Organelle morphology and positioning orchestrate physiological and disease-associated processes

Abstract

In cells, organelles are distributed nonrandomly to regulate cells’ physiological and disease-associated processes. Based on their morphology, position within the cell, and contacts with other organelles, they exert different biological functions. Endo-lysosomes are critical cell metabolism and nutrient-sensing regulators modulating cell growth and cellular adaptation in response to nutrient availability. Their spatial distribution is intimately linked to their function. In this review, we will discuss the role of endolysosomes under physiological conditions and in the context of cancer progression, with a special focus on their morphology, the molecular mechanisms determining their subcellular position, and the contacts they form with other organelles. We aim to highlight the relationship between cell architecture and cell function and its impact on maintaining organismal homeostasis.

Introduction

To preserve cell homeostasis, subcellular compartments are tightly regulated in size, shape, and number, and their activities must be coordinated in time and space. Vital cell processes are distributed among different organelles, requiring a complex cellular organization that brings them close, often through dynamic inter-organelle contact sites [1]. Among multiple organelles with respective positions and functions, endolysosomes are membrane-bound motile organelles classically known for their degradative functions. However, several recent studies report noncanonical functions for endolysosomes such as serving as metabolic signaling centers, facilitating cell migration, cell adhesion, plasma membrane repair, and antigen presentation. Recent evidence suggests that several of these cellular functions are linked to the specific spatial localization of endolysosomes in cells. We will first describe the molecular mechanisms driving the endolysosomal subcellular distribution, and then we will highlight studies exploring the implications of dynamic endolysosomal distribution in diverse biological processes. We will focus on the global subcellular changes in endolysosomes that exist in physiological processes such as cell migration and nutrient sensing and in the disease context of cancer progression and metastasis (Figure 1). Organelle morphology and spatial distribution are essential in maintaining cellular homeostasis in a rapidly changing environment.

Figure 1. Subcellular morphological changes in physiological processes and in cancer progression.

Top insets: schematic model showing the mechanism of endolysosome positioning in mammalian cells. Arrows show the direction of molecular motor movement along the microtubules. The main panel depicts the impact of various stimuli on cell ultrastructure and on its function in different biological processes. In yellow = factors promoting a given state, in green = mTORC1 activation, in red = mTORC1 inhibition, blue lines = microtubules, red dots = lysosomes, blue dots = late endosomes. Abbreviations. PM: plasma membrane; ECM: extracellular matrix.

Molecular mechanisms of endolysosomal positioning

To exert their function, endolysosomes are repositioned within the cell via motor proteins in response to environmental cues, such as the presence of growth factors or a lack of nutrients. Endolysosomes are transported along the microtubules towards the nucleus (retrograde transport) by dynein and towards the cell periphery (anterograde transport) by kinesin. Several molecular players are known to regulate both plus-end (kinesin) and minus-end-directed (dynein) movement on microtubule tracks (Figure 1, top inserts). First, small GTPases (Arl8b, Rab7) are recruited to the endolysosome membrane [2,3], promoting the recruitment of their effector proteins (RUFY3, JIP4, RILP, FYCO1, PLEKHM1, SKIP), which are often adapters for the motor proteins (kinesin, dynein), resulting in directed organelle movement. BORC complex [4,5] is essential for recruiting Arl8b onto the lysosomal membrane, which binds its downstream effectors, SKIP [6] and RUFY3 [7*,8], which further interact with kinesin and dynein motors, respectively. Small GTPase Rab7 recruits FYCO1 [9] and RILP [10], which bind kinesin and dynein, respectively, and thus regulate the spatial distribution of endolysosomes. In mammalian cells, lysosome docking to the cell periphery near the plasma membrane is known to be mediated by a tripartite complex between small GTPase Rab3a, synaptotagmin-like-4, and nonmuscle myosin heavy chain IIA [11]. Lysosome shuttling has been extensively studied [12–14], yet only recently have we started to uncover how the endo-lysosome position determines their function and regulates important cellular functions.

Cellular metabolism

Cellular metabolism is one of all living organisms’ most fundamental physiological processes. Catabolic reactions break down macromolecules to provide energy and building blocks, while anabolic reactions assemble macromolecules from available cellular resources [15]. Cellular metabolism is fine-tuned according to the cellular demands. Multiple studies have shown that lysosomes and late-endosomes play a central role in regulating cellular metabolism, and the process is conserved from yeast to mammals [16]. Endo-lysosomes degrade both intracellular and extracellular cargo and act as nutrient reservoirs. They either retain or release metabolites into the cytoplasm via membrane transporters in response to the cellular need [16]. Lysosomes gained much attention as metabolic centers of the cell after the discovery of Mechanistic Target of Rapamycin Complex 1 (mTORC1), the master growth regulator, being recruited and activated on the lysosomal membranes in response to amino acid and cholesterol availability [17]. Interestingly, lysosome-associated mTORC1 activity is also tightly linked to the organelle’s position. Nutrient-rich conditions lead to lysosome dispersion to the cell periphery and active mTORC1 signaling, further promoting anabolism and cell growth. However, nutrient depletion causes lysosome clustering in the perinuclear region, thus inactivating mTORC1 signaling, further leading to the upregulation of autophagy in these cells [18,19]. The molecular details of mTORC1 complex localization and activity have already been reviewed extensively [20–22].

However, the physiological implications of the association between mTORC1 activity and lysosome positioning in different cellular contexts are in the spotlight. For example, a recent study gives mechanistic details of how PI-3P (Phosphatidylinositol 3-phosphate) formed on lysosomes under nutrient-rich conditions promotes the recruitment of plus-end directed motor proteins, resulting in peripheral lysosomal distribution. Lysosomes stationed near the cell membrane bring mTORC1 close to nutrient signaling complexes, thus increasing its activity [23]. The peripheral distribution of lysosomes also regulates mTORC2 and AKT signaling activity in response to growth factor signaling at the plasma membrane. Genetically depleting myrlysin, a BORC complex subunit, and KIF1B-KIF5B motor proteins leads to lysosome clustering in cells and subsequent spatial separation of mTOR complexes and their corresponding growth factor signaling complexes at the plasma membrane [24]. Under hypoxia, cells undergo acidification as they adapt metabolically, resulting in the peripheral distribution of lysosomes. However, mTORC1 signaling activity is diminished under hypoxic conditions and affects the circadian clock circuit [25]. mTORC1 complex signaling regulation is diverse and has implications for both health and disease. Further dissection of its regulation in various cellular contexts will inform the behavior of cells in pathological conditions such as cancer.

Lysosomes also maintain cellular homeostasis by forming inter-organelle contacts, which are essential for exchanging amino acids, lipids, inorganic ions, fatty acids, and nucleotides. A recent study has shown that nutrient starvation repositions lysosomes towards the nucleus, where they fuse with mature autophagosomes [7*]. In addition, starvation increases lysosome size and promotes mitochondria-lysosome contacts. The mitochondria-lysosome contacts act as sites for metabolite exchange and regulate mitochondrial quality control [26,27]. The lysosome’s positioning, function, and interaction with other organelles are adjusted according to the nutrient status of the cell. Cholesterol depletion/sequestration increases lysosome contact with the endoplasmic reticulum (ER), further affecting ER morphology as it forms extended tubules [28]. ER-lysosome contacts exchange cholesterol, which is facilitated by mTORC1 signaling [29]. Lysosome positioning is intimately connected to mTORC1 signaling and to the inter-organelle contacts formed. It will be interesting to look at common molecular regulators of these events to gain further insight into the tight regulation of cellular metabolism.

Cell migration and polarity

Evolutionarily conserved, cell migration is a central and fundamental mechanism that modulates the development and functioning of unicellular and multicellular organisms. Cell migration shapes, for example, embryonic development, tissue morphogenesis, angiogenesis, wound healing, immune surveillance, and cancer metastasis. Cells can migrate individually or collectively, guided by chemical cues and physical forces. Mechanisms of cell migration involve the reorganization of the cell’s internal structure, the formation of protrusions like lamellipodia and filopodia, and interaction with the extracellular matrix [30]. Spatiotemporal dynamics of cytoskeletal elements, focal adhesions, integrins, and intracellular organelles such as endosomes, Golgi complexes, and nuclei, are essential for efficient cell migration [31*]. In this context, endolysosome redistribution during this process has gained particular attention as it performs crucial regulatory roles. During cell migration, late-endosomes carrying the p14-MP1 (LAMTOR2/3) complex move to the cell periphery of migrating fibroblasts, where they transiently interact with focal adhesions and facilitate the dissociation of IQGAP1, leading to their turnover [32]. Upon inflammation, dendritic cells undergo directed single-cell migration after exposure to chemokines, during which lysosomes polarize behind the nucleus in the direction opposite to that of migration. At the rear end of a migrating dendritic cell, lysosomes locally release calcium for myosin IIA activity and actin remodeling [33]. More recently, lysosome polarization to the cell periphery has been implicated in regulating collective cell migration by facilitating leader cell emergence. Leader cell formation is a critical aspect of collective cell migration, where a subset of cells assumes a pioneering role in guiding the movement of the collective. Leader cells often display dynamic protrusions such as lamellipodia and filopodia, that sense the extracellular environment, allowing them to respond to chemotactic gradients and physical barriers [34]. Peripheral lysosome polarization is crucial for lamellipodial extension in the leader cells during the collective movement of epithelia during wound healing [35*]. Lysosome positioning is also vital for the invasion of the vulval tissue by the anchor cell of C. elegans. Lysosomes polarize towards the invasion site and are transiently exocytosed, providing a membrane to the invasive protrusion [36]. During this nematode’s development, the intestinal epithelial cells undergo apicobasal polarization of the cytoplasmic components. While nuclei, early endosomes, late endosomes, and lysosomes become apically polarized, lysosome-related organelles/gut granules (LROs), yolk platelets and lipid droplets are basally localized. The basal localization of LROs is independent of the PAR polarity pathway but is regulated by gut granule-resident ABCG transporters and Rab GTPase signaling pathways [37]. Inter-organelle contacts formed by lysosomes are yet to be explored in the context of cell migration. Separate studies have shown that lysosome redistribution facilitates ER tubule extension into the cell periphery and that the dynamics of ER tubules contribute to focal adhesion assembly and disassembly, respectively [28,38]. It will be interesting to explore the sequence of events in this process. We speculate that lysosomes and ER tubules sequentially redistribute to the cell periphery while maintaining inter-organelle contact. Both organelles facilitate focal adhesion and actin cytoskeleton dynamics at the cell periphery and promote efficient cell migration. Altogether, these recent studies shed light on organelles’ essential role and adaptability to cellular needs (i.e. cell polarity and migration) by changing their spatial organization. Exploring multi-organelle localization and inter-organelle contacts and their conservation across evolution will be an exciting area of research in the future and could inform the controlled positioning of organelles at demand.

Cancer and metastasis

Cancer cells, which strongly exploit cell migration machineries, need to adapt to the changing environment in order to grow and disseminate in distal organs to form metastasis. In response to the environment, cells acquire metabolic and morphological changes driven by differential gene regulation. Not surprisingly, organelle positioning significantly impacts cancer progression. Very recent work demonstrates that cancer cells display a peculiar organelle morphology and positioning that can be phenotypically classified using artificial intelligence models [39]. In addition to opening new doors that the community might explore in terms of diagnostics, it confirmed how important organelle position and morphology are to the disease progression.

Among all the organelles, the endolysosomal system has shown multiple contributions to cancer, in particular to its migration-dependent invasive nature. Lysosomes regulate cell signaling and they contribute to extracellular matrix (ECM) degradation via the release of proteases through lysosomal exocytosis, promoting tumor invasion and metastasis. To secrete their content, lysosomes are transported along microtubules to the cell periphery by the Arl8b-SKIP-BORC-kinesin complex [4]. Peripherally localized lysosomes are a hallmark of cancer cells, as they are often present in aggressive and metastatic cancers including breast cancer [5], bladder cancer [40*], and melanoma [41,42*]. Peripheral lysosomes fuse with the plasma membrane and secrete various proteases (cathepsins, metalloproteases), resulting in ECM remodeling, cancer cell migration, and invasion [5,41,42*]. Melanoma cells show lower levels of Rab7a as compared to melanocytes, promoting the lysosomal peripheral localization [41]. Peripheral lysosomes are more mobile and resistant to damage than the highly acidic and immobile perinuclear pool [7*,41,43]. Lysosome motility is further restricted by the contacts of lysosomes with the ER [44]. Analysis of breast cancer patient samples showed that high ARL8b expression strongly correlates with poor patient prognosis [5]. Importantly, cells that survive radiation therapy reposition their lysosomes toward the cell periphery, increasing their invasiveness. Arl8b silencing decreased lysosomal exocytosis, invasion, and lung metastasis upon irradiation in a mouse xenograft model [5]. Forcing the perinuclear lysosomal localization in metastatic melanoma cells decreased lysosome secretion, ECM degradation, and cell invasion in a zebrafish model, suggesting an essential role of lysosome positioning in tumorigenesis [42*].

Similar to lysosomes, late endosomes display changes along the cancer progression. There are more late endosomes (CD63, Rab7-positive) in the tumor cells compared to the adjacent healthy tissue, and they are localized further away from the nucleus in the tumor cells [45]. Perinuclear endosomes fuse with lysosomes in a Homotypic fusion and Protein Sorting (HOPS) complex-dependent manner and are degraded, in contrast to the peripheral endosomes, which undergo exocytosis [46]. In hepatocellular carcinoma (HCC), changes in late endosome distribution elevate secretion levels of exosomes [45], which are known to promote premetastatic niche formation and thus metastasis outgrowth [47]. Disruption of the HOPS complex inhibits endosome-lysosome fusion and significantly increases exosome secretion [46].

Furthermore, lysosome contact sites with other organelles can regulate their morphology. For example, Rab7-mediated lysosome-mitochondria membrane contacts were shown to mark the sites for mitochondrial fission [27]. Interestingly, many RAS (Rat Sarcoma) gene-driven cancers have fragmented mitochondria, which are mainly driven by phospho-Drp1 (Dynamin-related protein 1) activity downstream of RAS signaling. Drp1 deletion leads to elongated mitochondria and reduces tumor volume in vivo in a xenograft model of human cutaneous SCC (Squamous Cell Carcinoma) cells [48]. Interestingly, fused mitochondria hinder efficient cell division [49], and they have been shown to induce cellular senescence [50], a tumor suppressive process that prevents cancer cell proliferation. In summary, cancer invasiveness and progression appear to be tightly orchestrated by a peripheral localization of the endolysosomal machinery and the contacts formed with other organelles.

Conclusion

Organelle positioning is tightly controlled within cells; it reacts and adapts to extracellular signals or to the inner cell’s needs. Organelles are often interconnected, forming homotypic or heterotypic interactions in order to exchange their content or to convey a signal. But how the organelles communicate and what are the exact implications of the inter-organelle contacts and the upstream regulators remain partially understood [51]. Such insights will inform how these shape physiological and pathological processes such as cancer progression. While preclinical and clinical studies provide valuable information to predict the tumor behavior or response to treatment, they lack ultrastructural resolution and therefore cannot inform on the morphological and spatial status of individual organelles. This is, however, required to establish causal links between organelle spatial distribution and disease progression in realistic preclinical settings. Recent advances in intravital imaging will help to address this issue in the future [52] and interrogate organelle position and dynamics at will in experimental models. Recently, landmark technological breakthroughs now allow for the study of the whole-cell organelle landscape using volume electron microscopy and multi-spectral imaging, which allows for studying several organelles’ morphology as well as interactions between organelles that are of great importance for the cell’s intrinsic properties [53,54]. This will provide an added value to classically used approaches (transcriptomics, proteomics, and gene knockouts), which usually probe the function of specific genes or proteins at a given time. In addition to improving in vivo organelle imaging, the emergence of artificial intelligence provides means for probing the spatio-temporal distribution of multiple organelles. Interestingly, such developments allow us to distinguish healthy cells from cancer cells based on their organelle spatial distribution, which could help in the future to achieve phenotypic classification of tissue samples [39], bringing hope to the improvement of cancer diagnostics.

Various diseases, including cancer, show differences in their subcellular architecture when compared to their healthy tissue counterparts. Although we do not fully understand what controls their increased expression levels, many proteins have now been described to control the subcellular morphological patterns, which can either i) allow the development of new diagnostic strategies [39] or ii) serve as interesting therapeutic targets. For example, peripheral lysosome positioning emerged as a hallmark of highly invasive types of cancer, but the molecular controls highly depend on the tissue of origin. Indeed, the RUFY3 protein, which promotes perinuclear lysosome localization, is, for example, often downregulated in many cancers, and its absence is connected to a bad prognosis in pancreatic and head and neck cancer. On the contrary, in liver cancer, RUFY3 downregulation is favorable for patient survival [55]. This suggests that a multifactor process is at play, which depends on the cell intrinsic properties, the genetic context, and the environment, accounting for the dramatic differences based on the tissue of origin. A proper understanding of the cell architecture can help identify proteins driving the subcellular organization. If combined with small-molecule inhibitor, screens, one could envision efficient repositioning of organelles within the cells to prevent disease progression. Several studies have been made along this line with the use of genetic tools, where lysosome repositioning curtails cancer-associated malignant phenotypes, which serve as a proof of principle for future studies [5,40*,42*]. In conclusion, further analyses of causal links between organelle position and important cellular functions will provide groundwork to better understand physiological and pathological processes and to provide new means to counteract disease progression.

Data availability

No data was used for the research described in the article.