Splitting up to heal: Mitochondrial shape regulates signaling for focal membrane repair

Abstract

Mitochondria are central to the health of eukaryotic cells. While commonly known for their bioenergetic role, mitochondria also function as signaling organelles that regulate cell stress responses capable of restoring homeostasis or leading the stressed cell to eventual death. Damage to the plasma membrane is a potentially fatal stressor incurred by all cells. Repairing plasma membrane damage requires cells to mount a rapid and localized response to injury. Accumulating evidence has identified a role for mitochondria as an important facilitator of this acute and localized repair response. However, as mitochondria are organized in a cell-wide, interconnected network, it is unclear how they collectively sense and respond to a focal injury. Here we will discuss how mitochondrial shape change is an integral part of this localized repair response. Mitochondrial fragmentation spatially restricts beneficial repair signaling, enabling a localized response to focal injury. Conservation of mitochondrial fragmentation in response to cell and tissue damage across species demonstrates that this is a universal pro-survival adaptation to injury and suggests that mitochondrial fragmentation may provide cells a mechanism to facilitate localized signaling in contexts beyond repairing plasma membrane injury.

Introduction

In order to maintain their healthy state, cells rely on adaptive and reparative responses elicited by cellular stress. Physical trauma, chemical exposure, and pathogens are all stressors that can acutely injure cells by damaging the plasma membrane. This damage exposes the cytoplasm to the extracellular environment, disrupting the tightly regulated ionic and biomolecular equilibrium maintained in the cytoplasm (1, 2). Influx of extracellular calcium represents a ubiquitous trigger for initiating plasma membrane repair (3); however, chronically elevated cytosolic calcium can lead to cell death (2, 4). To restore homeostasis and ensure cell survival, it is essential that plasma membrane damage is rapidly repaired. Injury to the plasma membrane can vary in size and location. Thus, successful repair requires the amplitude of the repair response to be proportional to size of the injury, and it requires the repair response to be polarized toward the site of injury. The repair response is mediated by proteins and organelles that work collectively to stabilize and reshape the wounded plasma membrane, reorganize the cortical cytoskeleton, and restore the ionic balance (3, 5). While our understanding of the machinery of plasma membrane repair continues to grow, we lack full understanding of how cells sense the scale of damage and direct the repair response toward the site of injury. Here, we will discuss how mitochondria generate signaling to enable a localized plasma membrane repair response.

Plasma membrane injury: Mitochondria send a message

Vesicle fusion and membrane trafficking have long been recognized for their role in repairing injured plasma membrane (1, 6–8). However, recent studies have added mitochondria as another critical component of the plasma membrane repair response (9–13). In healthy cells, normal mitochondrial function is maintained by mitochondria constantly undergoing the opposing processes of fusion and fragmentation (14, 15) (Figure 1). However, exposure of cells to a variety of stressors alters mitochondrial structure, modifying mitochondrial function and affecting their ability to respond to stress (14, 16) (Figure 1). While mitochondria remain best known for their role in energy production, they are increasingly being recognized for their role in signaling through the regulation or production of molecules including calcium, lipids, metabolites, and reactive oxygen species (ROS) (17–22). Production of many of these signals is regulated by the TCA cycle, highlighting the integration of the metabolic and signaling roles for mitochondria (17, 18).

Figure 1: Mitochondrial fragmentation is a beneficial adaptation to cell stress.

(A) Fusion and fragmentation are opposing structural modifications. Fusion and fragmentation machinery is shown in boxes that represent the dotted area above. (B) Mitochondrial fragmentation increases with cell stress. In response to increasing stress, transient and local fragmentation enables adaptation to stress; however, excess or chronic fragmentation leads to cell death. (C) Mitochondrial function is linked to morphology. Green, yellow, and red colors refer to the magnitude of stress shown in B.

Acute cell stress induced by plasma membrane injury can result in cell death due to influx of calcium from the extracellular space; however, presence of extracellular calcium is required for repair (1, 2, 7). Mitochondria respond to injury through rapid calcium uptake mediated by the mitochondrial calcium uniporter (MCU) complex (11), and MCU-mediated calcium uptake is required for successful repair (10, 11). Routine calcium handling in healthy cells allows mitochondria to manipulate cytosolic calcium transients and control cell signaling (23). Thus, mitochondrial calcium uptake in response to injury provides a mechanism for controlling reparative signaling in addition to buffering cytosolic calcium increase. Calcium uptake also stimulates oxidative phosphorylation through the activation of the TCA cycle (24), and therefore regulates ATP production. However, acute increase in de novo ATP production is not required for plasma membrane repair, which occurs within tens of seconds (1, 3, 10, 11). Instead, increased ATP production following cell injury facilitates slower repair processes such as axonal regeneration in damaged neurons (25). While de novo production of ATP is not needed for rapid membrane resealing, stimulation of oxidative phosphorylation by calcium uptake also generates ROS as a metabolic by-product (26).

While historically viewed solely as a detriment to cell health, ROS are now appreciated for their role in physiological signaling that is needed for normal cell and organismal function (19, 27). Unlike chronic and cell-wide ROS production, which is associated with cell damage and death, transient or local ROS production represents a homeostatic signaling mechanism (19, 27). Mitochondrial redox signaling, activated by calcium uptake, enables plasma membrane repair through the redox-mediated activation of the RhoA GTPase (11, 13). Redox activation of RhoA is independent of conventional guanine nucleotide exchange factor (GEF) regulation (28). This direct RhoA activation enables local F-actin accumulation at the site of injury (11, 13) – a process required for wound closure (29, 30). Thus, increased ROS production by mitochondria promotes plasma membrane repair while limiting the potentially detrimental effects of oxidative-stress induced damage. While this highlights the benefit of acute mitochondrial ROS signaling for wound repair, the question of how mitochondrial ROS signaling is localized to the injury site remains.

Plasma membrane repair: The dynamic mitochondria

Mitochondria are organized into a cell-wide, interconnected network that relies on continual fusion and fragmentation to maintain function in healthy and stressed cells (16). This cell-wide mitochondrial reticulum lends itself well to regulation of global processes such as metabolism or gene expression (15). However, the existence of a global mitochondrial network poses a challenge for generating local responses, such as the response needed to repair focal injury to the plasma membrane (3).

Mitochondrial form and function

The mitochondrial network is highly dynamic and trafficking of mitochondria has been proposed to localize mitochondrial signaling (31). In cells such as skeletal myofibers and neurons that extend over large distances, mitochondrial trafficking contributes to generating a localized response to injury by accumulating mitochondria near the site of damage (12, 32, 33). Defects in this response are observed in muscle diseases (34, 35). However, in other cells, such as fibroblasts and myoblasts, mitochondria do not accumulate at the site of injury (11). In these cells, a different strategy is needed to localize mitochondrial signaling. To this end, cells take advantage of the same processes reserved for maintenance of mitochondrial network function - fusion and fragmentation (15, 16). In uninjured cells, mitochondrial fusion enables the dilution of potentially deleterious DNA mutations and allows sharing constituents such as proteins and lipids (15, 16). In contrast, fragmentation spatially segregates dysfunctional mitochondria, allowing them to be marked for removal by mitophagy (14, 15). In this way, balancing fusion and fragmentation maintains mitochondrial health, and dysregulation of these processes can alter the mitochondrial network and result in disease (16, 36, 37).

Cells employ a dedicated machinery to precisely control mitochondrial fusion and fragmentation (15, 36) (Figure 1). Mitochondrial fusion is regulated by the Mitofusins (Mfn) and Optic-atrophy 1 (Opa1), which facilitate fusion of the outer and inner mitochondrial membranes, respectively (38). Fragmentation, on the other hand, requires Dynamin related protein 1 (Drp1) (39). Being a cytosolic protein, Drp1 must be recruited to mitochondria, a process that is controlled by post-translational modification in response to upstream signals such as increase in cytosolic calcium or ROS (15, 40, 41). Drp1 recruitment is facilitated by adaptor proteins, Fis1, Mff, MiD49 and MiD51, which are localized to the outer mitochondrial membrane (15, 36). Sites of mitochondrial fragmentation are marked by close contact between mitochondria and the endoplasmic reticulum (ER) (42, 43). Here, F-actin mediates constriction of mitochondria – a step that can precede Drp1-dependent fragmentation (15, 42).

Chronic or cell-wide mitochondrial fragmentation is detrimental as it contributes to mitochondrial dysfunction, results in excessive ROS production, and is associated with degenerative disease (15, 16). However, transient mitochondrial fragmentation is an adaptive stress response required for normal cell function (Figure 1). Mitochondrial fragmentation plays important roles in fundamental processes such as cell division and differentiation, and is a master regulator of normal immune responses (15, 16, 44). Thus, the outcomes associated with mitochondrial fragmentation are diverse and context-dependent. Recent studies have identified that in the context of injury, the ability of mitochondria to undergo local fragmentation is required to mount an efficient repair response and ensure survival (9, 45, 46).

Mitochondria divide to conquer repair

Plasma membrane injury causes cytosolic and mitochondrial calcium to increase throughout the cell; however, mitochondrial redox signaling that is dependent on calcium uptake is localized near the wound site (11). How do mitochondria achieve this localized signaling in the context of global calcium increase? An important observation in this regard is that mitochondria undergo rapid (within seconds) and spatially restricted fragmentation following injury in a calcium-dependent manner (9, 46). While calcium induced mitochondrial fragmentation can lead to cell death (2), injury-triggered mitochondrial fragmentation occurs in a manner such that only the injury-proximal mitochondria fragment, leaving the remaining mitochondrial network intact (9, 46). This suggests that precise regulation of mitochondrial fragmentation after injury may ensure cell survival.

The machinery for regulating mitochondrial dynamics is conserved across species with mitochondrial fragmentation driven by Drp1 and fusion driven by Mfns (47). Cytosolic calcium increase is a trigger for Drp1-dependent mitochondrial fragmentation and calcium is required for fragmentation in response to injury (9, 41, 46). While Drp1 is required for injury-triggered mitochondrial fragmentation in mammalian cells and Drosophila epidermis, mitochondrial fragmentation occurs independently of Drp1 after injury in C. elegans epithelium (9, 45, 46). In mammalian cells, lack of Drp1 or its adaptor protein MiD49 both lead to a hyper-fused mitochondrial network that fails to fragment and prevents successful repair (9). Similarly, lack of Drp1 and its adaptor Fis1 prevents wound closure in Drosophila (45). This supports a requirement of Drp1 and its adaptors in maintaining the health of organisms and may contribute to the mild to lethal phenotypes caused by their absence (47). In tissues such as skeletal muscle, where mechanical activity can induce membrane damage, lack of Drp1 or its adaptor MiD49 leads to increased muscle damage, leakage of muscle enzymes (e.g. creatine kinase) in the serum, as well as muscle degeneration (48, 49). These findings collectively attest to the in vivo relevance of regulating mitochondrial shape for effective repair and survival of injured cells.

While it is clear that biochemical signaling mediated by calcium is required for mitochondrial fragmentation, mitochondrial fragmentation can also be triggered by mechanical force (50). Given the physical changes to the plasma membrane and cytoskeleton that are associated with injury (11, 29, 51, 52), mechanical signaling may also contribute to local mitochondrial fragmentation during membrane repair. Combined with the effect of an injury-induced cytosolic calcium gradient, mechanical signaling proportional to the size of injury could ensure that mitochondrial fragmentation is localized to the injury-proximal region. In support of this, mitochondrial fragmentation in response to injury of C. elegans epithelium was found to depend on Miro-1, a mitochondria-associated protein that has a calcium binding motif and is involved in microtubule-based mitochondrial motility and shape transitions (46, 53). The sites of mitochondrial fragmentation are dictated in part by association with the ER (15, 43). Interestingly, ER also undergoes acute and localized fragmentation following plasma membrane injury and excessive ER fragmentation is associated with poor repair (4). The role of ER fragmentation, and how it may cooperate with mitochondrial fragmentation, has not been examined. However, given the functional coupling of these organelles in calcium handling and their parallel roles in membrane repair, it is likely that their response to cell injury is linked.

Divided mitochondria put the squeeze on wound closure

Although the precise mechanism regulating injury-induced mitochondrial fragmentation requires further elucidation, it is clear that fragmentation is required for successful repair (9, 45, 46). Fragmentation may promote repair by physically isolating injury-proximal mitochondria from the rest of the interconnected network. Unlike apoptosis, homeostatic fragmentation of mitochondria does not abolish its membrane potential - the driving force of calcium uptake (54, 55). In the context of membrane repair, where mitochondrial redox signaling depends on calcium uptake, the rate, in addition to the magnitude, of calcium import by mitochondria are both critical factors that determine the spatial and temporal profile of ROS production. Fragmentation may allow for increasing the initial rate of calcium uptake compared to tubular mitochondria upon stimulation (56). In response to injury, fragmented mitochondria that are proximal to the injury site accumulate significantly more calcium than the distal, tubular mitochondria (9). This is similar to the process of long-term potentiation in neurons, where mitochondrial fragmentation allows greater magnitude and duration of calcium increase (57). These observations suggest that fragmentation itself may facilitate acute elevations in mitochondrial calcium response. This is in contrast to chronic mitochondrial fragmentation, which reduces the rate and capacity of mitochondrial calcium uptake (58). Thus, the relationship between mitochondrial shape and calcium homeostasis is context-specific, and further work is needed to clarify how acute fragmentation alters the characteristics of calcium handling by mitochondria.

In the context of focal injury, fragmented mitochondria at the site of injury show greater, and sustained calcium increase, while distal mitochondria experience only transient and lower calcium increase (9, 11). This profile of calcium increase generates polarity with respect to the site of injury, and preventing mitochondrial fragmentation by Drp1 knockout significantly decreases the calcium load experienced by injury-proximal mitochondria (9, 45) (Figure 2). This may be explained by the observation that calcium diffuses more rapidly through electrically connected mitochondria than between physically separated mitochondria. This explanation is supported by overexpression of Drp1 or its receptor Fis1, which increase mitochondrial fragmentation and prevent rapid propagation of calcium between neighboring mitochondria (54, 56). Increased mitochondrial fragmentation in mammalian cells, using the alternate approach of blocking fusion through Mfn knockout, had no effect on mitochondrial calcium dynamics or membrane repair (9). Similarly, knockout of the mitochondrial fusion regulators in C. elegans did not compromise epithelial repair, and even showed a mild enhancement of repair (46). Together, the above findings indicate that mitochondrial fragmentation can facilitate enhanced focal calcium signaling by injury-proximal mitochondria. This allows for spatial polarization of the repair response, enabling localized signaling needed for repair. Lack of fragmentation allows mitochondrial calcium to spread throughout the mitochondrial network, preventing buildup of calcium in the injury-proximal mitochondria, ultimately leading to the loss of a polarized repair response (Figure 2).

Figure 2: Mitochondrial fragmentation generates a polarized repair response.

Focal plasma membrane (PM) injury results in influx of extracellular calcium (blue). Mitochondria take up this calcium and generate reactive oxygen species (ROS, green) that are necessary for repair. In the absence of mitochondrial fragmentation, diffusion of calcium throughout the matrix of the mitochondrial network results in insufficient local buildup of ROS, leading to poor focal repair response. However, local fragmentation of mitochondria physically separates the injury-proximal mitochondria from the distal, tubular mitochondrial network. This allows for greater and sustained redox signaling at the injury site (indicated by white arrows), which mediates the localized accumulation of F-actin (brown filaments) to successfully repair the focal wound.

Mitochondrial calcium uptake contributes to repair by increased ROS production, leading to F-actin accumulation that is needed to close the wound - a role conserved across cell types and species (11, 13, 45, 59, 60). By shaping the profile of mitochondrial calcium uptake, fragmentation regulates mitochondrial ROS production (Figure 2). Mitochondrial fragmentation is also a common response to injury in human cells (9, 46, 61). Despite the species and tissue-specific specialization of mechanisms that regulate fragmentation, in each case mitochondrial fragmentation provides a mechanism to localize the repair response by sensing damage and spatially restricting repair signaling to accumulate F-actin only at the site of injury.

Conclusion

Mitochondria respond to cellular stress and can modify their function to restore homeostasis. Unlike cell-wide or chronic stressors, plasma membrane injury is an acute form of stress that requires a localized response. Mitochondria respond to plasma membrane injury by generating beneficial redox signaling. Given their organization in a cell-wide, interconnected network, mitochondria must adapt in order to generate the localized signaling that is needed for repair. While trafficking of mitochondria has been proposed as a method of localized redox signaling, accumulation of mitochondria at the injury site is not required for successful repair. Instead, mitochondria undergo local fragmentation, which modifies calcium handling, ROS production, and enables a localized repair response. Mitochondrial fragmentation has been proposed as a universal stress response to cytotoxicity, and mitochondrial fragmentation occurs in response to a wide variety of stressors. The mitochondrial response to cell injury supports a role for mitochondrial fragmentation in environmental stress sensing. However, rather than leading to cell death, mitochondrial fragmentation in response to plasma membrane injury is a pro-survival adaptation that allows cells to mount a targeted repair response. Conservation of mitochondrial fragmentation as a beneficial response to cell and tissue injury across species indicates that this is a ubiquitous adaptation to damage that is required for survival. This suggests that fragmentation may be a general mechanism employed by mitochondria to facilitate local signaling in contexts beyond repair from cell damage. Future studies are needed to detail the machinery and mechanisms that regulate acute and localized mitochondrial fragmentation, and how this is different from broadly detrimental cell-wide and chronic fragmentation. These studies must continue across species to identify important tissue and organism-specific specialization in the machinery that regulates mitochondrial shape. Such studies to further elucidate the link between mitochondrial shape and signaling would reveal how defects in mitochondrial fragmentation lead to excess cell stress and damage observed in degenerative disease.

Perspectives.

• Importance of the field

The role of mitochondria in cells ranges from meeting energy demands to mediating cellular stress responses. Insights into the mechanisms that regulate these complementary roles of mitochondria are needed to understand their intersection and how disruption of normal mitochondrial function causes degenerative disease.

• Summary of current thinking

Mitochondria are dynamic organelles that undergo fusion and fragmentation. While excessive fusion or fragmentation of mitochondria contributes to cell stress, proper regulation of these opposing processes helps to maintain mitochondrial health and promotes the ability of cells to respond to stress and injury.

• Comment on future direction

While the link between chronic mitochondrial fragmentation and environmental stressors or disease is well described, further studies are needed to address how acute changes in mitochondrial shape are regulated and how these transient shape changes promote the ability of cells to respond to stress and damage.