Cytoskeleton: Recent work evidenced that cytoskeleton elements differentially modulate the mobility and shape of mitochondria ^[1][2][7,12]. Nocodazole, a drug that disrupts microtubule polymerization, reduces mitochondrial network cellular coverage, disrupts mitochondrial alignment with microtubules, and decreases mitochondrial mobility. In contrast, F-actin or intermediate filaments maintain mitochondria confined to the microtubule network, and their disruption alters mitochondrial shape ^[1][2][7,12]. Of note, it has been reported that ARP2/3 and Formin-dependent actin cycling (actin assembly and disassembly) occur through the mitochondrial network ^[3][1]. Furthermore, actin polymerization promotes mitochondrial fission, whereas F-actin disruption causes mitochondria fusion ^[3][1]. In this regard, F-actin is assembled into the outer mitochondrial membrane (OMM), thereby contributing to mitochondrial fission ^[3][4][1,13].

Mitochondrial dynamics: Continual shifting of mitochondrial network architecture is supported by mitochondrial dynamics proteins. In the last 20 years, several proteins that coordinate mitochondrial fission and fusion events have been defined ^{[5][6][7][8][9]}[14,15,16,17,18]. Moreover, recent studies have indicated that mitochondrial fission and fusion occur in the same site on the mitochondria ^[10][2], and also that ER wrapping around mitochondria is required to sustain the dynamics of these organelles ^[11][6]. A recent report describes two distinct types of mitochondrial fission. Peripheral fission sustains mitochondrial degradation mediated by mitophagy while midzone mitochondrial fission is required to maintain mitochondrial biogenesis and dynamics ^[12][19] (see Section 2).

Mitochondrial contacts with other organelles. A growing number of studies have demonstrated the interaction of mitochondria with different organelles. This is an emergent field of research that will permit understanding of how some metabolic and cell signaling mechanisms originate.

Mitochondria–endoplasmic reticulum contacts. Several investigations have reported that mitochondria–endoplasmic reticulum interactions are required to modulate the calcium signal. Close contact between mitochondria and endoplasmic reticulum plays a critical role in mitochondrial calcium uptake. For instance, the complex VDAC1-GRP75-PI3R mediates mitochondria–endoplasmic reticulum tethering and is required to sustain mitochondrial calcium homeostasis ^[13][20]. Moreover, mitochondria–endoplasmic reticulum interaction is involved in apoptosis and autophagy activation ^[14][15][16][21,22,23]. Overnutrition induced via a high fat diet in mice leads to alteration in calcium handling in the liver ^[17][18][24,25]. These studies have demonstrated that mitochondrial–endoplasmic reticulum contacts are disrupted upon ingestion of a high fat diet. On the other hand, the mechanism of phosphatidylethanolamine (PE) synthesis and shuttling requires mitochondrial–endoplasmic reticulum contacts. The enzymes required for phosphatidic acid (PA) conversion to PE are localized in mitochondrial–endoplasmic reticulum contact. Several proteins contribute to mitochondria–endoplasmic reticulum tethering and the maintenance of calcium transfer and lipid synthesis (revised by Wenzel et al., 2022) ^[19][26].

Mitochondria–lipid droplets contacts. Contacts between mitochondria and lipid droplets (LDs) are mediated by several proteins including PLIN1, PLIN5, MFN2, and MIGA2 ^[20][27] (see Section 4). The highly dynamic interactions between both organelles allow fatty acid migration from LD to mitochondria, where it is oxidized to produce ATP or heat. Furthermore, they are also needed for LD expansion, which stores fatty acids and lipid intermediates to avoid cellular lipotoxicity ^[21][22][28,29].

Mitochondria–peroxisome contacts. Mitochondrial–peroxisome interaction plays a relevant role in fatty acid metabolism and in cellular redox homeostasis; a coordinate function of these organelles is required to sustain mitochondrial activity. Studies have shown that peroxisomal protein (PEX5 or PEX16) ablation in mice livers led to mitochondrial dysfunction and fragmentation ^[23][24][30,31]. Moreover, PEX16 knockout mice showed that peroxisomes are required to sustain mitochondrial homeostasis upon metabolic stress induced by a high fat diet ^[24][31]. Two proteins involved in mitochondria–peroxisome tethering have been identified, enoyl-CoA-δ isomerase 2 (ECI2) and the lipid transport protein VPS13D ^[25][26][32,33]. Interestingly, knockdown of VPS13D expression promotes mitochondrial fragmentation, revealing the role of this protein in mitochondrial architecture ^[27][34]. Also interesting, a recent study of yeast demonstrated that PEX4 and Fzo1 (the yeast orthologue of MFN1 and MFN2 proteins) are involved in mitochondria–peroxisome tethering ^[28][35]. Further investigations are required to elucidate the role of this protein in mammalian cells.

Mitochondria–endosome contacts. A recent report has documented that VDAC2 tethers RAS-PI3K-positive early endosomes with mitochondria ^[29][36]. This researchtudy also shows that this interaction promotes endosome acidification and maturation ^[29][36]. Depletion of the mitochondrial fusion protein MFN1 promotes the association of endosomes and mitochondria through a process that requires Rab5C ^[30][37]. In addition, it has been described that endosomes are in close contact with mitochondria in axons of retinal ganglion cells ^[31][38]. Rab7a is localized in endosomes, and ribonucleoprotein particles and mitochondria are in contact in axons. In this way, these contacts are required for local translation of mRNA that encodes to mitochondrial proteins. Moreover, Rab7a mutations lead to alteration of mitochondrial morphology, decrease mitochondrial membrane potential, and modify the mitochondrial retrograde and anterograde transport in axons ^[31][38].

Mitochondria–lysosome contacts. Lysosome–mitochondrial tethering is mediated by Rab7, is localized in lysosomes, and is modulated by TBC1D15, a Rab7 GTPase activating protein, and Fis1, localized in mitochondria ^[32][39]. This researchtudy also documented that lysosomes localize in mitochondrial fission sites, suggesting that they modulate mitochondrial dynamics ^[32][39]. The functional role of mitochondrial–lysosome tethering on calcium homeostasis has recently been reported ^[33][40]. The cation channel TRPML1 localized in lysosomes and late endosomes mediates the direct calcium transfer into mitochondria. Moreover, TRPML1 loss-of-function impairs calcium transfer, and it results in alterations of tethering dynamics ^[33][40].

Mitochondrial architecture is altered by environmental stimuli. It has been widely described in cell models that metabolic stress, induced by OXPHOS inhibitors, nutrient starvation, endoplasmic reticulum (ER) stressors, protein and RNA synthesis suppressors, and UV irradiation, induce transient mitochondrial elongation upon acute treatment ^{[34][35][36][37]}[41,42,43,44]. Moreover, mitochondrial elongation improves the OXPHOS activity under these stimuli ^[34][38][41,45]. It has been demonstrated that the relationship between mitochondrial morphology and OXPHOS is complex. Given that, maximal mitochondrial respiration is carried out upon mitochondrial uncoupling agents (such as CCCP) treatment and these compounds promote mitochondrial depolarization and fragmentation. A simple relation between mitochondrial elongation and increased OXPHOS activity may not occur under all conditions. Thus, genetic ablation of the mitochondrial fusion protein Mitofusin 1, in which the mitochondrial network is fragmented, showed similar OXPHOS activity in glucose- or galactose-supplemented culture media ^[39][46]. In keeping with this, it has been reported that hepatic MFN1 ablation increases mitochondrial mass and OXPHOS activity ^[40][47]. Recent evidence has shed new light on the link between the morphology and function of mitochondria. Ngo et al. demonstrated that mitochondrial fragmentation enhances long-chain fatty acid oxidation. Moreover, mitochondrial fragmentation decreases the inhibition of malonyl-CoA-dependent carnitine palmitoyltransferase I. These data reveal that mitochondrial fragmentation upon lipid overload activates mitochondrial β-oxidation ^[41][48].

Mitochondrial shape alterations have been reported in conditions such as obesity, diabetes, ischemic–reperfusion, senescence, and cancer ^{[17][42][43][44][45][46][47]}[24,49,50,51,52,53,54]. Under metabolic stress induced by nutrient overload, mitochondrial network architecture is fragmented, and this alteration is associated with a metabolic dysfunction that results in a decrease in OXPHOS activity, apoptosis, or mitophagy ^[48][55]. These alterations have been evidenced in several cell models, including β-pancreatic cells, skeletal muscle cells, cardiomyocytes, and adipocytes ^{[49][50][51][52][53]}[56,57,58,59,60]. Similarly, disruption of the mitochondrial network architecture and mitochondrial dysfunction have been reported based on in vivo mouse models of diabetes, cardiomyopathy, non-alcoholic fatty liver disease, and obesity ^{[44][54][55][56]}[51,61,62,63]. Emergent evidence indicates that mitophagy is impaired in metabolic diseases. The perturbation of mitochondrial homeostasis that causes a decrease in mitochondrial membrane potential induces mitochondrial fragmentation and activates lysosome-dependent mitochondrial degradation ^[48][57][58][55,64,65]. Recently, Han and colleagues demonstrated the presence of distinct populations of mitochondria in non-small cell lung cancer tumors. They reported a peri-droplet mitochondrial network throughout the cytoplasm in oxidative lung adenocarcinoma (LUAD) cells that was accompanied by an increase in fatty acid oxidation. On the other hand, cells with higher glycolytic activity (lung squamous cell carcinoma) show perinuclear mitochondria. These exciting results support the importance of mitochondrial architecture in sustaining tumor metabolism ^[47][54]. Thus, modulation of mitochondrial architecture appears to play a crucial role in cellular responses leading to metabolic diseases.

The discovery of proteins that modulate mitochondrial architecture and the mechanism of action of the same has allowed intense research efforts regarding the link between metabolism and mitochondrial function. These efforts have revealed that changes in the expression or post-translational modification of mitochondrial dynamics proteins are associated with metabolic diseases. As a result of these investigations, new fields of drug discovery aiming to modulate mitochondrial dynamics and thereby redirect cellular metabolism have emerged.

Given the continuous and rapid changes in mitochondrial architecture, sophisticated microscopy approaches are required to elucidate the biological factors that regulate mitochondrial morphology in vivo. Recent studies using super-resolution microscopy have revealed the cellular mechanisms by which mitochondrial network architecture is modulated ^{[10][11][12][59]}[2,5,6,19]. In this regard, live-cell imaging has shown that ER–mitochondrial constriction sites prime and coordinate the mitochondrial fission and fusion protein machinery ^[10][2]. Remarkably, recent evidence using proximal proteomic and live-cell microscopy demonstrates that ABHD16A, a hydrolase enzyme that mediates the conversion of phosphatidylserine (PS) to lysophosphatidylserine (lysoPS), is localized at ER–mitochondria contacts and is required for mitochondrial fission and fusion ^[60][8].

The use of intravital multiphoton microscopy-based technology, which allows collection of cell images in live animals, has revealed that the mitochondrial network is disrupted upon pathological conditions in vivo. This woresearchk has demonstrated the transient disruption of the mitochondrial network upon brain ischemia. The mitochondria of dendrites at pyramidal neurons have an elongated shape, as revealed by intravital microscopy and electron microscopy. In contrast, upon transient brain ischemia, mitochondria undergo fragmentation and have a globular morphology, and “mitochondria-on-a-string” (MOAS) structures appear. Ischemic recovery rescues mitochondrial elongation and decreases globular mitochondria, and MOAS morphology is not observed in dendrites ^[61][66]. Taken together, these observations indicate that a transient and reversible alteration of mitochondrial morphology occurs under ischemia in vivo. Interestingly, MOAS morphology has also been described in neurons in cellular and mouse models of Alzheimer’s disease, aging, and hypoxia. It has been proposed that energetic collapse upon ischemic injury leads to unfinished mitochondrial fission ^[62][63][67,68]. Intravital microscopy has also been used to study live-cell mitochondrial dynamics and mitophagy in the livers of mice after acute ethanol treatment ^[63][68]. This reseaporch t demonstrated that this treatment disrupts mitochondrial membrane potential; however, only a subpopulation of mitochondria undergoes mitophagy. In addition, ethanol activates selective mitophagy instead of lipophagy in the livers of this model ^[64][69]. In this context, a novel state-of-the-art methodological approach will unravel the function and modification of the mitochondrial network under physiological and pathological conditions (Figure 1 and Table 1).