseline values in time periods varying from seconds to minutes. [44,45] Associated with these cytosolic changes, mitochondrial free calcium ion values are estimated to reach almost millimolar levels. [46,47]

The mechanism by which mitochondria respond to the calcium ion variations just noted has recently been elucidated. The porins comprising the outer mitochondrial membrane admit many small molecules, including calcium ions, into the intermembranous space of the organelle. [33–35] In turn, the highly selective calcium channel, the MCU, located in the cristae, transports calcium ions across this membrane into the intramitochondrial matrix, as also noted earlier [Figure 2]. [38,39] This channel consists of the oligomeric protein (MCU) and other transmembrane proteins, including the MCU regulatory subunit b (MCUb) and the essential MCU regulatory element (EMRE) together with multiple regulatory EF-hand proteins (MCU1-3). [38,39,48] Specificity of the complex is mediated by the ratio of the MCU to each of the regulatory units. [49] Possibly, it is this specificity that reflects differences in the calcium ion accumulating properties of the mitochondria of a particular tissue and certainly it would not be surprising to find that the MCU activity of bone or dentin is greater than that of non-mineralizing tissues. [50]

The ability of the MCU to bind calcium ions and transport them into the intramitochondrial matrix is dependent on two factors: a negative electrochemical gradient generated as protons (H⁺) extruded through the ETC oxidative phosphorylation complexes I-IV (with the mitochondrial membrane potential, ΔΨm, being 150–180 mV) and a tightly coupled pH gradient (ΔpH). [38,49,51] MCU calcium ion uptake is not linear, despite the high ΔΨm of the inner mitochondrial membrane. The MCU is activated when the cytosolic calcium ion concentration rises above 400 nM and is possibly initiated at sites of contact between the mitochondria and the endoplasmic reticulum [38,49], as discussed in greater detail below.

Calcium ions moved into the intramitochondrial matrix affect the oxidative activity of the organelle. An increase of calcium ions promotes the production of NADH through the TCA cycle and NADH subsequently interacts with complex I of the ETC to generate additional protons. [52] Protons now liberated from the respiratory substrates flow through the inner membrane-bound F₁F₀ ATP synthase enzyme system to generate ATP as a major source of cell energy [Figure 1]. [41,53] The utilization of the released protons for ATP synthesis results in an increase in the internal pH of the mitochondrion. [54] The possible effect of changes in mitochondrial pH on mineral formation is noted later in this review.

Mitochondria as a mineral source for cells and extracellular matrices: The organelle as a repository for calcium phosphate granules

Compared to understanding the role of mitochondria as an energy source for cells, the concept that these organelles may serve as a mineral source for cells and extracellular matrices is infrequently considered and less well detailed. To this point, as mentioned previously in this paper, several investigations have reported the presence of dense granules in the matrix of mitochondria from various tissues [Figure 2]. [6,9–17] Electron microscopy has established such granules as generally round or oblate-shaped electron opaque bodies, ranging in diameter from a few hundred Ångstroms to a micron or more. [17] In the early literature on this subject, such granules were described in the hepatopancreas of the blue crab, Callinectes sapidus, by Becker, Chen and others in the Lehninger laboratory [6,11] as well as in the mitochondria of osteoblasts and osteocytes, [10,12,13,15] chondrocytes, [9,10] and the cells of additional mineralizing and non-mineralizing vertebrate tissues. [17]

Although the studies of granules were numerous and yielded similar ultrastructural results in different tissues, many of the investigations were seriously questioned as to whether the microscopic methods of sample preparation were optimized to preserve the tissues, mineral and granules faithfully. [12,13,55] However, subsequent work with improved techniques also demonstrated high calcium and phosphate ion content and discrete granules in mitochondrial matrices of vertebrate tissues. [15–17] In this context, Mahamid et al. reported that frozen thin sections of developing parietal bone contained intracellular vesicles sequestering calcium phosphate globules 80 nm in diameter comprised of smaller 10 nm electron dense globules. [56,57] Electron diffraction and energy dispersive x-ray spectroscopic analyses of the granules respectively produced a diffuse amorphous pattern with a low calcium:phosphorus ratio. [56,57] In embryonic chicken bone, Kerschnitzki et al. found membrane-bound particle-containing vesicles, similar to those reported by Mahamid et al., [56,57] as well as “mineral particles in mitochondrial networks.” [58] Likewise, Boonrungsiman and colleagues used high-pressure freezing and freeze substitution with high angle-annular dark-field scanning electron microscopy and electron energy-loss spectroscopy to confirm that granules containing calcium and phosphorus were present within mitochondria of cultured mouse osteoblast bone and marrow stromal cells. [15] Calcium signals were also observed in granule-free regions of the mitochondrial matrix and in vesicles closely associated with these mitochondria. [15] The observation that the calcium distribution varied from cell to cell and from mitochondrion to mitochondrion suggested that mitochondria are functionally heterogeneous in a single cell or tissue. [59]

A mechanism of granule formation in the mitochondrial matrix is not clearly understood. One possible explanation alluded to earlier is that the solubility product of the calcium and phosphate ions taken up by mitochondria is exceeded and mineral is precipitated as a result. In this context, the pH of the intramitochondrial matrix critically influences the solubility product of the mineral. Llopis et al. report that the resting pH in mitochondria is 8-8.5 [60] while Poburko et al. note a value of 7.9. [61] Mitochondrial pH would be expected to vary with calcium ion uptake, ΔΨm and the hydrogen (H⁺) concentration. Thus, when ΔΨm is high, the steady state calcium ion concentration regulates mitochondrial ATP generation through dehydrogenase activity and oxidative phosphorylation. [62] The flow of protons through the enzyme system, F₁F₀ ATP synthase, favors alkalization. While there is some uncertainty concerning the extent of alkalinization, [61] under conditions of increasing ionic strength and pH, the least soluble calcium phosphate mineral species (hydroxyapatite) would be formed. As the concentration of calcium ions in the mitochondrion increases, it interferes with organelle function: the activity of the TCA cycle activity is inhibited, resulting in a fall in ΔΨm and decreased ATP synthase activity. In this circumstance, the H⁺ concentration in the mitochondrial matrix would drop from high to neutral or to a slightly acidic pH. [61,62] This change in pH favors formation of mineral phases which are intermediate in the transitions between an amorphous mineral and crystalline hydroxyapatite. Higher pH would therefore promote hydroxyapatite formation and lower pH would enhance the possible presence of mineral phases such as brushite or octacalcium phosphate that may comprise mitochondrial granules.

There are other aspects of the intramitochondrial environment to be considered regarding the formation and mineral composition of mitochondrial granules. Shifts in ion species or their charge would influence the formation of the phase present in the intramitochondrial environment at a given time. Accompanying the presence of free calcium (Ca²⁺) and quadrivalent phosphate (PO₄³⁻) ions in this milieu are the common species, H₂PO₄⁻ and HPO₄²⁻, together with a possible early forming mineral phase, amorphous calcium phosphate, whose composition is proposed as Ca₉(PO₄)₆. [55,63,64] The latter is suggested to consist of aggregates of roughly spherical close-packed (Posner) clusters having only short-range order of its composite calcium and phosphate ions. [63–65] Indeed, the order is so short that these ions produce only diffuse (amorphous) rings rather than sharp reflections on electron or X-ray diffraction, a result thereby identifying the phase as an amorphous calcium phosphate. [64] In point of fact, electron diffraction of mitochondrial granules in osteoblasts and chondrocytes from tissue prepared under conditions that minimize alterations in the mineral yields amorphous calcium phosphate patterns. [12,13] Depending on the influx of calcium ions into the cell and their subsequent effects on the mitochondrial environment, as noted above, changes may occur in amorphous calcium phosphate, H₂PO₄⁻ and HPO₄²⁻, or other mineral species to reduce free H+ and raise the intramitochondrial pH. These events would then become important factors driving transitions of amorphous calcium phosphate or intermediate mineral phases more stable at higher pH toward the formation of hydroxyapatite as the final mineral phase appearing in mitochondria in the form of discrete granules. [66,67] In summary, it is plausible to consider that shifts in intramitochondrial pH directly influence the mineral species present in mitochondria and would explain why granule phases comprised of amorphous calcium phosphate, brushite, octacalcium phosphate and/or hydroxyapatite are reported in respiring mitochondria. [66–68] Whatever the composition of the granules, they consist of calcium and phosphate ions and are a potential source of these ions for other mitochondrial, cellular or extracellular processes, including matrix mineralization.

As an additional matter related to this topic of mitochondrial granules, it is important to recognize that these structures are also present in the mitochondria of cells of non-mineralizing tissues. For example, Wolf et al. reported that mitochondria of cells of several different non-mineralizing tissues contained calcium phosphate granules. [16] The granules were present as non-crystalline deposits and their sizes in these examples varied from 20-100 nm in diameter. [16] Based on this finding, it is likely that mitochondria have a general homeostatic function serving to prevent swings in calcium ion concentrations which would damage cytosolic enzyme systems and induce other adverse effects on cells. In cells from mineralizing vertebrate tissues, granules formed from the ionic fluxes and mineral phase transitions described above would have a second fate, in that they could be utilized as a source of calcium and phosphate ions for the mineralization of the extracellular matrix. This concept will be elaborated in the following aspects of this paper.

Pathways to granule formation: Mechanisms of calcium and phosphate ion accumulation by mitochondria

A characteristic of the mitochondria in cells of mineralizing vertebrate tissues is the presence of calcium phosphate granules, as noted previously. Since the formation of such granules relies on the transport of ions into the mitochondrion, it is relevant to consider how this process is accomplished and regulated [Figure 2]. A priori, it is important to state that the calcium ion concentration of the cytosol is very low (nanomolar) [44] and reflects the powerful barrier function of the plasma membrane of the cell. Thus, a concentration gradient exists between the high (millimolar) calcium ion levels in the extracellular fluid [47] and the very low calcium ion content of the cytosol. Contained within the membrane barrier are numerous and different channels which permit the controlled and very carefully regulated entry of calcium ions into the cell. These ion influx pathways, some of which were mentioned briefly above, include voltage-operated calcium channels, receptor-operated calcium channels, members of the transient receptor potential family of channels, and channels linking the plasma membrane with the endoplasmic reticulum. [69] Opening of these various channels and the concomitant rise in cytoplasmic calcium ion levels activate mitochondria, which take up calcium ions to restore or maintain normal calcium homeostasis. [69] As described earlier, the response to rapid spikes or changes in cytoplasmic calcium ion concentration is principally mediated by the MCU embedded in the mitochondrial cristae. [38,49] This uniporter, directed by the negative electrochemical gradient of the oxidative phosphorylation pathway and coupled pH gradient also noted above, admits the calcium ions into the intramitochondrial matrix, where they are now isolated from the cytoplasm and sequestered and may undergo the interactions putatively leading to calcium phosphate granule formation [Figure 2]. [38,39,49,50]

Besides respiratory substrates, mitochondrial function is influenced by oxygen tension and the availability of calcium and phosphate ions in the organelle. [70] An adequate oxygen supply serves to maintain the electrochemical proton and pH gradients across the mitochondrial inner membrane, thereby promoting calcium uptake into the mitochondrion. If the oxygen supply is limited, a condition (hypoxia) evident in many mineralized tissues, the cytochrome system is compromised, an effect leading to a decrease in ΔΨm, a reduced rate of ATP synthesis, and an accumulation of protons. [71] In this case, as noted previously, the mitochondrial pH shifts from high to neutral. [62] The change in pH may be consequential to calcium phosphate granule formation and composition, as also mentioned above.

In addition to sequestering calcium and phosphate ions and mediating granule development, mitochondria may significantly influence events associated with the extracellular mineralization of vertebrate hard tissues. Increases in intramitochondrial calcium ion levels enhance the activities of a number of TCA cycle dehydrogenase enzymes which in turn promote oxidative phosphorylation and the synthesis of ATP [Figure 2]. [72] Stimulation of the TCA cycle generates metabolic intermediates of the cycle and products of glycolysis that are known to affect the mineralization process in bone. Of these products, pyruvate, citrate, glutamate, and α-ketoglutarate levels are elevated during bone fracture healing, and there is a significant increase in the level of succinate synthesized during the healing process. [73] The rise in the levels of these molecules is linked to an increase in the activity of isocitrate dehydrogenase 1, citrate synthase and one of the rate-limiting enzymes of glycolysis, phosphofructokinase. [73]

With respect to citrate, this TCA cycle intermediate is formed in mitochondria by the condensation of oxaloacetate and acetyl-CoA, a reaction catalyzed by citrate synthase. Bone tissue is a major reservoir of citrate, containing 80% of the total body content. [74] Functions attributed to citrate include stabilization of the nanocrystals of hydroxyapatite and regulation of mineralization. [74,75] Szeri et al. reported that the membrane receptor, ANKH, mediated citrate export and, in its absence, bone exhibited reduced mechanical strength. [76] Dirckx et al. showed that osteoblasts expressed high levels of the citrate transporter solute carrier family 13 member 5 (SLC13A5) gene. [77] If this gene is deleted in mice, enamel and bone development is impaired. [77,78] In the latter case, citrate uptake by osteoblasts is slowed in the animals and leads to decreased mineralization of cell nodules as well as a loss in cortical bone thickness with compromised mechanical strength. [77] A clinical problem linked to a mutation of SLC13A5 is that primary teeth of children display enamel and dentin lesions. [77] This discussion highlights the fact that mitochondria are important mediators of extracellular mineral deposition in vertebrate tissues. Other examples of this role for mitochondria will be detailed below.

As recounted earlier in the context of calcium ions and mitochondria, calcium ions transported into the mitochondrial matrix stimulate dehydrogenase activity and the production of NADH and FADH₂ during the TCA cycle. This result ultimately drives the synthesis of ATP through the ETC [Figure 1]. In this process, protons (H⁺) translocate across the cristae to the intermembranous space and a rise in pH occurs in the mitochondrial matrix. As intramitochondrial calcium ion content increases, oxidative phosphorylation is depressed, an event likely enhancing interactions between intramitochondrial calcium and phosphate ions and the subsequent formation of ion clusters and mitochondrial granules. [68] Generation of calcium phosphate granules would probably compromise mitochondrial function and possibly lead to a change described as a “mitochondrial permeability transition.” [79,80] In this state, there are dramatic shifts in mitochondrial activity and structure along with membrane rupture and loss of mitochondrial contents. [80–82] The damaged mitochondria would then become enveloped in a membrane vesicle and degraded by mitophagy, a form of autophagy [Figure 3]. [17,83] An additional factor driving a mitophagic response is that an excess of calcium ions would block the TCA cycle and lower ΔΨm by inhibiting complex I of the cytochrome system. [82] The resulting decrease in ATP and the subsequent increase in the AMP/ATP ratio in the mitochondria are recognized by AMP kinase, the cell energy sensor, and these changes would activate mitophagy. [84] The importance of mitophagy in mediating a mineralization response is considered by Pei et al. [17] and discussed in more detail later in this review.

Figure 3.

Schematic of possible pathways in which mitochondria may be involved in the efflux of calcium and phosphate to the extracellular matrix for subsequent mineralization events. A segment of a mitochondrion, a portion of a cell and its plasma membrane (PM), and aspects of the mitochondrial, intracellular and extracellular matrices are shown in the diagram. Mitochondrial granules in the intramitochondrial matrix may liberate calcium (Ca²⁺) and phosphate (Pi) ions with changes in the mitochondrial pH. Ca²⁺ ions exit mitochondria through NCLX/VDAC channels embedded in the inner (IMM) and outer (OMM) mitochondrial membrane, respectively. Pi exits mitochondria by unknown means (dashed line across the IMM and OMM). Intact mitochondrial granules may interact with the IMM and OMM and disrupt the membranes, an event leading to mitophagy. This process initially involves the formation of mitophagic vesicles enveloping the damaged mitochondria, their Ca²⁺ and Pi ions and any other compromised cellular components. Vesicle contents may be enzymatically digested and recycled in the cell or exported to the extracellular matrix. Ca²⁺ and Pi ions passed through the mitochondrial membrane to the intracellular matrix may follow different pathways that include uptake by endosomes, autophagosomes (APS), and lysosomes. Other Ca²⁺ and Pi ions may be released to the extracellular matrix by passage respectively through NCLX or XPR1 channels located in the PM of the cell. Endosomes and APS may form amphisomes carrying the Ca²⁺ and Pi ions as well as their small aggregates (Ca-P). Amphisomes traverse the PM and may make their way into the extracellular as mineralizing exosomes or mineralizing vesicles (matrix vesicles). Lysosomes and APS form autophagolysosomes (APLS) enclosing Ca-P and these organelles may pass the PM through lysosomal exocytosis to enter the extracellular matrix. Pi in the extracellular matrix appears in the form of HPO₄ ²⁻ and H₂PO₄ ⁻.

Unlike calcium ion transport, far less is known of phosphate ion movement intracellularly and specifically into mitochondria. Phosphate ions are utilized for the synthesis of nucleoside triphosphates and thus regulation of phosphate ion transport is critically important: the presence of this negatively charged anion influences the conformational state of many membrane receptors, downstream signaling pathways, and the targeting of proteins involved with a myriad of events governing cell function. [85] Of the relatively limited information available with respect to phosphate ion entry into the cells of vertebrate mineralized tissues, three plasma membrane exchanger/transporter proteins, members of the solute carrier (SLC) family of molecules, have been identified in dental tissues (SLC20A1, SLC20A2 and SLC34A2) [86] and five in osteoblasts (SLC20A1, SLC20A2, SLC34A1, SLC34A2 and SLC34A3). [87] Phosphate ion efflux from the cells appears to be regulated by the plasma membrane transporter protein, XPR1, together with exosomes/matrix vesicles which independently traffic phosphate ions to extracellular domains [Figure 3]. [88–90] In a recent study, Lopez-Sanchez and colleagues reported that SLC20A2 and XPR1 regulated intracellular phosphate homeostasis where both of them together were required to maintain constant levels of intracellular phosphate and ATP levels. [90] Loss of either of these transporters resulted in primary familial brain calcification, a disease characterized by aberrant mineral deposition in the cerebrum. [90] Other possible phosphate ion carriers include the uncoupling proteins encoded by SLC25A14 and SLC25A30. [91–93] The solute carrier gene SLC25A3 is reported to encode a protein that promotes phosphate entry into mitochondria [Figure 2]. [94,95] This protein is electroneutral in that it can serve as a symporter to transport phosphate ions with protons or as an antiporter to exchange phosphate for hydroxyl ions across the mitochondrial cristae. [94,95]

It is interesting in the context of intracellular phosphate regulation that multilamellar organelles have been reported recently to modulate cytosolic inorganic phosphate in enterocytes populating the midgut of the fruit fly, Drosophila melanogaster. [96] In this instance, the inorganic phosphate transporter, PXo, was localized to these organelles (PXo bodies), which themselves were found to be critically important in enterocyte phosphate maintenance and control. [96] How the discovery of such PXo bodies in Drosophila relates to the cells of vertebrate hard tissues is not clear, but the finding provides an impetus for uncovering new information about possible phosphate signaling and trafficking in the process of mineralization in higher order species.

Certainly, a critical and central question in mineralization research concerns the source of phosphate ions for apatite formation. While it is likely that the source is dependent on the activities of the phosphate transporters discussed earlier, there remains the possibility that it is derived indirectly from the cleavage and breakdown of ATP. Almost all the ATP synthesized in the mitochondrion is exported into the cytosol or trafficked into the endoplasmic reticulum by means of the mitochondrial associated membrane (MAM) complex (details of this complex are discussed below). [97–99] Yong and coworkers reported that this interaction was sensitive to the calcium status of the cell and that a negative relationship existed between the cytosolic calcium ion concentration and ATP import into the lumen of the endoplasmic reticulum. [97] These investigators also showed that an increase in the cytosolic calcium ion concentration inhibited ATP uptake by the endoplasmic reticulum. [97] Thus, mitochondria provide ATP to the endoplasmic reticulum only when the cytosolic calcium ion store is low and refilling of the endoplasmic reticulum is necessary. [97]

It is important to recognize that the ATP requirements of the endoplasmic reticulum are very high since this organelle is the site of synthesis of both housekeeping and exported proteins. [99] Cleavage of ester phosphate groups from ATP and release of inorganic phosphate ions would be expected to cause an accumulation of the anion within the membranes of the endoplasmic reticulum. Based on this observation, it is suggested here that, besides maintaining calcium homeostasis, mitochondria provide the endoplasmic reticulum with a source of inorganic phosphate derived from the metabolism and hydrolysis of the high energy intermediate, ATP. Furthermore, it is proposed that this product of ATP metabolism may be utilized for mineral formation.

While the cleavage of ATP may possibly provide a source of phosphate ions for mineralization, its levels may be insufficient for biosynthetic functions in tissues that are rapidly synthesizing proteins for extracellular matrices. In this regard, the limited vascularization and the corresponding decrease in oxygen tension present in forming hard tissues would be unable to support mitochondrial respiration. As a result, mitochondrial ATP generation would be depressed and inadequate to meet cellular demands. Recently reviewing this problem, Flood et al. commented that other cellular processes must come into play when ATP requirements are high. [71] In these instances, mechanisms that may provide and channel ATP to sites of high demand include HIF-1-dependent upregulation of mitochondrial movement regulator (HUMMR), which influences the anterograde transport of mitochondria. [100] This movement would deliver mitochondrial ATP directly to organelles like the endoplasmic reticulum, and complications associated with passive loss of ATP by diffusion would thereby be obviated.

Other factors that could facilitate intracellular ATP delivery include survivin-dependent mitochondrial trafficking to the cortical cytoskeleton [101] and the formation of mobile glycolytic “metabolons” and liquid-liquid phase condensates of glycolytic substrates and enzymes. [102,103] Such delivery vehicles could compensate for intrinsic problems related to hypoxic inhibition of oxidative metabolism. Finally, changes at the level of the cytoskeleton could enhance the transport capabilities of mitochondrial and glycolytic sources of ATP to subcellular components. [104] It would not be surprising to learn that one or all of these possible sources of ATP are activated during synthesis of the extracellular matrix of hard tissues although whether these same sources serve to provide phosphate ions for the mineralization process is currently unknown.

As an additional comment concerning calcium ion transit in mitochondria, it should be noted that phosphoenolpyruvate, an intermediate of the glycolytic pathway which provides reducing power for gluconeogenesis, is tightly linked to mitochondrial function. [105] Besides the fact that this high energy compound promoted adenine nucleotide efflux from mitochondria, [105] Chudapongse reported that phosphoenolpyruvate also stimulated calcium ion efflux from the organelle. [106] This finding was further developed by Shapiro and Lee, who noted that phosphoenolpyruvate inhibited calcium ion uptake in the absence of added ATP and released more than 50% of the calcium ions from chondrocyte mitochondria, an effect that was maximal in the presence of an NADH-linked substrate. [107] Further analysis of phosphoenolpyruvate in the cartilaginous growth plate showed that its highest levels were present in the most hypoxic zones of this tissue. [107] The combination of inhibition of mitochondrial NADH-linked dehydrogenase activity and enhanced activity of the glycolytic pathway within a hypoxic environment would be expected to drive calcium ion release from mitochondrial calcium stores. [107]

To summarize the discussion above, while the precise sources of calcium and phosphate ions imported into mitochondria have yet to be determined, the effects of these ions on mitochondrial function are profound. Thus, as levels of organelle calcium ions are increased, there is a shift in intramitochondrial pH, increased production of citrate and other TCA cycle intermediates, and generation of a mitophagic response from damaged mitochondria. Moreover, interactions between calcium and phosphate ions in the intramitochondrial matrix would likely foster ion clustering and granule formation. Each of these events impacts maintenance of calcium ions and energy homeostasis, inter-organelle transport of calcium and other ions, and subsequently the mineralization of the extracellular matrix.

Cytosolic calcium ions and the linkage between endoplasmic reticulum calcium efflux and mitochondrial calcium influx

The initial studies described by Lehninger and co-workers were performed on isolated mitochondria and, while the information gathered from these investigations was robust, the findings could not be related to other cellular processes. Technological advances now permit contemporary research work to be carried out on intact cells in situ. The uses of calcium-sensitive fluorescent proteins targeted in situ to intracellular organelles, cryomicroscopy combined with electron tomography to visualize the metabolic state of a cell with minimal ion movement and re-distribution, and other techniques have provided new understanding of calcium ion homeostasis, mitochondrial function, and relationships between mitochondria and other components and molecules that crowd the cytoplasm of a cell. [16,44,108,109] In this respect, such methods have also identified the endoplasmic reticulum as a major repository of calcium ions in the cell and a principal source of these ions for intracellular signaling and additional activities. [110–112] The uptake of calcium ions by the endoplasmic reticulum occurs when the intracellular calcium ion load falls, an event which initiates a store-operated calcium entry (SOCE) response. [113] The low levels of calcium ions are sensed by an endoplasmic reticulum membrane protein, stromal interaction molecule (STIM), which undergoes a conformational change leading to coupling with the plasma membrane calcium channel protein, ORAI. [114,115] As a result of STIM-ORAI interaction, an ion-selective channel is formed which allows refilling of the cytosol with calcium ions from the extracellular tissue fluid. [114]

A sarcoendoplasmic reticulum calcium ATPase (SERCA) pumps calcium ions from the cytosol into the endoplasmic reticulum; another P-type ATPase, secretory pathway calcium ATPase (SPCA1), transports calcium ions into the Golgi apparatus. [116,117] Storage in these organelles is transient and dependent on the status of the calcium ion level in the cytosol. Receptor proteins (inositol 1,4,5-trisphosphate, IP3R, and ryanodine receptors, RyR) in the endoplasmic reticulum membrane bind ligands that then release the sequestered calcium ions into the cytosol or promote their transport into mitochondria, lysosomes, autophagosomes and endosomes. [118,119] The resulting oscillations and spikes in the concentration of cytosolic calcium ions mediate a spectrum of downstream cellular responses including changes in oxidative metabolism, gene expression, and cell proliferation. [120] The free calcium ions liberated from the endoplasmic reticulum into the cytosol do not diffuse extensively as they interact with intracellular calcium binding proteins to form microdomains known as calcium puffs. [109,118,121] It has been suggested by Raturi and Simmen that the actual position of mitochondria in the cell relates to the location of the microdomains of calcium ions. [121] In other words, the endoplasmic reticulum and calcium binding proteins of the cytosol dictate positional data to mitochondria.

There is a very close anatomical and physiological relationship between the endoplasmic reticulum and mitochondria which provides an active and preferential avenue for passage of lipids and calcium ions between these two organelles [Figure 2]. [98] These cellular components are tethered together by membrane proteins to form attachment complexes (mitochondria associated membranes, MAMs), 10–30 nm in width [Figure 2]. [122–124] Among several important molecules identified in the complex, the MAM contains mitofusin-2, a dynamin-related GTPase, and Grp75 (75kDa glucose-regulated protein), which promotes the stability of the complex and increases the efficiency of lipid and ion transfer [Figure 2]. [124–126] Calcium ions are pumped into the mitochondrion as a result of the interaction between the endoplasmic reticulum proteins and the mitochondrial outer membrane VDAC (voltage-dependent anion channel) and inner membrane calcium uniporter [Figure 2]. [48,127,128] This activity leads to mitochondrial calcium loads that reach values 5- to 10-fold higher than those in the cytosol. [109,127–129] As a related counterpart to the mammalian MAM, yeast maintains a heterotrimeric tethering complex, the endoplasmic reticulum-mitochondrial encounter structure (ERMES), for lipid transfer. [130–132] Membranes of the ERMES contain the endoplasmic reticulum protein, Mmm1p, and the mitochondrial distribution and morphology proteins, Mdm10p and Mdm34p, to facilitate lipid movement between the organelles. [131–133] The ERMES, then, like the MAM, is an example of a physical coupling and membrane connection that provides a pathway for the transport of lipids between organelles and an important molecular pore for the passage of calcium ions between the endoplasmic reticulum and the outer mitochondrial membrane VDAC and cristae MCU [Figure 2]. [121,123,132,134] The linkage in the case of the MAM involves endoplasmic reticulum calcium efflux and mitochondrial calcium influx as noted above and in the following paragraphs.

With respect to calcium ion interorganelle transport, Tang et al. examined the development of mouse parietal bone cells and found calcium and phosphorus ion clusters present on endoplasmic reticulum membranes. [135] Subsequently, such electron dense clusters were observed on or close to mitochondrial membranes. [135] This finding supported the notion that calcium ions were transferred directly from the endoplasmic reticulum to the mitochondrion. [135] Additionally, the immediate involvement of the endoplasmic reticulum in the mineralization process is implied by the observation that a mutation in STIM1 is associated with defective enamel mineralization. [136] Further, knockout of ORAI1 causes loss of bone trabecula and trabecular thinning with a decreased ratio of bone volume to tissue volume. [137,138]

These numerous studies suggest it is plausible to consider that the endoplasmic reticulum and mitochondria together play a critical role in cellular calcium homeostasis. Mitochondria buffer changes in cytosolic calcium ion levels and function through molecular complexes to maintain levels of calcium ions in the endoplasmic reticulum. Although the route and trafficking of phosphate ions are not as well understood compared to the movement of calcium ions in the cell cytoplasm, phosphate ion homeostasis and entry into mitochondria are regulated by a mitochondrial proton/phosphate symporter (PiC) [Figure 2]. [139] Moreover, it is plausible that the mitochondria also provide the endoplasmic reticulum with phosphate ions. The mitochondrial reservoir of stored ions, particularly that of calcium, directly influences energy metabolism and alters the pH of the mitochondrial matrix. [60,61] Both of these metabolic and pH changes may favor ion clustering and calcium phosphate granule formation. Details of granule structure and the subsequent transport of clustered ions or granules originating in mitochondria as a possible means of mediating mineral deposition in the extracellular matrix are explored further below.

Mitochondrial granules and extracellular mineralization

There are a number of ways in which the granules of calcium phosphate sequestered in mitochondria can be utilized by the cell. The first is simply that granules are maintained and stored in the organelle, having no further significance other than to protect the cell from shifts in cytosolic calcium ion levels. Second, the granules promote mitochondrial autophagy. Packaged as cargo in autophagic vesicles, the granules can be trafficked to the plasma membrane and exocytosed into the extracellular matrix. In a collagenous or amelogenin-rich environment, the calcium phosphate granules could then enhance mineral growth and development. Third, as discussed earlier, in a low pH (4.0-6.5) environment, the mitochondrial granules could dissociate into ions or ion clusters. Such free ions or small ion clusters, enclosed like intact granules as cargo in the multitude of vesicles residing in the cytoplasm or associated with mitochondria, would transit the cell to reach the extracellular milieu following vesicle exocytosis. These concepts warrant further discussion.

Regarding the relationship between mitochondria and cytoplasmic vesicles and their sequestered mineral ions or granules, it is possible that the inorganic cargo becomes crowded in the volume-limited environment of these organelles. Increasing mineral ion content and concentration would favor ion clustering and granule formation and serve as a driving force for nucleation of amorphous calcium phosphate or other early mineral phases which are precursors to apatite. Transit of the vesicles and release of their preformed mineral phases into the external milieu of the cell could promote and enhance matrix mineralization, as just noted.

There is growing experimental support for the concept that intact mitochondrial granules may find their way into the extracellular matrix of mineralized vertebrate tissues through the cellular process of autophagy or mitophagy, in the specific case of mitochondria. Most commonly, autophagy serves to remove misfolded or damaged proteins, dysfunctional cellular organelles and spent membranes from the cell by enclosing them in cytoplasmic vesicles which then fuse with lysosomes. [140] The hydrolytic enzymes secreted by lysosomes and their acidic (pH 4.5-5.0) environment provided by cytosolic proton-pumping V-type ATPase digest vesicle cargo, which can then be recycled and used as a source of energy for the cell. [140–142] Mitophagy is not a unique or solitary event but part of a process common to all cells and tissues. As a survival mechanism, the autophagic system permits cells to adapt to metabolic stress and thereby maintain cellular and tissue homeostasis. [140] Indeed, the actual mitophagic process stimulates mitochondrial fission and with it the genesis of replacement mitochondria, [143] which can continue the events of ion accumulation, granule formation and transport of mineral to the external environment of the cell.

It is conceivable that the presence of granules in mitochondria suggests that the organelle is metabolically degenerating or dysfunctional and signals its impending senescence. Perhaps the granules also exert stress within or upon the cristae or matrix of the mitochondrion, damaging or disrupting membranes and their many pumps and ion channels. Such an impaired mitochondrion would be a likely candidate for mitophagic activity. In this case, following an initial envelopment in a phagophore, the compromised mitochondrion with granules would fuse with lysosomes or endolysosomes to form an autophagolysosome or amphisome, respectively. The low pH of these organelles could solubilize the mitochondrion and its mineral particles and liberate the residual calcium and phosphate ions into the extracellular milieu as described above. Alternatively, the cargo of intact granules might be delivered by exocytosis from the cell to its surrounding extracellular matrix [Figure 3]. [140]

Autophagy appears to play a role in mediating mitochondrial ion cluster and particle transport. In osteoblasts, Nollet et al. showed that autophagic vacuoles, positive for autophagic marker proteins, autophagy related 7 (ATG7) and beclin 1 (BECN1), contained mineral crystals. [144] Confocal time-lapse microscopy indicated that these autophagosomes moved to the plasma membrane and fused with the cell membrane to provide a potential mechanism by which their contents could exit the cell. [144] Additionally, inhibition of autophagy resulted in a decrease in the mineralization capacity of the osteoblasts being examined. [144] Tang and colleagues reported that the mitochondria containing calcium phosphate granules in cultured bone cells underwent mitophagy and the mineral deposits were identified in autolysosomes. [135] These organelles are involved in pathways leading to the movement and export of cargo to extracellular matrices, and nanosized biomineral precursors (~30 nm in diameter), originating from mitochondrial granules, were found to initiate intrafibrillar mineralization of collagen. [135]

It should be noted that mitophagy represents only one possible fate of mitochondrial granules. Another possibility includes the release of granule-related mitochondrial calcium and phosphate ions into vesicles that could prime mineralization activities in the extracellular matrix. Granule solubilization would result from an increase in TCA cycle oxidative activity and a corresponding decrease in intramitochondrial pH. While the mechanism of release of phosphate ions or small clusters of calcium phosphate from solubilized mineral granules is currently unknown, calcium ions would be liberated from mitochondria by a sodium/calcium exchanger (three sodium ions for one calcium ion) [145] and a mitochondrial hydrogen/calcium exchanger (two hydrogen ions for one calcium ion) [146] working together with VDAC proteins embedded in the outer mitochondrial membrane [Figure 3]. [147] The calcium ions could be released into vesicles or specialized regions of the plasma membrane [148] and then channeled to the extracellular matrix. [57,88,141,149]

Mitochondria are not entirely free-moving structures in the cell cytoplasm. They are, as noted above, in direct contact with membranes of the endoplasmic reticulum through (MAMs), and these two interconnected organelles thereby allow the passage of calcium ions between them. [98,121] Whether MAMs also transit phosphate ions, calcium phosphate ion clusters, granules or selected molecules remains to be studied. The MAMs pathway would help maintain endoplasmic reticulum levels of calcium ions, which can then be used to load endolysosomal vesicles destined for transport to the plasma membrane. Wong and colleagues recently reported that dynamic attachments were also formed between mitochondria and lysosomes (similar to MAMs). [150] These investigators found that contact formation was promoted by GTP-bound lysosomal RAB7 and that mitochondria directly contacting lysosomes did not undergo mitophagy. [150] Thus, the possibility exists that mitochondria could discharge their cargo into lysosomes without loss of function and the induction of mitophagy.

An additional consideration regarding mitochondria and mineralization is that ions could be released to vesicles derived from the mitochondria, themselves. Mitochondrial-derived vesicles (MDVs), between 70 and 150 nm in diameter, have been reported, comprised of outer/inner mitochondrial membranes and certain mitochondrial matrix molecules. [151,152] MDVs are targeting to late endosome/multivesicular bodies and lysosomes. [151,152] Those MDVs destined for lysosomes require the protein kinase, PINK1, and the cytosolic ubiquitin E3 ligase, Parkin. [153] The uptake of calcium ions into MDVs, lysosomal, endosomal or autophagic vacuoles would likely promote ion clustering and mineral formation as noted earlier and thus provide a direct mechanism for transporting and then exocytosing these inorganic components to the mineralization front of bone, dentin, cartilage or other hard tissues [Figure 3]. [57,88,141,149] Conjecture aside, the current research literature as yet offers no definitive answers to whether ions or granules of calcium phosphate are discharged from calcium-loaded mitochondria and whether mitophagy is a contributor to vertebrate mineralization. Additionally, information concerning phosphate efflux from mitochondria and transport into vesicles remains incomplete.

Although much is to be learned about the transport of cargo among various intracellular organelles and the plasma membrane and beyond, there is little doubt that mitochondria are themselves dynamic structures that could influence the transit of mineral ions and granules. [154] That mitochondria are dynamic is underscored by observations that they vary in their location in a cell, being close to both calcium ion microdomains (puffs) and specific proteins of the endoplasmic reticulum and plasma membrane. [109,121,155,156] Moreover, there is clear evidence of movement of the organelle during both mitophagy and the normal function of mitochondria. [154,157,158] In this regard, it is interesting to discuss the intracellular transit of mitochondria and their possible relation to extracellular mineral deposition.

Changes in the location of mitochondria are mediated principally by two proteins of the cytoskeleton, microtubules and actin. [158,159] Microtubules are heterodimers of two proteins, α- and β-tubulin, which form long linear polymers ranging in length from <1 μm to >100 μm and having a diameter of about 25 nm. [159] Microtubules create tracks or pathways along which kinesins and dynein–dynactin complexes move mitochondria, vesicles and other subcellular structures. [158] Actin filaments have a diameter of about 7 nm, consist of polar linear molecules that are polymerized by the protein, inverted formin 2 (INF2), and serve to enhance the movement of calcium ions from the endoplasmic reticulum to mitochondria, a transport process dependent on MCU activity. [160] More relevant to the mineralization process, the release of matrix vesicles from hard tissue cells is also dependent on filamentous actin and its reorganization. [161] Actin has another principal function in that it participates in the regulation of the activity of key enzymes of the glycolytic pathway, aldolase and glyceraldhyde-3-phosphate dehydrogenase, which provide metabolic substrates for the TCA cycle. [162] Moreover, actin activates and stabilizes phosphofructokinase-1 and additionally protects this enzyme from downstream inhibitory compounds like citrate and lactate. [163] In these ways, cytoskeletal proteins not only govern movement of mitochondria with respect to their intracellular location, particularly in relation to the endoplasmic reticulum, but also influence mitochondrial energy generation, synthesis of TCA intermediates and calcium ion accumulation. If elements of the cytoskeleton are disturbed, there is a direct influence on the deposition of mineral in the extracellular matrix. [164] These considerations lead to the conclusion that, while mitochondria store calcium and phosphate ions in the form of granules, they are more than just passive elements in a highly dynamic process. Tethered to other organelles, including the endoplasmic reticulum, lysosomes and the actin cytoskeleton, mitochondria are part of a complex regulatory system that governs both intracellular calcium ion accumulation and granule formation and mediates cargo transport to sites of extracellular mineralization.

Summary

It is important to stress that mitochondria are part of an intricate subcellular membrane system that functions in an intimate manner with the endoplasmic reticulum and the autophagic system to facilitate calcium ion homeostasis in the cell. The question may be asked as to why organelles like the mitochondria and the endoplasmic reticulum are involved in the mineralization process when SOCE activity at the plasma membrane alone produces an increase in cytoplasmic calcium ions. [113] The answer likely lies in the fact that organellar structures like mitochondria and the endoplasmic reticulum mutually raise concentrations of calcium ions to achieve two principal results. One is to provide essential protection to the cytoplasm from excessively high and damaging calcium ion concentrations as the ions are removed from intracellular domains and sequestered within these organelles. The other, as proposed here and elsewhere, [141] is to bind and effectively neutralize the calcium ions with counterpart phosphate ions to form ion clusters that lower the free energy barrier to the formation of stable nuclei of a calcium phosphate mineral species. While the manner of sequestration of phosphate ions in mitochondria is not understood, this change in free energy in the confines of the organelle is conceptually one of the most important events in the mineralization process. Once mineral nuclei are formed and transported to the extracellular matrix, the high calcium and phosphate ion levels in the extracellular tissue fluid ensures their subsequent growth and development.

There are sound reasons for considering that mitochondria play a critical role in promoting events that result in mineral formation. First and foremost, mitochondria are in intimate contact with the endoplasmic reticulum and receive calcium ions through MAMs. Cytosolic calcium ions are imported into the organelle by MCU activity. Within the mitochondrial matrix, the imported calcium ions enhance electron transport and oxidative phosphorylation, activities that initially promote ATP synthesis; high levels of calcium ions inhibit these processes. These calcium-dependent effects on ATP synthesis influence the intramitochondrial matrix pH, and it is suggested that the pH changes in turn influence mitochondrial granule formation, composition and dissolution. The physical nature of these deposits also modifies the mechanism of their liberation from the mitochondria and transport ultimately to the extracellular milieu. This paper together with a recent review of mineralization by Yan et al. [165] proposes that one or more conduits exist to release mitochondrial stores of calcium and phosphate ions, small ion clusters or larger, intact mineral granules. This paper and Yan et al. [165] also suggest that the mitochondrial supply of calcium and phosphate ions, ion clusters or granules is taken up by cytoplasmic vesicles originating principally in the endoplasmic reticulum and Golgi apparatus of the cell. Those vesicles traffic their calcium and phosphate cargo to the plasma membrane where it is destined for extracellular deposition. That modes of transport may differ are not by themselves surprising since profound differences are well documented in patterns of tissue mineralization among vertebrate hard tissues. For example, while (matrix) vesicles appear to be required for the formation of endochondral bone, a different process involving collagen molecules, fibrils and fibers appears to be necessary for the mineralization of lamellar bone. In either case, it might be expected that mitochondrial activity supports the mineralization process by exporting stored ions or granules to extracellular matrices while maintaining critical cellular activities. A more complete understanding of the factors that regulate and coordinate these putative mitochondrial functions and interdependent cellular and extracellular activities represents a new perspective toward elucidating the mechanisms of vertebrate mineralization.

In his presentation to the British Biochemical Society in 1970, Albert Lehninger commented that, “We now see mitochondria as something more than phosphorylating power plants, as we first viewed them 20 years ago. What is remarkable is that the same molecular apparatus of the electron transport chain that generates the driving force for oxidative phosphorylation may also be used to transport ions against gradients, to change the conformation of mitochondria and to carry out reductive biosynthesis.… and harbour the secret as to why we do not all turn into stone.” [5] Since his talk, extraordinary progress has been made in understanding the biology of the mineralization process in skeletal and dental cells. However, still urgently required is information concerning factors that mediate and coordinate the mitochondrial functions and interdependent cellular and extracellular transit activities Lehninger noted. When available, that new knowledge would provide a more complete perspective toward elucidating the role of mitochondria in regulating cell function in relation to shifts in intracellular calcium and the mineralization of vertebrate hard tissues.

Highlights.

1. Mitochondria play an important role in intracellular calcium ion homeostasis.

2. Mitochondria of vertebrate mineralized tissues contain calcium phosphate granules comprised of amorphous calcium phosphate or other possible calcium phosphate mineral phases.

3. ATP generated by mitochondrial oxidative phosphorylation may provide phosphate ions for mineral formation.

4. Mitochondrial granule presence may be linked to the extracellular matrix mineralization of vertebrate hard tissues.

5. Intracellular vesicles originating from mitochondria and the endoplasmic reticulum convey calcium and phosphate ions or mineral particles to the extracellular matrix.