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Lysosomes and plasma membrane repair
CHAPTER ONE
Lysosomes and plasma membrane repair Matthias Corrottea, Thiago Castro-Gomesb,*
a Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, United States b Department of Parasitology, Federal University of Minas Gerais, Belo Horizonte, Brazil *Corresponding author: e-mail address: [email protected]
The authors dedicate this manuscript to professor Norma Windsor Andrews from the University of Maryland-USA who leaded the team that made most of the discoveries described in this book chapter.
Contents 1. Plasma membrane repair—A Ca2 +-dependent physiological cellular response of fundamental importance 2. Lysosomes—Not just dealing with the trash 3. Linking exocytosis and endocytosis—The need to remove damage from the plasma membrane 4. Understanding the cell machinery that repairs the plasma membrane by endocytosis 5. Lysosome positioning, dynamics and exocytosis during plasma membrane repair 6. Lysosomal enzymes as mediators of plasma membrane repair References
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Abstract The ability of repairing damages on the plasma membrane is crucial for cell survival. When damaged, eukaryotic cells are able to recover plasma membrane integrity within a few seconds, thus avoiding cytoplasm leakage and cell death. The process is driven by the influx of extracellular calcium which triggers a multitude of intracellular effects that participate in the process of plasma membrane resealing. One of the landmarks of plasma membrane repair is the triggering of intracellular vesicles recruitment and their exocytosis at damage sites. Since lysosomes are able to respond to calcium influx and that some of the lysosomal enzymes exocytosed after plasma membrane permeabilization are essential to restore cell integrity, these organelles have emerged as essential for the maintenance of plasma membrane integrity. Here we summarize the scientific evidences showing the involvement of lysosomes in plasma membrane repair that allowed researchers to propose a totally different function for this famous organelle. Current Topics in Membranes, Volume 84 ISSN 1063-5823 https://doi.org/10.1016/bs.ctm.2019.08.001
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2019 Elsevier Inc. All rights reserved.
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1. Plasma membrane repair—A Ca2+-dependent physiological cellular response of fundamental importance Maintenance of cell homeostasis, the process by which cells balance intra and extracellular compositions, is a necessary function for cell survival and is dependent on the PM’s selective permeability. Therefore, restoration of plasma membrane (PM) integrity after injury is of crucial importance, notably for cells under constant physical stress such as muscle, skin and gut cells or cells subjected to bacterial attacks during infection (Bhakdi & Tranum-Jensen, 1988; McNeil & Khakee, 1992). Upon injury, nucleated cells have been shown to reseal their PM within 30 s, thus avoiding leakage of cytoplasm, ion efflux and cell death. Early studies have shown that sea urchin eggs injured in the presence of Ca2+ are able to rapidly reseal their PM, maintaining their capacity to be fertilized and normally divide after injury recovery (PMR) (Heilbrunn, 1956). By contrast, when eggs were placed in Ca2+-free water instead, injury led to cytosol leakage and cell death, demonstrating the need for Ca2+ for proper PM resealing (Heilbrunn, 1956). The importance of Ca2+ in this process is certainly the main consensus among researchers and has been the central discovery in the field of plasma membrane repair (PMR), guiding the majority of advances made in the following decades. Fig. 1 shows that nucleated cells are able to reseal the plasma membrane, excluding propidium iodide marker when in the presence of Ca2+ and after plasma membrane injury by both mechanical (glass beads) and molecular (SLO pores) driven wounding. Ca2+ influx through the wounded membrane can lead to several cell responses, from cytoskeleton reorganization to exocytosis of intracellular vesicles. Imaging experiments of cells undergoing injury recovery led to the detection of rapid vesicle recruitment and fusion to the site of PM wounding, which became another landmark result in the field of PMR (Bi, Alderton, & Steinhardt, 1995; Miyake & McNeil, 1995). The importance of intracellular vesicles for a proper PM repair (PMR) has been known since the early 1990s and led to the idea that intracellular vesicles facilitates PM resealing by reducing plasma membrane tension. While Ca2+-dependent exocytosis of intracellular vesicles had been mostly described for specialized secretory cells, it was later discovered
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Fig. 1 Fluorescence microscopy images of propidium iodide (PI) staining illustrating the permeability of the PM in wounded cells. NRK cells were wounded by contact with glass beads (A) or by permeabilization with the pore-forming toxin SLO (B). Glass beads lead to larger mechanical wounding but affect a smaller fraction of the cell population when compared to SLO (capable of forming a 30 nm wide pore through the PM) as indicated by the number of PI-positive cells in calcium-free conditions. In both cases, the presence of calcium in the medium leads to extensive cell resealing and exclusion of PI. Bars ¼ 50 μm. Adapted from Corrotte, M., Castro-Gomes, T., Koushik, A. B., & Andrews, N. W. (2015). Approaches for plasma membrane wounding and assessment of lysosome-mediated repair responses. In: Methods in cell biology (Vol. 126, pp. 139–158). https://doi.org/ 10.1016/bs.mcb.2014.11.009.
(between the late 1990s and the early aughts) that conventional lysosomes were actually the major population of Ca2+-sensitive intracellular vesicles able to fuse with the PM and playing a central role in PMR in various cell types such as fibroblasts and epithelial cells (Rodrı´guez, Webster, Ortego, & Andrews, 1997). Fig. 2A shows the lumenal epitope of the lysosomal protein Lamp-1 exposed on the surface of cells mechanically wounded, indicating that lysosomes undergo exocytosis when cells are injured. Fig. 2B shows the activity of the lysosomal protease cathepsin B secreted in cell supernatant after streptolysin-o (SLO) permeabilization.
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Fig. 2 (A) The lumenal domain of Lamp-1 is exposed on the surface of wounded cells. NRK, 3T3, L6E9, and CHO cells were wounded by physically scratching cell monolayers in the presence of Texas-Red dextran, and labeled at 4 °C with anti-Lamp-1 lumenal domain mAbs. Red staining corresponds to the influx of dextran into the cytosol of wounded cells, green to the punctate anti-Lamp-1 surface labeling, and blue to DAPI staining of nuclei. Arrows point to wounded cells positive for surface Lamp-1; arrowheads point to nonwounded cells in the same microscopic field. The interrupted line arrow indicates where scratching occurred. (B) Detection of lysosomal enzymes secreted after SLO permeabilization. Kinetics of degradation of a substrate specific for cathepsin B by the supernatant of NRK cells collected from untreated control cells, or 5 s after permeabilization with SLO. Panel A: Adapted from Reddy, A., Caler, E. V., & Andrews, N. W. (2001). Plasma membrane repair is mediated by Ca(2 +)-regulated exocytosis of lysosomes. Cell, 106(2), 157–169. Retrieved from http://www.ncbi.nlm. nih.gov/pubmed/11511344. Panel B: Adapted from Corrotte, M., Castro-Gomes, T., Koushik, A. B., & Andrews, N. W. (2015). Approaches for plasma membrane wounding and assessment of lysosome-mediated repair responses. In: Methods in cell biology (Vol. 126, pp. 139–158). https://doi.org/10.1016/bs.mcb.2014.11.009.
2. Lysosomes—Not just dealing with the trash Lysosomes are membrane-enclosed acidic organelles typical of eukaryotic cells and are responsible for the intracellular digestion and degradation of a multitude of biomolecules such as proteins, lipids and nucleic acids (Schr€ oder, Wrocklage, Hasilik, & Saftig, 2010). Degradation is achieved by more than 60 hydrolases with different substrate specificity such as proteases, lipases, glucosidases and nucleases, all enzymes contained in the lumen of the organelle (Schr€ oder et al., 2010). Because lysosomes are charged with the degradation of materials taken up by endocytosis, phagocytosis or produced by autophagic pathways, the organelle is crucial for cell homeostasis and defense (Tardieux, Nathanson, & Andrews, 1994). These well-characterized intracellular lysosomal functions coupled to early observations that lysosomal enzymes seemed confined to the lumen of lysosomes, led researchers to consider those organelles only as the final degradative
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compartment for the endocytic pathway. However, this perception has changed in recent decades and today, lysosomes are considered to take part in many other cellular functions and processes such as antigen presentation, cell migration, adhesion, exosome release, cancer metastasis, apoptosis, pathogen invasion and, as mentioned here, PMR (Cabukusta & Neefjes, 2018; Tardieux et al., 1992). Additionally, and even though lysosomes are usually involved in intracellular processes, secreted lysosomal enzymes have also been shown to function extracellularly where they mediate important cellular processes such as bone reabsorption and PMR (Castro-Gomes, Corrotte, Tam, & Andrews, 2016). Indeed, one of the most unexpected and interesting functions of lysosomes is their involvement in PMR, a story initiated in the late 1990s by the group of Dr. Norma Andrews at Yale University. Researchers observed lysosomal fusion at the site of invasion of intracellular parasite Trypanosoma cruzi, a process essential for infection efficiency (Tardieux et al., 1992). Later work demonstrated that parasite entry actually induced PM wounds in host cells, leading to Ca2+ influx and subsequent lysosomal exocytosis. Thus, it was the study of cellular mechanisms of invasion of an intracellular pathogen that allowed researchers to gain insights into the nature of the exocytic vesicles involved in PMR.
3. Linking exocytosis and endocytosis—The need to remove damage from the plasma membrane However, one aspect of PMR remained elusive: how exactly are the lesions resealed or removed and how do cells manage to restore membrane integrity to block cytoplasmic leakage within a few seconds after wounding? Originally, two major hypothesis were trying to answer those questions. First, the “patch hypothesis” stipulated that vesicles would accumulate at the wounding site, fuse together and form a type of clog or patch that would fill the wound, fuse with the PM and avoid continued cell leakage. Second, the “tension release hypothesis” explained that vesicles fusing with the PM during repair would decrease PM tension, thereby facilitating resealing by the thermodynamically-favored partitioning of hydrophobic phospholipid groups away from the aqueous environment, similarly to what happen to erythrocyte ghosts. However, these two hypothesis could not explain how cells recover from pore-induced injuries (such as those made by pore-forming proteins) since, in both cases, a stable protein-lined pore would remain on the PM, maintaining PM permeability (Browne et al., 1999;
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Morgan & Campbell, 1985; Walev et al., 2001). To allow PMR of this type of wounding, the physical pore would have to be removed by a mechanism that had not yet been described, but seemed to involve the same steps as repair of mechanical wounding. Indeed, experiments using the bacterial poreforming toxin streptolysin-O and the complement membrane attack complex showed that both are repaired in a Ca2+-dependent fashion (Idone et al., 2008; Morgan & Campbell, 1985). Interestingly, even though the presence of Ca2+ was a common requirement, the recovery from mechanical wounds and pore-based lesions were originally considered to be two distinct instances governed by different cellular machineries. Reinforcing this distinction was the perception that recovery from mechanical wounds was a fast process while removal of transmembrane pores was assumed to require more time. However, experiments using streptolysin-O showed that the repair of mechanical wounds and resealing after pore-induced injuries have exactly the same fast kinetics (Idone et al., 2008). Of further importance though was the finding that both methods of wounding led to the appearance of Ca2+ and cholesterol-dependent endosomes in the cytosol that were essential to promote PM resealing (Idone et al., 2008). These were the first demonstrations that, along with Ca2+-induced exocytosis of lysosomes, a form of massive endocytosis was necessary to remove wounded membrane and SLO pores from the PM.
4. Understanding the cell machinery that repairs the plasma membrane by endocytosis For a long time the vesicles visualized near the wounding site during PMR were considered to be a patch of vesicles that accumulated close to the damage to fix it. However, more accurate experiments using BSA-gold added extracellularly before wounding showed that those vesicles contained BSA-gold and therefore, were originated from invaginations of the PM during PMR (Fig. 3) (Idone et al., 2008). Corroborating the findings described above, independent experiments by Lariccia and cols. (Lariccia et al., 2011) using electrophysiological techniques demonstrated that the exocytosis triggered by Ca2+ influx in eukaryotic cells was immediately followed by massive endocytosis. Interestingly, the ability to repair damaged membranes using endocytosis does not seem to involve the actin cytoskeleton nor dynamin-dependent vesicular fission. Similarly, classic endocytic mechanisms such as clatrin-mediated endocytosis are also not involved in PMR. On the other hand, Corrotte and cols. showed that PMR involves caveolins, a group of proteins that forms
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Fig. 3 Diagram showing that addition of extracellular tracers identifies the vesicles accumulating next to wound sites as endocytic, and not an exocytic “patch.” (A) In earlier experiments performed without endocytic tracers in the medium, the numerous vesicles observed next to wound sites were thought to undergo Ca2+-dependent homotypic fusion and form a large exocytic patch to reseal the membrane. (B) When wounding experiments were performed in the presence of BSA-gold the vesicles next to wound sites incorporated the extracellular tracer, showing that they originated by endocytosis. These endocytic vesicles subsequently merge generating larger endosomes that join the endocytic pathway, trafficking deeper into the cells and fusing with lysosomes. Adapted from Andrews, N. W., Corrotte, M., & Castro-Gomes, T. (2015). Above the fray: Surface remodeling by secreted lysosomal enzymes leads to endocytosis-mediated plasma membrane repair. Seminars in Cell & Developmental Biology, 45, 10–17. https:// doi.org/10.1016/j.semcdb.2015.09.022.
membrane invaginations named caveolae (Corrotte et al., 2013). Endocytosis of these membrane invaginations was shown to mediate PMR seconds after Ca2+ influx and lysosomal exocytosis. After PMR, endocytosed SLO pores were detected along the endosomal pathway and ended up degraded by lysosomes (Corrotte, Fernandes, Tam, & Andrews, 2012). Interestingly, and confirming the importance of endocytosis for both mechanical and SLO induced wounding, the authors found that caveolar endocytosis was triggered by both, mechanical injury with glass beads and poreformation induced by streptolysin-O. The model presented in Fig. 4 illustrates how caveolar endocytosis could mediate PMR.
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Fig. 4 Model for PM repair mediated by caveolar endocytosis. Permeabilization with transmembrane toxin pores (A) or mechanical wounding (B) triggers Ca2+ influx, exocytosis of lysosomes, release of ASM, and generation of ceramide at the PM outer leaflet, a process that promotes caveolae internalization and fusion. Toxin pores would be removed from the PM by caveolar endocytosis (A), while larger breaches on the lipid bilayer would be gradually constricted and resealed as a results of forces generated on the PM by the intracellular clustering, fusion and internalization of merged/branched caveolar structures (B). Adapted from Corrotte, M., Almeida, P. E., Tam, C., Castro-Gomes, T., Fernandes, M. C., Millis, B. A., et al. (2013). Caveolae internalization repairs wounded cells and muscle fibers. eLife, 2, e00926. https://doi.org/10.7554/eLife.00926.
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Lysosomes and plasma membrane repair
5. Lysosome positioning, dynamics and exocytosis during plasma membrane repair Lysosomes are present everywhere in the cytosol of the cell. In nonpolarized cells these organelles are typically accumulated around the nucleus where they form the “perinuclear cloud” but they are also found throughout the cytoplasm and reach as far as the plasma membrane (Cabukusta & Neefjes, 2018) and studies have shown that they can be found predocked to the PM, possibly to allow a fast response to intracellular Ca2+ elevation after membrane injuries ( Jaiswal, Andrews, & Simon, 2002). The positioning of lysosomes can also change due to several alterations suffered by cells such as acidification, alkalinization or ionic imbalance. Lysosomes travel along microtubules both to cell periphery (anterograde direction) and toward cell nuclei (retrograde direction) in a stop-and-go manner that can be regulated by the cells (Figs. 5 and 6).
Lysosome
MTOC ER Nucleus
Perinuclear lysosomes
Peripheral lysosomes
Cortical actin
Fig. 5 Intracellular distribution of lysosomes. Lysosomes can be found in two intracellular locations: a relatively immobile pool of perinuclear lysosomes clustered around the MTOC and a set of peripheral lysosomes moving fast along microtubules in a stop-and-go manner or are found attached to peripheral actin networks. Adapted from Cabukusta, B., & Neefjes, J. (2018). Mechanisms of lysosomal positioning and movement. Traffic, 19(10), 761–769. https://doi.org/10.1111/tra.12587.
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Lysosome
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B
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D
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Ca2+ BO
Rab7 RILP
ALG2
PI(3,5)P2 TRPML1
SKIP ArI8
JIP4
RC
PI(3)P
Rab7
FYCO1
TMEM55B
Dynein/Dynactin Kinesin-1
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Microtubule
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Fig. 6 Many mechanisms of lysosomal transport. Various mechanisms of lysosomal recruitment of motor proteins are reported. (A) The small GTPase Rab7 recruits effector RILP and the dynein/dynactin motor to lysosomes for minus-end transport. (B) Calcium plays a crucial role in lysosomal positioning. PI(3,5)P2 on lysosomaldelimiting membranes activates the TRPML1 channel to stimulate calcium efflux. Then, the cytosolic calcium sensor ALG2 recruits the dynein/dynactin complex to TRPML1-containing lysosomes. (C) Transmembrane protein TMEM55B interacts with the dynein adaptor JIP4 and facilitates minus-end transport. TMEM55B levels are controlled transcriptionally: depletion of nutrients or cholesterol upregulates TMEM55B transcription via autophagy-associated transcription factors. (D) BORC, a multisubunit complex on the lysosomal membrane, recruits Arl8b to lysosomes. The small GTPase Arl8b interacts with effector SKIP for lysosomal localization of kinesin-1. (E) The kinesin adaptor FYCO1 interacts with active Rab7 and PI(3)P on lysosomal membranes to recruit kinesin-1 to lysosomes. Adapted from Cabukusta, B., & Neefjes, J. (2018). Mechanisms of lysosomal positioning and movement. Traffic, 19(10), 761–769. https://doi.org/ 10.1111/tra.12587.
Lysosome positioning and their ability to respond to Ca2+ influx is crucial for PMR. Lysosomal sensitivity to calcium seems to be mediated through the presence of an isoform of synaptotagmin VII (Syt VII), a ubiquitous Ca2+-sensor, on the membrane of lysosomes. Syt VII is present on lysosomes in mammalian cells but was also observed ubiquitously expressed in non-synaptic intracellular vesicles in Drosophila tissue (Martinez et al., 2000). Synaptotagmins are a family of calcium sensors mostly known for their involvement in the exocytosis of synaptic vesicles. The regulation of lysosomal exocytosis by Syt VII involves specific SNARE complex
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interactions between the lysosomal v-SNARE and VAMP7 and the plasma membrane t-SNARE syntaxin 4 and SNAP-23. During PMR, Ca2+ influx through PM wounds provokes a rapid Ca2+ increase in the cytoplasm which can then bind to the C2A and C2B domains of Syt VII leading to an interaction of the C2 domains with plasma membrane phospholipids and SNARE molecules. This interaction then drives the formation of a fusion pore and the lysosomal content is delivered to the extracellular media (Andrews, 2005). A diagram of this process is shown in Fig. 7.
6. Lysosomal enzymes as mediators of plasma membrane repair These combined findings describe a sequence of events that ultimately lead to PMR, from Ca2+ entry, recruitment and fusion of Ca2+-sensitive exocytic lysosomes to the PM followed by the subsequent endocytosis of caveolae to promote the removal of PM lesions. With the lack of involvement of the cytoskeleton and other classical mediators of endocytosis in the endocytic mechanisms observed during PMR, the molecular mechanism driving the process remained unknown. Furthermore, due to the amount of enzymes that would be delivered extracellularly during PM repair, lysosomes were long perceived as organelles too dangerous to be used in the process. Also, since lysosomal hydrolases are acidic enzymes, researchers usually considered that they likely would be inactivated upon exocytosis into the neutral extracellular environment. However, since PM repair happens within 30 s, it was also reasonable to assume that some amount of enzymatic activity would remain in this time frame and could plausibly take part in the process. Interestingly, the morphology of endosomes generated after PM repair was very similar to those found in J774 macrophages incubated with bacterial sphingomyelinase (Zha, 1998). Acid sphingomyelinase (ASM) is actually a lipase found in the lumen of lysosomes and is able to cleave the abundant PM lipid sphingomyelin to generate ceramide. Ceramide itself had been shown previously to induce membranes to coalescence and form large inward budding structures in cell membranes (Holopainen, Angelova, & Kinnunen, 2000). Expanding onto these findings, further studies of PMR showed that the secretion of lysosomal ASM is essential for PM repair by acting extracellularly to mediate wound removal by endocytosis (Tam et al., 2010). Interestingly, the enzyme is so important for PM repair that its addition in Ca2+-free conditions, thus without lysosomal exocytosis, induces cell
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Fig. 7 (A) Diagram of the general structure of members of the synaptotagmin family. Synaptotagmins are transmembrane proteins, with a short N-terminal luminal domain and a large cytosolic region containing the tandem Ca2+-binding C2 domains. The C2A and C2B domains are responsible for the Ca2+-dependent interactions with t-SNAREs and phospholipids, which are thought to be essential for the regulation of exocytosis. This is the Ca2+ sensor that endows lysosomes with the ability to perform Ca2+dependent exocytosis. (B) Model of the interactions between synaptotagmin and components of the SNARE fusion complex during Ca2+-triggered exocytosis. Cytosolic Ca2+ binds to the C2A and C2B domains of synaptotagmin on the membrane of exocytic vesicles. Ca2+ binding induces the association of synaptotagmin C2 domains with plasma membrane phospholipids and SNARE molecules. Synaptotagmin C2 domains penetrate the membrane bilayer and SNARE four-helix bundles form, driving the formation of a fusion pore. Syt I, the synaptotagmin isoform involved in synaptic vesicle exocytosis, interacts with the v-SNARE synaptobrevin and with the plasma membrane t-SNARES syntaxin 1 and SNAP-25. Syt VII, the synaptotagmin isoform regulating lysosomal exocytosis, interacts with the v-SNARE VAMP7 and with the t-SNARES syntaxin 4 and SNAP-23. Adapted from Andrews, N. W., Chakrabarti, S., There’s more to life than neurotransmission: the regulation of exocytosis by synaptotagmin VII, Trends in Cell Biology 15(11), 2005, 626–631. Epub 2005 Sep 15.
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resealing after SLO wounding. Additionally, cells treated with siRNA against ASM, cells from patients afflicted with Nieman-Pick Type A (NPA) Disease, a condition characterized by a lack of ASM, or the reduction of ASM activity by specific inhibitors all presented with a strong PM repair deficiency. As explained previously, lysosomes carry a number of enzymes of various functions and with affinities to different substrates. Lysosomal proteases of the cathepsin family have been also shown to be regulators of PM repair (Castro-Gomes et al., 2016). Experiments using siRNA silencing and inhibitors of aspartyl and cysteine proteases showed that the two have opposite effects on PM repair mechanisms: cysteine cathepsins improve PM repair, while aspartyl cathepsin D functions as a down regulator of PM repair. Interestingly, extracellular proteolysis was detected shortly after cell wounding and lysosomal exocytosis, and inhibition of this process impairs PM repair. On the other hand, the removal of proteins from the cell surface by unspecific proteases, facilitates cell resealing. Another interesting finding showed that ASM is itself regulated by lysosomal proteases after its secretion in the extracellular media, indicating that the scenario described here is multifactorial and may include both specific and unspecific proteolytic events (Fig. 8). Overall, while further research on the specific functions of lysosomal exocytosis and its numerous enzymes in plasma membrane repair is necessary to refine our understanding of the process, a clearer picture of this complex process has started to emerge in recent years, building on established findings. Lysosomes and their enzymes both play an upstream role and a downstream role in PMR. Upstream, by responding to calcium influx through PM wounds by fast and localized recruitment to and fusion with the PM. And downstream, by allowing the release of a number of enzymes, either directly involved in stimulating endocytosis, an essential step in the removal of PM lesions, through ASM or indirectly by regulating other aspects of the process through various cathepsins. The balance between up-regulation and down-regulation of enzymatic activities and PM repair steps observed in this latest study may be a function of the amount or size of wounding or other factors. But, importantly, it indicates the presence, within lysosomes, of a fine tuned machinery of regulatory effectors, many probably still unknown, which allow for the possibility that lysosomes may be involved in many more cellular functions than originally envisioned, may be especially in light of their capacity to be secreted in response to extracellular stimulus.
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CATB and CATL (Cysteine Proteases) CATD (Aspartyl Proteases)
Extracellular Activity of Lysosomal Hidrolases
ASM (Acid Sphingomyelinase)
5s ASM release /activation ? PM Wounding
15 s
30 s
Cleavage of extracellular proteins facilitates ASM binding to PM Ceramide generation
60 s
90 s
CATD-mediated ASM downregulation Ceramide-mediated CATD activation
PM
SLO Inactive ASM
Ca2+ Influx Exocytosis of Lysosomes
Endocytosis of Lesions PM Resealing
Active ASM Cysteine Proteases PM protein Ceramide Cathepsin D (CATSD) CeramideActivated CATSD
Fig. 8 Proposed mechanism for rapid modulation of PM repair by secreted lysosomal proteases. Ca2+ influx through wounds in the PM rapidly triggers exocytosis of lysosomes, releasing the proteases cathepsins B, L and D along with the lipase ASM. Cathepsins B and L are active extracellularly 5–10 s after PM wounding, while cathepsin D activity appears after a delay of 30–60 s. At early time points, cathepsins B and L (and possibly additional cysteine proteases) may cleave cell surface proteins and contribute to membrane access and/or activation of ASM. ASM hydrolizes sphingomyelin on the outer leaflet of the PM generating ceramide, which promotes lesion endocytosis and PM repair. ASM-generated ceramide may enhance cathepsin D activity, which plays a role in down-regulating ASM. Around 1 min after wounding the PM integrity is restored, and lysosomal hydrolases are no longer active extracellularly. Adapted from Castro-Gomes, T., Corrotte, M., Tam, C., & Andrews, N. W. (2016). Plasma membrane repair is regulated extracellularly by proteases released from lysosomes. PLoS One, 11(3), e0152583. https://doi.org/10.1371/journal.pone.0152583.
References Andrews, N. W. (2005). Membrane resealing: Synaptotagmin VII keeps running the show. Science Signaling, 2005(282) pe19. https://doi.org/10.1126/stke.2822005pe19. Bhakdi, S., & Tranum-Jensen, J. (1988). Damage to cell membranes by pore-forming bacterial cytolysins. Progress in Allergy, 40, 1–43. Retrieved from http://www.ncbi.nlm.nih. gov/pubmed/2451254. Bi, G. Q., Alderton, J. M., & Steinhardt, R. A. (1995). Calcium-regulated exocytosis is required for cell membrane resealing. The Journal of Cell Biology, 131(6), 1747–1758. https://doi.org/10.1083/jcb.131.6.1747. Browne, K. A., Blink, E., Sutton, V. R., Froelich, C. J., Jans, D. A., & Trapani, J. A. (1999). Cytosolic delivery of granzyme B by bacterial toxins: Evidence that endosomal disruption, in addition to transmembrane pore formation, is an important function of perforin. Molecular and Cellular Biology, 19(12), 8604–8615. https://doi.org/10.1128/mcb.19.12.8604.
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Cabukusta, B., & Neefjes, J. (2018). Mechanisms of lysosomal positioning and movement. Traffic, 19(10), 761–769. https://doi.org/10.1111/tra.12587. Castro-Gomes, T., Corrotte, M., Tam, C., & Andrews, N. W. (2016). Plasma membrane repair is regulated extracellularly by proteases released from lysosomes. PLoS One, 11(3), e0152583. https://doi.org/10.1371/journal.pone.0152583. Corrotte, M., Almeida, P. E., Tam, C., Castro-Gomes, T., Fernandes, M. C., Millis, B. A., et al. (2013). Caveolae internalization repairs wounded cells and muscle fibers. eLife, 2, e00926. https://doi.org/10.7554/eLife.00926. Corrotte, M., Fernandes, M. C., Tam, C., & Andrews, N. W. (2012). Toxin pores endocytosed during plasma membrane repair traffic into the lumen of MVBs for degradation. Traffic, 13(3), 483–494. https://doi.org/10.1111/j.1600-0854.2011.01323.x. Heilbrunn, L. V. (1956). The dynamics of living protoplasm. Elsevier. https://doi.org/10.1016/ C2013-0-12539-1. Holopainen, J. M., Angelova, M. I., & Kinnunen, P. K. (2000). Vectorial budding of vesicles by asymmetrical enzymatic formation of ceramide in giant liposomes. Biophysical Journal, 78(2), 830–838. Idone, V., Tam, C., Goss, J. W., Toomre, D., Pypaert, M., & Andrews, N. W. (2008). Repair of injured plasma membrane by rapid Ca2+-dependent endocytosis. The Journal of Cell Biology, 180(5), 905–914. https://doi.org/10.1083/jcb.200708010. Jaiswal, J. K., Andrews, N. W., & Simon, S. M. (2002). Membrane proximal lysosomes are the major vesicles responsible for calcium-dependent exocytosis in nonsecretory cells. The Journal of Cell Biology, 159(4), 625–635. https://doi.org/10.1083/jcb.200208154. Lariccia, V., Fine, M., Magi, S., Lin, M. J., Yaradanakul, A., & Llaguno, M. C. (2011). Massive calcium-activated endocytosis without involvement of classical endocytic proteins. The Journal of General Physiology, 137(1), 111–132. https://doi.org/10.1085/jgp.201010468. Martinez, I., Chakrabarti, S., Hellevik, T., Morehead, J., Fowler, K., & Andrews, N. W. (2000). Synaptotagmin VII regulates Ca(2 +)-dependent exocytosis of lysosomes in fibroblasts. The Journal of Cell Biology, 148(6), 1141–1149. Retrieved from http:// www.ncbi.nlm.nih.gov/pubmed/10725327. McNeil, P. L., & Khakee, R. (1992). Disruptions of muscle fiber plasma membranes. Role in exercise-induced damage. The American Journal of Pathology, 140(5), 1097–1109. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/1374591. Miyake, K., & McNeil, P. L. (1995). Vesicle accumulation and exocytosis at sites of plasma membrane disruption. The Journal of Cell Biology, 131(6), 1737–1745. https://doi.org/ 10.1083/jcb.131.6.1737. Morgan, B. P., & Campbell, A. K. (1985). The recovery of human polymorphonuclear leucocytes from sublytic complement attack is mediated by changes in intracellular free calcium. Biochemical Journal, 231(1), 205–208. https://doi.org/10.1042/bj2310205. Rodrı´guez, A., Webster, P., Ortego, J., & Andrews, N. W. (1997). Lysosomes behave as Ca2+regulated exocytic vesicles in fibroblasts and epithelial cells. The Journal of Cell Biology, 137(1), 93–104. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2139854/ pdf/18982.pdf. Schr€ oder, B. A., Wrocklage, C., Hasilik, A., & Saftig, P. (2010). The proteome of lysosomes. Proteomics, 10(22), 4053–4076. https://doi.org/10.1002/pmic.201000196. Tam, C., Idone, V., Devlin, C., Fernandes, M. C., Flannery, A., He, X., et al. (2010). Exocytosis of acid sphingomyelinase by wounded cells promotes endocytosis and plasma membrane repair. The Journal of Cell Biology, 189(6), 1027–1038. https://doi.org/ 10.1083/jcb.201003053. Tardieux, I., Nathanson, M. H., & Andrews, N. W. (1994). Role in host cell invasion of Trypanosoma cruzi-induced cytosolic-free Ca2+ transients. The Journal of Experimental Medicine, 179(3), 1017–1022. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/ 8113670.
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Tardieux, I., Webster, P., Ravesloot, J., Boron, W., Lunn, J. A., Heuser, J. E., et al. (1992). Lysosome recruitment and fusion are early events required for trypanosome invasion of mammalian cells. Cell, 71(7), 1117–1130. https://doi.org/10.1016/S0092-8674(05) 80061-3. Walev, I., Bhakdi, S. C., Hofmann, F., Djonder, N., Valeva, A., Aktories, K., et al. (2001). Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O. Proceedings of the National Academy of Sciences of the United States of America, 98(6), 3185–3190. https://doi.org/10.1073/pnas.051429498. Zha, X., Pierini, L. M., Leopold, P. L., Skiba, P. J., Tabas, I., & Maxfield, F. R. (1998). Sphingomyelinase treatment induces ATP-independent endocytosis. The Journal of Cell Biology, 140, 39–47. https://doi.org/10.1083/jcb.140.1.39.
Lysosomes and plasma membrane repair Matthias Corrottea, Thiago Castro-Gomesb,*
a Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, United States b Department of Parasitology, Federal University of Minas Gerais, Belo Horizonte, Brazil *Corresponding author: e-mail address: [email protected]
The authors dedicate this manuscript to professor Norma Windsor Andrews from the University of Maryland-USA who leaded the team that made most of the discoveries described in this book chapter.
Contents 1. Plasma membrane repair—A Ca2 +-dependent physiological cellular response of fundamental importance 2. Lysosomes—Not just dealing with the trash 3. Linking exocytosis and endocytosis—The need to remove damage from the plasma membrane 4. Understanding the cell machinery that repairs the plasma membrane by endocytosis 5. Lysosome positioning, dynamics and exocytosis during plasma membrane repair 6. Lysosomal enzymes as mediators of plasma membrane repair References
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Abstract The ability of repairing damages on the plasma membrane is crucial for cell survival. When damaged, eukaryotic cells are able to recover plasma membrane integrity within a few seconds, thus avoiding cytoplasm leakage and cell death. The process is driven by the influx of extracellular calcium which triggers a multitude of intracellular effects that participate in the process of plasma membrane resealing. One of the landmarks of plasma membrane repair is the triggering of intracellular vesicles recruitment and their exocytosis at damage sites. Since lysosomes are able to respond to calcium influx and that some of the lysosomal enzymes exocytosed after plasma membrane permeabilization are essential to restore cell integrity, these organelles have emerged as essential for the maintenance of plasma membrane integrity. Here we summarize the scientific evidences showing the involvement of lysosomes in plasma membrane repair that allowed researchers to propose a totally different function for this famous organelle. Current Topics in Membranes, Volume 84 ISSN 1063-5823 https://doi.org/10.1016/bs.ctm.2019.08.001
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2019 Elsevier Inc. All rights reserved.
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1. Plasma membrane repair—A Ca2+-dependent physiological cellular response of fundamental importance Maintenance of cell homeostasis, the process by which cells balance intra and extracellular compositions, is a necessary function for cell survival and is dependent on the PM’s selective permeability. Therefore, restoration of plasma membrane (PM) integrity after injury is of crucial importance, notably for cells under constant physical stress such as muscle, skin and gut cells or cells subjected to bacterial attacks during infection (Bhakdi & Tranum-Jensen, 1988; McNeil & Khakee, 1992). Upon injury, nucleated cells have been shown to reseal their PM within 30 s, thus avoiding leakage of cytoplasm, ion efflux and cell death. Early studies have shown that sea urchin eggs injured in the presence of Ca2+ are able to rapidly reseal their PM, maintaining their capacity to be fertilized and normally divide after injury recovery (PMR) (Heilbrunn, 1956). By contrast, when eggs were placed in Ca2+-free water instead, injury led to cytosol leakage and cell death, demonstrating the need for Ca2+ for proper PM resealing (Heilbrunn, 1956). The importance of Ca2+ in this process is certainly the main consensus among researchers and has been the central discovery in the field of plasma membrane repair (PMR), guiding the majority of advances made in the following decades. Fig. 1 shows that nucleated cells are able to reseal the plasma membrane, excluding propidium iodide marker when in the presence of Ca2+ and after plasma membrane injury by both mechanical (glass beads) and molecular (SLO pores) driven wounding. Ca2+ influx through the wounded membrane can lead to several cell responses, from cytoskeleton reorganization to exocytosis of intracellular vesicles. Imaging experiments of cells undergoing injury recovery led to the detection of rapid vesicle recruitment and fusion to the site of PM wounding, which became another landmark result in the field of PMR (Bi, Alderton, & Steinhardt, 1995; Miyake & McNeil, 1995). The importance of intracellular vesicles for a proper PM repair (PMR) has been known since the early 1990s and led to the idea that intracellular vesicles facilitates PM resealing by reducing plasma membrane tension. While Ca2+-dependent exocytosis of intracellular vesicles had been mostly described for specialized secretory cells, it was later discovered
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Fig. 1 Fluorescence microscopy images of propidium iodide (PI) staining illustrating the permeability of the PM in wounded cells. NRK cells were wounded by contact with glass beads (A) or by permeabilization with the pore-forming toxin SLO (B). Glass beads lead to larger mechanical wounding but affect a smaller fraction of the cell population when compared to SLO (capable of forming a 30 nm wide pore through the PM) as indicated by the number of PI-positive cells in calcium-free conditions. In both cases, the presence of calcium in the medium leads to extensive cell resealing and exclusion of PI. Bars ¼ 50 μm. Adapted from Corrotte, M., Castro-Gomes, T., Koushik, A. B., & Andrews, N. W. (2015). Approaches for plasma membrane wounding and assessment of lysosome-mediated repair responses. In: Methods in cell biology (Vol. 126, pp. 139–158). https://doi.org/ 10.1016/bs.mcb.2014.11.009.
(between the late 1990s and the early aughts) that conventional lysosomes were actually the major population of Ca2+-sensitive intracellular vesicles able to fuse with the PM and playing a central role in PMR in various cell types such as fibroblasts and epithelial cells (Rodrı´guez, Webster, Ortego, & Andrews, 1997). Fig. 2A shows the lumenal epitope of the lysosomal protein Lamp-1 exposed on the surface of cells mechanically wounded, indicating that lysosomes undergo exocytosis when cells are injured. Fig. 2B shows the activity of the lysosomal protease cathepsin B secreted in cell supernatant after streptolysin-o (SLO) permeabilization.
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Fig. 2 (A) The lumenal domain of Lamp-1 is exposed on the surface of wounded cells. NRK, 3T3, L6E9, and CHO cells were wounded by physically scratching cell monolayers in the presence of Texas-Red dextran, and labeled at 4 °C with anti-Lamp-1 lumenal domain mAbs. Red staining corresponds to the influx of dextran into the cytosol of wounded cells, green to the punctate anti-Lamp-1 surface labeling, and blue to DAPI staining of nuclei. Arrows point to wounded cells positive for surface Lamp-1; arrowheads point to nonwounded cells in the same microscopic field. The interrupted line arrow indicates where scratching occurred. (B) Detection of lysosomal enzymes secreted after SLO permeabilization. Kinetics of degradation of a substrate specific for cathepsin B by the supernatant of NRK cells collected from untreated control cells, or 5 s after permeabilization with SLO. Panel A: Adapted from Reddy, A., Caler, E. V., & Andrews, N. W. (2001). Plasma membrane repair is mediated by Ca(2 +)-regulated exocytosis of lysosomes. Cell, 106(2), 157–169. Retrieved from http://www.ncbi.nlm. nih.gov/pubmed/11511344. Panel B: Adapted from Corrotte, M., Castro-Gomes, T., Koushik, A. B., & Andrews, N. W. (2015). Approaches for plasma membrane wounding and assessment of lysosome-mediated repair responses. In: Methods in cell biology (Vol. 126, pp. 139–158). https://doi.org/10.1016/bs.mcb.2014.11.009.
2. Lysosomes—Not just dealing with the trash Lysosomes are membrane-enclosed acidic organelles typical of eukaryotic cells and are responsible for the intracellular digestion and degradation of a multitude of biomolecules such as proteins, lipids and nucleic acids (Schr€ oder, Wrocklage, Hasilik, & Saftig, 2010). Degradation is achieved by more than 60 hydrolases with different substrate specificity such as proteases, lipases, glucosidases and nucleases, all enzymes contained in the lumen of the organelle (Schr€ oder et al., 2010). Because lysosomes are charged with the degradation of materials taken up by endocytosis, phagocytosis or produced by autophagic pathways, the organelle is crucial for cell homeostasis and defense (Tardieux, Nathanson, & Andrews, 1994). These well-characterized intracellular lysosomal functions coupled to early observations that lysosomal enzymes seemed confined to the lumen of lysosomes, led researchers to consider those organelles only as the final degradative
Lysosomes and plasma membrane repair
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compartment for the endocytic pathway. However, this perception has changed in recent decades and today, lysosomes are considered to take part in many other cellular functions and processes such as antigen presentation, cell migration, adhesion, exosome release, cancer metastasis, apoptosis, pathogen invasion and, as mentioned here, PMR (Cabukusta & Neefjes, 2018; Tardieux et al., 1992). Additionally, and even though lysosomes are usually involved in intracellular processes, secreted lysosomal enzymes have also been shown to function extracellularly where they mediate important cellular processes such as bone reabsorption and PMR (Castro-Gomes, Corrotte, Tam, & Andrews, 2016). Indeed, one of the most unexpected and interesting functions of lysosomes is their involvement in PMR, a story initiated in the late 1990s by the group of Dr. Norma Andrews at Yale University. Researchers observed lysosomal fusion at the site of invasion of intracellular parasite Trypanosoma cruzi, a process essential for infection efficiency (Tardieux et al., 1992). Later work demonstrated that parasite entry actually induced PM wounds in host cells, leading to Ca2+ influx and subsequent lysosomal exocytosis. Thus, it was the study of cellular mechanisms of invasion of an intracellular pathogen that allowed researchers to gain insights into the nature of the exocytic vesicles involved in PMR.
3. Linking exocytosis and endocytosis—The need to remove damage from the plasma membrane However, one aspect of PMR remained elusive: how exactly are the lesions resealed or removed and how do cells manage to restore membrane integrity to block cytoplasmic leakage within a few seconds after wounding? Originally, two major hypothesis were trying to answer those questions. First, the “patch hypothesis” stipulated that vesicles would accumulate at the wounding site, fuse together and form a type of clog or patch that would fill the wound, fuse with the PM and avoid continued cell leakage. Second, the “tension release hypothesis” explained that vesicles fusing with the PM during repair would decrease PM tension, thereby facilitating resealing by the thermodynamically-favored partitioning of hydrophobic phospholipid groups away from the aqueous environment, similarly to what happen to erythrocyte ghosts. However, these two hypothesis could not explain how cells recover from pore-induced injuries (such as those made by pore-forming proteins) since, in both cases, a stable protein-lined pore would remain on the PM, maintaining PM permeability (Browne et al., 1999;
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Morgan & Campbell, 1985; Walev et al., 2001). To allow PMR of this type of wounding, the physical pore would have to be removed by a mechanism that had not yet been described, but seemed to involve the same steps as repair of mechanical wounding. Indeed, experiments using the bacterial poreforming toxin streptolysin-O and the complement membrane attack complex showed that both are repaired in a Ca2+-dependent fashion (Idone et al., 2008; Morgan & Campbell, 1985). Interestingly, even though the presence of Ca2+ was a common requirement, the recovery from mechanical wounds and pore-based lesions were originally considered to be two distinct instances governed by different cellular machineries. Reinforcing this distinction was the perception that recovery from mechanical wounds was a fast process while removal of transmembrane pores was assumed to require more time. However, experiments using streptolysin-O showed that the repair of mechanical wounds and resealing after pore-induced injuries have exactly the same fast kinetics (Idone et al., 2008). Of further importance though was the finding that both methods of wounding led to the appearance of Ca2+ and cholesterol-dependent endosomes in the cytosol that were essential to promote PM resealing (Idone et al., 2008). These were the first demonstrations that, along with Ca2+-induced exocytosis of lysosomes, a form of massive endocytosis was necessary to remove wounded membrane and SLO pores from the PM.
4. Understanding the cell machinery that repairs the plasma membrane by endocytosis For a long time the vesicles visualized near the wounding site during PMR were considered to be a patch of vesicles that accumulated close to the damage to fix it. However, more accurate experiments using BSA-gold added extracellularly before wounding showed that those vesicles contained BSA-gold and therefore, were originated from invaginations of the PM during PMR (Fig. 3) (Idone et al., 2008). Corroborating the findings described above, independent experiments by Lariccia and cols. (Lariccia et al., 2011) using electrophysiological techniques demonstrated that the exocytosis triggered by Ca2+ influx in eukaryotic cells was immediately followed by massive endocytosis. Interestingly, the ability to repair damaged membranes using endocytosis does not seem to involve the actin cytoskeleton nor dynamin-dependent vesicular fission. Similarly, classic endocytic mechanisms such as clatrin-mediated endocytosis are also not involved in PMR. On the other hand, Corrotte and cols. showed that PMR involves caveolins, a group of proteins that forms
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Fig. 3 Diagram showing that addition of extracellular tracers identifies the vesicles accumulating next to wound sites as endocytic, and not an exocytic “patch.” (A) In earlier experiments performed without endocytic tracers in the medium, the numerous vesicles observed next to wound sites were thought to undergo Ca2+-dependent homotypic fusion and form a large exocytic patch to reseal the membrane. (B) When wounding experiments were performed in the presence of BSA-gold the vesicles next to wound sites incorporated the extracellular tracer, showing that they originated by endocytosis. These endocytic vesicles subsequently merge generating larger endosomes that join the endocytic pathway, trafficking deeper into the cells and fusing with lysosomes. Adapted from Andrews, N. W., Corrotte, M., & Castro-Gomes, T. (2015). Above the fray: Surface remodeling by secreted lysosomal enzymes leads to endocytosis-mediated plasma membrane repair. Seminars in Cell & Developmental Biology, 45, 10–17. https:// doi.org/10.1016/j.semcdb.2015.09.022.
membrane invaginations named caveolae (Corrotte et al., 2013). Endocytosis of these membrane invaginations was shown to mediate PMR seconds after Ca2+ influx and lysosomal exocytosis. After PMR, endocytosed SLO pores were detected along the endosomal pathway and ended up degraded by lysosomes (Corrotte, Fernandes, Tam, & Andrews, 2012). Interestingly, and confirming the importance of endocytosis for both mechanical and SLO induced wounding, the authors found that caveolar endocytosis was triggered by both, mechanical injury with glass beads and poreformation induced by streptolysin-O. The model presented in Fig. 4 illustrates how caveolar endocytosis could mediate PMR.
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Fig. 4 Model for PM repair mediated by caveolar endocytosis. Permeabilization with transmembrane toxin pores (A) or mechanical wounding (B) triggers Ca2+ influx, exocytosis of lysosomes, release of ASM, and generation of ceramide at the PM outer leaflet, a process that promotes caveolae internalization and fusion. Toxin pores would be removed from the PM by caveolar endocytosis (A), while larger breaches on the lipid bilayer would be gradually constricted and resealed as a results of forces generated on the PM by the intracellular clustering, fusion and internalization of merged/branched caveolar structures (B). Adapted from Corrotte, M., Almeida, P. E., Tam, C., Castro-Gomes, T., Fernandes, M. C., Millis, B. A., et al. (2013). Caveolae internalization repairs wounded cells and muscle fibers. eLife, 2, e00926. https://doi.org/10.7554/eLife.00926.
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Lysosomes and plasma membrane repair
5. Lysosome positioning, dynamics and exocytosis during plasma membrane repair Lysosomes are present everywhere in the cytosol of the cell. In nonpolarized cells these organelles are typically accumulated around the nucleus where they form the “perinuclear cloud” but they are also found throughout the cytoplasm and reach as far as the plasma membrane (Cabukusta & Neefjes, 2018) and studies have shown that they can be found predocked to the PM, possibly to allow a fast response to intracellular Ca2+ elevation after membrane injuries ( Jaiswal, Andrews, & Simon, 2002). The positioning of lysosomes can also change due to several alterations suffered by cells such as acidification, alkalinization or ionic imbalance. Lysosomes travel along microtubules both to cell periphery (anterograde direction) and toward cell nuclei (retrograde direction) in a stop-and-go manner that can be regulated by the cells (Figs. 5 and 6).
Lysosome
MTOC ER Nucleus
Perinuclear lysosomes
Peripheral lysosomes
Cortical actin
Fig. 5 Intracellular distribution of lysosomes. Lysosomes can be found in two intracellular locations: a relatively immobile pool of perinuclear lysosomes clustered around the MTOC and a set of peripheral lysosomes moving fast along microtubules in a stop-and-go manner or are found attached to peripheral actin networks. Adapted from Cabukusta, B., & Neefjes, J. (2018). Mechanisms of lysosomal positioning and movement. Traffic, 19(10), 761–769. https://doi.org/10.1111/tra.12587.
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–
+
Lysosome
A
B
C
D
E
Ca2+ BO
Rab7 RILP
ALG2
PI(3,5)P2 TRPML1
SKIP ArI8
JIP4
RC
PI(3)P
Rab7
FYCO1
TMEM55B
Dynein/Dynactin Kinesin-1
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Microtubule
+
Fig. 6 Many mechanisms of lysosomal transport. Various mechanisms of lysosomal recruitment of motor proteins are reported. (A) The small GTPase Rab7 recruits effector RILP and the dynein/dynactin motor to lysosomes for minus-end transport. (B) Calcium plays a crucial role in lysosomal positioning. PI(3,5)P2 on lysosomaldelimiting membranes activates the TRPML1 channel to stimulate calcium efflux. Then, the cytosolic calcium sensor ALG2 recruits the dynein/dynactin complex to TRPML1-containing lysosomes. (C) Transmembrane protein TMEM55B interacts with the dynein adaptor JIP4 and facilitates minus-end transport. TMEM55B levels are controlled transcriptionally: depletion of nutrients or cholesterol upregulates TMEM55B transcription via autophagy-associated transcription factors. (D) BORC, a multisubunit complex on the lysosomal membrane, recruits Arl8b to lysosomes. The small GTPase Arl8b interacts with effector SKIP for lysosomal localization of kinesin-1. (E) The kinesin adaptor FYCO1 interacts with active Rab7 and PI(3)P on lysosomal membranes to recruit kinesin-1 to lysosomes. Adapted from Cabukusta, B., & Neefjes, J. (2018). Mechanisms of lysosomal positioning and movement. Traffic, 19(10), 761–769. https://doi.org/ 10.1111/tra.12587.
Lysosome positioning and their ability to respond to Ca2+ influx is crucial for PMR. Lysosomal sensitivity to calcium seems to be mediated through the presence of an isoform of synaptotagmin VII (Syt VII), a ubiquitous Ca2+-sensor, on the membrane of lysosomes. Syt VII is present on lysosomes in mammalian cells but was also observed ubiquitously expressed in non-synaptic intracellular vesicles in Drosophila tissue (Martinez et al., 2000). Synaptotagmins are a family of calcium sensors mostly known for their involvement in the exocytosis of synaptic vesicles. The regulation of lysosomal exocytosis by Syt VII involves specific SNARE complex
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interactions between the lysosomal v-SNARE and VAMP7 and the plasma membrane t-SNARE syntaxin 4 and SNAP-23. During PMR, Ca2+ influx through PM wounds provokes a rapid Ca2+ increase in the cytoplasm which can then bind to the C2A and C2B domains of Syt VII leading to an interaction of the C2 domains with plasma membrane phospholipids and SNARE molecules. This interaction then drives the formation of a fusion pore and the lysosomal content is delivered to the extracellular media (Andrews, 2005). A diagram of this process is shown in Fig. 7.
6. Lysosomal enzymes as mediators of plasma membrane repair These combined findings describe a sequence of events that ultimately lead to PMR, from Ca2+ entry, recruitment and fusion of Ca2+-sensitive exocytic lysosomes to the PM followed by the subsequent endocytosis of caveolae to promote the removal of PM lesions. With the lack of involvement of the cytoskeleton and other classical mediators of endocytosis in the endocytic mechanisms observed during PMR, the molecular mechanism driving the process remained unknown. Furthermore, due to the amount of enzymes that would be delivered extracellularly during PM repair, lysosomes were long perceived as organelles too dangerous to be used in the process. Also, since lysosomal hydrolases are acidic enzymes, researchers usually considered that they likely would be inactivated upon exocytosis into the neutral extracellular environment. However, since PM repair happens within 30 s, it was also reasonable to assume that some amount of enzymatic activity would remain in this time frame and could plausibly take part in the process. Interestingly, the morphology of endosomes generated after PM repair was very similar to those found in J774 macrophages incubated with bacterial sphingomyelinase (Zha, 1998). Acid sphingomyelinase (ASM) is actually a lipase found in the lumen of lysosomes and is able to cleave the abundant PM lipid sphingomyelin to generate ceramide. Ceramide itself had been shown previously to induce membranes to coalescence and form large inward budding structures in cell membranes (Holopainen, Angelova, & Kinnunen, 2000). Expanding onto these findings, further studies of PMR showed that the secretion of lysosomal ASM is essential for PM repair by acting extracellularly to mediate wound removal by endocytosis (Tam et al., 2010). Interestingly, the enzyme is so important for PM repair that its addition in Ca2+-free conditions, thus without lysosomal exocytosis, induces cell
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Fig. 7 (A) Diagram of the general structure of members of the synaptotagmin family. Synaptotagmins are transmembrane proteins, with a short N-terminal luminal domain and a large cytosolic region containing the tandem Ca2+-binding C2 domains. The C2A and C2B domains are responsible for the Ca2+-dependent interactions with t-SNAREs and phospholipids, which are thought to be essential for the regulation of exocytosis. This is the Ca2+ sensor that endows lysosomes with the ability to perform Ca2+dependent exocytosis. (B) Model of the interactions between synaptotagmin and components of the SNARE fusion complex during Ca2+-triggered exocytosis. Cytosolic Ca2+ binds to the C2A and C2B domains of synaptotagmin on the membrane of exocytic vesicles. Ca2+ binding induces the association of synaptotagmin C2 domains with plasma membrane phospholipids and SNARE molecules. Synaptotagmin C2 domains penetrate the membrane bilayer and SNARE four-helix bundles form, driving the formation of a fusion pore. Syt I, the synaptotagmin isoform involved in synaptic vesicle exocytosis, interacts with the v-SNARE synaptobrevin and with the plasma membrane t-SNARES syntaxin 1 and SNAP-25. Syt VII, the synaptotagmin isoform regulating lysosomal exocytosis, interacts with the v-SNARE VAMP7 and with the t-SNARES syntaxin 4 and SNAP-23. Adapted from Andrews, N. W., Chakrabarti, S., There’s more to life than neurotransmission: the regulation of exocytosis by synaptotagmin VII, Trends in Cell Biology 15(11), 2005, 626–631. Epub 2005 Sep 15.
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resealing after SLO wounding. Additionally, cells treated with siRNA against ASM, cells from patients afflicted with Nieman-Pick Type A (NPA) Disease, a condition characterized by a lack of ASM, or the reduction of ASM activity by specific inhibitors all presented with a strong PM repair deficiency. As explained previously, lysosomes carry a number of enzymes of various functions and with affinities to different substrates. Lysosomal proteases of the cathepsin family have been also shown to be regulators of PM repair (Castro-Gomes et al., 2016). Experiments using siRNA silencing and inhibitors of aspartyl and cysteine proteases showed that the two have opposite effects on PM repair mechanisms: cysteine cathepsins improve PM repair, while aspartyl cathepsin D functions as a down regulator of PM repair. Interestingly, extracellular proteolysis was detected shortly after cell wounding and lysosomal exocytosis, and inhibition of this process impairs PM repair. On the other hand, the removal of proteins from the cell surface by unspecific proteases, facilitates cell resealing. Another interesting finding showed that ASM is itself regulated by lysosomal proteases after its secretion in the extracellular media, indicating that the scenario described here is multifactorial and may include both specific and unspecific proteolytic events (Fig. 8). Overall, while further research on the specific functions of lysosomal exocytosis and its numerous enzymes in plasma membrane repair is necessary to refine our understanding of the process, a clearer picture of this complex process has started to emerge in recent years, building on established findings. Lysosomes and their enzymes both play an upstream role and a downstream role in PMR. Upstream, by responding to calcium influx through PM wounds by fast and localized recruitment to and fusion with the PM. And downstream, by allowing the release of a number of enzymes, either directly involved in stimulating endocytosis, an essential step in the removal of PM lesions, through ASM or indirectly by regulating other aspects of the process through various cathepsins. The balance between up-regulation and down-regulation of enzymatic activities and PM repair steps observed in this latest study may be a function of the amount or size of wounding or other factors. But, importantly, it indicates the presence, within lysosomes, of a fine tuned machinery of regulatory effectors, many probably still unknown, which allow for the possibility that lysosomes may be involved in many more cellular functions than originally envisioned, may be especially in light of their capacity to be secreted in response to extracellular stimulus.
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CATB and CATL (Cysteine Proteases) CATD (Aspartyl Proteases)
Extracellular Activity of Lysosomal Hidrolases
ASM (Acid Sphingomyelinase)
5s ASM release /activation ? PM Wounding
15 s
30 s
Cleavage of extracellular proteins facilitates ASM binding to PM Ceramide generation
60 s
90 s
CATD-mediated ASM downregulation Ceramide-mediated CATD activation
PM
SLO Inactive ASM
Ca2+ Influx Exocytosis of Lysosomes
Endocytosis of Lesions PM Resealing
Active ASM Cysteine Proteases PM protein Ceramide Cathepsin D (CATSD) CeramideActivated CATSD
Fig. 8 Proposed mechanism for rapid modulation of PM repair by secreted lysosomal proteases. Ca2+ influx through wounds in the PM rapidly triggers exocytosis of lysosomes, releasing the proteases cathepsins B, L and D along with the lipase ASM. Cathepsins B and L are active extracellularly 5–10 s after PM wounding, while cathepsin D activity appears after a delay of 30–60 s. At early time points, cathepsins B and L (and possibly additional cysteine proteases) may cleave cell surface proteins and contribute to membrane access and/or activation of ASM. ASM hydrolizes sphingomyelin on the outer leaflet of the PM generating ceramide, which promotes lesion endocytosis and PM repair. ASM-generated ceramide may enhance cathepsin D activity, which plays a role in down-regulating ASM. Around 1 min after wounding the PM integrity is restored, and lysosomal hydrolases are no longer active extracellularly. Adapted from Castro-Gomes, T., Corrotte, M., Tam, C., & Andrews, N. W. (2016). Plasma membrane repair is regulated extracellularly by proteases released from lysosomes. PLoS One, 11(3), e0152583. https://doi.org/10.1371/journal.pone.0152583.
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