MIT. DYNAMICS mit. are highly dynamic, constantly undergoing fission and fusion. Fission results in the production of short mit. rods or spheres. In contrast, fusion promotes a long, filamentous morphology of mit. In healthy cells, the frequencies of fusion and fission events are finely tuned and balanced to maintain mit. homeostasis. Mit fission & fusion -> function, inheritance, quality control Under intracellular or extracellular stresses, this balance is disrupted, resulting in the cleavage of mit. from an elongated network into small spheres or short rods, i.e., mit. fragmentation. fusion : Mfn1, Mfn2, and OPA1, fission : Drp1 and Fis1. Defects in these proteins lead to severely altered mit. morphology as well as impaired mit. function, loss of mit. DNA (mtDNA) integrity, and eventually cell death In mammals, developmental defects neurological anomalies neurodegenerative diseases l Mit. fission machinery Drp1 a large GTPase essential role in mit. fission in mammalian cells In unstressed cells, Drp1 is mainly cytosolic. Upon stimulation, activated and translocates to the scission sites of MOM through interaction with Fis1, where they oligomerize and form spirals to constrict MOM through GTP hydrolysis, resulting in mit. fission. After fission, Drp1 returns to the cytosol Activation, translocation are regulated by mechanisms of posttranslational modifications including phosphorylation, ubiquitylation, sumoylation Blockade of Drp1 can suppress mit. fission, leading to elongated mit.; conversely, overexpression of Drp1 results in mit. fragmentation Fis1 mammalian cells small membrane protein evenly anchored on MOM through a C-terminal transmembrane domain N-terminal region facing the cytosol The cytosolic domain facilitate specific protein–protein interactions. Fis1 act as a receptor protein for Drp1 on the outer membrane of mit. Overexpression of Fis1 promoted mit. fission resulting in mit. fragmentation, followed by the release of cytochrome c and ultimately apoptosis transmembrane (TM) domain, which facilitate Fis1 anchoring on the outer mit. membrane (OMM). l Mit. fusion machinery Mfn1, Mfn2, OPA1 all large GTPases Mfn1 and Mfn2 are localized to the MOM OPA1 is localized MIM Successful mit. fusion requires a coordinated action of Mfns and OPA1, although it is unclear how the molecular events at MOM are coordinated with those at MIM fusion starts from the MOM fusion, followed by MIM fusion Mfn1/2 contain GTPase domain, transmembrane (TM) domains, (HR1 and HR2). HR2 functions in mit. tethering. interaction of adjacent HR2 regions-> mitofusin oligomerization(homodimers or heterodimers) through GTP hydrolysis->MOM fusion Mfn1 is the main tethering protein, whereas Mfn2 may have a regulatory role. OPA1 -maintenance of the cristae structure -preservation of cytochrome c in mit. -maintenance of mit. electron transport chain -mtDNA stability impaired mit. fusion links to severe defects in cell respiration in neurodegenerative disorders MECHANISMS LEADING TO MIT. FRAGMENTATION IN CELL INJURY cell injury and apoptosis-> mit. fragmentation combined result of the activation of fission and the suppression of fusion. involvement of Bcl-2 family proteins, Drp1, and Ca2+/calcineurin-mediated dephosphorylation. Reactive oxygen species (ROS) and mit. permeability transition (MPT) Bcl-2 family proteins Characterized by the presence of Bcl-2 homology (BH) domain (a) multi-BH domain/ antiapoptotic Bcl-2 and Bcl-xL; (b) multi-BH domain proapoptotic Bax and Bak (c) BH3-only proteins(only one BH domain) Bid and Bad Bcl-2 family proteins are recognized as the central regulators of mit. integrity during cell injury and death. Bax and Bak provide the requisite gateway of mit. outer membrane permeabilization (MOMP), which is inhibited by Bcl-2/Bcl-xL under n ormal conditions and activated by BH3-only proteins during apoptosis. n In unstressed cells Bak interacts with both Mfn1/2 to maintain a filamentous mit. network Bax inactivated in the cytosol n apoptotic stress, Bak dissociates from Mfn2 increases its association with Mfn1 à suppression of mit. fusion à mit. fragmentation Bax activated and translocates to mit. Fragmented mit. are sensitized to Bax insertion and oligomerization, leading to the formation of pathological pores for the release of apoptogenic factors such as cytochrome c. Drp1 activation posttranslational modifications, including phosphorylation, dephosphorylation, ubiquitination, sumoylation, and S-nitrosylation have been implicated in Drp1 regulation under various physiological and pathological conditions Thus, depending on the experimental condition, Drp1 may be phosphorylated at multiple sites by different protein kinases, resulting in either inhibition or activation of mit. fission. Ex) Ca2+/calmodulindependent protein kinase-a (CaMKI) activates mit. fission Rho-associated coiled coil–containing protein kinase 1 phosphorylates Drp1 under high-glucose stimulation, resulting in Drp1 translocation and mit. fission. dephosphorylation of Drp1 at Ser637 by calcineurin promotes Drp1 translocation to mit. and mit. fragmentation, sensitizing cells to apoptosis. n neurons, PKCd can phosphorylate Drp1 at Ser579 inducing mit. fragmentation to contribute to cell death under oxidative stress. , ubiquitination of Drp1 by MARCH5 resulting in mit. fission.73 Sumoylation of Drp1 induce mit. fragmentation and apoptosis. S-nitrosylation has been shown to increase Drp1 dimer formation and GTPase activity, leading to mit. fission or fragmentation during nitric oxide–induced neuronal synaptic injury and death, suggesting a novel pathogenic mechanism of neurodegeneration. Ca2+/calcineurin Perturbation of Ca2+ homeostasis has a crucial role in cell injury and death. Physiologically, mit. are involved in the maintenance of Ca2+ homeostasis by buffering cytosolic Ca2+ when it rises to high levels. However, excessive Ca2+ overload in mit. m ay lead to mit. damage, resulting in cell injury and death. sustained Ca2+ rise in cytosol can induce mit. fragmentation in a Drp1-dependent manner. Calcineurin a Ca2+-activated phosphatase calcineurin may promote apoptosis through at least two distinct mechanisms, 1. Drp1 high cytosolic Ca2+ was shown to activate calcineurin to directly dephosphorylate Drp1 at Ser637, leading to Drp1 activation and translocation to the mit. to induce mit. fission and fragmentation. 2. dephosphorylation of the proapoptotic Bcl-2 family protein Bad Reactive oxygen species Mit. are known to be a major intracellular source of ROS. Under pathological conditions, uncoupling of oxidative phosphorylation and loss of mit. membrane integrity induce excessive ROS production from the respiratory chain, especially at the Complex I and III. In contrast, mit. are also a critical target of the damaging effects of ROS. Oxidative damage leads to mit. dysfunction and disruption, triggering MPT and/or the release of proapoptotic proteins like cytochrome c to induce cell death. neurons exposed to oxidative stress an increased ROS production was shown to be accompanied by mit. fragmentation and cell death Thus, there is a complex interaction between ROS and mit. dynamics. In some experimental models, ROS may be the triggering event for mit. fragmentation, but in others mit. fragmentation may lead to mit. dysfunction and ROS production. under certain conditions, these two events may exacerbate each other to form a vicious cycle ultimately resulting in cell injury and death. MECHANISMS WHEREBY MIT. DYNAMICS OR ITS DISRUPTION CONTRIBUTES TO MIT. DAMAGE AND CELL DEATH MTP (mit. 투과성 변화) fragmentation has a crucial role in mit. injury during cell death, especially in the MOMP how the fragmentation, a seemingly morphological change, contributes to MOMP and loss of mit. integrity ‘two-hit’ hypothesis Bak triggers the first hit, mit. fragmentation, second hit that involves Bax activation and formation of pathological pores in the MOM fragmentation is likely process that facilitates Bax activation in mit. l membrane it emphasizes on a collaborative action of Bak and Bax in promoting MOMP preservation of filamentous mit. by either blocking mit. fission or enhancing mit. fusion can suppress Bax insertion and oligomerization in mit. membrane. Moreover, mitofusin-null cells with fragmented mit. are highly sensitive to Bax insertion and oligomerization in mit. membrane resulting in increased MOMP and apoptosis Together, these results suggest that the alteration of mit. dynamics and consequent mit. fragmentation contribute to MOMP at least partially by facilitating Bax insertion and activation in mit. membrane - during apoptosis. Drp1 contributes to MOMP Drp1 promotes Bax oligomerization by triggering membrane tethering and hemifusion. Importantly, during apoptosis associated mit. fragmentation, Drp1 may constrict the MOM resulting in the formation of similar intermediate membrane structures to induce Bax oligomerization Cristae remodeling the release of apoptogenic factors from mit. also depends on the remodeling of mit. cristae, the inner membrane structure This is particularly true for cytochrome c because ~85% of the molecule is sequestered within the mit. cristae and as a result, its release requires the opening of the cristae. (complete release of cytochrome c was induced by the combination of cristae remodeling, mit. fragmentation, and Bax activation) remodeling of mit. cristae fusion of individual cristae & widening of cristae junctions resulting in the opening into the intermembrane space for cytochrome c release OPA1 existed as both long and short forms which interacted and oligomerized to tighten the cristae junctions. Downregulation of OPA1 or disruption of OPA1 oligomers not only caused mit. fragmentation but also disrupted normal cristae structure OPA1 may govern the remodeling of mit. cristae independently from its regulation of mit. fusion Drp1 has also been implicated in the remodeling and opening of mit. cristae complete release of cytochrome c was induced by the combination of cristae remodeling, mit. fragmentation, and Bax activation inhibition of mit. Fission(Drp1) may block the release of soluble OPA1, which is required for cristae remodeling and cytochrome c release from mit. mit. fragmentation during apoptosis may affect cristae remodeling cristae remodeling is a key event induced by mit. fragmentation for the release of apoptogenic factors, such as cytochrome c. the molecular basis linking these structural changes is largely unknown and deserves further in-depth investigation. Respiration and ATP production Thus, suppression of either fusion or fission results in mit. dysfunction, manifested by a reduction in respiration and ATP production. dynamic balance, and not fission or fusion alone, is important for the maintenance of mit. function and long-term health.