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Fundamental roles for inter-organelle communication in aging

Advances in public health have nearly doubled life expectancy over the last century, but this demographic shift has also changed the landscape of human illness. Today, chronic and age-dependent diseases dominate the leading causes of morbidity and mortality worldwide. Targeting the underlying molecular, genetic and cell biological drivers of the aging process itself appears to be an increasingly viable strategy for developing therapeutics against these diseases of aging. Towards this end, one of the most exciting developments in cell biology over the last decade is the explosion of research into organelle contact sites and related mechanisms of inter-organelle communication. Identification of the molecular mediators of inter-organelle tethering and signaling is now allowing the field to investigate the consequences of aberrant organelle interactions, which frequently seem to correlate with age-onset pathophysiology. This review introduces the major cellular roles for inter-organelle interactions, including the regulation of organelle morphology, the transfer of ions, lipids and other metabolites, and the formation of hubs for nutrient and stress signaling. We explore how these interactions are disrupted in aging and present findings that modulation of inter-organelle communication is a promising avenue for promoting longevity. Through this review, we propose that the maintenance of inter-organelle interactions is a pillar of healthy aging. Learning how to target the cellular mechanisms for sensing and controlling inter-organelle communication is a key next hurdle for geroscience.


Introduction
Over the course of evolution, cellular pathways and processes have become highly compartmentalized. Cell biologists have spent years mapping the molecular constituents, metabolic pathways, and cellular roles of each of these compartments, typically organelles enclosed by membrane barriers. These cataloging approaches provide simple and foundational models of how cells distribute tasks among their many organelles but rely upon isolating and purifying each compartment to study it discretely from its neighbors. Today, fueled by a combination of enabling technologies and recent discoveries in cell biology, the field is now circling back to understand how the many compartments of cells are connected. Through these efforts, new levels of complexity and logic are emerging to explain how interactions between organelles control cell and organismal homeostasis.
Compartmentalization of metabolic processes within organelles enables cells to concentrate substrates and insulate these pathways from undesirable inputs or external interference. Compartmentalization also brings new vulnerabilities, however. Because each organelle network performs unique roles in the cell, they must function cohesively to perform the complete set of tasks required for life. This community structure increases complexity and creates new dependencies, as organelles must efficiently share metabolic substrates and coordinate functional states with distal partners. As appreciation grows for how interconnected this web of organelles really is, indeed we are finding more signs that miscommunication between these compartments is an important source of disease etiology and progression [1]. This interdependency raises fundamental questions in aging and stress biology relating to how aberrant connections between organelles may act as mechanisms of aging and/or therapeutic targets for promoting healthspan and protecting against specific age-onset diseases.
This review focuses primarily on inter-organelle communication associated with organelle contact sites and how these processes relate to aging biology. However, the breadth of cellular processes that involve functional or signaling coordination between organelles independently of contact sites is both fascinating and staggering [2], and we will include some examples that are proving foundational to the aging field. We also limit discussion of organelle-localized sensors activating retrograde signaling and transcriptional responses in the nucleus some of the earliest and best understood examples of inter-organelle communication. These organelle stress response pathways are indeed critical in aging biology and well-covered in other recent reviews. [3][4][5] Finally, many intriguing connections are emerging involving inter-organelle communication and specific age-onset pathophysiologies [6], but here we strive to take more of a geroscience perspective on how inter-organelle communication may serve as a fundamental driver of the aging process itself.

Overview of inter-organelle interactions
Given the immense complexity of the organelle interactome, we will discuss an overview of the established roles and purposes of these interactions and highlight examples with special relevance to aging biology. Broadly these roles include the coordination of organelle dynamics and cellular distributions, flux of ions and metabolites, lipid and membrane transfer, and integration of signal transduction pathways. Several recent in-depth reviews cover more of the specific genetic and molecular mediators of organelle crosstalk in various contexts [6][7][8][9][10].

Structural interactions
Organelles physically interact as a means to spatially coordinate organelle morphology and dynamics [11,12]. The endoplasmic reticulum (ER) plays an especially central role in these dynamics, as ER tubules mediate the fission of endosomes [13,14], mitochondria [15,16] and biomolecular condensates [17]. The role of ER in controlling mitochondrial dynamics is currently the best understood and most complex example of these organelle structural interactions. ER tubules form relatively stable associations with mitochondrial membranes. At a subset of these contact sites, mitochondrial constrictions occur and eventually develop into complete fission events through a mechanism that involves ER-assisted recruitment of core fission and actin-remodeling machineries [15,16,18] (Figure 1A). Evidence is emerging to suggest that ER-mitochondrial contacts are not only facilitating fission, but mitochondrial fusion as well, thus playing a role in coordinating the balance between these opposing processes [19]. Given the many links between aberrant mitochondrial morphology and age-related diseases [20], exploring the role of these contact sites as mitochondrial dynamics 'hotspots' is an exciting avenue moving forward. Importantly, ER influence over mitochondrial homeostasis extends deeper than the outer mitochondrial membrane. Some of the more stable ER-mitochondrial contact sites correlate with internal mitochondrial functions and properties, including membrane potential [19,21,22]. Sites of ER-directed mitochondrial fission are also spatially and functionally associated with mtDNA replication [16,[21][22][23][24][25], thereby linking the ER to control of mitochondrial genome segregation and copy number control ( Figure 1A). Through these contacts, the ER is thus a key mediator of mitochondrial 'structure-function,' and with analogous roles for the ER in mediating fission and distribution of other non-mitochondrial organelle networks just beginning to emerge, it will be exciting to learn if the ER is as deeply involved in their functional outputs as well. As the most expansive and inter-connected organelle network of the cell, the ER is certainly well-positioned to function as a primary sensor and coordinator of organelle dynamics.
Because every organelle network is constantly responding to unique and fluctuating functional demands, the availability of cellular resources must be similarly flexible. The logic is straightforward then for each organelle to have an input into mitochondria, the central bioenergetic and metabolic hub of the cell, as a way to coordinate the availability of energy and metabolic substrates. Consistent with this logic, diverse organelles beyond the ER play roles in controlling mitochondrial dynamics. Lysosomes also form contacts with mitochondria for bi-directional regulation of organelle fission and distribution through a mechanism centering on the Rab7 GTPase [26] ( Figure 1B). More recently, studies have shown a role for vesicles from the trans-Golgi network in mitochondrial dynamics [27]. On these vesicles, ADP-ribosylation factor 1 (Arf1) and PI(4)KIIIβ convert phosphatidylinositol to PI(4)P, which is required for membrane remodeling in the late steps of mitochondrial fission ( Figure 1C). Thus, ER, lysosomes and Golgi networks all seem to play roles in controlling mitochondrial  fission. How many organelles need to cooperate to execute a single fission event is not consistently clear, but one recent study demonstrated that over 80% of mitochondrial fission sites have four-way structural interactions with the ER, lysosomes, and Golgi-derived vesicles [27]. Intriguingly, spatially distinct regions of the mitochondrion may be differentially targeted for certain types of fission events [21]. Fission events in the peripheral regions of tubular mitochondria are linked more strongly to lysosomal contacts and lead to mitophagy, whereas midzone fission events are mediated by the ER. Consistent with the association of ER contacts with replicating nucleoids, these ER-mediated midzone fission events lead to mitochondrial biogenesis [21]. Overall, the increasing number of distinct molecular and organelle players that are important for maintaining mitochondrial dynamics may begin to explain why aberrant mitochondrial morphology is so consistently associated with age-onset disease. Given the apparent sensitivity of mitochondrial shape to the fitness and inputs of so many distal organelles, changes in fission/fusion processes may represent an early warning sign that healthy inter-organelle interactions are beginning to break down.

Transfer of ions and polar metabolites
Ions and polar metabolites are transported between organelles for multiple purposes, including both signaling and fueling metabolic pathways with essential substrates. Reaching back nearly three decades now, the 'quasisynaptic' connections formed between the ER and mitochondria and their utility as a mechanism to funnel calcium are perhaps the best-studied example of inter-organelle communication [28][29][30]. The more recent molecular identification of the mitochondrial calcium uniporter complex [31][32][33][34] and various tethers [35,36] has greatly enhanced genetic tractability to probe questions in this area. The release of ER calcium into the mitochondrial matrix potently stimulates ATP production by coordinately activating several key metabolic enzymes controlling the mitochondrial uptake of fuel and reducing equivalents, the TCA cycle, and the electron transport chain [37]. The transfer of calcium from the ER to mitochondria thus acts as a rheostat-like control for cells to tune mitochondrial bioenergetic tone, but is also stimulated in specific contexts to provide acute bioenergetic support for recovery [38,39]. For example, ER stress promotes ER-mitochondrial coupling, potentially through mechanisms involving calcium leakage from the ER. After recruitment of mitochondria, ER calcium released by the inositol triphosphate receptor (InsP3R) flows directly into the matrix to acutely activate respiration, and this burst of ATP production is important for the resolution and recovery from perturbation to the ER [39] ( Figure 1D). Both too little and too much ER-mitochondrial coupling increases cell dysfunction and death upon ER stress, which is notably a chronic feature of aging [39][40][41]. Thus, cells must keep the number and activities of these contact sites under tight regulatory control for a healthy aging process, and the mechanisms cells use to sense and regulate the amount of these contacts remain a fundamentally important question as the field moves forward. Dysregulation of ER-mitochondrial contacts and/or calcium signaling is now extensively implicated in diverse age-onset pathophysiologies and diseases, including cancer, metabolic disease and neurodegeneration [6,40,[42][43][44]. Despite increasingly ubiquitous correlations with disease, however, more direct roles of ER-mitochondrial contacts and communication in driving cell and organismal aging at foundational levels remain less thoroughly explored. Recent links between lifespan and ER-mitochondrial communication have emerged from experiments in both worms and flies when contact sites or inter-organelle calcium flux are modulated [45,46], altogether suggesting that ER-mitochondrial miscommunication may not simply be a common proximal step in disease pathogenesis, but an underlying driver of age-dependent dysfunction.
A compartmentalized architecture requires cells to connect metabolic modules between organelles [9], and many of the emerging links between organelle communication and the aging process arise from these metabolic connections. Pioneering work in yeast models revealed that mitochondrial declines during aging are at least partly the result of failures in another distal organelle, the vacuole/lysosome [47]. In replicatively aged cells, vacuoles lose the ability to maintain low pH as a relatively early step in the aging process. Surprisingly, the loss of canonical degradative functions of the vacuole is not the trigger for mitochondrial dysfunction; rather, failure in vacuolar compartmentalization of amino acids is the cause. In particular, free cysteine serves as a mitochondrial toxin in aging cells by interfering with iron and redox homeostasis when vacuolar storage fails and cysteine levels increase in other compartments [47,48] ( Figure 1E). The damage triggered by defects in vacuolar/lysosomal compartmentalization is not limited to mitochondria either. Although the mechanism of amino acid toxicity appears quite different, failures in lysosomal amino acid storage are also recently linked to stress and dysfunction in the ER [49] ( Figure 1E). These results perhaps suggest that early, age-dependent defects in the lysosomal compartment initiate a much broader cascade of inter-compartmental metabolic dysfunction.
While the vacuole-mitochondrial interaction in yeast typifies how a failure in one organelle can instigate dysfunction in another, there are also contrasting scenarios where different compartments provide metabolic resiliency. For example, peroxisomes and mitochondria can function in redundant or complementary roles in regulating cellular redox control and fatty acid oxidation [50][51][52]. Supporting this concept, simultaneous inhibition of mitochondrial fission and fusion factors extends lifespan in C. elegans through a mechanism that depends entirely upon a functional peroxisomal network [53]. Metabolic profiling of these long-lived, mitochondrial 'adynamic' worms revealed that fatty acid oxidation capacities and very long-and long-chain acylcarnitines are both elevated, suggesting that peroxisomal networks confer metabolic adaptability when mitochondria lose their morphological plasticity [53] ( Figure 1F). How mitochondrial and peroxisome networks signal and coordinate this adaptation is not yet clear, but both networks are major players in cellular redox balance and considerable crosstalk can occur via reactive oxygen species (ROS) signaling and oxidative modifications [52]. Furthermore, many of the calcium transport machineries of the ER and mitochondria are redox sensitive [54], suggesting that local ROS production could serve to convey the functional status of several key metabolic hubs. Given the many complex links between redox biology and aging, the use of reactive oxygen species as a line of communication between metabolic organelles is likely an exciting area of future investigation.

Lipid and membrane transfer
Inter-organelle interactions are critical for effectively distributing hydrophobic lipid and membrane components between compartments and throughout the cell. While membrane fusion events necessary for the vesicular traffic of the endomembrane system promote sharing of membrane lipids, there have long been clues suggesting that non-vesicular modes of lipid transport are physiologically important. Such examples include neurons, in which many subcellular compartments are distant from the Golgi complex [55], and mitochondrial networks with membranes that do not fuse with the endomembrane system. In fact, Vance's pioneering work to understand how ER and mitochondria coordinate phospholipid biosynthesis was the first discovery of specific cellular functions occurring at organelle contact sites [56]. Today, we appreciate that the combination of lipid transfer proteins (LTPs) with the close proximity of membranes at organelle contact sites does facilitate a fusion-independent, high-volume and precisely targeted flux of lipids between organelles [57,58]. Recent examples have demonstrated that cells coordinate both pathways of lipid transport, i.e. vesicular trafficking and nonvesicular LTP-mediated flux [58][59][60][61], but how cells manage these lipid dynamics at a broader systems level is a complex and important question moving forward. These mechanisms represent the essential pathways by which cells regulate the varied compositions of organelle membranes, and modulation of lipid compositions is an emerging node for the regulation of lifespan [62].
Mitochondria-associated ER membranes (MAMs) serve as a classic example of an inter-organelle pathway mediating membrane homeostasis. MAMs are contact sites between ER and mitochondria that are so tightly linked that they can be biochemically fractionated and purified [56], and at these sites the subsequent enzymes of a lipid biosynthetic pathway are split between each organelle. Phosphatidylserine synthase is enriched in the ER at MAMs and promotes efficient transfer of new phosphatidylserine from the ER to the mitochondrial membrane [58,63]. After phosphatidylserine enters the mitochondrial membrane, phosphatidylserine decarboxylase converts it into phosphatidylethanolamine [63][64][65] (Figure 1G). Demonstrating the potential for cells to tune lipid trafficking by regulating contact site formation, genetic enrichment of phosphatidylserine synthase at MAMs or increased tethering between ER and mitochondria enhances the flux of phosphatidylserine to mitochondria [58,63]. Recent studies have revealed more nuanced dynamics of this spatial organization by showing that altered metabolic demands can promote ER-targeting of phosphatidylserine decarboxylase, which was previously thought to be purely mitochondrial [66]. The fates of the phosphatidylethanolamine produced in each compartment seem to be unique [66,67], adding another layer of regulation for cells to control membrane lipid distributions. Intriguingly, phosphatidylethanolamine levels decline in aged eukaryotes, and both exogenous administration and enhanced production through phosphatidylserine decarboxylase overexpression enhances autophagy and extends lifespan in yeast and Drosophila [68].
While the example above focuses on lipids involved in membrane composition, organelle interactions also regulate neutral lipids involved in energy homeostasis [69]. Lipid droplets store triacylglycerol and sterol esters, and a number of enzymes exploit the unique properties of lipid droplets and their monolayer membrane to both sense and regulate the amount and composition of lipids stored within [70]. A recent burst of research has revealed that lipid droplets are functionally and spatially heterogeneous, and excitingly, modulating specific subpopulations of lipid droplets prolongs organismal lifespan [71][72][73]. Specifically, some lipid droplets are spatially segregated to the cell periphery, where they form contacts with plasma membrane and are linked with lipid trafficking mechanisms distinct from those found in perinuclear lipid droplets. Sorting nexin Snazarus is enriched at the lipid droplet-plasma membrane contact, and expanding this peripheral lipid droplet pool through Snazarus overexpression enhances starvation resistance and lifespan in Drosophila [72] (Figure 1H). While the mechanisms of lifespan extension in this model remain unclear, enriching this peripheral lipid droplet pool is sufficient to shift the lipidome composition towards unsaturated species correlated with longevity [62,72]. Given the fundamental links between lifespan and cell/organismal energy homeostasis [62,74,75], the mechanisms by which cells sense, store and mobilize lipid fuels is an active area of research that will likely become even more fertile for identifying therapeutic targets relevant to aging biology.
During fluctuations in nutrient conditions, cells spatially remodel their interior compartments in ways that facilitate the flux of lipid substrates between lipid droplets, sites of oxidation, i.e. peroxisomes and mitochondria, and sites of membrane synthesis [76,77]. Metabolic plasticity depends upon efficient and precise mobilization of lipids, which ensures that substrate is available for either membrane synthesis or energy production at the right time and place in the cell [78]. By spatially linking lipid droplets with specific target organelles during periods of high metabolic demand, cells also avoid the lipotoxic effects of free fatty acids in the cytosol. Towards this end, mitochondria can directly associate with lipid droplets through multiple mechanisms, tending to involve conserved perilipin proteins [69,79,80]. Intriguingly, this association with lipid droplets defines a unique, functional subpopulation of mitochondria [79]. While tempting to expect that these droplet-associated mitochondria are simply poised for delivery and β-oxidation of fatty acids, instead this mitochondrial subpopulation is supporting lipid droplet expansion via local, pyruvate-fueled ATP production for triacylglyceride synthesis [79]. Notably, these studies were performed in adipose cells, and how interactions between lipid droplets and mitochondria affect mitochondrial specialization may be specific to cell type and context. During fasting or nutrient scarcity, cells can also mobilize fatty acid fuels rapidly and in bulk via lipophagy, and in some cases lipid droplet-lysosomal contacts facilitate the direct delivery of storage lipids to lysosomal lipases without requiring the autophagosome intermediates [81]. In contrast, other studies reveal that cytoplasmic lipases are responsible for release and delivery of fatty acids to mitochondria during periods of acute nutrient restriction, while autophagic machineries instead recycle organelle membranes to refill lipid droplets with substrate [76] (Figure 1I). A final example of inter-organelle lipid transfer illustrates how rewiring these lipid flux pathways can link directly to longevity. Specifically, the genetic activation of a lysosomal lipase, LIPL-4, extends lifespan in C. elegans by releasing lipid signals that trigger both a transcriptional response via nuclear hormone receptors and a shift in mitochondrial metabolic functions [82,83] (Figure 1J). In this case, oleoylethanolamine is the lipid signal and the specificity of the signaling is mediated in part by cytosolic lipid chaperones. Overall, this wide variety of cellular strategies to orient and facilitate lipid mobilization highlights an exciting frontier in understanding the contextual importance of remodeling organelle interactions to adapt to stress and metabolic fluctuations.
Finally, one of the most basic and vital roles for inter-organelle interactions is the de novo generation of organelles. Relating back to the central role of the ER in mediating structural dynamics between organelle networks, it is also a nexus of membrane biogenesis. Lipid droplets are a key example of this process, born solely from ER tubules [84][85][86]. De novo lipid droplet formation begins between the ER membrane bilayer with formation of an oil lens by local triacylglycerol synthesis [70]. As the lens fills with triacylglycerols, it buds outwards from the ER membrane and recruits specific lipid droplet-associated proteins before release into the cytoplasm, generating the unique monolayer membrane described above. Other organelles require more complex coordination. Peroxisomes arise from two compartments, the ER and mitochondria [87] (Figure 1K), while autophagosome biogenesis is linked to several organelles, likely depending on the spatial or environmental context [7,88,89]. The ER is a primary contributor to autophagosome initiation and maturation, and ER contact sites with other organelles appear to be hotspots for initiating autophagosome formation. For example, starvation causes ER-resident mediators of autophagy formation, syntaxin-17 (STX17) and autophagy-related 14 (ATG14), to concentrate at MAMs, where they recruit autophagy-related 5 (ATG5) and initiate formation of the omegasome, the membrane responsible for autophagosome formation [89,90] (Figure 1L). Intriguingly, autophagosomes formed at ER-mitochondrial contacts seem to recruit the necessary machineries for mitochondrial turnover via mitophagy [91]. Golgi vesicles, endosomes, and lipid droplets have all been shown to regulate autophagosome initiation or maturation, suggesting cells may promote accurate autophagic targeting by using the same organelle to scaffold the autophagosome [90]. Given the strong associations between autophagy, metabolism, and age-related disease, understanding the role of inter-organelle communication in mediating subcellular turnover will be an exciting area of investigation moving forward.

Organelle interfaces as signaling hubs
Finally, we increasingly understand that organelle contact sites serve as central hubs for signal transduction pathways. Nutrient-signaling pathways possess the deepest ties to the aging process, and from these pathways the mechanistic target of rapamycin (mTOR) is emerging as a major reader and regulator of inter-organelle communication. The first direct link between mTOR and contact sites was the finding that mTORC2 presence at ER-mitochondrial contact sites is dynamic and dependent on growth factor signaling [92]. Furthermore, mTORC2-Akt signaling coordinates contact site functions by phosphorylating distinct mediators of ER-mitochondrial tethering, signaling and metabolism ( Figure 1M). While it remains technically challenging to differentiate mTORC2 activity at the ER-mitochondrial contact site from its function at other cell locales, this model provides an example of a mechanism to couple metabolic sensing with local regulation of contact site activities. Similarly, pharmacological studies with rapamycin suggested that mTORC1 function may also be linked to the positioning and coupling of ER and mitochondria, though it is not yet clear if mTORC1 directly regulates contact site residents [93].
Of course, the organelles most canonically linked to mTOR regulation are lysosomes, and a series of studies between yeast and mammalian models have linked contact site-dependent regulation of vacuolar/lysosomal membrane compositions to the activation state of mTOR. In yeast, sterol transport proteins, specifically Ltc1 and Ltc3/4, act as tethers at membrane contact sites between ER-vacuole and ER-plasma membrane, respectively [94]. In an example of cells exploiting contact sites to spatially segregate signaling pathways, Ltc1 at the ER-vacuole regulates mTORC1 activation, while Ltc3/4 at the plasma membrane contact is linked instead to mTORC2. At each of these contact sites, the sterol transport function of these proteins promotes the local formation of sterol-enriched membrane microdomains that are important for concentrating mTOR signaling partners, including components of the Ragulator complex upstream of mTORC1 [94]. In the case of Ltc1, which interacts with vacuole membrane protein Vac8 at ER-vacuole contacts, these vacuolar microdomains play a critical role in driving recruitment of GTPase activating proteins to ultimately inactivate the Ragulator complex and reduce mTORC1 activation [94] (Figure 1N). Sterols are also linked to mTORC signaling in mammalian cells, where oxysterol binding protein serves the lipid transfer role analogous to Ltc1, and VAMP-associated protein (VAP)-A/VAP-B act to tether ER and lysosomes [95]. In mammalian cells, high levels of lysosomal cholesterol are sufficient to activate mTORC1. In the context of Niemann-Pick lysosomal storage disease, where lysosomal cholesterol levels are chronically elevated, mTORC1 is constitutively hyperactive. Furthermore, the hyperactive mTORC1 signaling in this context plays a central role in promoting mitochondrial dysfunction and neurodegenerative phenotypes associated with Niemann-Pick disease [95,96]. Given how tightly linked mTOR, mitochondrial dysfunction, and neurodegeneration are with aging, these results suggest an intriguing model where aberrant inter-organelle sterol trafficking disrupts core nutrient-signaling pathways to exacerbate the cascade of cellular pathophysiologies correlated with aging [97].
Similar to nutrient signaling, stress response pathways are important for a number of pro-longevity interventions, and organelle contact sites are also emerging as hubs for launching and integrating these responses. In addition to the InsP3R-mediated links between ER stress and mitochondria described above, canonical mediators of the ER stress response, termed the unfolded protein response (UPR), also localize to ER-mitochondrial contact sites. Inositol-requiring enzyme (IRE)1 is a dominant player in the UPR, normally responding to unfolded proteins in the ER lumen by oligomerizing and activating splicing and/or digestion of ER-associated mRNAs [3]. Independent of its enzymatic activity, however, IRE1 promotes mitochondrial bioenergetics by acting as a scaffold for the InsP3R at ER-mitochondrial contacts. Localization at ER-mitochondrial contacts also allows IRE1 to receive inputs back from mitochondria. Specifically, mitochondrial ubiquitin ligase MITOL ubiquitylates IRE1 at contact sites, attenuating UPR activation by preventing IRE1-mediated splicing of XBP1 mRNA [98] (Figure 1O). This crosstalk is physiologically important in contexts of unresolvable or excessive ER stress, upon which cells switch from a survival response to actively inducing apoptosis. ER stress promotes apoptosis partly by overloading mitochondria with calcium, and it thus it appears that IRE1 presence at the ER-mitochondrial contact sites is a key mechanism to control this survival switch. A second major UPR mediator, PKR-like endoplasmic reticulum kinase (PERK), is also linked to this survival switch [99,100]. Also found at contact sites, PERK strengthens ER-mitochondrial tethering by mechanisms that are not yet clear, but in doing so propagates apoptosis-associated redox signaling between organelles [101]. The extent to which the ER responds to mitochondrial stress is less clear, but in one example, disruption of mitochondrial dynamics via Mitofusin 2 ablation triggers widespread ER disruption and enhanced UPR activation [99] ( Figure 1O). The ER response in this context appears maladaptive, as inhibition of PERK in Mitofusin 2-depleted cells surprisingly rescues some aspects of mitochondrial dysfunction. Lastly, there are no reports of the third major UPR branch, activating transcription factor (ATF)-6, at contact sites, but a recent study in C. elegans revealed a link between atf-6 and lifespan through mechanisms involving mitochondria [46]. Specifically, aging animals appear to possess hyperactivated UPR signaling through ATF-6, which leads to impaired ER calcium release through the InsP3R and downstream defects in mitochondrial function. Taken together, these stress response pathways illustrate how organelle contact sites can act both as avenues for stressed organelles to receive support or transmit damage to neighbors. Understanding how to target the signaling at contact sites and shift the balance towards beneficial outcomes will be an exciting pursuit in geroscience going forward.

Conclusions and future directions
Our perspective on physiology is intrinsically linked to anatomy. At the subcellular level, however, organelle anatomy is complicated by the fact that very little is fixed in place. Organelles are motile and their morphologies and distributions within the cell are highly dynamic. Thus, the mechanisms by which these organelle compartments signal and connect to each other are similarly dynamic. The discoveries of diverse molecular signaling and tethering mechanisms linking organelles together is therefore opening entirely new perspectives and frontiers in cell physiology. As the manipulation of inter-organelle interactions grows increasingly tractable, the field is shifting towards understanding the cellular and organismal roles of these interactions. The observation that a growing proportion of chronic, age-related diseases features signs of aberrant inter-organelle communication compels a deeper look into whether these lines of communication might play a more fundamental role in the aging process.
The number and nature of inter-organelle interactions are highly diverse, but a few key themes emerge ( Figure 1). First, in multiple cases organelle morphology and dynamics rely on specific interactions with other organelles. Age-related deficiencies in one network can therefore drive improper shape, positioning and quality control of another at this structural level. Because the ER appears to direct fission of multiple other organelles, it may play an especially important role in maintaining organelle architecture during aging. Second, organelle interactions enable the precise and efficient transfer of ions and polar metabolites. In cases such as ER-mitochondrial calcium flux, the ions serve as a mechanism for one organelle to recruit and regulate the metabolic functions of another. Alternatively, age-onset defects in lysosomal amino acid storage drive declines in multiple other organelles, revealing the importance of metabolite targeting and compartmentalization during aging. Third, organelle contact sites mediate precise, high-volume flux of hydrophobic lipids and membranes. By coupling lipid droplets with different partners, cells can control the pathways and fates of their lipid fuels.
Artificially rewiring lipid storage and release is proving to extend lifespan in multiple contexts, which is exciting considering that modulation of energy metabolism is central to multiple conserved longevity paradigms. Finally, inter-organelle contact sites provide local hubs for integration of signaling pathways. Local nutrient flux at organelle contact sites fine-tune the activation of nutrient sensors, such as mTORC1. And in examples involving ER and mitochondria, ER stress sensors directly regulate the mediators of inter-organelle signaling, providing a pathway to promote functional resilience in cells by enabling a compromised organelle to receive support.
Inter-organelle interactions appear to play roles in the aging process that are perhaps as broad as traditional "hallmarks of aging" [97] (Figure 2). In this current framework, interconnectivity between the hallmarks is a central theme. For example, age-dependent damage at the molecular level (i.e. the genome, proteome and the networks that maintain them) is the primary instigator of declines that grow in scale, from mitochondria to cells, tissues, and ultimately systemic physiology [97]. Consistent with this theme of damage crossing scales, organelle communication plays a key role in how dysfunction is transmitted within and between cells. For instance, the ER is a key link between molecular damage and mitochondrial function, as proteostasis defects or metabolic dysfunction in the ER leads to remodeling of its contacts and signaling with mitochondria [40,46,102] (Figure 2A,B). Mitochondrial function is also highly dependent on lysosomes, not only due to lysosomal roles in mitophagic turnover, but also because age-onset impairment of lysosomal amino acid compartmentalization causes pronounced toxicity to mitochondria [47,48]. Similarly, lysosomal homeostasis depends on contacts with ER and mitochondria, and age-related declines in lysosomal processes can cascade into impaired nutrient signaling through direct links to mTOR [26,[94][95][96] (Figure 2C). Both mitochondrial and lysosomal/autophagosomal function are also intimately linked to another hallmark, cell senescence [103,104]. Given the key roles of inter-organelle contacts in maintaining mitochondrial and lysosomal health, studies into the roles of inter-organelle communication in cell senescence are an intriguing future pursuit ( Figure 2D). Similarly, mitochondrial fission/fusion dynamics and function are important determinants for stem cell fate decisions and proliferative capacities, leading to intriguing suggestions that age-related alterations in ER-mitochondrial sites may underlie stem cell exhaustion [105] (Figure 2E). Experimental links between altered organelle communication and systemic pathology are also emerging. Altered trafficking of lipids along the chain from lipid droplets to lysosomes and mitochondria can promote altered intercellular lipid signaling and cell nonautonomous impacts on aging [76,83,106,107] (Figure 2F). Similarly, intercellular propagation of ER stress signals within the liver promotes ER dysfunction and insulin resistance [108]. In obesity, excess nutrients and cell nonautonomous signaling promote chronic hepatic ER stress, which triggers a vicious cycle of aberrant MAM formation, mitochondrial dysfunction and systemic metabolic defects [40] (Figure 2G).
Moving forward, many exciting challenges await both in our basic understanding of how cells sense and regulate the dynamics of communication between organelle pairs and how these dynamics impact the aging process. The microscopy needed for quantitative analyses of contact site dynamics continues to evolve and propel the field forward. Trade-offs between spatial and temporal resolution and the limited ability to visualize multiple cell structures have traditionally limited advancement, but these barriers continue to fall [109][110][111]. One of the key hurdles is our limited ability to simultaneously visualize both a physical contact site and aspects of its function or composition. Towards this end, emerging mass spectrometry imaging technologies will also begin to provide additional information on the molecular mediators and metabolic activity at these tiny organelle interfaces [112,113]. Already, the ability to subclassify individual organelles based on their contact sitepartners [21,79] and/or location in the cell [72] has revealed unexpected functional heterogeneity within an organelle network. As we continue to identify the mediators of this intra-organelle heterogeneity, new avenues open for precision-targeting of maladaptive cell biological and metabolic processes. Given the growing body of evidence for altered inter-organelle interactions in aging and age-dependent disease, experimentally assigning causality between specific inter-organelle interactions will unlock their potential as therapeutic targets.

Perspectives
• Aging is characterized by a progressive inability to maintain homeostasis and resilience.
Several modes of inter-organelle communication are emerging as key mechanisms by which cells adapt to changing or stressful conditions and are thus likely to provide new therapeutic targets for the diseases of aging.
• Remodeling of multiple organelle contact sites is increasingly associated with age-onset diseases, but evidence that aberrant inter-organelle communication is a systemic driver of aging is just emerging.
• As the technology for quantitatively assessing both the physical and functional interactions between organelles continues expanding our insights into how cells regulate the dynamics of inter-organelle communication, we can experimentally manipulate these processes to test for causal roles in aging and age-related disease.