Cell Biology of the Mitochondrion

Abstract

Mitochondria are best known for harboring pathways involved in ATP synthesis through the tricarboxylic acid cycle and oxidative phosphorylation. Major advances in understanding these roles were made with Caenorhabditiselegans mutants affecting key components of the metabolic pathways. These mutants have not only helped elucidate some of the intricacies of metabolism pathways, but they have also served as jumping off points for pharmacology, toxicology, and aging studies. The field of mitochondria research has also undergone a renaissance, with the increased appreciation of the role of mitochondria in cell processes other than energy production. Here, we focus on discoveries that were made using C. elegans, with a few excursions into areas that were studied more thoroughly in other organisms, like mitochondrial protein import in yeast. Advances in mitochondrial biogenesis and membrane dynamics were made through the discoveries of novel functions in mitochondrial fission and fusion proteins. Some of these functions were only apparent through the use of diverse model systems, such as C. elegans Studies of stress responses, exemplified by mitophagy and the mitochondrial unfolded protein response, have also benefitted greatly from the use of model organisms. Recent developments include the discoveries in C. elegans of cell autonomous and nonautonomous pathways controlling the mitochondrial unfolded protein response, as well as mechanisms for degradation of paternal mitochondria after fertilization. The evolutionary conservation of many, if not all, of these pathways ensures that results obtained with C. elegans are equally applicable to studies of human mitochondria in health and disease.

Keywords: WormBook; biogenesis; electron transport chain; mitochondrial morphology; quality control; respiration, free radicals.

Figure 1

A schematic representation of components of oxidative phosphorylation within the inner mitochondrial membrane. Complex specific substrates like malate (complex I;CI) or succinate (complex II;CII) donate electrons that are ultimately accepted by oxygen to produce water. In the process, protons are pumped out of the mitochondrion to generate an electrochemical force that drives ATP synthesis. For clarity, each complex is shown as an individual entity. Ordinarily complexes I, III, and IV are part of a larger supercomplex that functions as a respirasome. There is little complex I found by itself on native gels from C. elegans mitochondria. (Figure courtesy of Ernst-Bernhard Kayser, PhD.)

Figure 2

(A, B) Representative tracings of complex I-dependent oxidative phosphorylation in a Clark electrode. Oxygen concentration is plotted on the y-axis, time on the x-axis. In N2, (A) initially, mitochondria are placed in a chamber and allowed to respire. (1) Addition of ADP (2) does not increase the slow rate of oxygen consumption until malate is added (3) as a substrate for complex I respiration. Over time, ADP is consumed, and oxygen depletion slows until ADP is added a second time (4). Addition of DNP (5) dissipates the proton gradient normally present across the inner mitochondrial membrane, and allows oxygen to be reduced without linkage to production of ATP. This allows any complex I transport electrons at maximum rates when provided with the proper substrate. In the absence of DNP, providing a substrate for complex IV, TMPD ascorbate (6), bypasses the rate limiting step of complex I in the ETC, and rapidly depletes all oxygen within the chamber. (B) In gas-1, a mutant which causes a defect in the 49 kDa subunit of complex I (Kayser et al. 1999, 2001), it can be seen that even the addition of large amounts of ADP (5) does not increase the very slow rate of oxygen consumption when mitochondria are supplied with a complex I substrate, nor does addition of DNP. Bypassing complex I respiration completely by adding TMPD ascorbate (7) rapidly depletes oxygen. In both (A) and (B) an ADP/O ratio is calculated by measuring the amount of oxygen consumed when mitochondria are supplied with a known amount of ADP. (C) ADP is assumed to be converted to ATP when the rate of oxygen consumption abruptly slows. This “knee” in the curve is sometimes referred to as the state 3 to state 4 transition. (Figure courtesy of Ernst-Bernhard Kayser, PhD.)

Figure 3

Representative tracing of respiration in a Seahorse Analyzer. Capable of measuring mitochondrial respiration in cells in culture, oxygen consumption can be measured in very small volumes, as in 96-well plates. OCR is measured over time, fueled often by glucose. However, whole nematode respiration, respiration of purified mitochondria, synaptosomes, etc., can also be assayed. Oligomycin is a specific inhibitor of the ATPase complex V, and prevents protons from crossing the membrane through this complex to phosphorylate ADP. Oligomycin is used in these experiments to completely inhibit respiration. FCCP is a mitochondrial uncoupler (similar to DNP discussed in the legend to Figure 2) which allows protons to leak freely across the mitochondrial membrane and uncouples oxygen consumption from phosphorylation of ADP. Antimycin A is a specific inhibitor of complex III, and rotenone is a specific inhibitor of complex I. They are used together to block all mitochondrial respiration even in the presence of an uncoupler such as FCCP. At the same time the Seahorse can measure acidification of the medium, providing an estimate of glycolysis. This measurement is termed ECAR. (Figure courtesy of Ernst-Bernhard Kayser, PhD.)

Figure 4

(A) A representation of assays of enzymatic steps within electron transfer. Electron donors and acceptors can be added to permeabilized mitochondria to isolate specific steps of the ETC. Their reduction is followed by a color change of an electron acceptor in a spectrophotometer. For example, the electron donor NADH is oxidized by complex I. All subsequent flow of electrons within the complex is blocked by rotenone. Ferricyanate is the electron acceptor for the first part of complex I (termed NFR activity and taken as a measure of the amount of complex I, rather than the activity of complex I); decylQ is the acceptor when electrons are allowed to pass through the entirety of complex I (a measure of complex I activity). However, electrons from NADH can, in the absence of rotenone, flow through Q and through complex III, to reduce the electron acceptor, oxidized cytochrome c (a measure of I–III activity). Further flow is blocked by potassium cyanide. Other individual steps are indicated by their respective electron donors and acceptors. (B) An example of a battery of ETC enzymatic assays used to investigate knockdown of two different subunits of complex IV. COX, flow of electrons through complex IV; CI, flow of electrons through complex I; NFR, NADH-ferricyanide reductase, the first step of NADH oxidation; I–III, flow of electrons through complex I and complex III; II–III, flow of electrons through complex II and complex III; CIII, flow of electrons through complex III. In this case, knockdown of complex IV subunits reduced enzymatic activity of complex IV, but also affected electron flow within complex I, reflecting the significance of allosteric interactions between components of respiratory chain supercomplexes. Values for N2 are in blue, knockdown of COX subunits are in red or yellow. [Figure originally published in Suthammarak et al. (2009)]. (Figure 4A courtesy of Ernst-Bernhard Kayser, PhD.)

Figure 5

Mitochondrial morphologies in different C. elegans cell types. (A) Mitochondria in larval body wall muscles detected with mitochondrial GFP under control of the myo-3 promoter. (B) The same, but in a young adult animal. (C) Mitochondria in a neuronal process detected with mitochondrial GFP under control of the mec-7 promoter. (D) Mitochondria in hypodermal cells detected with mitochondrial GFP under control of the col-12 promoter. (E) Mitochondria in intestinal cells detected with mitochondrial GFP under control of the ges-1 promoter. (F) Mitochondria in dissected gonad detected by staining with Mitotracker. (A–E) are conventional epifluorescence images. (F) is a confocal image.

Figure 6

Control of mitochondrial inner and outer membrane morphologies. (A) Cristae shapes are determined by the MICOS protein complex, which provide negative curvature at cristae junctions and dimers of ATP-synthase, which have pair at an angle providing positive curvature for tube shaped cristae. (B) Mitochondrial fusion is mediated by FZO-1 on the outer membranes of opposing mitochondria, and by the inner membrane protein EAT-3. Mitochondrial fission is mediated by DRP-1, which is largely cytosolic but can be recruited to mitochondria, where it forms a large spiral that constricts mitochondrial membranes for fission. Transport along microtubules is mediated by kinesins and dynein, which are coupled to mitochondria through interactions with MIRO-1, -2 or -3.

Figure 7

Overview of mitochondrial protein import pathways. Most proteins enter the intermembrane space through the Tom40 complex, followed by further targeting to their final destination. Proteins with an N-terminal targeting sequence (1) are sent to the mitochondrial matrix through the Tim23 complex, or if they have a single transmembrane segment they become anchored in the inner membrane (2). Multispanning proteins are also first translocated into the intermembrane space, but, once inside, they are sequestered by small TIM proteins and then inserted into the inner membrane via the Tim22 complex (3). Some remain in the intermembrane space, where they can be trapped by protein modifications, such as disulfide bonds or covalent heme attachment (4). Other proteins are returned to the outer membrane for folding into β-barrel proteins through the SAM pathway (5). Mitochondrial outer membrane proteins with α−helical transmembrane segments are inserted through other pathways outside of the Tom40 complex (6).

Figure 8

Outline of mitophagy pathways. (A) Mitochondria with low membrane potential, as might result from mutations in mitochondrial DNA or by damage from ROS, accumulate PINK-1 on their surface. PINK-1 then recruits the E3 ubiquitin ligase parkin (PDR-1) from the cytosol. Parkin ubiquitinates mitochondrial membrane proteins, such as Miro, and triggers autophagy through Ulk1 (C. elegans UNC-51). These events lead to the formation of an autophagosome encapsulating the damaged mitochondrion with membrane, followed by fusion with lysosomes. This specialized form of autophagy ensures that damaged mitochondria are quickly removed from the cell. (B) Paternal mitochondria are eliminated from zygotes by mitophagy. Soon after fertilization, the mitochondrial inner membrane becomes permeable, allowing entry of the intermembrane space endonuclease, CPS-6, into the matrix, where it degrades mtDNA. Further degradation of paternal mitochondria is mediated PINK-1 and PDR-1/parkin induced mitophagy.

Figure 9

Outline of pathways controlling the mitochondrial unfolded protein response in C. elegans. (A) Cell autonomous pathways controlling UPR^mt within a cell include signaling through the ATFS-1 transcription factor, which is normally targeted to mitochondria and degraded, but can get routed to the nucleus when mitochondrial protein import is blocked. The other pathway involves activation of GCN-2, which inhibits cytosolic protein synthesis. (B) Cell nonautonomous UPR^mt is triggered in intestinal cells by neurotransmitters (serotonin and FLP-2) that are secreted by stressed neurons.