F₁F_o-ATP synthase is a highly conserved enzyme that catalyzes the production of ATP from ADP and Pi. The structure of this multiprotein complex comprises two functional domains: a membrane-spanning subunit (F_o) and a soluble subunit (F₁) that generates ATP through the action of a rotational mechanism (Davies et al., 2012). ATP synthesis is powered by a proton transmembrane gradient in mitochondria, which is established by pumping protons to the IMS against their electrochemical gradient across the IMM by the electron transfer chain complexes. The protons passively pass from the IMS to the matrix through Fo, which transfers the stored energy created by the proton electrochemical gradient to F1, causing a conformational change in F1Fo-ATP synthase so that ADP can be phosphorylated to form ATP.

In 1995, Allen proposed a model suggesting that the association of ATP synthase dimers promotes a distortion of the inner mitochondrial membrane plane (Allen, 1995). Analysis of 2D membrane crystals discloses that a monomeric mitochondrial F₁Fo-ATP synthase is sufficient to bend a lipid bilayer in vitro. This local membrane curvature is likely to be a prerequisite for the formation of ATP synthase dimers and dimer rows, which are necessary for shaping mitochondrial cristae (Jiko et al., 2015). A later study found that deficiency in either subunit e or g of yeast ATP synthase caused abnormal onion-like or separated cristae corresponding to an uncontrolled remodeling of the inner mitochondrial membrane (Figure 3) (Paumard et al., 2002). This study demonstrated that subunits e and g of the yeast F₁F_o-ATP synthase, which are non-essential components of ATP synthase but required for the dimerization and oligomerization of F₁F_o-ATP synthase, are involved in generating mitochondrial membrane curvature. A mutation in the ɑ subunit of yeast ATP synthase causes remarkably aberrant cristae membrane structure while having less effect on ATP production (Kucharczyk et al., 2009). Electron cryo-tomography study with mammalian mitochondria further reveals that the F1Fo-ATP synthase is arranged in rows of dimeric supercomplexes, localized to regions with a pronounced positive curvature such as ridges and rims as well as tubular cristae (Strauss et al., 2008). The structure of the ATP synthase dimer from bovine heart mitochondria shows that the enzyme is overall cone-shaped (Minauro-Sanmiguel et al., 2005). The spontaneous curvature of the mitochondria ATP synthase dimers can introduce positive curvature into the IMM and stabilize the rims of cristae (Paumard et al., 2002; Strauss et al., 2008; Dudkina et al., 2010; Blum et al., 2019). Thereby, these dimer rows are important determinants of membrane bending in the formation of crista tips (Strauss et al., 2008; Davies et al., 2012; Blum et al., 2019; Muhleip et al., 2019).

MICOS is a conserved hetero-oligomeric membrane protein complex. All subunits of MICOS are named as Mic'X' where ‘X' represents the approximate molecular weight in kilodalton (kDa) as determined for the yeast homologs of the complex (Kozjak-Pavlovic, 2017; van der Laan et al., 2016; Pfanner et al., 2014; Stephan et al., 2020). The MICOS complex was originally discovered in budding yeast (Harner et al., 2011; Hoppins et al., 2011b; von der Malsburg et al., 2011), an organism in which MICOS has been the most researched. Downregulation of MICOS subunits results in drastic changes in the IMM morphology and leads to reduced crista junction numbers (Figure 3). Consequently, cristae membranes are detached from the IMM and appear as stacks within the mitochondrial matrix underlining the important role of MICOS in the formation of crista junctions (Alkhaja et al., 2012; Harner et al., 2011; Hoppins et al., 2011b; John et al., 2005; von der Malsburg et al., 2011). However, a molecular understanding of how membrane sculpting occurs at the crista junctions is just emerging.

In mammals, eight MICOS subunits have been identified so far (Kozjak-Pavlovic, 2017), namely Mic10 (MINOS1, yeast Mic10), Mic13 (QIL1, yeast Mic12), Mic14 (CHCHD10), Mic19 (CHCHD3, yeast Mic19), Mic25 (CHCHD6, yeast Mic19), Mic26 (Mic23 or APoO, yeast Mic26), Mic27 (APoOL, yeast Mic26), and Mic60 (Mitofilin, yeast Mic60) (John et al., 2005; Darshi et al., 2011; Alkhaja et al., 2012; An et al., 2012; Weber et al., 2013; Guarani et al., 2015; Koob et al., 2015; Genin et al., 2016; Kozjak-Pavlovic, 2017). These subunits form two distinct subcomplexes, Mic10/13/23/27 and Mic60/19/25, bridged together by Mic19 (Friedman et al., 2015; Guarani et al., 2015). However, the exact functions of these two MICOS subcomplexes, particularly how they bend the membrane to the degree observed at crista junctions, are yet to be defined. Recently, the two MICOS subcomplexes are suggested to have different functions in human cells. The Mic60 subcomplex, which is stable in the absence of the Mic10-subcomplex, is critical for the maintenance of crista junctions and the stability of the holo-MICOS complex. In contrast, the Mic10-subcomplex is essential for the formation of lamellar cristae (Stephan et al., 2020). Mic10 and Mic60 are the core components of the MICOS complex, which are highly conserved during evolution, required for the stability of the MICOS complex (Harner et al., 2011; Hoppins et al., 2011b; von der Malsburg et al., 2011). Mic10 can control the spatial distribution of Mic60 and the formation of Mic60 assemblies (Stephan et al., 2020). Depletion of Mic10 or Mic60 caused dramatically altered cristae ultrastructure. Importantly, Mic10 and Mic60 possess membrane-shaping activities (Barbot et al., 2015; Bohnert et al., 2015; Hessenberger et al., 2017; Tarasenko et al., 2017). Purified Mic10 (MINOS1, yeast Mic10) reconstituted into proteoliposomes induces high-degree membrane curvature in model membranes, generating tubules with a diameter between 15 and 25 nm (Barbot et al., 2015). The membrane-shaping activity of Mic10 relies on the formation of homo-oligomers via conserved glycine motifs in the transmembrane segments of Mic10 (Barbot et al., 2015; Bohnert et al., 2015) because loss of the conserved glycine motifs abolishes its membrane-shaping activity both in vivo and in vitro. While the oligomerization mutants fail to induce curvature in model membranes overexpression of Mic10 in budding yeast promotes the formation of Mic10 oligomers, which leads to a substantial extension and deformation of cristae membranes and crista junctions (Bohnert et al., 2015). A molecular mechanism underlying the process of the IMM sculpting by Mic10 has been proposed, by which Mic10 adopts a wedge-like structure in the lipid bilayer that can generate curved membranes at the crista junction (Barbot et al., 2015), a similar membrane-shaping model demonstrated for the BAR domain family proteins (Mim and Unger, 2012). Recently, Mic10 was found associated with the ATP synthase dimers, which facilitates the formation of ATP synthase oligomers, suggesting that it may link the two major membrane shaping machineries required for the formation of crista tips and crista junctions, respectively (Rampelt et al., 2017).

Mic60 (mitofilin, yeast Fcj1) is anchored in the inner membrane by a single transmembrane segment at the N terminus (Rabl et al., 2009), forming homo-oligomers. Mic60 associates with at least five other MICOS proteins, namely Mic27, Mic26, Mic19, Mic12, and Mic10. Depletion of Mic60, but not of Mic10, results in the absence of all MICOS subunits (Stephan et al., 2020). Furthermore, loss of Mic60 results in loss of crista junctions and the formation of onion ring-like cristae in Saccharomyces cerevisiae and mammalian cell lines (John et al., 2005; Li et al., 2016; von der Malsburg et al., 2011). Compared to WT Hela cells, the number of CJs was close to zero in Mic60-KO cells. In contrast, the occurrence of CJs is reduced by about 25% in Mic26-KO cells and by more than 70% in Mic10-, Mic13-, and Mic19-KO cells. Overexpression of Mic60 in yeast cells and Gram-negative bacterium E. coli caused additional branching of cristae membranes and formation of plasma membrane invaginations that are reminiscent of mitochondrial cristae, respectively (Rabl et al., 2009; Bohnert et al., 2015). Importantly, Mic60 displays a direct membrane-shaping activity in vitro. Purified yeast Mic60 reconstituted into proteoliposomes induces a high degree of membrane curvature, generating long and branched membrane tubules with diameters between 10 and 20 nm (Tarasenko et al., 2017). In addition, purified soluble forms of Mic60 from the thermophilic fungus Chaetomium thermophilum and Arabidopsis thaliana lacking the amino-terminal transmembrane domain cause extensive deformation of liposomes (Michaud et al., 2016; Hessenberger et al., 2017). Importantly, Mic60 plays a prominent role in connecting the inner and outer membranes of mitochondria. The major segment of Mic60 localizing in the intermembrane space associates with several outer membrane proteins involved in mitochondrial protein import and assembly, including the protein translocase of the outer membrane (TOM), the sorting and assembly machinery (SAM), porin (VDAC), metaxins 1 and 2, as well as SLC25A46 (yeast Ugo1) involved in mitochondrial dynamics (Bohnert et al., 2012; Darshi et al., 2011; Harner et al., 2011; Hoppins et al., 2011b; von der Malsburg et al., 2011; Ott et al., 2012; Xie et al., 2007; Zerbes et al., 2012). Moreover, the Mic60 subcomplex bridges the MICOS and SAM complexes in the IMM and OMM, respectively. Depletion of mammalian Sam50 impairs the cristae architecture and assembly of respiratory chain complexes, suggesting an intricate interplay between the structural and functional organization of both mitochondrial membrane systems mediated by the SAM and MICOS complexes (Ott et al., 2012). Mic60 also interacts with disrupted-in-schizophrenia 1 (DISC1) (Park et al., 2010), DNAJC11 (Guarani et al., 2015), TMEM11 (Guarani et al., 2015), and OPA1(Barrera et al., 2016). Recently, Mic60 is found to form clusters preferentially localized in the inner membrane at two opposing sides of the mitochondrial tubules with the twisted and helical arrangements (Bohnert et al., 2015). Interestingly, the Mic60 distribution bands are largely independent of the cristae morphology.

Mic13 (Qil1 or C19orf70), a small inner membrane protein with an amino-terminal transmembrane segment and a carboxy-terminal domain facing the intermembrane space, physically interacts with Mic60 and Mic10 (Guarani et al., 2015). Depletion of Mic13 caused onion-like cristae membranes and a complete loss of crista junctions suggesting that Mic13 is strictly required for the formation of crista junctions (Anand et al., 2016). Furthermore, Mic13 facilitates the efficient assemblies of other subunits including Mic10, Mic26, and Mic27 into the MICOS complex (Guarani et al., 2015). Depletion of Mic13 not only leads to reduced biogenesis of the Mic10 subcomplex but also the dissociation of two MICOS subcomplexes Mic60-Mic19-Mic25 and Mic10-Mic13-Mic26-Mic27. Thereby, Mic13 is also required to stabilize the full MICOS complex by holding two subcomplexes together (Anand et al., 2016).

Mic14 (CHCHD10 or C22orf16) encodes a mitochondrial intermembrane space protein that is enriched at crista junctions residing with Mic60/mitofilin, Mic19/CHCHD3, and Mic25/CHCHD6 (Bannwarth et al., 2014; Genin et al., 2016). Expression of Mic14/CHCHD10 mutant alleles leads to abnormal crista structures with loss of crista junctions (Bannwarth et al., 2014), triggered by partial disassembly of the Mic60/Mic19/Mic25/Mic14 complex upon Mic14 mutations. In addition, mutations of Mic14 cause reductions in nucleoid number and nucleoid disorganization (Genin et al., 2016).

Comparably little is known about the peripheral MICOS protein MIC19 (CHCHD3, yeast Mic19 or Aim13), having a coiled-coil-helix-coiled-coil-helix (CHCH) domain that is essential for the import of MIC19 into mitochondria (Darshi et al., 2011, 2012; Modjtahedi et al., 2016). Human MIC19 possesses five cysteine residues, four of which in the CHCH domain form two disulfide bonds (Sakowska et al., 2015). In contrast to human MIC19, yeast Mic19 does not possess a standard twin Cys-X9-Cys motif but has a simplified Cys-X10-Cys motif that also forms a disulfide bond corresponding to the inner disulfide bond of human MIC19 between Cys193 and Cys204. MIC19 appears to be a regulatory subunit that acts as a redox-sensor of the MICOS complex (Sakowska et al., 2015), which is important for the formation of the MICOS complex and regulation of the mitochondrial membrane architecture. However, MIC19 oxidation is dispensable for its integration into the MICOS complex. MIC19 associates directly with Mic60 (Xie et al., 2007). Downregulation of Mic60/Mitofilin or MIC19 results in disorganized mitochondrial cristae, disassembly of MICOS complex, and reduced number of crista junctions (Darshi et al., 2011; von der Malsburg et al., 2011). However, MIC19 (yeast Aim13) deficiency displays less severe effects compared to Mic60 indicating that MIC19 may regulate the complex stability and/or subunit assembly of the complex. Furthermore, MIC19 mediates mitochondrial outer- and inner-membrane contact by directly interacting with mitochondrial outer-membrane protein Sam50 and Mic60 to form the Sam50-Mic19-Mic60 axis (Tang et al., 2020). In contrast, loss of another CHCH-domain containing protein Mic25 (CHCHD6, yeast Mic19 or Aim13) from mammalian mitochondria has only minor effects on MICOS integrity and cristae morphology, but the specific role of this MICOS subunit remains elusive (Tang et al., 2020).

Mic26 (Mic23, ApoO) and Mic27 (ApoOL or FAM121A) are apolipoprotein-O-like proteins sharing substantial sequence similarity. Both subunits are required for the maintenance of the cristae ultrastructure and assembly of the F1 subunit into monomeric F₁F_o-ATP synthase (Anand et al., 2020). Depletion of Mic26 and Mic27 caused aberrant onion-like crista structures with loss of crista junctions (Weber et al., 2013; Koob et al., 2015). In addition, single and double deletion of Mic26 and Mic27 in human cells lead to more concentric onion-like cristae with loss of crista junctions than any single deletion, demonstrating overlapping roles of Mic26 and Mic27 in the formation of crista junctions. Furthermore, Mic26 and Mic27 are cooperatively required for global integrity and stability of multimeric OXPHOS complexes by modulating the cardiolipin level (Koob et al., 2015; Anand et al., 2020). Unlike the loss of Mic60, Mic10, or Mic13 that resulted in destabilization of either the whole or part of the MICOS subcomplex, Mic26 and Mic27, however, were dispensable for the stability of the remaining subunits of the MICOS complex and their incorporation into higher molecular weight complexes (Anand et al., 2020).

Besides the MICOS-associated OMM proteins involved in protein import and assembly as discussed above, interaction and function links between the MICOS and the F₁F_o-ATP synthase have been demonstrated (Rabl et al., 2009; Eydt et al., 2017; Rampelt et al., 2017). A fraction of Mic10 physically interacts with the ATP synthase dimers and overexpression of Mic10 stabilizes the ATP synthase oligomers (Eydt et al., 2017; Rampelt et al., 2017). In contrast, no direct interaction has been demonstrated between Mic60 and any ATP synthase subunits although a pronounced increase of ATP synthase oligomers is observed upon deletion of Mic60 (Rabl et al., 2009). The dimeric F1Fo-ATP synthase, however, can affect the Mic60 distribution (Stephan et al., 2020). Importantly, Mic60 and the F₁F_O-ATP synthase subunits e or g act antagonistically to control the oligomerization of F₁F_O-ATP synthase (Rabl et al., 2009). Overall, MICOS promotes the formation of crista junctions, whereas the oligomeric F₁F_o-ATP synthase is crucial for shaping the crista rims. The interplay between the MICOS complex and F₁F_o-ATP synthase highlights a remarkable molecular mechanism by which they cooperatively play critical roles in shaping the inner membrane to regulate the formation of crista junctions and rims.

Optic atrophia 1 (OPA1), the homolog of S. cerevisiae Mgm1p, plays essential roles in mitochondrial dynamics, cristae integrity, respiratory capacity, and mtDNA maintenance (Cipolat et al., 2006; Serrano Cardona and Muñoz Mata, 2013; Patten et al., 2014). Loss of OPA1 causes mitochondrial fragmentation, abnormal cristae architecture and dynamics (Amutha et al., 2004; Hu et al., 2020; Sesaki et al., 2003; Song et al., 2007) (Figure 3), leading to impaired cell proliferation, mitochondrial membrane potential, respiratory capacity (Olichon et al., 2003; Song et al., 2007; Mishra et al., 2014; Patten et al., 2014), and depletion of mtDNA (Chen et al., 2010; Elachouri et al., 2011). Structurally, OPA1 contains an N-terminal mitochondrial targeting sequence (MTS), followed by a transmembrane domain (TM), a coiled-coil domain, a highly conserved gtpase domain, a middle region, and a C-terminal gtpase effector domain (GED). Eight OPA1 variants are present in humans, which are ubiquitously expressed but their levels display great variability in different human tissues (Olichon et al., 2007). Cleavage of the MTS leads to long isoforms (L-OPA1) anchored to the inner mitochondrial membrane by the transmembrane domain, with the other domains exposed to the inner mitochondrial membrane space. Additional cleavage by OMA1 and YME1L proteases at sites S1 (exon 5) or S2 (exon 5b) results in soluble short isoforms (S-OPA1) devoid of the transmembrane segment in the intermembrane spaces (MacVicar and Langer, 2016; Del Dotto et al., 2017, 2018). The long membrane-bound isoforms L-OPA1 are shown to be required for mitochondrial fusion while short forms S-OPA1 facilitate mitochondrial fission (Ishihara et al., 2006; Griparic et al., 2007; Song et al., 2007). Later experiments, however, demonstrate that expression of any OPA1 isoform is able to restore the cristae morphology, preserve the assembly of respiratory complexes and mtDNA content (Del Dotto et al., 2017). The maintenance of mitochondrial network morphology requires at least two OPA1 isoforms with a specific balance of L- and S-forms and an adequate amount of proteins (Del Dotto et al., 2017).

Importantly, OPA1 oligomers tight the crista junctions and maintain a negative crista junction curvature. OPA1 also physically interacts with the MICOS core component MIC60 to stabilize the curvature of crista junctions (Barrera et al., 2016; Glytsou et al., 2016). Mild OPA1 overexpression can inhibit apoptotic crista remodeling and correct altered cristae shape even if MICOS are altered in dysregulated mitochondria, indicating that OPA1 lies upstream of Mic60 in regulating the number and stability of crista junctions (Glytsou et al., 2016). Furthermore, OPA1 and Mic10 antagonistically influence the size and the distribution of Mic60 assemblies (Stephan et al., 2020). OPA1 can stabilize tubular crista junctions in Mic10-KO cells suggesting that OPA1 works in concert with the Mic10-subcomplex to maintain the tubular crista junctions. Additionally, OPA1 associates with the F₁F_o-ATP synthase, and its function relies on the oligomerization of ATP synthase (Frezza et al., 2006; Patten et al., 2014; Quintana-Cabrera et al., 2018; Quintana-Cabrera et al., 2021). Both S-OPA1 and S-Mgm1 are able to induce membrane curvature as observed by tubulation of cardiolipin-containing liposomes, revealing the molecular mechanism by which they regulate the IMM morphology (Tadato et al., 2010; Rujiviphat et al., 2015). Recently, electron cryotomography structural studies of the OPA1 homologue Mgm1 on reconstituted membrane tubes show that Mgm1 from Chaetomium thermophilum can assemble into a helical filament on positively and negatively curved membranes (Faelber et al., 2019). Consistently, a structural study with S-OPA1 reveals that S-OPA1 can assemble onto membranes in a helical array with a dimer building block, revealing a power stroke membrane remodeling mechanism during mitochondrial inner membrane fusion (Zhang et al., 2020).

Mitochondrial calcium uniporter (MCU) complex consists of two pore-forming proteins MCU and EMRE (essential MCU regulator). The activity of the MCU-Complex is controlled by MICU1 (mitochondrial calcium uptake 1) and its paralog MICU2 (Stefani et al., 2016). In contrast to the homogeneous distribution of MCU and EMRE at the IMM, MICU1 forms dimer or hexamer via its C-terminal oligomerization site and exclusively localizes at the IBM through electrostatic interaction of its polybasic domain. Loss of MICU1 causes strong widening of the crista junctions suggesting that MICU1 contributes to the structural integrity of the crista junctions (Figure 3). In contrast, knockdown of MCU or EMRE or both has no effect on the morphology of crista junctions. Hence, as a regulator of the MCU complex MICU1 also plays an essential role in the stabilization of crista junctions (Gottschalk et al., 2019).

Mitochondrial prohibitins consist of two subunits PHB-1 (32 KDa) and PHB-2 (34 KDa), belonging to the SPFH (stomatin/prohibitin/flotillin/HflKC) family. Proteins in the SPFH family often are membrane-anchored and perform diverse cellular functions in different organelles (Tavernarakis et al., 1999). Within mitochondria, approximately 14 heterodimers composed of PHB1 and PHB2 assemble into a ring-like macromolecular structure at the inner membrane, which is involved in diverse cellular processes, such as maintenance of cristae structure and assembly of the OXPHOS complexes (Ou et al., 2010; Merkwirth et al., 2012). Deletion of PHB2 leads to specific loss of L-OPA1, resulting in an aberrant cristae morphogenesis, suggesting that prohibitin is required for the formation of mitochondrial cristae by stabilizing a long form of the dynamin-like gtpase OPA1. Furthermore, expression of L-OPA1 in PHB2-deficient cells suppresses this defect indicating that impaired OPA1 processing is the primary cellular defect in the absence of prohibitin. Prohibitin can prevent the hydrolysis of L-OPA1 into S-OPA1 thereby enhancing the effect of OPA1 on the morphology of mitochondrial cristae (Merkwirth et al., 2008). In addition, prohibitins genetically interact with genes modulating the biosynthesis of mitochondrial phospholipids, in particular for cardiolipin and PE, suggesting that the prohibitin complex also acts as a membrane organizer affecting the distribution of cardiolipin and PE by clustering them at distinct sites of the IMM (Osman et al., 2009; Hernando-Rodríguez and Artal-Sanz, 2018). Due to the critical roles of cardiolipin and PE in the regulation of cristae membrane curvature, prohibitin is proposed to maintain the proper ultrastructural organization of the IMM. Furthermore, PHB proteins interact with ATAD3 in human cells (He et al., 2012) that has been shown to control cristae structure (Gilquin et al., 2010; Cooper et al., 2017).

MCL-1 is a member of the anti-apoptotic BCL-2 family and plays a critical role in the survival of multiple cell lineages. MCL-1 is proteolyzed at the N-terminus to generate three different MCL-1 species, including the full-length (40 kD), two truncated proteins cleaved at Ile 10 (38 kD) or Leu 33 (MW 36 kD). Within mitochondria, MCL-1 proteins with sizes of 40 kD and 38 kD are enriched in the OMM, whereas the 36 kD form localizes to the IMM and matrix, where they perform distinct functions at different mitochondrial subcompartments. The OMM localized MCL-1 proteins exert their anti-apoptotic activity by antagonizing the BAX and BAK activation to maintain mitochondrial integrity. In contrast, the IMM and matrix localized MCL-1 maintains normal IMM architecture through facilitating mitochondrial fusion and promoting the assembly of ATP synthase oligomers (Perciavalle et al., 2012).

MICOS complex and OPA1 are critical for the formation of crista junctions and F₁F_o-ATP synthase is essential for the generation of crista tips (Davies et al., 2012; Ding et al., 2015; Friedman et al., 2015; Guarani et al., 2015; Harner et al., 2011; Hoppins et al., 2011b; Hu et al., 2020; van der Laan et al., 2016; Milenkovic and Larsson, 2015; Mühleip et al., 2019; Rabl et al., 2009; Strauss et al., 2008). However, it is yet to be defined how the deeply curved inner membrane invaginations are formed and in particular whether dedicated membrane-shaping proteins are involved in the process. FAM92A1 is a BAR domain protein localizes to the matrix side of the IMM and the majority of FAM92A1 resides in the lamella segments of cristae (Figures 1C, 3C). Loss of FAM92A1 caused a severe disruption to mitochondrial morphology and ultrastructure underlying its critical role in mitochondrial membrane morphogenesis (Wang et al., 2019) (Figure 3). FAM92A1 preferentially interacts with negatively charged phospholipids such as phosphatidylinositol 4,5-bisphosphate and cardiolipin. Importantly, FAM92A1 possesses a membrane-remodeling activity, inducing a high degree of positive membrane curvature in vitro suggesting that FAM92A1 is involved in generating and maintaining the curved membrane along the lamella regions (Figure 3). However, the cooperative interplays between FAM92A1 and the other regulators of cristae membrane curvature including MICOS, ATP synthase, and OPA1 remain to be defined.

ATAD3A (atpase family AAA-domain containing protein 3 A) is a nuclear-encoded protein in mitochondria, which is anchored in the IMM by a central transmembrane domain, with the N-terminal region associating with the inner surface of OMM and the C-terminal AAA+ atpase domain residing in the matrix, thus connecting both the inner and outer mitochondrial membranes. ATAD3 is a component of mtDNA nucleoids and plays a role in mtDNA maintenance (Gerhold et al., 2015). In addition, ATAD3A regulates mitochondrial morphology and controls cholesterol trafficking at the ER-mitochondria contact sites (Gilquin et al., 2010; Gerhold et al., 2015; Issop et al., 2015; Baudier, 2018). Loss of ATAD3A causes abnormal cristae morphology and dynamics (Gilquin et al., 2010; Cooper et al., 2017). Moreover, ATAD3A knockout resulted in increased mitochondrial crista-IBM and crista-crista contacts indicating that ATAD3A negatively regulates the membrane contacts. Furthermore, depletion of ATAD3A remarkably reduced the levels of OPA1, Yme1L, and Mic60, suggesting that ATAD3A may cooperate with OPA1, Yme1L, and MICOS to regulate mitochondrial cristae morphology (Hu et al., 2020). The molecular mechanisms underlying the role of ATAD3A in membrane remodeling and association with other membrane-shaping proteins, however, are unknown and warrant further investigation.

Besides the membrane-remodeling activities of the above-mentioned proteins and protein complexes, membrane lipids and their organization in a lipid bilayer play crucial roles in the generation of membrane curvature. A eukaryotic cell contains more than 1,000 different lipid species that are not homogenously distributed among intracellular membranes, but instead each organelle has a characteristic lipid composition that is required for its proper function. The specific distribution of lipid species across the different cellular membranes/leaflets determines membrane physical properties, such as permeability, membrane curvature, or membrane surface charge, which regulates protein binding and organelle functions. In addition, protein-membrane interaction can cause dynamic rearrangement of lipid and proteins in the membranes, for example, inducing the formation of membrane microdomains and protein oligomerization, consequently modulating protein function, membrane curvature, and dynamics of both protein and lipids (Epand et al., 2007; Zhao et al., 2013; Anand et al., 2020). On the other hand, membrane curvature can control the distribution of membrane lipids and proteins. For example, cardiolipin sorting is observed with increasing membrane curvature (Beltrán-Heredia et al., 2019). Cardiolipin and PE residing in the IMM play critical roles in mitochondrial membrane morphogenesis. The IMM comprises ∼18 and 34% cardiolipin and PE, respectively (Horvath and Daum, 2013). Cardiolipin, an acidic phospholipid with negative charges, plays essential role in the characteristic shape of the mitochondrial inner membrane (Ikon and Ryan, 2017b). Different from the other glycerophospholipids, cardiolipin consists of two phosphatidyl moieties that share a glycerol head group. Thus, the combination of a compact anionic polar head group and four esterified fatty acyl chains results in a distinctly conical lipid molecule (Schlame, 2008). Based on its molecular geometry, cardiolipin is classified as a “non-bilayer” phospholipid. Usually, cardiolipin forms clusters with a certain size by itself or together with another conical lipid PE, promoting negative curvature in biomembranes where headgroups face the concave surface. Due to their specific cone shape, they are segregated into the negatively curved inner monolayer leaflet of the IMM. This lipid asymmetry can reduce membrane tension at the curved region and is required for the generation and maintenance of membrane curvature at the crista tip. Furthermore, the local enrichment of cardiolipin or PE can lower the energetic cost of negative membrane curvature originating from lipid asymmetry between leaflets and distribution of membrane remodeling enzymes, such as F₁F_o-ATP synthase and MICOS (Iovine et al., 2021). Genetic disruption of cardiolipin or PE biosynthesis mediates the impairment of cristae morphology, demonstrating the indispensable roles of cardiolipin and PE in the formation of cristae membrane curvature (Gonzalvez et al., 2013; Tasseva et al., 2013). Moreover, cardiolipin interacts with proteins in the IMM regulating their localization and activities. Cardiolipin can directly bind the mitochondrial F₁F_o-ATP synthase and the dimer interface to stabilize the structure of the enzyme, promoting the long distance ribbon-like assembly of F₁F_o-ATP synthase dimers and affecting the lateral ultrastructural organization of the cristae membrane (Duncan et al., 2016; Mühleip et al., 2019). The phospholipid PE is also a conical-shaped, non-bilayer phospholipid, which is the most autonomously synthesized phospholipid in the mitochondrial membranes. It is synthesized from PS by the enzyme phosphatidylserine decarboxylase (PSD) through the CDP-ethanolamine pathway (Kennedy and Weiss, 1956). Genetic deletion of the PSD enzyme leads to swollen, rounded, and fragmented mitochondria, indicating that PE is critical for mitochondrial morphology (Steenbergen et al., 2005). Several excellent reviews summarize our current understanding of lipid signaling and its scaffolding role (Tatsuta et al., 2014; Aufschnaiter et al., 2017; Dudek, 2017; Nielson and Rutter, 2018; Royes et al., 2020).

Taken together, factors regulating membrane curvature includes lipid composition and asymmetry, nanoscopic scaffolding by oligomerized protein domains, insertion of transmembrane protein domain that have an intrinsic conical or inverted conical shape, or penetration of amphipathic helices like a wedge into one leaflet of a bilayer (Jarsch et al., 2016). The mechanisms that generate membrane curvature and remodel cristae membranes, however, have only been unraveled in part. In particular, we are only beginning to understand how dynamic the inner membrane is and how the various determinants of membrane shape may communicate to generate both cristae architecture and dynamics. The cooperative interplay of the above-mentioned protein and protein complexes in the regulation of mitochondrial membrane architecture and dynamics is not fully understood. Furthermore, mitochondrial lipids such as cardiolipin and PE are emerging as important players in regulating mitochondrial membrane morphology. Whether they modulate functions of the membrane shaping proteins or protein complexes or whether the nonbilayer lipids directly influence the curvature of the IMM by lipid sorting requires further studies. Answering these questions will allow us to understand the basis of human diseases associated with abnormal IMM morphology, which may promote the identification of potential targets for developing therapeutics for mitochondrial diseases.