How does the extensive folding of the inner mitochondrial membrane benefit a eukaryotic cell?

Abstract

Mitochondria, cellular organelles of respiration and adenosine triphosphate (ATP) production, are found in almost all eukaryotic cells. Eukaryotes that live under anaerobic conditions do not have conventional mitochondria but instead contain structurally and functionally reduced mitochondria (mitochondrion-related organelles). The mitochondrion has a primary role in energy metabolism, a role that is intimately connected with its double-membrane structure (outer and inner, each comprising a lipid bilayer). Formation of mitochondria (mitochondrial biogenesis) is under the dual control of the nuclear and mitochondrial genetic systems. The presence of functional DNA in the mitochondrion reflects its evolutionary descent from an endosymbiotic bacterial ancestor.

Abstract

Mitochondria are essential organelles involved in energy production, cell signaling, and cell fate. They represent a node for numerous signaling pathways involved both in the processes of cell survival and cell death. Thus, aged and/or dysfunctional mitochondria need to be selectively removed to protect the cells from excessive oxidative stress and cell death. Moreover, the biogenesis of new mitochondria should be compensated by the elimination of the oldest ones. Thus a mitochondrial quality control system is needed for achieving such a task. The selective degradation of mitochondria by autophagy (mitophagy) is a process whereby damaged mitochondria are sequestered into double-membrane vesicles called autophagosomes and transported to lysosomes for degradation. In physiological conditions mitophagy contributes to the maturation of different cellular subsets like red blood cells, the cardiomyocytes adaptation to hypoxia as well as to the selective transmission of the maternal mitochondrial genome (mitochondrial inheritance). Alteration of mitophagy is also associated to pathological situations such as Parkinson’s and Alzheimer’s diseases. Indeed, mutations in the key constituents of the mitophagy’s core machinery have been associated with these neurodegenerative diseases. Hence, understanding the mechanisms of mitophagy regulation and signaling not only would bring a new vision for the comprehension of the physiological adaptation in multicellular organisms but also might help in the development of effective clinical strategies against neurodegenerative diseases. This chapter aims to underline the current advances in the study of mitophagy in physiological and pathological situations.

Structure

The mitochondrion has two bounding membranes, outer and inner, which are structurally and functionally distinct. One major difference is their permeability properties: the outer membrane permits free passage of most molecules of molecular weight less than about 10 000 daltons, whereas the inner membrane forms an effective barrier to even small molecules and ions. The inner and outer membranes define two submitochondrial soluble compartments, the intermembrane space and the matrix (the latter enclosed by the inner membrane). The inner membrane is highly invaginated, folded into cristae that greatly increase the membrane's surface area.

As isolated or as viewed in electron micrograph thin sections, mitochondria often appear round or oblong in shape. However, in a living cell, mitochondria may actually comprise a dynamic interconnected network, or syncytium, pieces of which are constantly breaking off and re-fusing.

Background on Mitochondria

Mitochondria play a pivotal role in cell homeostasis, housing multiple metabolic pathways (including the tricarboxylic acid-, β-oxidation- and urea cycle) and the oxidative phosphorylation machinery that generates energy in the form of ATP. Research during the last decade has extended the prevailing view of mitochondrial function well beyond their critical role in supplying energy. Mitochondria synthesize heme and steroid hormones; are important for intracellular Ca2+ metabolism and signaling; generate the majority of cellular reactive oxygen species (ROS); and serve as the cell's gatekeeper for apoptosis. They contain multiple copies of circular mitochondrial DNA (mtDNA) and function under dual genetic control. Both nuclear and mtDNA encode genes that modulate mitochondrial protein synthesis and import, enzymatic activities, biogenesis, and apoptosis.

Most of the ATP necessary to supply energy to tissues and organs is generated by the mitochondrial oxidative phosphorylation system (OXPHOS). Under aerobic conditions, electrons derived from the oxidation of glucose or fatty acids are transferred through the respiratory chain complexes I–IV. At each of the enzymatic complexes I, III, and IV, protons are pumped out of the mitochondrial matrix into the intermembrane space, generating an electrochemical gradient used by the fifth enzymatic complex (ATP synthase) to drive ATP synthesis.

Mitochondria consume more than 90% of the cell's oxygen and are therefore the main site of ROS production. ROS, including superoxide, hydrogen peroxide, and hydroxyl radicals, are generated as a by-product of mitochondrial activity, primarily at complexes I, II, and III of the respiratory chain. Normally, 2–5% of O2 used in the mitochondria is metabolized into superoxide. The superoxide radicals are converted to hydrogen peroxide by superoxide dismutase (SOD), which can further form the highly reactive hydroxyl radical. Mitochondrial (Mn-SOD and glutathione peroxidase) and cytosolic (Cu-SOD and catalase) antioxidant enzymes help to scavenge the ROS and limit their toxicity.

ROS play a significant role in the regulation of cell-signaling processes and in cytoprotection; thus their controlled generation is necessary for cell survival. On the other hand, when produced in excess of cellular antioxidant reserves, lipid peroxidation, mtDNA damage, OXPHOS dysfunction, and damage to Fe–S-containing enzymes ensue. Oxidative stress to nuclear or mtDNA results in strand breaks and base modifications, followed by cellular dysfunction, mutagenesis, and carcinogenesis.

Mitochondria are involved in cell death by at least three general mechanisms: the release of proteins that trigger the activation of the caspase family of proteases, the disruption of oxidative phosphorylation and ATP production, and the modification of the cellular reduction–oxidation (redox) potential. Each of those mechanisms can result in programmed cell death (apoptosis) or cell necrosis. The initiation of apoptosis occurs via the opening of the permeability transition pore (mtPTP) located in the inner mitochondrial membrane. The mtPTP is composed of the adenine nucleotide translocator (ANT), voltage-dependent anion channel (VDAC), Bax, Bcl2, cyclophilin D, and benzodiazepine receptor. The opening of the mtPTP leads to swelling of the mitochondrial inner membrane and the rupture of the outer membrane, followed by the release of cytochrome c, procaspases 2,3,4, and caspase-activated deoxyribonuclease (CAD) into the cytoplasm. Cytochrome c activates apoptotic protease activating factor (Apaf-)1, which first activates procaspase 2 and 9, and subsequently caspases 3, 6, and 7, inducing apoptosis. Another caspase-activating protein released by mitochondria is the apoptosis-inducing factor (AIF). AIF and endonuclease G are transported to the nucleus and initiate chromatin condensation and degradation. Depending on the amount of cytochrome c and caspase inhibitors available, the cell undergoes either necrosis or apoptosis. The Bcl-2 family proteins exhibit pro- and/or anti-apoptotic properties (Figure 1).

Given their critical role in cell physiology, it is obvious that mitochondria are among the first responders to various stressful stimuli challenging cell homeostasis. They are responsible for meeting the enormous energy demands of the fight-or-flight response in vital tissues. Among other equally important effects, they control the fever response by the modulation of thermogenesis, balance the host immune response during infection by deciding on the fate of the affected cells, and adjust the oxidative stress response for cytoprotective and signaling purposes.

1.1 Definition

Mitochondria from even a single region of the brain are highly heterogeneous based on their morphological, histochemical, and enzymatic characteristics. Most methods are designed to isolate three distinct populations of mitochondria from rat brain: (a) non-synaptic mitochondria, the so-called “free mitochondria” (FM); (b) synaptosomal mitochondria (synaptic), which can be further subdivided into two fractions based on sedimentation properties, heavy (HM) and light (LM) (Reijnierse, Veldstra, & Van den Berg, 1975; Van den Berg, 1973). Synaptosomal mitochondria are involved in regulating neurotransmitter release and synaptic vesicle formation. In contrast, non-synaptic mitochondria derive from multiple cell types and from neuronal soma and are involved in microRNA regulation and energy production (Ly & Verstreken, 2006; Vos, Lauwers, & Verstreken, 2010; Wang, Sullivan, & Springer, 2017).

Abstract

Mitochondria and endoplasmic reticulum (ER) are fundamental in the control of cell physiology regulating several signal transduction pathways. They continuously communicate exchanging messages in their contact sites called MAMs (mitochondria-associated membranes). MAMs are specific microdomains acting as a platform for the sorting of vital and dangerous signals.

In recent years increasing evidence reported that multiple scaffold proteins and regulatory factors localize to this subcellular fraction suggesting MAMs as hotspot signaling domains.

In this review we describe the current knowledge about MAMs' dynamics and processes, which provided new correlations between MAMs' dysfunctions and human diseases. In fact, MAMs machinery is strictly connected with several pathologies, like neurodegeneration, diabetes and mainly cancer. These pathological events are characterized by alterations in the normal communication between ER and mitochondria, leading to deep metabolic defects that contribute to the progression of the diseases.

Mitochondria Import Lactic Acid, Then Metabolize it; This Forms a Carrier System for NADH Oxidation

Mitochondria possess a monocarboxylic acid transporter (MCT1) that allows lactic acid to enter the mitochondria. Mitochondria also possess lactic dehydrogenase, LDH, which converts lactic acid to pyruvate. The pyruvate is then oxidized by the TCA cycle. The ability of lactic acid to enter the mitochondria and be converted back to pyruvate forms a pathway for the oxidation of cytoplasmic NADH, as shown in Figure 3.7.4. Lactic acid is not a shuttle system in that it is consumed by the mitochondria. Thus it is more like a carrier of reducing equivalents.

Abstract

Mitochondria are cellular organelles with roles in adenosine triphosphate production, cytosolic calcium buffering and reactive oxygen species production. A complex molecular motor system mediates the transport and positioning of mitochondria along axons. Recent studies have identified the transport and positioning of axonal mitochondria as crucial to multiple aspects of nervous system development including the developmental and regenerative extension of axons and the formation of axon collateral/interstitial branches, which underlie the formation of complex circuitry. Advances had been made possible by the development of novel experimental methods for targeting mitochondria and importantly for imaging mitochondria in living axons and in vivo. This chapter reviews the current understanding of the role of mitochondria in axon development.

2.2 Mitochondria

Mitochondria are the cellular sites of respiration, energy production and some aspects of calcium handling. Mitochondria are unique organelles as they contain a fraction of the genetic information required for their maintenance, and must also import many proteins that are encoded by nuclear DNA (Ernster and Schatz, 1981). Import of proteins into the mitochondria was thought to occur posttranslationally as proteins translated in vitro are capable of import into the mitochondria (Neupert and Herrmann, 2007). However, ribosomes are present on the surface of yeast mitochondria, suggesting that there may be co-translational import of some proteins into the mitochondria (Kellems et al., 1974, 1975). Genome-wide studies of purified yeast mitochondria have demonstrated that several hundred mRNAs copurify with mitochondria-bound ribosomes (Gadir et al., 2011; Saint-Georges et al., 2008). Interestingly, the majority of the mitochondria-localized mRNAs code for proteins that function in the mitochondria, consistent with a role for co-translational protein import. Localization of a subset of these mitochondria-localized mRNAs has revealed that translation of the N-terminal mitochondrial targeting sequence (MTS), as well as specific RNA elements both in the coding regions and 3′UTRs are important for mRNA targeting to mitochondria (Corral-Debrinski et al., 2000).

Globally, the majority of mitochondria-localized transcripts depend on translation to target to the mitochondria, but as is the case with the ER, there is a class of mRNAs that localize independently of translation (Saint-Georges et al., 2008). Two studies have shown the Puf3 RNA-binding protein is important for localization of a subset of transcripts to the mitochondria: Puf3 associates primarily with transcripts that function at the mitochondria (Gerber et al., 2004) and is required for the localization of these transcripts to the organelle (Gadir et al., 2011; Saint-Georges et al., 2008). Interestingly, deletion of mitochondria-targeting elements within the ATP2 mRNA resulted in defects in mitochondrial respiration and defects in protein import into the mitochondria, demonstrating that mRNA localization to mitochondria is critical for the proper function of the organelle (Margeot et al., 2002). In addition, a recent study of in Xenopus cultured neurons identified a nuclear protein, Lamin B2, as being translated in the axon. Surprisingly, the axonal pool of translated LB2 protein localized to mitochondria where it was required for proper mitochondrial function (Yoon et al., 2012). It will be interesting to determine how some messages localize independently of translation and what role those messages play in mitochondrial function.

3.2.5 The role of fusion and fission for mtDNA integrity

Mitochondria contain their own DNA (mtDNA), which is organized in hundreds of nucleoids per cell. Nucleoids are of varying size and each contains several mtDNA molecules.224–226 Within a mitochondrion, these nucleoids are almost static, most probably because they are bound to IMM proteins,225 they do not appear to exchange DNA among each other, but heterologous nucleoids can complement each other apparently by diffusion of mtDNA transcripts.227 Nucleoids have been observed to divide160,224,228 and move apart from each other with a speed comparable to separation of cristae.229 In many mitochondria, nucleoids show preferred distances to each other. This is in favor of the hypothesis that each group of mtDNA serves for a certain region by transcript diffusion surrounding its confined position. With age, mtDNA is subjected to deletions and to mutations, which in part can be eliminated by repair mechanisms (for review see Ref. 230), but in particular a 4977 bp deletion is a common event in aged cells.20,231 The continuous fusion and fission processes redistribute mtDNA throughout the chondriome. This dynamics has been considered an important factor in delaying senescence due to the delayed accumulation of mutant or truncated mtDNA.182,232 Tissue-specific dominant mtDNA mutations obviously arise from single mutation expansion and not from ongoing mutational events.15 The mathematical models describing different scenarios do not explain how a single mutation becomes dominant within a tissue. Fission of mitochondria may produce tubules lacking mtDNA,224 which in a following fusion process again will be connected to mtRNA supply. In fibroblasts lacking this way, most mitochondria become devoid of nucleoids.219 Fusion of cells with mitochondria lacking mtDNA with those bearing nucleoids results in the homogenous distribution of nucleoids throughout the chondriome after 12–24 h what has been interpreted as intramitochondrial mobility of nucleoids by the authors,226 but could just be a result of several fusion and fission cycles. In Drp1-deficient cells or cells undergoing terminal differentiation, nucleoids are enlarged and clustered in hyperfused mitochondria.224 The same happens if in fission-deficient mitochondria, mtDNA accumulates in bulb-like structures, and apoptotic events are delayed in these cells.233 ER supports fission close to sites of mtDNA within the matrix resulting in nucleoid separation157 (Fig. 4.3). Nucleoid separation seems to be a process preceding mitochondrial fission,160 this is the reason why Drp1 does not colocalize with mtDNA. As discussed earlier, fission can result in very heterogenous mitochondrial tubules (Fig. 4.5). In the case of a confined zone of mtDNA-controlled protein synthesis, deficient DNA could be responsible for the synthesis of dysfunctional proteins which then are removed by fission together with the DNA tethered to IMM proteins. This speculation fits to all the experimental observations but still needs to be proven.