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Pairing phosphoinositides with calcium ions in endolysosomal dynamics: Phosphoinositides control the direction and specificity of membrane trafficking by regulating the activity of calcium channels in the endolysosomes
Abstract
The direction and specificity of endolysosomal membrane trafficking is tightly regulated by various cytosolic and membrane-bound factors, including soluble NSF attachment protein receptors (SNAREs), Rab GTPases, and phosphoinositides. Another trafficking regulatory factor is juxta-organellar Ca(2+) , which is hypothesized to be released from the lumen of endolysosomes and to be present at higher concentrations near fusion/fission sites. The recent identification and characterization of several Ca(2+) channel proteins from endolysosomal membranes has provided a unique opportunity to examine the roles of Ca(2+) and Ca(2+) channels in the membrane trafficking of endolysosomes. SNAREs, Rab GTPases, and phosphoinositides have been reported to regulate plasma membrane ion channels, thereby suggesting that these trafficking regulators may also modulate endolysosomal dynamics by controlling Ca(2+) flux across endolysosomal membranes. In this paper, we discuss the roles of phosphoinositides, Ca(2+) , and potential interactions between endolysosomal Ca(2+) channels and phosphoinositides in endolysosomal dynamics.
... Consistent with this localization, the loss of Fab1 causes defects in retrograde traffic from vacuoles to the Golgi (Dove et al., 2004), vacuole membrane fission (Duex et al., 2006b) and vacuole acidification . Similarly, mammalian PI(3,5)P 2 -deficient cells from Fig4-or Vac14-knockout mice, exhibit defects in multiple LEL-associated cellular processes, including enlarged vacuoles/LELs (de Lartigue et al., 2009;Jefferies et al., 2008;Zhang et al., 2012b;Zhang et al., 2007a), defects in retrograde traffic from early endosomes to trans-Golgi network (Rutherford et al., 2006), impaired degradation of EGF receptor (de Lartigue et al., 2009), defects in autophagy (Ferguson et al., 2009;Rusten et al., 2007) and disrupted ionic homeostasis in LELs (Shen et al., 2011). These phenotypes are reminiscent of the cellular defects observed in TRPML1-deficient cells (Cheng et al., 2010), suggesting TRPML1 may functionally interact with the PI(3,5)P 2 signaling pathway and mediate LEL-associated functions, which would be discussed later as part of my thesis work. ...
... The concentration of Ca 2+ stores associated with each vesicle type is also indicated. Figure is adapted from reference (Shen et al., 2011). ...
... Like Na V and Ca V channels, they contain multiple positively charged amino acid residues in their voltage sensor domains and negatively-charged amino acid residues in their pore domains, but their intracellular localization has prevented characterization of basic channel properties such as selectivity and gating. PI(3,5)P 2 is an endolysosome-specific phosphoinositide (PIP) of low abundance (Dove et al., 2009;Shen et al., 2011). Upon cellular stimulation, PI 5-kinase PIKfyve/Fab1 phosphorylates PI(3)P to increase PI(3,5)P 2 from low nM to µM concentrations (Dove et al., 2009;Shen et al., 2011). ...
Lysosomes primarily serve as the cell???s ???garbage disposal and recycling center???, and are recently found to be involved in many important cellular functions. Lysosomes are also ion stores enriched with H+, Ca2+, and Na??+. While it???s well known that the lysosomal ionic homeostasis is essential for its proper functions, the properties of ion transporters and channels residing on lysosomal membranes are barely understood, largely due to the lack of a reliable functional assay. Recently our lab has established a unique lysosomal patch-clamp method to directly record from native lysosomal membrane. Taking advantage of the technique, I discovered two novel lysosomal Na+-selective channels (Two-Pore-Channels TPC1 and TPC2), which are previously thought to be Ca2+ release channels, triggered by the second messenger NAADP. Using an integrative approach, I further demonstrated that TPCs are not activated by NAADP, but instead by PI(3,5)P2, a lysosome-specific phosphoinositide that regulates lysosomal ion homeostasis and membrane trafficking. TPCs represent the first intracellular Na+-selective channels, although their functions are not characterized. In addition, my colleagues and I found that PI(3,5)P2???also activates TRPML1, a principle lysosomal Ca2+ channel. Loss-of-function mutations in human TRPML1 cause type IV Mucolipidosis (ML4), a childhood neurodegenerative disease. My results showed that increasing TRPML1???s activity alleviated lysosomal trafficking defects in PI(3,5)P2-deficient cells, suggesting that PI(3,5)P2 controls Ca2+-dependent membrane trafficking by regulating TRPML1. To study the role of TRPML1 in membrane trafficking, I focused on the involvement of TRPML1 in Ca2+-dependent lysosomal exocytosis, a universal process important for many cellular functions, including cellular clearance, plasma membrane repair and phagocytosis. I found that gain-of-function mutations of TRPML1 caused a dramatic increase in lysosomal exocytosis. During particle uptake in macrophages, lysosomal exocytosis is required to provide membrane supplies to facilitate phagosome formation. By whole-cell recordings and newly developed whole-phagosome recordings, I found that upon particle binding, TRPML1-associated lysosomes are delivered to the newly-formed phagosomes via lysosomal exocytosis in a Ca2+-dependent manner. Overall, my thesis work has characterized two types of important channels (TPCs and TRPMLs) in the lysosome, identified their first endogenous activator PI(3,5)P2, and explored their functions in lysosomal biology.
... Ca 2+ is hypothesized to be another important regulator of endolysosomal membrane trafficking (Peters and Mayer 1998;Burgoyne and Clague 2003;Hay 2007). Similar to the well-established role of Ca 2+ as a key regulator of synaptic vesicle fusion during neurotransmitter release (Suudhof 2008), the transient rise of the Ca 2+ concentration near the endolysosomal fusion site is considered as the final trigger of intracellular membrane fusion (Cheng, Shen et al. 2010;Shen, Wang et al. 2011). In vitro membrane fusion assays using cell extracts from yeast or mammalian cells have shown that both homotypic and heterotypic fusion events between LELs are inhibited by BAPTA, but not by EGTA (Peters and Mayer 1998;Pryor, Mullock et al. 2000). ...
... In NP fibroblasts, accumulation of SMs or cholesterol in LELs causes LEL-to-Golgi trafficking defects. Same defects are also observed in PI(3,5)P 2 -deficient cells (Zhang, Zolov et al. 2007), ML4 fibroblasts (Chen, Bach et al. 1998;Shen, Wang et al. 2011), as well as fibroblasts from several other sphingolipid LSDs (Chen, Patterson et al. 1999;Puri, Watanabe et al. 1999), which suggest that SMs, cholesterol, PI(3,5)P 2 and TRPML1 may function in the same pathway that regulates the LEL-to-Golgi trafficking process. ...
... Cells that lack mucolipin transient receptor potential (TRP) channel 1 (TRPML1) exhibit lysosomal defects similar to those seen in NP cells: defective autophagosome-lysosome fusion or lysosome reformation, abnormal lipid trafficking, and altered Ca 2+ and Fe 2+ homeostasis (Pryor, Reimann et al. 2006;Vergarajauregui and Puertollano 2006;Thompson, Schaheen et al. 2007;Dong, Cheng et al. 2008;Venkatachalam, Long et al. 2008;Cheng, Shen et al. 2010;Dong, Shen et al. 2010;Shen, Wang et al. 2011). Furthermore, mutations in the human Trpml1 gene cause mucolipidosis type IV (ML4) LSD, which exhibits membrane trafficking defects and excessive lysosomal storage (Bargal, Avidan et al. 2000;Bassi, Manzoni et al. 2000;Sun, Goldin et al. 2000). ...
TRPML1 is an inwardly-rectifying Ca2+-permeable channel in late endosomes and lysosomes (LELs). Loss-of-function mutations on the human TRPML1 gene cause a devastating pediatric neurodegenerative disease called type IV Mucolipidosis (ML4). Although it is well established that TRPML1 is involved in multiple late endocytic membrane trafficking processes, the regulatory mechanism for TRPML1 remains elusive. By directly patch-clamping endolysosomal membranes, we found that PI(3,5)P2, a low abundance endolysosome-specific phosphoinositide, potently and specifically activates TRPML1 in both heterologous and endogenous systems (Chapter 2). Lipid-protein binding assays showed that PI(3,5)P2 binds directly to the poly-basic region of the N terminus of TRPML1. Overexpression of TRPML1 rescued the enlarged vacuole phenotype in PI(3,5)P2-deficient cells, suggesting that TRPML1 is a downstream effector of PI(3,5)P2. Notably, this PI(3,5)P2-dependent regulation of TRPML1 is evolutionarily conserved. In budding yeast, the activity of a yeast functional TRPML homologue is also PI(3,5)P2-dependent. These results indicate that lysosomal PI(3,5)P2 is a physiological regulator of TRPML1, providing a previously unknown link between these two important regulators of intracellular membrane trafficking.
In Chapter 3, a membrane-permeable small-molecule synthetic agonist for TRPMLs, Mucolipin-Synthetic Agonist 1 (ML-SA1), was identified. Electrophysiological results showed that ML-SA1 potently and specifically activates recombinant TRPMLs, as well as endogenous TRPML-like currents in many cell types. In addition, ML-SA1 evoked TRPML1-dependent Ca2+ release from endolysosomes in intact cells. ML-SA1 can therefore serve as a valuable tool for studying intracellular functions of TRPMLs.
By taking advantage of ML-SA1, the activity of TRPML1 was examined under a pathological context (Chapter 4). TRPML1-mediated lysosomal Ca2+ release, measured using a genetically-encoded Ca2+ indicator (GCaMP3) attached directly to TRPML1, was dramatically reduced in Niemann-Pick (NP) disease cells. Patch-clamp analyses revealed that TRPML1 channel activity was inhibited by sphingomyelin, but potentiated by spingomyelinase. Importantly, increasing the expression/activity of TRPML1 was able to alleviate the lipid accumulation and trafficking defects in NPC cells. Our findings suggest that compromised channel activity of TRPML1 is the pathogenic cause for secondary lysosomal storage seen in many lysosomal storage disorders (LSDs). Thus manipulating TRPML1 channel activity by chemical agonists may provide therapeutic approaches not only for ML4, but also for other LSDs.
... Consistent with previous studies showing that uptake of large particles is a Ca 2+ -dependent process in BMMs (Czibener et al., 2006), and consistent with ML1 being a lysosomal Ca 2+ -permeable channel , pretreatment of BMMs with a membrane-permeable, fast Ca 2+ chelator BAPTA-AM (Shen et al., 2011) inhibited particle uptake in WT macrophages but to a lower degree in ML1 KO cells ( Figure 2F). In contrast, a membrane-permeable, slow Ca 2+ chelator EGTA-AM had little or no effect ( Figure S2B), suggesting a local source of Ca 2+ (Shen et al., 2011). ...
... Consistent with previous studies showing that uptake of large particles is a Ca 2+ -dependent process in BMMs (Czibener et al., 2006), and consistent with ML1 being a lysosomal Ca 2+ -permeable channel , pretreatment of BMMs with a membrane-permeable, fast Ca 2+ chelator BAPTA-AM (Shen et al., 2011) inhibited particle uptake in WT macrophages but to a lower degree in ML1 KO cells ( Figure 2F). In contrast, a membrane-permeable, slow Ca 2+ chelator EGTA-AM had little or no effect ( Figure S2B), suggesting a local source of Ca 2+ (Shen et al., 2011). ...
... Lysosomal membrane fusion events are presumed to be dependent on [Ca 2+ ] i increase in the close vicinity of fusion spots, though direct evidence has not been reported (Pryor et al., 2000;Piper and Luzio 2004;Shen et al., 2011). Because ML1-GFP was colocalized with Lamp1 in the tips of pseudopods in RAW cells ( Figure S3C), we used a lysosome-targeted genetically encoded Ca 2+ Indicator, GCaMP3-ML1, to specifically measure lysosomal Ca 2+ release in intact cells . ...
Phagocytosis of large extracellular particles such as apoptotic bodies requires delivery of the intracellular endosomal and lysosomal membranes to form plasmalemmal pseudopods. Here, we identified mucolipin TRP channel 1 (TRPML1) as the key lysosomal Ca(2+) channel regulating focal exocytosis and phagosome biogenesis. Both particle ingestion and lysosomal exocytosis are inhibited by synthetic TRPML1 blockers and are defective in macrophages isolated from TRPML1 knockout mice. Furthermore, TRPML1 overexpression and TRPML1 agonists facilitate both lysosomal exocytosis and particle uptake. Using time-lapse confocal imaging and direct patch clamping of phagosomal membranes, we found that particle binding induces lysosomal PI(3,5)P2 elevation to trigger TRPML1-mediated lysosomal Ca(2+) release specifically at the site of uptake, rapidly delivering TRPML1-resident lysosomal membranes to nascent phagosomes via lysosomal exocytosis. Thus phagocytic ingestion of large particles activates a phosphoinositide- and Ca(2+)-dependent exocytosis pathway to provide membranes necessary for pseudopod extension, leading to clearance of senescent and apoptotic cells in vivo.
... TRPML1 plays an important role in Ca 2+ -dependent lysosomal trafficking processes, including maturation of late endosomes, formation of autophagosomes, retrograde trafficking from late endosomes and lysosomes to the trans-Golgi network (TGN), and lysosomal exocytosis (Chen et al. 1998;LaPlante et al. 2006; Thompson et al. 2007;Vergarajauregui et al. 2008;Dong et al. 2009;Curcio-Morelli et al. 2010;Shen et al. 2011a;Medina et al. 2011;Wong et al. 2012;Xu and Ren 2015). ...
... By placing GECIs on the cytosolic surface of endo-lysosomal vesicles, researchers can develop sophisticated methods for detecting Ca 2+ release from endo-lysosomes in a more localized way around the peri-endolysosomal surface of the vesicle, without disrupting lysosomal pH. To record peri-vesicular Ca 2+ release, Shen et al. (Shen et al. 2011a) fused GCaMP3 to the cytosolic N-terminus of TRPML1 to anchor the sensor to the cytosolic surface of vesicles. The idea is to monitor cytosolic Ca 2+ while simultaneously activating endo-lysosomal channels or rapidly discharging intracellular Ca 2+ stores with agents that selectively target endo-lysosomal stores, as described above (GPN, bafilomycin A1, monensin and nigericin). ...
Endo-lysosomes are membrane-bound acidic organelles that are involved in endocytosis, recycling, and degradation of extracellular and intracellular material. The membranes of endo-lysosomes express several Ca2+-permeable cation ion channels, including two-pore channels (TPC1-3) and transient receptor potential mucolipin channels (TRPML1-3). In this chapter, we will describe four different state-of-the-art Ca2+ imaging approaches, which are well-suited to investigate the function of endo-lysosomal cation channels. These techniques include (1) global cytosolic Ca2+ measurements, (2) peri-endo-lysosomal Ca2+ imaging using genetically encoded Ca2+ sensors that are directed to the cytosolic endo-lysosomal membrane surface, (3) Ca2+ imaging of endo-lysosomal cation channels, which are engineered in order to redirect them to the plasma membrane in combination with approaches 1 and 2, and (4) Ca2+ imaging by directing Ca2+ indicators to the endo-lysosomal lumen. Moreover, we will review useful small molecules, which can be used as valuable tools for endo-lysosomal Ca2+ imaging. Rather than providing complete protocols, we will discuss specific methodological issues related to endo-lysosomal Ca2+ imaging.
... 3). In particular it is clear that Ca 2 þ release from endosomes and lysosomes is important for fission and fusion events occurring in the endo-lysosomal pathway; endosomes and lysosomes represent an important source of Ca 2 þ because of the high concentration of this cation in the lumen (B0.5 mM in lysosomes and between 0.003 and 2 mM in the endosome) (Shen et al., 2011). Furthermore, an anion binding site was found in transferrin demonstrating that also Cl À has an essential role: indeed, increasing concentrations of this anion, in addition to a low pH, play a role in the release of iron from transferrin in EEs (Byrne et al., 2010). ...
... After this stable four helix bundle complex is formed, the bilayer mixing reaction starts. Heterotypic fusion is not driven only by SNAREs, as also Rab effectors and Ca 2 þ released from endolysosomes are essential regulators (Luzio et al., 2007;Shen et al., 2011). After fusion the NSF ATPase and its cofactor SNAP cause the recycle of SNAREs (Balderhaar and Ungermann, 2013). ...
Endosome to lysosome transport is a highly regulated process important for targeting molecules to degradation. Proteins destined to degradation are transported from early endosomes to late endosomes and lysosomes, where degradation takes place. Sorting mechanisms and acidification drive proteins in this pathway. Rab proteins and their effectors play a key regulatory role together with molecular motors and cytoskeletal elements, which are required for the correct movement and positioning of organelles within this pathway. Here we review the molecular mechanisms responsible for this transport and we focus on regulation elements that control the different steps.
... The above-mentioned components important for trafficking through the endolysosomal system undergo additional complex and mutual regulation in various ways. For example, SNAREs can regulate Rab effectors and Ca 2+ channels, and PIPs can modulate SNAREs as well as Ca 2+ release channels (Shen et al., 2011). In addition, there is intricate crosstalk between PIPs and Rab GTPases. ...
... Other reported interactors such as Rab8 and Rab11, or syntaxin 12/16, play roles in the endocytic recycling compartment. Whilst not acidic in nature, this compartment has been shown to contain Ca 2+ as well as TRPML2 (Shen et al., 2011), raising the possibility that TPCN2 may also play a role in vesicular trafficking to and from the recycling compartment (Ruas et al., 2010). Finally, the TPCN2 interactor Rab14 is known to be involved in the recycling pathway between the Golgi and endosomal compartments, whilst other interactors such as Rab7, VAMP7, syntaxins 7 and 8 are localized to late endosomes and lysosomes, thus overlapping with the most prominent steady-state localization of TPCN2 (Fig. 1). ...
Two-pore channels are endolysosomal Ca(2+) release channels involved in proper trafficking to and from those organelles. They are the likely targets for the Ca(2+)-mobilizing messenger NAADP, and are further regulated by a variety of mechanisms including phosphoinositide levels and Rab proteins. As discussed here, recent studies highlight a role for these channels in the pathomechanism(s) underlying Parkinson's disease, with important implications for possible alternative treatment strategies.
... 84 Phosphoinositides (PIPs), specifically PtdIns(3,5)P2, can determine the vesicular identity and the direction of membrane trafficking, both of them essential functions for endolysosomal trafficking. 85 Shen et al. 85 demonstrated that PI(3,5)P 2 -deficient cells are characterized for enlarged endolysosomes/vacuoles and trafficking defects in the late endocytic pathways; however, it was not until 2012 when, based on patchclamp studies, in the fibroblast-like cell line derived from monkey kidney tissue (COS-1) endolysosomes, and in TPC1 and TPC2 KO mice, was proposed the hypothesis that the TPCs are Na C channels activated by PtdIns(3,5)P 2 . 5 Metabolism/nutrient sensing Cang et al. 6 showed in HEK293 cells and in TPC1/2 KO mice that mTOR is capable of associating and controlling the activation of an endolysosomal Na C channel adenosine triphosphate (ATP) (lysoNa ATP ) sensitive, formed by TPCs and other proteins. ...
... 84 Phosphoinositides (PIPs), specifically PtdIns(3,5)P2, can determine the vesicular identity and the direction of membrane trafficking, both of them essential functions for endolysosomal trafficking. 85 Shen et al. 85 demonstrated that PI(3,5)P 2 -deficient cells are characterized for enlarged endolysosomes/vacuoles and trafficking defects in the late endocytic pathways; however, it was not until 2012 when, based on patchclamp studies, in the fibroblast-like cell line derived from monkey kidney tissue (COS-1) endolysosomes, and in TPC1 and TPC2 KO mice, was proposed the hypothesis that the TPCs are Na C channels activated by PtdIns(3,5)P 2 . 5 Metabolism/nutrient sensing Cang et al. 6 showed in HEK293 cells and in TPC1/2 KO mice that mTOR is capable of associating and controlling the activation of an endolysosomal Na C channel adenosine triphosphate (ATP) (lysoNa ATP ) sensitive, formed by TPCs and other proteins. ...
Two-pore channels (TPC1-3) comprise a subfamily of the eukaryotic voltage-gated ion channels (VGICs) superfamily that are mainly expressed in acidic stores in plants and animals. TPCS are widespread across the animal kingdom, with primates, mice and rats lacking TPC3, and mainly act as Ca(+) and Na(+) channels, although it was also suggested that they could be permeable to other ions. Nowadays, TPCs have been related to the development of different diseases, including Parkinson´s disease, obesity or myocardial ischemia. Due to this, their study has raised the interest of the scientific community to try to understand their mechanism of action in order to be able to develop an efficient drug that could regulate TPCs activity. In this review, we will provide an updated view regarding TPCs structure, function and activation, as well as their role in different pathophysiological processes.
... The various PtdInsPs, and the mono-phosphorlyated forms, in particular, are very short lived, and can be metabolized within minutes under normal physiologic conditions (Michell, Heath et al. 2006). Of the PtdInsPs, PtdIns(3,5)P 2 is one of the least abundant, comprising only ~0.05% of the total population of PtdInsPs (Shen, Wang et al. 2011). Flux between PtdIns(3)P and PtdIns(3,5)P 2 regulates processes including endocytosis, endosomal trafficking, and autophagy (Itoh and Takenawa 2002, Payrastre 2004, Di Paolo and De Camilli 2006, Michell, Heath et al. 2006, Lecompte, Poch et al. 2008). ...
... In addition, work from the Xu lab has discovered a very important role for PtdIns(3,5)P 2 in the activation of lysosomal ion channels (Shen, Wang et al. 2011). Several studies have also described a role of PtdIns(3,5)P 2 in the activation of the ryanodine receptor in cardiac (Touchberry, Bales et al. 2010) and skeletal muscle (Shen, Yu et al. 2009). ...
X-linked myotubular myopathy (MTM) is a severe centronuclear myopathy, characterized by profound weakness and hypotonia at birth, early mortality, and significant morbidities. MTM results from mutations in the phosphoinositide phosphatase myotubularin (MTM1), which dephosphorylates the lipid substrates phosphatidylinositol 3-phosphate (PtdIns(3)P) and phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2). We propose that dysregulation of PtdIns(3)P and PtdIns(3,5)P2 leads to the disease state in MTM, and that these lipids play a significant role in muscle development and homeostasis. To elucidate the effect of PtdIns(3)P dysregulation in MTM, we examined several mouse models with targeted inactivation of phosphoinositide phosphatases (Mtm1 and Fig4) and kinases (Pik3c3 and Pik3c2??).
Previous studies showed that tissue-specific neuronal rescue of the spontaneously occurring Fig4 mutation of pale tremor (plt) mice prevented overt muscle weakness and the development of obvious muscle abnormalities. Our studies confirmed that the changes in skeletal muscle observed in the plt mice were primarily related to denervation. Fig4 deletion had previously been shown to rescue aspects of Mtmr2-dependent neuropathy, so we evaluated the effect of Fig4 haploinsufficiency on the myopathy of Mtm1-knockout mice. Compared with Mtm1???/Y males, mice with the genotype Fig4+/???/Mtm1???/Y displayed no improvement in muscle histology, muscle size, or longevity, indicating that reduction of FIG4 does not ameliorate the Mtm1-knockout phenotype.
To further investigate the function of PtdIns(3)P in skeletal muscle, we focused on the primary kinase responsible for its production, and created a muscle-specific conditional knockout of the class III PI 3-kinase, Pik3c3. This resulted in progressive disease characterized by reduced activity and death by two months of age. Histopathology demonstrated changes consistent with a murine muscular dystrophy. Mechanistic evaluations revealed significant alterations in the autophagolysosomal pathway with mislocation of known dystrophy proteins to the lysosomal compartment. We present the first analysis of Pik3c3 in skeletal muscle, and report a novel association between deletion of Pik3c3 and muscular dystrophy.
In contrast, muscle-specific deletion of Pik3c2?? caused no overt physiological defects by 11 months of age, other than impaired glucose tolerance. These studies demonstrate the critical role of PIK3C3 in muscle physiology and give insight to some of the mechanisms disrupted in muscle diseases.
... 3). In particular it is clear that Ca 2 þ release from endosomes and lysosomes is important for fission and fusion events occurring in the endo-lysosomal pathway; endosomes and lysosomes represent an important source of Ca 2 þ because of the high concentration of this cation in the lumen (B0.5 mM in lysosomes and between 0.003 and 2 mM in the endosome) (Shen et al., 2011). Furthermore, an anion binding site was found in transferrin demonstrating that also Cl À has an essential role: indeed, increasing concentrations of this anion, in addition to a low pH, play a role in the release of iron from transferrin in EEs (Byrne et al., 2010). ...
... After this stable four helix bundle complex is formed, the bilayer mixing reaction starts. Heterotypic fusion is not driven only by SNAREs, as also Rab effectors and Ca 2 þ released from endolysosomes are essential regulators (Luzio et al., 2007;Shen et al., 2011). After fusion the NSF ATPase and its cofactor SNAP cause the recycle of SNAREs (Balderhaar and Ungermann, 2013). ...
Endosome to lysosome transport is a highly regulated process important for targeting molecules to degradation. Proteins destined to degradation are transported from early endosomes to late endosomes and lysosomes, where degradation takes place. Sorting mechanisms and acidification drive proteins in this pathway. Rab proteins and their effectors play a key regulatory role together with molecular motors and cytoskeletal elements, which are required for the correct movement and positioning of organelles within this pathway. Here we review the molecular mechanisms responsible for this transport and we focus on regulation elements that control the different steps.
... A stringent requirement for a functional luminal acid hydrolases is low pH L, of 4.5 to 5.0, maintained by ATP-mediated proton pump and leak hypothesis. Either pump in or leak out of H + is electrogenic requiring a counter mechanism (ML1/TPC2-mediated cation efflux or anion influx that may not be the rheogenic Cl _ antiporter) to banish a positive net charge inside the lumen generated by ATP hydrolysis to regulate V-ATPase, the principle regulator of pH L in membrane trafficking, accumulation of macromolecules and altered Ca 2+ homeostasis are common typical features in NP form of LSD [4,5,20,34]. For the AD and as observed in Fig. 3, and illustrated in Fig. 2, Aβ42 [33] and tau accumulate in the endolytic pathway. ...
... This documentation precisely defined the source of Ca 2+ and repudiates a previous report [22], that implicated only lysosomal Ca 2+ stores as the source of Ca 2+ signaling selectively compromised in NP. The source of the unidentified Ca 2+ channels from the lumen of acidic organelles and vesicles [46], that can mediate Ca 2+ release is the ML1 [4,5,34]. Though considering how difficult it may be to clamp intracellular Ca 2+ , a perfectly firm condition to rule out the participation of Ca 2+ from the lysosomal Ca 2+ stores may have not been adequately ensured in the [20] documentation. ...
Studies have reported typically biophysical lysosomal cation channels including TPCs. Their plausible biological roles are being elucidated by pharmacological, genetic and conventional patch clamp procedures. The best characterized so far among these channels is the ML1 isoform of TRP. The reported TRPs and TPCs are bypass for cation fluxes and are strategic for homeostasis of ionic milieu of the acidic organelles they confine to. Ca(2+) homeostasis and adequate acidic pHL are critically influential for the regulation of a plethora of biological functions these intracellular cation channels perform. In lysosomal ion channel biology, we review: ML1 and TPC2 in Ca(2+) signaling, ML1 and TPC2 in pHL regulation. Using Aβ42 and tau proteins found along clathrin endolysosomal internalization pathway (Fig. 3), we proffer a mechanism of abnormal pHL and ML1/TPC2-dependent cation homeostasis in AD.
... In the case of metabolic receptors, their crosstalk with regulatory transport proteins is mediated by small signaling sequences in their cytoplasmic domains [2,3]. A number of ion channels and transporters also become endosome components [4][5][6] actively participating in organization of sorting processes. ...
... Since initially Ca 2+ concentration in the endosome lumen corresponds to a high extracellular concentration, the endosome per se is a calcium depot, and the presence of calcium channels of several types (including pH-sensitive) provides local increases in the level of this ion within a limited area at the cytoplasmic side of the endosome membrane. Such local increases in calcium, without affecting overall cell processes, are important for the regulation of fusions, can modulate the properties of cytoskeleton associated with endosomes, and can regulate the activity of numerous proteins in the "near-endosome" region [4,5]. ...
Receptor-mediated endocytosis is the most specific pathway for macromolecules and macromolecular complexes generally designated as ligands to enter cells. Upon binding to their transmembrane receptors, the ligands enter endocytic vesicles that fuse with each other giving rise to the so-called early endosomes. The sorting of ligand-receptor complexes internalized in these endosomes depends on their nature: metabolic receptors are recycled back to the plasma membrane, while signaling receptors and their ligands (e.g. receptor tyrosine kinases or receptors associated with tyrosine kinase) are delivered to internal vesicles of the multivesicular late endosomes and finally are degraded after interaction with lysosomes. During these processes, endosomes undergo translocation from the cell periphery to the juxtanuclear region, which is accompanied by multiple fusion, invagination, tabulation, and membrane fission events. This review considers modern concepts of the sorting mechanisms of ligand-receptor complexes, the crosstalk between endosomes, microtubules, and actin, and the role of this crosstalk in endosome maturation.
... PI(3,5)P 2 is a key regulator of membrane trafficking (membrane fusion and fission) in LELs, and PI(3,5)P 2 deficiency causes endolysosomal enlargement due to trafficking defects (7). Because PI(3,5)P 2 directly activates lysosomal TRPML1 channels, it was hypothesized that PI(3,5)P 2 elevation may induce lysosomal Ca 2+ release to trigger endolysosomal membrane fusion/ fission (13,20,29). To visualize PI(3,5)P 2 dynamics in relation to membrane fusion/fission in live cells, we performed time-lapse confocal imaging in COS1 cells that were dually transfected with GFP-ML1N*2 and Lamp1-mCherry, with Lamp1-mCherry revealing the fusion events and GFP-ML1N*2 tracking PI(3,5)P 2 dynamics. ...
... By using the PI(3,5)P 2 probe that we have developed and characterized, we have shown that PI(3,5)P 2 elevation occurs immediately before endolysosomal fusion, highlighting the role of PI(3,5)P 2 in regulating endolysosomal fusion. TRPML1 is a lysosomal channel that mediates Ca 2+ release from the lysosomal lumen (13) to trigger membrane fusion in LELs (29). Because PI (3,5)P 2 elevation may activate TRPML1-mediated Ca 2+ release (13,20), it is likely that PI(3,5)P 2 is the trigger for fusion between lysosomes and various other compartments (e.g., late endosomes, autophagosomes, phagosomes, and plasma membrane). ...
Significance
Phosphatidylinositol polyphosphates (PIPs) are transiently generated at specific membrane subdomains. Changes of PIP levels regulate the trafficking of vesicles and the activity of membrane transport proteins. To directly visualize the intracellular dynamics of phosphatidylinositol 3,5-bisphosphate [PI(3,5)P 2 ], a key phosphoinositide in the endosome and lysosome, we have engineered a PI(3,5)P 2 probe by fusing fluorescent proteins directly to the lipid-binding domain of TRPML1, a lysosomal ion channel that is potently and specifically activated by PI(3,5)P 2 . This PI(3,5)P 2 probe binds to PI(3,5)P 2 with biochemical specificity in vitro and responds quickly to changes in the intracellular PI(3,5)P 2 level in living cells. With this biosensor, rapid changes of PI(3,5)P 2 on single vesicle membranes are captured prior to membrane fusion of two vesicles.
... On the one hand, released Ca 2+ regulates membrane fusion events through Ca 2+ sensors (i.e., synaptotagmins) involving the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors. On the other hand, it controls the direction and specificity of trafficking processes via Rab effectors and (Ca 2+ sensors) synaptotagmins [115,116]. ...
Astrocytes are increasingly recognized as important viral host cells in the central nervous system. These cells can produce relatively high quantities of new virions. In part, this can be attributed to the characteristics of astrocyte metabolism and its abundant and dynamic cytoskeleton network. Astrocytes are anatomically localized adjacent to interfaces between blood capillaries and brain parenchyma and between blood capillaries and brain ventricles. Moreover, astrocytes exhibit a larger membrane interface with the extracellular space than neurons. These properties, together with the expression of various and numerous viral entry receptors, a relatively high rate of endocytosis, and morphological plasticity of intracellular organelles, render astrocytes important target cells in neurotropic infections. In this review, we describe factors that mediate the high susceptibility of astrocytes to viral infection and replication, including the anatomic localization of astrocytes, morphology, expression of viral entry receptors, and various forms of autophagy.
... Animal TPCs, in contrast to their plant counterparts can make use of phosphoinositol 3,5bisphosphate (PI(3,5)P2) to promote activation in either a voltage dependent (TPC1-like subfamily) or a voltage independent (TPC2-like subfamily) manner (Wang et al., 2012;Cang et al., 2014b;Boccaccio et al., 2014). PI(3,5)P2 produced by PIKfyve (FYVE finger-containing phosphoinositide kinase) can impact vesicular membrane trafficking, while cells which are PI(3,5)P2-deficient are characterized by enlarged endolysosomal compartments (Shen et al., 2011;Zolov et al., 2012). Animal TPC PI(3,5)P2 binding motifs vary structurally. ...
The following chapter summarizes the current knowledge about the slow vacuolar (SV) cation current and two-pore channels (TPCs) in plants and animals. TPCs are ubiquitously expressed in plant vacuoles and the animal endolysosomal system. Evolutionary aspects of TPCs in the plant and animal kingdoms will be discussed. Differences in plant and animal TPC regulation and the main functional domains in both homologs are compared. Finally, the long-lasting cross kingdom debates over the contribution of TPCs to calcium signalling is summarized.
... Canonical endolysosomal trafficking is mediated by Endosomal Sorting Complexes Required for Transport (ESCRT) complexes and small Rab GTPases (Weeratunga et al., 2020). Regulation of how ESCRT machinery sorts endocytic cargo for recycling and degradation continues to be an active area of discovery and has revealed lipid mediators such as phosphoinositides and lipid transporters (Shen et al., 2011;Stalder and Gershlick, 2020). In addition to the ESCRT-mediated exosome biogenesis pathway, there is also an ESCRT-independent biogenesis pathway that relies on neutral sphingomyelinase (nSMase; Trajkovic et al., 2008;Menck et al., 2017). ...
Many neurodegenerative diseases are characterized by abnormal protein aggregates, including the two most common neurodegenerative diseases Alzheimer’s disease (AD) and Parkinson’s disease (PD). In the global search to prevent and treat diseases, most research has been focused on the early stages of the diseases, including how these pathogenic protein aggregates are initially formed. We argue, however, that an equally important aspect of disease etiology is the characteristic spread of protein aggregates throughout the nervous system, a key process in disease progression. Growing evidence suggests that both alterations in lipid metabolism and dysregulation of extracellular vesicles (EVs) accelerate the spread of protein aggregation and progression of neurodegeneration, both in neurons and potentially in surrounding glia. We will review how these two pathways are intertwined and accelerate the progression of AD and PD. Understanding how lipid metabolism, EV biogenesis, and EV uptake regulate the spread of pathogenic protein aggregation could reveal novel therapeutic targets to slow or halt neurodegenerative disease progression.
... 147,156 The role played by endo-lysosomal Two-Pore Channels (TPCs) on CoV biology and the feasibility of blocking the intracellular pathway of the virus by inhibiting these channels can be of significance to impede virus trafficking. 141,142,144,[198][199][200][201][202] Interestingly, NAR can inhibit the activity of TPC1 and TPC2, both in humans and plants. 141 NAR is a hydrophilic substance with a higher affinity for the cytoplasmic membrane, generating intracellular accumulation of NAR. ...
Naringenin, widely distributed in fruits and vegetables, is endowed with antiviral and other health beneficial activities, such as immune-stimulating and anti-inflammatory actions that could play a role in contributing, to some extent, to either preventing or alleviating coronavirus infection. Several computational studies have identified naringenin as one of the prominent flavonoids that can possibly inhibit internalization of the virus, virus-host interactions that trigger the cytokine storm, and replication of the virus. This review highlights the antiviral potential of naringenin in COVID-19 associated risk factors and its predicted therapeutic targets against SARS-CoV-2 infection.
... Another possible mechanism is the inhibition of the twopore ionic channel (TPC1 and TPC2) (92). Inhibition of TPC1 and TPC2 reduces MERS-CoV infectivity, intracellular traffic (93), and viral replication (93,94). ...
Coronavirus disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), was declared a pandemic by the World Health Organization in March 2020. Severe COVID-19 cases develop severe acute respiratory syndrome, which can result in multiple organ failure, sepsis, and death. The higher risk group includes the elderly and subjects with pre-existing chronic illnesses such as obesity, hypertension, and diabetes. To date, no specific treatment or vaccine is available for COVID-19. Among many compounds, naringenin (NAR) a flavonoid present in citrus fruits has been investigated for antiviral and anti-inflammatory properties like reducing viral replication and cytokine production. In this perspective, we summarize NAR potential anti-inflammatory role in COVID-19 associated risk factors and SARS-CoV-2 infection.
... Rab7, a small GTPase, and lysosomal Ca 2+ -sensor ALG-2 (also known as PDCD6) are known to play essential roles in the lysosome coupling process [15]. Belonging to the large family of transient receptor potential ion channels, lysosome Transient receptor potential mucolipin 1 (encoded by the Mcoln1 gene, also known as TRPML1) [16,17] permeates Ca 2+ , Na + , and other cations [18][19][20]. Mutations in the TRPML1 gene lead to mucolipidosis type IV (ML4) disease in humans, which is characterized by defects in membrane tra cking in the late endocytic pathway due to enlarged lysosomes [16,21] and impaired lysosome biogenesis [22,23]. ...
Arterial calcification is a highly regulated cellular process that is characterized by the pathological mineral deposition leading to reduced arterial compliance. The aim of the present study was to investigate whether mucolipin 1 gene contributes to lysosomal positioning and exosome secretion which may results in phenotype change in SMCs during arterial medial calcification (AMC). In mucolipin 1 gene knock out (KO) mice, a high dose of Vit D (500,000 IU/kg/day) resulted in increased aortic calcification compared to their wild type littermates, which was accompanied by downregulation of SM22‐a and upregulation of RUNX2 and osteopontin in the arotic media, indicating phenotypic switch to osteogenic. Furthermore, we observed decreased colocalization of lysosome marker (Lamp‐1) with lysosome coupling marker (Rab 7 and ALG‐2) in the arterial wall of mucolipin 1 KO mice as compared to their wild type littermates. In addition, mucolipin 1 KO mice showed increased expression of exosome markers, CD63 and annexin‐II (AnX2), in the arterial medial wall, which was associated with reduced co‐localization of lysosome marker (Lamp‐1) with multivesicular body (MVB) marker (VPS16). This suggests reduction of the lysosome‐MVB interactions. Furthermore, we found significantly increased exosomes in the plasma of mucolipin 1 KO mice as compared to the wild type littermates. Functionally, in mucolipin 1 KO mice pulse wave velocity (PWV) significantly increased and Vit D treatment further enhance such stiffening. All these data indicate that mucolipin‐1 gene deletion leads to abnormal lysosome positioning and exosome secretion in the arterial wall, which may contribute to the arterial stiffness during the development of AMC.
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(Supported by NIH grants HL057244, HL075316 and DK120491)
... Rab7, a small GTPase, and lysosomal Ca 2+ -sensor ALG-2 (also known as PDCD6) are known to play essential roles in the lysosome coupling process [15]. Belonging to the large family of transient receptor potential ion channels, lysosome Transient receptor potential mucolipin 1 (encoded by the Mcoln1 gene, also known as TRPML1) [16,17] permeates Ca 2+ , Na + , and other cations [18][19][20]. Mutations in the TRPML1 gene lead to mucolipidosis type IV (ML4) disease in humans, which is characterized by defects in membrane trafficking in the late endocytic pathway due to enlarged lysosomes [16,21] and impaired lysosome biogenesis [22,23]. ...
Recent studies have shown that arterial medial calcification is mediated by abnormal release of exosomes/small extracellular vesicles from vascular smooth muscle cells (VSMCs) and that small extracellular vesicle (sEV) secretion from cells is associated with lysosome activity. The present study was designed to investigate whether lysosomal expression of mucolipin-1, a product of the mouse Mcoln1 gene, contributes to lysosomal positioning and sEV secretion, thereby leading to arterial medial calcification (AMC) and stiffening. In Mcoln1−/− mice, we found that a high dose of vitamin D (Vit D; 500,000 IU/kg/day) resulted in increased AMC compared to their wild-type littermates, which was accompanied by significant downregulation of SM22-α and upregulation of RUNX2 and osteopontin in the arterial media, indicating a phenotypic switch to osteogenic. It was also shown that significantly decreased co-localization of lysosome marker (Lamp-1) with lysosome coupling marker (Rab 7 and ALG-2) in the aortic wall of Mcoln1−/− mice as compared to their wild-type littermates. Besides, Mcoln1−/− mice showed significant increase in the expression of exosome/ sEV markers, CD63, and annexin-II (AnX2) in the arterial medial wall, accompanied by significantly reduced co-localization of lysosome marker (Lamp-1) with multivesicular body (MVB) marker (VPS16), suggesting a reduction of the lysosome-MVB interactions. In the plasma of Mcoln1−/− mice, the number of sEVs significantly increased as compared to the wild-type littermates. Functionally, pulse wave velocity (PWV), an arterial stiffening indicator, was found significantly increased in Mcoln1−/− mice, and Vit D treatment further enhanced such stiffening. All these data indicate that the Mcoln1 gene deletion in mice leads to abnormal lysosome positioning and increased sEV secretion, which may contribute to the arterial stiffness during the development of AMC.
... The EALP is a complex pathway regulated by a number of kinases, membrane proteins, transport machinery, signaling membrane phospholipids and cations, such as Ca 2+ , Mg 2+ , Fe 2+ and Zn 2+ [73][74][75][76][77]. It is not an easy task to determine which step is most critically affected in a particular neurodegenerative condition. ...
Abstract A number of neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis, share intra- and/or extracellular deposition of protein aggregates as a common core pathology. While the species of accumulating proteins are distinct in each disease, an increasing body of evidence indicates that defects in the protein clearance system play a crucial role in the gradual accumulation of protein aggregates. Among protein degradation systems, the endosome-autophagosome-lysosome pathway (EALP) is the main degradation machinery, especially for large protein aggregates. Lysosomal dysfunction or defects in fusion with vesicles containing cargo are commonly observed abnormalities in proteinopathic neurodegenerative diseases. In this review, we discuss the available evidence for a mechanistic connection between components of the EALP-especially lysosomes-and neurodegenerative diseases. We also focus on lysosomal pH regulation and its significance in maintaining flux through the EALP. Finally, we suggest that raising cAMP and free zinc levels in brain cells may be beneficial in normalizing lysosomal pH and EALP flux.
... Calcium enters the cytoplasm through calcium channels found on the plasma membrane, on intracellular vesicles such as endosomes and the endoplasmic reticulum. The change in cytoplasmic calcium concentration is detected by sensing proteins that modify cell behavior including endosome trafficking and cytoskeletal remodeling (Abe and Puertollano 2011;Shen et al. 2011;Gandini and Felix 2015). Using siRNA screening, host factors important for cell entry of pseudotypes carrying EBOV GP, calcium sensing proteins including calmodulin and Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) were identified (Kolokoltsov et al. 2009). ...
Filovirus entry into cells is complex, perhaps as complex as any viral entry mechanism identified to date. However, over the past 10 years, the important events required for filoviruses to enter into the endosomal compartment and fuse with vesicular membranes have been elucidated (Fig. 1). Here, we highlight the important steps that are required for productive entry of filoviruses into mammalian cells.
... TRPML1 (transient receptor potential mucolipin 1) (10,(13)(14)(15), belongs to the large family of transient receptor potential (TRP) ion channels that permeates Ca 2+ , Na + and other cations (10,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). Mutations in TRPML1 gene lead to Mucolipidosis type IV (ML4) disease which is characterized with defects in membrane trafficking in the late endocytic pathway (10,29), enlarged lysosomes (10,29), and impaired lysosome biogenesis (30)(31)(32). ...
Intracellular lysosomal membrane trafficking, including fusion and fission, is crucial for cellular homeostasis and normal cell function. Both fusion and fission of lysosomal membrane are accompanied by lysosomal Ca2+ release. We recently have demonstrated that the lysosomal Ca2+ release channel P2X4 regulates lysosome fusion through a calmodulin (CaM)-dependent mechanism. However, the molecular mechanism underlying lysosome fission remains uncertain. In this study, we report that enlarged lysosomes/vacuoles induced by either vacuolin-1 or P2X4 activation are suppressed by upregulating the lysosomal Ca2+ release channel transient receptor potential mucolipin 1 (TRPML1), but not the lysosomal Na+ release channel two pore channel 2 (TPC2). Activation of TRPML1 facilitated the recovery of enlarged lysosomes/vacuoles. Moreover, the effects of TRPML1 on lysosome/vacuole size regulation were eliminated by Ca2+ chelation, suggesting a requirement for TRPML1-mediated Ca2+ release. We further demonstrate that the prototypical Ca2+ sensor CaM is required for the regulation of lysosome/vacuole size by TRPML1, suggesting that TRPML1 may promote lysosome fission by activating CaM. Given that lysosome fission is implicated in both lysosome biogenesis and reformation, our findings suggest that TRPML1 may function as a key lysosomal Ca2+ channel controlling both lysosome biogenesis and reformation.
... Phagolysosome fusion in vitro has been reported to require PtdIns4P in addition to PtdIns3P, but the effectors of PtdIns4P have not been identified ( Jeschke et al. 2015). PtdIns(3,5)P 2 on late endosomes has also been proposed to modulate Ca 2+ release from TRPML1 channels in endolysosome membranes and thereby promote vesicle fusion mediated by Ca 2+ -sensitive fusion regulators (Shen et al. 2011). ...
Most functions of eukaryotic cells are controlled by cellular membranes, which are not static entities but undergo frequent budding, fission, fusion, and sculpting reactions collectively referred to as membrane dynamics. Consequently, regulation of membrane dynamics is crucial for cellular functions. A key mechanism in such regulation is the reversible recruitment of cytosolic proteins or protein complexes to specific membranes at specific time points. To a large extent this recruitment is orchestrated by phosphorylated derivatives of the membrane lipid phosphatidylinositol, known as phosphoinositides. The seven phosphoinositides found in nature localize to distinct membrane domains and recruit distinct effectors, thereby contributing strongly to the maintenance of membrane identity. Many of the phosphoinositide effectors are proteins that control membrane dynamics, and in this review we discuss the functions of phosphoinositides in membrane dynamics during exocytosis, endocytosis, autophagy, cell division, cell migration, and epithelial cell polarity, with emphasis on protein effectors that are recruited by specific phosphoinositides during these processes. Expected final online publication date for the Annual Review of Cell and Developmental Biology Volume 32 is October 06, 2016. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
... The term 'endolysosomal organelles' describes acidic intracellular organelles, including early, late and recycling endosomes and lysosomes; secretory vesicles are also acidic, lysosomal-like organelles (Morgan et al., 2011, Shen et al., 2011. Early endo-somes are formed by plasma membrane endocytotic events; their lipid constituents may return to the plasma membrane via recycling endosomes, or proceed through the endolysosomal system, forming late endosomes and lysosomes (Fig. 1). ...
Ca2+ signals regulate a wide range of physiological processes. Intracellular Ca2+ stores can be mobilized in response to extracellular stimuli via a range of signal transduction mechanisms, often involving recruitment of diffusible second messenger molecules. The Ca2+ mobilizing messengers InsP3 and cADPR release Ca2+ from the endoplasmic reticulum via InsP3 and ryanodine receptors, respectively, while a third messenger, NAADP, releases Ca2+ from acidic endosomes and lysosomes. Bidirectional communication between the ER and acidic organelles has functional relevance for endolysosomal function as well as for the generation of Ca2+ signals. The two-pore channels (TPCs) are currently strong candidates for being key components of NAADP-regulated Ca2+ channels. Ca2+ signals have been shown to play important roles in embryonic development and cell differentiation; however, much remains to be established about the exact signalling mechanisms involved. Investigation of the role of NAADP and TPCs in development and differentiation is still at an early stage, but recent studies have suggested that they play important roles at key developmental stages in vivo and are important mediators of differentiation of neurons, skeletal muscle cells and osteoclasts in vitro. NAADP signals and TPCs have also been implicated in autophagy, an important process in differentiation. Moreover, potential links between TPC2 and cancer have been recently identified. Further studies will be required to identify the precise mechanisms of action of TPCs and their link with NAADP signalling, and to relate these to their roles in differentiation and other key developmental processes in the cell and organism.
... Growing evidence support a direct role of TRPML1 in lysosomal exocytosis. The permeation and gating properties of TRPML1 suggest that the channel function of TRPML1 is to release Ca 2+ from the lysosome lumen in response to various cellular cues [53,63]. Consistent with a role of TRPML1 in lysosomal trafficking, several fusion defects were observed in TRPML1 knockout cells [64,65]. ...
Resealing allows cells to mend damaged membranes rapidly when plasma membrane (PM) disruptions occur. Models of PM repair mechanisms include the "lipid-patch", "endocytic removal", and "macro-vesicle shedding" models, all of which postulate a dependence on local increases in intracellular Ca(2+) at injury sites. Multiple calcium sensors, including synaptotagmin (Syt) VII, dysferlin, and apoptosis-linked gene-2 (ALG-2), are involved in PM resealing, suggesting that Ca(2+) may regulate multiple steps of the repair process. Although earlier studies focused exclusively on external Ca(2+), recent studies suggest that Ca(2+) release from intracellular stores may also be important for PM resealing. Hence, depending on injury size and the type of injury, multiple sources of Ca(2+) may be recruited to trigger and orchestrate repair processes. In this review, we discuss the mechanisms by which the resealing process is promoted by vesicular Ca(2+) channels and Ca(2+) sensors that accumulate at damage sites.
... Recently, several Ca 2+ -permeable channels have been shown to localize to membranes of late endosome and lysosome (LEL). These include TRPML1 (transient receptor potential mucolipin 1; Dong et al., 2008Dong et al., , 2010Cheng et al., 2010;Shen et al., 2011), TRPM2 (transient receptor potential melastatin 2; Lange et al., 2009;Sumoza-Toledo et al., 2011), TPC2 (two-pore channel 2; Calcraft et al., 2009;Grimm et al., 2014), and P2X4 purinoceptor Huang et al., 2014). Both TRPML1 and TRPM2 belong to the superfamily of transient receptor potential cation channels. ...
Intra-endolysosomal Ca(2+) release is required for endolysosomal membrane fusion with intracellular organelles. However, the molecular mechanisms for intra-endolysosomal Ca(2+) release and the downstream Ca(2+) targets involved in the fusion remain elusive. Previously, we demonstrated that endolysosomal P2X4 forms channels activated by luminal adenosine triphosphate in a pH-dependent manner. In this paper, we show that overexpression of P2X4, as well as increasing endolysosomal P2X4 activity by alkalinization of endolysosome lumen, promoted vacuole enlargement in cells and endolysosome fusion in a cell-free assay. These effects were prevented by inhibiting P2X4, expressing a dominant-negative P2X4 mutant, and disrupting the P2X4 gene. We further show that P2X4 and calmodulin (CaM) form a complex at endolysosomal membrane where P2X4 activation recruits CaM to promote fusion and vacuolation in a Ca(2+)-dependent fashion. Moreover, P2X4 activation-triggered fusion and vacuolation were suppressed by inhibiting CaM. Our data thus suggest a new molecular mechanism for endolysosomal membrane fusion involving P2X4-mediated endolysosomal Ca(2+) release and subsequent CaM activation.
© 2015 Cao et al.
... channel [88]), as well as several RAB proteins on the surfaces of the lysosomes [89]. Although very limited information is available on the temporal events of Ca 2 þ -regulated lysosomal exocytosis, MCOLN1 is postulated to regulate plasma membrane delivery by releasing intralysosomal Ca 2 þ upon membrane depolarization, me- chanical force, and/or rapid changes in phosphoinositide con- centrations [90]. Vesicle SNAREs and target SNAREs cooperate to form helix coiled-coil bundles (SNARE complexes) in microdomains of the plasma membrane that are enriched with cholesterol, sphingolipids, and acidic phospholipids ("lipid/membrane rafts") [91,92] and may provide the force necessary to bring opposing bi- layers together and trigger membrane fusion [93]. ...
A unifying feature in the pathogenesis of aging, neurodegenerative disease, and lysosomal storage disorders is the progressive deposition of macromolecular debris impervious to enzyme catalysis by cellular waste disposal mechanisms (e.g., lipofuscin). Aerobic exercise training (AET) has pleiotropic effects and stimulates mitochondrial biogenesis, anti-oxidant defense systems, and autophagic flux in multiple organs and tissues. Our aim was to explore the therapeutic potential of AET as an ancillary therapy to mitigate autophagic buildup and oxidative damage, and rejuvenate the mitochondrial-lysosomal axis in Pompe disease (GSD II/PD).
Fourteen weeks of combined recombinant acid α-glucosidase (rhGAA) and AET polytherapy attenuated mitochondrial swelling, fortified anti-oxidant defense systems, reduced oxidative damage, and augmented glycogen clearance and removal of autophagic debris/lipofuscin in fast-twitch skeletal muscle of GAA-KO mice. Ancillary AET potently augmented the pool of PI4KA transcripts and exerted a mild restorative effect on Syt VII and VAMP-5/myobrevin, collectively suggesting improved endosomal transport and Ca(2+)- mediated lysosomal exocytosis. Compared to traditional rhGAA monotherapy, AET and rhGAA polytherapy effectively mitigated buildup of protein carbonyls, autophagic debris/lipofuscin, and P62/SQSTM1, while enhancing MnSOD expression, nuclear translocation of Nrf-2, muscle mass, and motor function in GAA-KO mice.
Combined AET and rhGAA therapy reactivates cellular clearance pathways, mitigates mitochondrial senescence, and strengthens antioxidant defense systems in GSD II/PD. Aerobic exercise training (or pharmacologic targeting of contractile activity-induced pathways) may have therapeutic potential for mitochondrial-lysosomal axis rejuvenation in lysosomal storage disorders and other related conditions (e.g., aging and neurodegenerative disease).
Copyright © 2015. Published by Elsevier Inc.
... The permeation and gating properties of TRPML1 suggest that the channel function of TRPML1 is to release Ca 2+ from the LEL lumen in response to various cellular cues (79,94), such as an increase in lysosomal PI(3,5)P 2 . TRPML1 may regulate the LE maturation process involving LE-lysosome fusion, as the lysosomal delivery of endocytosed proteins, such as plasma membrane growth factor receptors, is delayed in TRPML1 −/− cells (95,96). ...
Lysosomes are acidic compartments filled with more than 60 different types of hydrolases. They mediate the degradation of extracellular particles from endocytosis and of intracellular components from autophagy. The digested products are transported out of the lysosome via specific catabolite exporters or via vesicular membrane trafficking. Lysosomes also contain more than 50 membrane proteins and are equipped with the machinery to sense nutrient availability, which determines the distribution, number, size, and activity of lysosomes to control the specificity of cargo flux and timing (the initiation and termination) of degradation. Defects in degradation, export, or trafficking result in lysosomal dysfunction and lysosomal storage diseases (LSDs). Lysosomal channels and transporters mediate ion flux across perimeter membranes to regulate lysosomal ion homeostasis, membrane potential, catabolite export, membrane trafficking, and nutrient sensing. Dysregulation of lysosomal channels underlies the pathogenesis of many LSDs and possibly that of metabolic and common neurodegenerative diseases.
... PI(3,5)P 2 may regulate lysosomal traffi cking via three potential mechanisms. First, PI(3,5)P 2 may increase the fusogenic potential of lysosomes by affecting the properties of the lipid bilayer, especially at the fusion spot ( 101 ). At the plasma membrane, PI(4,5)P 2 helps generate the membrane curvature necessary for membrane fusion ( 97 ). ...
Lysosomes are acidic compartments in mammalian cells that are primarily responsible for the breakdown of endocytic and autophagic substrates such as membranes, proteins, and lipids into their basic building blocks. Lysosomal storage diseases (LSDs) are a group of metabolic disorders caused by genetic mutations in lysosomal hydrolases required for catabolic degradation, mutations in lysosomal membrane proteins important for catabolite export or membrane trafficking, or mutations in non-lysosomal proteins indirectly affecting these lysosomal functions. A hallmark feature of LSD is the primary and secondary excessive accumulation of undigested lipids in the lysosome, which causes lysosomal dysfunction and cell death, and subsequently pathological symptoms in various tissues and organs. There are more than 60 types of LSDs, but an effective therapeutic strategy is still lacking for most of them. Several recent in vitro and in vivo studies suggest that induction of lysosomal exocytosis could effectively reduce the accumulation of the storage materials. Meanwhile, the molecular machinery and regulatory mechanisms for lysosomal exocytosis are beginning to be revealed. In this paper, we first discuss these recent developments with the focus on the functional interactions between lipid storage and lysosomal exocytosis. We then discuss whether lysosomal exocytosis can be manipulated to correct lysosomal and cellular dysfunction caused by excessive lipid storage, providing a potentially general therapeutic approach for LSDs.
... It should be noted that the previous observations on calcium-regulated autophagy were mainly focused on Ca 2+ release from the ER or the intracellular Ca 2+ . However, homotypic and heterotypic fusion events between organelles within the endolysosomal system that are required for trafficking are sensitive to Ca 2+ release from lysosome [47], indicative of spatially restricted Ca 2+ elevations in close proximity to the fusion machinery. Recently, the lysosome has emerged as one of the most important intracellular Ca 2+ storage compartments, and the release of Ca 2+ from the lysosome can be triggered by the calcium-mobilizing messenger, NADDP [48,49]. ...
Podocytes are highly differentiated glomerular epithelial cells that contribute to the glomerular barrier function of kidney. A role for autophagy has been proposed in maintenance of their cellular integrity, but the mechanisms controlling autophagy in podocytes are not clear. The present study tested whether CD38-mediated regulation of lysosome function contributes to autophagic flux or autophagy maturation in podocytes. Podocytes were found to exhibit a high constitutive level of LC3-II, a robust marker of autophagosomes (APs), suggesting a high basal level of autophagic activity. Treatment with the mTOR inhibitor, rapamycin, increased LC3-II and the content of both APs detected by Cyto-ID Green staining and autophagolysosomes (APLs) measured by acridine orange staining and colocalization of LC3 and Lamp1. Lysosome function inhibitor bafilomycin A1 increased APs, but decreased APLs content under both basal and rapamycin-induced conditions. Inhibition of CD38 activity by nicotinamide or silencing of CD38 gene produced the similar effects to that bafilomycin A1 did in podocytes. To explore the possibility that CD38 may control podocyte autophagy through its regulation of lysosome function, the fusion of APs with lysosomes in living podocytes was observed by co-transfection of GFP-LC3B and RFP-Lamp1 expression vectors. A colocalization of GFP-LC3B and RFP-Lamp1 upon stimulation of rapamycin became obvious in transfected podocytes, which could be substantially blocked by nicotinamide, CD38 shRNA, and bafilomycin. Moreover, blockade of the CD38-mediated regulation by PPADS completely abolished rapamycin-induced fusion of APs with lysosomes. These results indicate that CD38 importantly control lysosomal function and influence autophagy at the maturation step in podocytes.
Two-pore channels, TPC1 and TPC2, are Ca2+- and Na+-permeable cation channels expressed on the membranes of endosomes and lysosomes in nearly all mammalian cells. These channels have been implicated in Ca2+ signaling initiated from the endolysosomes, vesicular trafficking, cellular metabolism, macropinocytosis, and viral infection. Although TPCs have been shown to mediate Ca2+ release from acidic organelles in response to NAADP (nicotinic acid adenine dinucleotide phosphate), the most potent Ca2+ mobilizing messenger, questions remain whether NAADP is a direct ligand of these channels. In whole-endolysosomal patch clamp recordings, it has been difficult to detect NAADP-evoked currents in vacuoles that expressed TPC1 or TPC2, while PI(3,5)P2 (phosphatidylinositol 3,5-bisphosphate) activated a highly Na+-selective current under the same experimental configuration. In this chapter, we summarize recent progress in this area and provide our observations on NAADP-elicited TPC2 currents from enlarged endolysosomes as well as their possible relationships with the currents evoked by PI(3,5)P2. We propose that TPCs are channels dually regulated by PI(3,5)P2 and NAADP in an interdependent manner and the two endogenous ligands may have both distinguished and cooperative roles.
Lysosomes are acidic membrane-bound organelles that use hydrolytic enzymes to break down material through pathways such as endocytosis, phagocytosis, mitophagy, and autophagy. To function properly, intralysosomal environments are strictly controlled by a set of integral membrane proteins such as ion channels and transporters. Potassium ion (K+) channels are a large and diverse family of membrane proteins that control K+ flux across both the plasma membrane and intracellular membranes. In the plasma membrane, they are essential in both excitable and non-excitable cells for the control of membrane potential and cell signaling. However, our understanding of intracellular K+ channels is very limited. In this review, we summarize the recent development in studies of K+ channels in the lysosome. We focus on their characterization, potential roles in maintaining lysosomal membrane potential and lysosomal function, and pathological implications.
PC-1 and PC-2 form a heteromeric ion channel complex (hereafter called the Polycystin complex) that is abundantly expressed on primary cilia of renal epithelial cells. Mutations within the polycystin complex cause Autosomal Dominant Polycystic Kidney Disease (ADPKD). The Polycystin complex forms a non-selective cation channel, yet the spatial and temporal regulation of the polycystin complex within the ciliary membrane remains poorly understood, partially due to technical limitations posed by the tiny ciliary compartment. Here, we employ our novel assays to functionally reconstitute the polycystin complex in the plasma membrane. Using whole-cell and ciliary patch-clamp recordings we identified a ciliary enriched oxysterol, 7β,27-DHC, as a critical component required for activation of the polycystin complex. We identified a novel oxysterol binding pocket in PC-2 using molecular docking simulation. We also identified two amino acids within the PC-2 oxysterol binding pocket, E208 and R581, to be critical for 7β,27-DHC dependent polycystin activation in both the plasma membrane and ciliary compartment. Further, we can show that the pharmacological and genetic inhibition of oxysterol synthesis by carbenoxolone (CNX) reduces channel activity in primary cilia. Our findings identified a unique second messenger that regulates the polycystin complex. We hypothesize that cilia-enriched lipids license the polycystin complex to be functional only in the ciliary organelle, thus providing novel insights into the spatial regulation of the polycystin complex. Our results also establish a framework to target the same allosteric regulatory site in the polycystin complex to identify activators of the polycystin channels as novel therapeutic strategies for ADPKD.
The lysosome is an important membrane-bound acidic organelle that is regarded as the degradative center as well as multifunctional signaling hub. It digests unwanted macromolecules, damaged organelles, microbes, and other materials derived from endocytosis, autophagy, and phagocytosis. To function properly, the ionic homeostasis and membrane potential of the lysosome are strictly regulated by transporters and ion channels. As the most abundant cation inside the cell, potassium ions (K⁺) are vital for lysosomal membrane potential and lysosomal calcium (Ca²⁺) signaling. However, our understanding about how lysosomal K⁺ homeostasis is regulated and what are the functions of K⁺ in the lysosome is very limited. Currently, two lysosomal K⁺ channels have been identified: large-conductance Ca²⁺-activated K⁺ channel (BK) and transmembrane Protein 175 (TMEM175). In this review, we summarize recent development in our understanding of K⁺ homeostasis and K⁺ channels in the lysosome. We hope to guide the readers into a more in-depth discussion of lysosomal K⁺ channels in lysosomal physiology and human diseases.
Macroautophagy/autophagy is elevated to ensure the high demand for nutrients for the growth of cancer cells. Here we demonstrated that MCOLN1/TRPML1 is a pharmaceutical target of oncogenic autophagy in cancers such as pancreatic cancer, breast cancer, gastric cancer, malignant melanoma, and glioma. First, we showed that activating MCOLN1, by increasing expression of the channel or using the MCOLN1 agonists, ML-SA5 or MK6-83, arrests autophagic flux by perturbing fusion between autophagosomes and lysosomes. Second, we demonstrated that MCOLN1 regulates autophagy by mediating the release of zinc from the lysosome to the cytosol. Third, we uncovered that zinc influx through MCOLN1 blocks the interaction between STX17 (syntaxin 17) in the autophagosome and VAMP8 in the lysosome and thereby disrupting the fusion process that is determined by the two SNARE proteins. Furthermore, we demonstrated that zinc influx originating from the extracellular fluid arrests autophagy by the same mechanism as lysosomal zinc, confirming the fundamental function of zinc as a participant in membrane trafficking. Last, we revealed that activating MCOLN1 with the agonists, ML-SA5 or MK6-83, triggers cell death of a number of cancer cells by evoking autophagic arrest and subsequent apoptotic response and cell cycle arrest, with little or no effect observed on normal cells. Consistent with the in vitro results, administration of ML-SA5 in Patu 8988 t xenograft mice profoundly suppresses tumor growth and improves survival. These results establish that a lysosomal cation channel, MCOLN1, finely controls oncogenic autophagy in cancer by mediating zinc influx into the cytosol.
Podocytes are visceral epithelial cells covering the outer surface of glomerular capillaries in the kidney. Blood is filtered through the slit diaphragm of podocytes to form urine. The functional and structural integrity of podocytes is essential for the normal function of the kidney. As a membrane-bound organelle, lysosomes are responsible for the degradation of molecules via hydrolytic enzymes. In addition to its degradative properties, recent studies have revealed that lysosomes may serve as a platform mediating cellular signaling in different types of cells. In the last decade, increasing evidence has revealed that the normal function of the lysosome is important for the maintenance of podocyte homeostasis. Podocytes have no ability to proliferate under most pathological conditions; therefore, lysosome-dependent autophagic flux is critical for podocyte survival. In addition, new insights into the pathogenic role of lysosome and associated signaling in podocyte injury and chronic kidney disease have recently emerged. Targeting lysosomal functions or signaling pathways are considered potential therapeutic strategies for some chronic glomerular diseases. This review briefly summarizes current evidence demonstrating the regulation of lysosomal function and signaling mechanisms as well as the canonical and noncanonical roles of podocyte lysosome dysfunction in the development of chronic glomerular diseases and associated therapeutic strategies.
The accumulation of abnormal protein aggregates contributes to the pathological progression of diverse neurodegenerative diseases. An increasing body of evidence indicates that defects in the protein clearance system play a crucial role in this process. Cargoes delivered via endosomes, phagosomes, and autophagosomes converge on lysosomes for degradation, which process is collectively called “the endosome-autophagosome-lysosome pathway” or EALP. As such, dysfunction of lysosomes may result in the accumulation of all these upstream vesicles/cargoes and may play a key role in diverse neurodegenerative conditions. Over the years, we found that Zn-metallothionein-3 (MT3), the brain-enriched form of metallothionein, regulates lysosomal functions in cortical astrocytes. Zn-MT3 appears to interact with β-actin and activate c-Abl kinase. As a result, Zn-MT3 plays a role in maintaining lysosomal acidity, a prerequisite for vesicle fusion as well as cargo degradation. The reported downregulation of MT3 in Alzheimer’s disease (AD), hence, may contribute to lysosomal dysfunction in AD. Of interest, raising intracellular free zinc levels also caused lysosomal acidification and normalization of degradation, even in the context of arrested autophagy. Pending further research on the mechanisms of these effects, we propose that measures increasing Zn-MT3 and/or intracellular/lysosomal free zinc may be useful in normalizing lysosomal functions in neurodegenerative conditions.
Niemann–Pick type C disease (NPCD) was first described in 1914 and affects approximately 1 in 150 000 live births. It is characterized clinically by diverse symptoms affecting liver, spleen, motor control, and brain; premature death invariably results. Its molecular origins were traced, as late as 1997, to a protein of late endosomes and lysosomes which was named NPC1. Mutation or absence of this protein leads to accumulation of cholesterol in these organelles. In this review, we focus on the intracellular events that drive the pathology of this disease. We first introduce endocytosis, a much‐studied area of dysfunction in NPCD cells, and survey the various ways in which this process malfunctions. We briefly consider autophagy before attempting to map the more complex pathways by which lysosomal cholesterol storage leads to protein misregulation, mitochondrial dysfunction, and cell death. We then briefly introduce the metabolic pathways of sphingolipids (as these emerge as key species for treatment) and critically examine the various treatment approaches that have been attempted to date.
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Middle East Respiratory Syndrome coronavirus (MERS-CoV) infections are associated with a significant mortality rate, and existing drugs show poor efficacy. Identifying novel targets/pathways required for MERS infectivity is therefore important for developing novel therapeutics. As an enveloped virus, translocation through the endolysosomal system provides one pathway for cellular entry of MERS-CoV. In this context, Ca2+-permeable channels within the endolysosomal system regulate both the luminal environment and trafficking events, meriting investigation of their role in regulating processing and trafficking of MERS-CoV. Knockdown of endogenous two-pore channels (TPCs), targets for the Ca2+ mobilizing second messenger NAADP, impaired infectivity in a MERS-CoV spike pseudovirus particle translocation assay. This effect was selective as knockdown of the lysosomal cation channel mucolipin-1 (TRPML1) was without effect. Pharmacological inhibition of NAADP-evoked Ca2+ release using several bisbenzylisoquinoline alkaloids also blocked MERS pseudovirus translocation. Knockdown of TPC1 (biased endosomally) or TPC2 (biased lysosomally) decreased the activity of furin, a protease which facilitates MERS fusion with cellular membranes. Pharmacological or genetic inhibition of TPC1 activity also inhibited endosomal motility impairing pseudovirus progression through the endolysosomal system. Overall, these data support a selective, spatially autonomous role for TPCs within acidic organelles to support MERS-CoV translocation.
Blockade of lysosomal calcium release due to lysosomal lipid accumulation has been shown to inhibit mTORC1 signaling. However, the mechanism by which lysosomal calcium regulates mTORC1 has remained undefined. Herein we report that proper lysosomal calcium release through the calcium channel TRPML1 is required for mTORC1 activation. TRPML1 depletion inhibits mTORC1 activity, while overexpression or pharmacologic activation of TRPML1 has the opposite effect. Lysosomal calcium activates mTORC1 by inducing association of calmodulin (CaM) with mTOR. Blocking the interaction between mTOR and CaM by antagonists of CaM significantly inhibits mTORC1 activity. Moreover, CaM is capable of stimulating the kinase activity of mTORC1 in a calcium-dependent manner in vitro. These results reveal that mTOR is a new type of CaM-dependent kinase, and TRPML1, lysosomal calcium and CaM play essential regulatory roles in the mTORC1 signaling pathway.
The central or lytic vacuole is the largest intracellular organelle in plant cells, but we know unacceptably little about the mechanisms regulating its function in vivo. The underlying reasons are related to difficulties in accessing this organelle without disrupting the cellular integrity and to the dynamic morphology of the vacuole, which lacks a defined structure. Among such morphological changes, vacuolar convolution is probably the most commonly observed event, reflected in the (reversible) transformation of a large central vacuole into a structure consisting of interconnected bubbles of a smaller size. Such behaviour is observed in plant cells subjected to hyperosmotic stress but also takes place in physiological conditions (e.g. during stomatal closure). Although vacuolar convolution is a relatively common phenomenon in plants, studies aimed at elucidating its execution mechanisms are rather scarce. In the present review, we analyse the available evidence on the participation of the cellular cytoskeleton and ion transporters in vacuolar morphology dynamics, putting special emphasis on the available evidence of the role played by phosphatidylinositol 3,5-bisphosphate in this process.
Lysosomes and lysosome-related organelles are emerging as intracellular Ca²⁺ stores and play important roles in a variety of membrane trafficking processes, including endocytosis, exocytosis, phagocytosis and autophagy. Impairment of lysosomal Ca²⁺ homeostasis and membrane trafficking has been implicated in many human diseases such as lysosomal storage diseases (LSDs), neurodegeneration, myopathy and cancer. Lysosomal membrane proteins, in particular ion channels, are crucial for lysosomal Ca²⁺ signaling. Compared with ion channels in the plasma membrane, lysosomal ion channels and their roles in lysosomal Ca²⁺ signaling are less understood, largely due to their intracellular localization and the lack of feasible functional assays directly applied to the native environment. Recent advances in biomedical methodology have made it possible to directly investigate ion channels in the lysosomal membrane. In this review, we provide a summary of the newly developed methods for monitoring lysosomal Ca²⁺ and ion channels, as well as the recent discovery of lysosomal ion channels and their significances in intracellular Ca²⁺ signaling. These new techniques will expand our research scope and our understanding of the nature of lysosomes and lysosome-related diseases.
The transient receptor potential mucolipin (TRPML) subfamily of transient receptor potential cation channels consists of three members (TRPML1-3) that function at various stages of endocytosis. Conventional research in the TRPML field suggests that dysfunction along these endocytic stages underlies the severe psychomotor impairment in mucolipidosis type IV (MLIV). However, recent studies intimate that TRPMLs may be implicated in other neuropathological disorders as well. This review follows the historical development of TRPML research from the clinical description of the first MLIV patient until present-day characterization of TRPML1-based defects in MLIV on the molecular and cell biological levels. In addition, the aberrant role of TRPML3 in varitint-waddler mouse pathology is elucidated and the normal function of the protein and its paralogs are described. TRPML electrophysiology, pharmacology, and animal models are discussed and TRPML-associated systemic and neurological disorders, not including MLIV, are also addressed. Recently, a number of prospective TRPML-based therapies have been proposed in the treatment of these disorders. These prospects are carefully considered here as well. Altogether, the aforementioned descriptions aim to highlight the transformation of TRPML research from a single discipline, single gene, and single disorder field into a multidisciplinary, multigene endeavor with wide application to therapeutic treatment of several neurobiological disorders.
The physiology and functions of ion channels have been major topics of interest in biomedical research. Patch clamping is one of the most powerful techniques used in the study of ion channels and has been widely applied to the investigation of electrical properties of ion channels on the plasma membrane in a variety of cells. A number of ion channels have been found in intracellular lysosomal membranes. However, their properties had been difficult to study due to the lack of a direct patch-clamping methodology on lysosomal membranes. Past attempts to record lysosomal channels that were forced to express on the plasma membrane or reconstituted into lipid bilayers have largely generated inconclusive and conflicting results. Recently, a novel lysosome patch-clamping technique has been developed, making it possible to examine lysosomal channels under near physiological conditions. This chapter provides a detailed description of this technique, which has been successfully applied in several studies concerning lysosomal ion channels. This technique will expand our understanding of the nature of lysosomes and lysosome-related diseases.
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The large Trp gene family encodes transient receptor potential (TRP) proteins that form novel cation-selective ion channels. In mammals, 28 Trp channel genes have been identified. TRP proteins exhibit diverse permeation and gating properties and are involved in a plethora of physiologic functions with a strong impact on cellular sensing and signaling pathways. Indeed, mutations in human genes encoding TRP channels, the so-called "TRP channelopathies," are responsible for a number of hereditary diseases that affect the musculoskeletal, cardiovascular, genitourinary, and nervous systems. This review gives an overview of the functional properties of mammalian TRP channels, describes their roles in acquired and hereditary diseases, and discusses their potential as drug targets for therapeutic intervention.
The first member of the mammalian mucolipin TRP channel subfamily (TRPML1) is a cation-permeable channel that is predominantly localized on the membranes of late endosomes and lysosomes (LELs) in all mammalian cell types. In response to the regulatory changes of LEL-specific phosphoinositides or other cellular cues, TRPML1 may mediate the release of Ca(2+) and heavy metal Fe(2+)/Zn(2+)ions into the cytosol from the LEL lumen, which in turn may regulate membrane trafficking events (fission and fusion), signal transduction, and ionic homeostasis in LELs. Human mutations in TRPML1 result in type IV mucolipidosis (ML-IV), a childhood neurodegenerative lysosome storage disease. At the cellular level, loss-of-function mutations of mammalian TRPML1 or its C. elegans or Drosophila homolog gene results in lysosomal trafficking defects and lysosome storage. In this chapter, we summarize recent advances in our understandings of the cell biological and channel functions of TRPML1. Studies on TRPML1's channel properties and its regulation by cellular activities may provide clues for developing new therapeutic strategies to delay neurodegeneration in ML-IV and other lysosome-related pediatric diseases.
Ca(2+) signals regulate a wide range of physiological processes. Intracellular Ca(2+) stores can be mobilized in response to extracellular stimuli via a range of signal transduction mechanisms, often involving recruitment of diffusible second messenger molecules. The Ca(2+) mobilizing messengers InsP3 and cADPR release Ca(2+) from the endoplasmic reticulum via the InsP3 and ryanodine receptors, respectively, while a third messenger, NAADP, releases Ca(2+) from acidic endosomes and lysosomes. Bidirectional communication between the ER and acidic organelles may have functional relevance for endolysosomal function as well as for the generation of Ca(2+) signals. The two-pore channels (TPCs), are currently strong candidates for being key components of NAADP-regulated Ca(2+) channels. Ca(2+) signals have been shown to play important roles in differentiation; however, much remains to be established about the exact signalling mechanisms involved. The investigation of the role of NAADP and TPCs in differentiation is still at an early stage but recent studies have suggested that they are important mediators of differentiation of neurons, skeletal muscle cells, and osteoclasts. NAADP signals and TPCs have also been implicated in autophagy, an important process in differentiation. Further studies will be required to identify the precise mechanism of TPC action and their link with NAADP signalling, as well as relating this to their roles in differentiation and other key processes in the cell and organism. This article is protected by copyright. All rights reserved.
Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+ mobilizing endogenous compound known to date. Although its Ca2+releasing activity has been demonstrated in many cell types and in response to different extracellular stimuli, several aspects of NAADP signaling are unclear. This overview focuses on the controversial aspects and reviews NAADP´s role as second messenger: endogenous concentrations and its receptor-mediated alterations, metabolism, and potential organelle and ion channel targets. Finally, the role of NAADP as Ca2+ trigger is discussed by reviewing the development of local into global Ca2+ signals evoked by NAADP.
Gene mutations in the phosphoinositide-metabolizing enzymes are linked to various human diseases. In mammals, PIKfyve synthesizes
PtdIns(3,5)P2 and PtdIns5P lipids that regulate endosomal trafficking and responses to extracellular stimuli. The consequence of pikfyve gene ablation in mammals is unknown. To clarify the importance of PIKfyve and PIKfyve lipid products, in this study, we have
characterized the first mouse model with global deletion of the pikfyve gene using the Cre-loxP approach. We report that nearly all PIKfyveKO/KO mutant embryos died before the 32–64-cell stage. Cultured fibroblasts derived from PIKfyveflox/flox embryos and rendered pikfyve-null by Cre recombinase expression displayed severely reduced DNA synthesis, consistent with impaired cell division causing
early embryo lethality. The heterozygous PIKfyveWT/KO mice were born at the expected Mendelian ratio and developed into adulthood. PIKfyveWT/KO mice were ostensibly normal by several other in vivo, ex vivo, and in vitro criteria despite the fact that their levels of the PIKfyve protein and in vitro enzymatic activity in cells and tissues were 50–55% lower than those of wild-type mice. Consistently, steady-state levels
of the PIKfyve products PtdIns(3,5)P2 and PtdIns5P selectively decreased, but this reduction (35–40%) was 10–15% less than that expected based on PIKfyve protein
reduction. The nonlinear decrease of the PIKfyve protein versus PIKfyve lipid products, the potential mechanism(s) discussed herein, may explain how one functional allele in PIKfyveWT/KO mice is able to support the demands for PtdIns(3,5)P2/PtdIns5P synthesis during life. Our data also shed light on the known human disorder linked to PIKFYVE mutations.
Phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P(2)] regulates several vacuolar functions, including acidification, morphology, and membrane traffic. The lipid kinase Fab1 converts phosphatidylinositol-3-phosphate [PtdIns(3)P] to PtdIns(3,5)P(2). PtdIns(3,5)P(2) levels are controlled by the adaptor-like protein Vac14 and the Fig4 PtdIns(3,5)P(2)-specific 5-phosphatase. Interestingly, Vac14 and Fig4 serve a dual function: they are both implicated in the synthesis and turnover of PtdIns(3,5)P(2) by an unknown mechanism. We now show that Fab1, through its chaperonin-like domain, binds to Vac14 and Fig4 and forms a vacuole-associated signaling complex. The Fab1 complex is tethered to the vacuole via an interaction between the FYVE domain in Fab1 and PtdIns(3)P on the vacuole. Moreover, Vac14 and Fig4 bind to each other directly and are mutually dependent for interaction with the Fab1 kinase. Our observations identify a protein complex that incorporates the antagonizing Fab1 lipid kinase and Fig4 lipid phosphatase into a common functional unit. We propose a model explaining the dual roles of Vac14 and Fig4 in the synthesis and turnover of PtdIns(3,5)P(2).
Phosphatidylinositol 4,5-bisphosphate (PI 4,5-P(2)) on the plasma membrane is essential for vesicle exocytosis but its role in membrane fusion has not been determined. Here, we quantify the concentration of PI 4,5-P(2) as approximately 6 mol% in the cytoplasmic leaflet of plasma membrane microdomains at sites of docked vesicles. At this concentration of PI 4,5-P(2) soluble NSF attachment protein receptor (SNARE)-dependent liposome fusion is inhibited. Inhibition by PI 4,5-P(2) likely results from its intrinsic positive curvature-promoting properties that inhibit formation of high negative curvature membrane fusion intermediates. Mutation of juxtamembrane basic residues in the plasma membrane SNARE syntaxin-1 increase inhibition by PI 4,5-P(2), suggesting that syntaxin sequesters PI 4,5-P(2) to alleviate inhibition. To define an essential rather than inhibitory role for PI 4,5-P(2), we test a PI 4,5-P(2)-binding priming factor required for vesicle exocytosis. Ca(2+)-dependent activator protein for secretion promotes increased rates of SNARE-dependent fusion that are PI 4,5-P(2) dependent. These results indicate that PI 4,5-P(2) regulates fusion both as a fusion restraint that syntaxin-1 alleviates and as an essential cofactor that recruits protein priming factors to facilitate SNARE-dependent fusion.
Gene mutations in the phosphoinositide-metabolizing enzymes are linked to various human diseases. In mammals, PIKfyve synthesizes
PtdIns(3,5)P2 and PtdIns5P lipids that regulate endosomal trafficking and responses to extracellular stimuli. The consequence of pikfyve gene ablation in mammals is unknown. To clarify the importance of PIKfyve and PIKfyve lipid products, in this study, we have
characterized the first mouse model with global deletion of the pikfyve gene using the Cre-loxP approach. We report that nearly all PIKfyveKO/KO mutant embryos died before the 32–64-cell stage. Cultured fibroblasts derived from PIKfyveflox/flox embryos and rendered pikfyve-null by Cre recombinase expression displayed severely reduced DNA synthesis, consistent with impaired cell division causing
early embryo lethality. The heterozygous PIKfyveWT/KO mice were born at the expected Mendelian ratio and developed into adulthood. PIKfyveWT/KO mice were ostensibly normal by several other in vivo, ex vivo, and in vitro criteria despite the fact that their levels of the PIKfyve protein and in vitro enzymatic activity in cells and tissues were 50–55% lower than those of wild-type mice. Consistently, steady-state levels
of the PIKfyve products PtdIns(3,5)P2 and PtdIns5P selectively decreased, but this reduction (35–40%) was 10–15% less than that expected based on PIKfyve protein
reduction. The nonlinear decrease of the PIKfyve protein versus PIKfyve lipid products, the potential mechanism(s) discussed herein, may explain how one functional allele in PIKfyveWT/KO mice is able to support the demands for PtdIns(3,5)P2/PtdIns5P synthesis during life. Our data also shed light on the known human disorder linked to PIKFYVE mutations.
Until recently, the mechanisms that regulate endolysosomal calcium homoeostasis were poorly understood. The discovery of the molecular target of NAADP (nicotinic acid-adenine dinucleotide phosphate) as the two-pore channels resident in the endolysosomal system has highlighted this compartment as an important calcium store. The recent findings that dysfunctional NAADP release leads to defective endocytic function which in turn results in secondary lipid accumulation in the lysosomal storage disease Niemann-Pick type C, is the first evidence of a direct connection between a human disease and defective lysosomal calcium release. In the present review, we provide a summary of the current knowledge on mechanisms of calcium homoeostasis within the endolysosomal system and how these mechanisms may be affected in human metabolic disorders.
Phosphatidylinositol 3-phosphate (PI(3)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) are essential for rapid SNARE-dependent fusion of yeast vacuoles and other organelles. These phosphoinositides also regulate the fusion of reconstituted proteoliposomes. The reconstituted reaction allows separate analysis of phosphoinositide-responsive subreactions: fusion with SNAREs alone, with the addition of the HOPS tethering factor, and with the further addition of the SNARE complex disassembly chaperones Sec17p and Sec18p. Using assays of membrane tethering, trans-SNARE pairing, and lipid mixing, we found that PI(3)P and PI(4,5)P(2) have distinct functions that are asymmetric with respect to R-SNARE (Nyv1p) and the 3Q-SNAREs (Vam3p, Vti1p, and Vam7p). Fusion reactions with the Q-SNAREs and R-SNARE on separate membranes showed that PI(3)P has two distinct functions. PI(3)P on Q-SNARE proteoliposomes promoted Vam7p binding and association with the other two Q-SNAREs. PI(3)P on R-SNARE proteoliposomes was recognized by the PX domain of Vam7p on Q-SNARE proteoliposomes to promote tethering, although this function could be supplanted by the tethering activity of HOPS. PI(4,5)P(2) stimulated fusion when it was on R-SNARE proteoliposomes, apposed to Q-SNARE proteoliposomes bearing PI(3)P. These functions are essential for the phosphoinositide-dependent synergy between HOPS and Sec17p/Sec18p in promoting rapid fusion.
Membrane fusion and fission events in intracellular trafficking are controlled by both intraluminal Ca(2+) release and phosphoinositide (PIP) signalling. However, the molecular identities of the Ca(2+) release channels and the target proteins of PIPs are elusive. In this paper, by direct patch-clamping of the endolysosomal membrane, we report that PI(3,5)P(2), an endolysosome-specific PIP, binds and activates endolysosome-localized mucolipin transient receptor potential (TRPML) channels with specificity and potency. Both PI(3,5)P(2)-deficient cells and cells that lack TRPML1 exhibited enlarged endolysosomes/vacuoles and trafficking defects in the late endocytic pathway. We find that the enlarged vacuole phenotype observed in PI(3,5)P(2)-deficient mouse fibroblasts is suppressed by overexpression of TRPML1. Notably, this PI(3,5)P(2)-dependent regulation of TRPML1 is evolutionarily conserved. In budding yeast, hyperosmotic stress induces Ca(2+) release from the vacuole. In this study, we show that this release requires both PI(3,5)P(2) production and a yeast functional TRPML homologue. We propose that TRPMLs regulate membrane trafficking by transducing information regarding PI(3,5)P(2) levels into changes in juxtaorganellar Ca(2+), thereby triggering membrane fusion/fission events.
“Spontaneous” Release Trigger
Synaptic vesicle release occurs in different phases that can be tightly coupled to action potentials (synchronous), immediately following action potentials (asynchronous), or as stochastic events not triggered by action potentials (spontaneous). The vesicle protein synaptotagmin is thought to act as the Ca ²⁺ sensor in the synchronous phase, but for the other two phases, Ca ²⁺ sensors have not been identified. Groffen et al. (p. 1614 , published online 11 February) now show that cytoplasmic proteins known as Doc2 (double C2 domain) proteins are required for spontaneous release. Doc2 proteins promote membrane fusion in response to exceptionally low increases in Ca ²⁺ , and are several orders of magnitude more sensitive to Ca ²⁺ than synaptotagmin. Doc2 and synaptotagmin compete for SNARE-complex binding during membrane fusion. A mutation that abolishes the Ca ²⁺ dependence of Doc2b also abolishes the Ca ²⁺ dependence of spontaneous release. Thus, Doc2 is a high-affinity Ca ²⁺ sensor for spontaneous release that competes with synaptotagmin for SNARE complex binding.
Loss of function mutations in mucolipin-1 (MCOLN1) have been linked to mucolipidosis type IV (MLIV), a recessive lysosomal
storage disease characterized by severe neurological and ophthalmological abnormalities. MCOLN1 is an ion channel that regulates
membrane transport along the endolysosomal pathway. It has been suggested that MCOLN1 participates in several Ca2+-dependent processes, including fusion of lysosomes with the plasma membrane, fusion of late endosomes and autophagosomes
with lysosomes, and lysosomal biogenesis. Here, we searched for proteins that interact with MCOLN1 in a Ca2+-dependent manner. We found that the penta-EF-hand protein ALG-2 binds to the NH-terminal cytosolic tail of MCOLN1. The interaction
is direct, strictly dependent on Ca2+, and mediated by a patch of charged and hydrophobic residues located between MCOLN1 residues 37 and 49. We further show that
MCOLN1 and ALG-2 co-localize to enlarged endosomes induced by overexpression of an ATPase-defective dominant-negative form
of Vps4B (Vps4BE235Q). In agreement with the proposed role of MCOLN1 in the regulation of fusion/fission events, we found that overexpression
of MCOLN1 caused accumulation of enlarged, aberrant endosomes that contain both early and late endosome markers. Interestingly,
aggregation of abnormal endosomes was greatly reduced when the ALG-2-binding domain in MCOLN1 was mutated, suggesting that
ALG-2 regulates MCOLN1 function. Overall, our data provide new insight into the molecular mechanisms that regulate MCOLN1
activity. We propose that ALG-2 acts as a Ca2+ sensor that modulates the function of MCOLN1 along the late endosomal-lysosomal pathway.
Autophagy or "self-eating" is a highly conserved pathway that enables cells to degrade pieces of themselves in autolysosomes to enable their survival in times of stress, including nutrient deprivation. The formation of these degradative compartments requires cytosolic proteins, some of which are autophagy specific, as well as intracellular organelles, such as the ER and Golgi, and the endosome-lysosome system. Here we discuss the cross talk between autophagy and intracellular compartments, highlighting recent exciting data about the role and regulation of the Vps34 class III phosphatidylinositol (PI) 3-kinase in autophagy.
Mutations affecting the conversion of PI3P to the signaling lipid PI(3,5)P(2) result in spongiform degeneration of mouse brain and are associated with the human disorders Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis (ALS). We now report accumulation of the proteins LC3-II, p62 and LAMP-2 in neurons and astrocytes of mice with mutations in two components of the PI(3,5)P(2) regulatory complex, Fig4 and Vac14. Cytoplasmic inclusion bodies containing p62 and ubiquinated proteins are present in regions of the mutant brain that undergo degeneration. Co-localization of p62 and LAMP-2 in affected cells indicates that formation or recycling of the autolysosome is impaired. These results establish a role for PI(3,5)P(2) in autophagy in the mammalian central nervous system (CNS) and demonstrate that mutations affecting PI(3,5)P(2) can contribute to inclusion body disease.
Autophagy is a process of self-degradation of cellular components in which double-membrane autophagosomes sequester organelles or portions of cytosol and fuse with lysosomes or vacuoles for breakdown by resident hydrolases. Autophagy is upregulated in response to extra- or intracellular stress and signals such as starvation, growth factor deprivation, ER stress, and pathogen infection. Defective autophagy plays a significant role in human pathologies, including cancer, neurodegeneration, and infectious diseases. We present our current knowledge on the key genes composing the autophagy machinery in eukaryotes from yeast to mammalian cells and the signaling pathways that sense the status of different types of stress and induce autophagy for cell survival and homeostasis. We also review the recent advances on the molecular mechanisms that regulate the autophagy machinery at various levels, from transcriptional activation to post-translational protein modification.
Insulin-regulated stimulation of glucose entry and mobilization of fat/muscle-specific glucose transporter GLUT4 onto the cell surface require the phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P(2)) pathway for optimal performance. The reduced insulin responsiveness observed under ablation of the PtdIns(3,5)P(2)-synthesizing PIKfyve and its associated activator ArPIKfyve in 3T3L1 adipocytes suggests that dysfunction of the PtdIns(3,5)P(2)-specific phosphatase Sac3 may yield the opposite effect. Paradoxically, as uncovered recently, in addition to turnover Sac3 also supports PtdIns(3,5)P(2) biosynthesis by allowing optimal PIKfyve-ArPIKfyve association. These opposing inputs raise the key question as to whether reduced Sac3 protein levels and/or hydrolyzing activity will produce gain in insulin responsiveness. Here we report that small interfering RNA-mediated knockdown of endogenous Sac3 by approximately 60%, which resulted in a slight but significant elevation of PtdIns(3,5)P(2) in 3T3L1 adipocytes, increased GLUT4 translocation and glucose entry in response to insulin. In contrast, ectopic expression of Sac3(WT), but not phosphatase-deficient Sac3(D488A), reduced GLUT4 surface abundance in the presence of insulin. Endogenous Sac3 physically assembled with PIKfyve and ArPIKfyve in both membrane and soluble fractions of 3T3L1 adipocytes, but this remained insulin-insensitive. Importantly, acute insulin markedly reduced the in vitro C8-PtdIns(3,5)P(2) hydrolyzing activity of Sac3. The insulin-sensitive Sac3 pool likely controls a discrete PtdIns(3,5)P(2) subfraction as the high pressure liquid chromatography-measurable insulin-dependent elevation in total [(3)H]inositol-PtdIns(3,5)P(2) was minor. Together, our data identify Sac3 as an insulin-sensitive phosphatase whose down-regulation increases insulin responsiveness, thus implicating Sac3 as a novel drug target in insulin resistance.
The intracellular Ca(2+) concentration ([Ca(2+)](i)) in skeletal muscles must be rapidly regulated during the excitation-contraction-relaxation process. However, the signalling components involved in such rapid Ca(2+) movement are not fully understood. Here we report that mice deficient in the newly identified PtdInsP (phosphatidylinositol phosphate) phosphatase MIP/MTMR14 (muscle-specific inositol phosphatase) show muscle weakness and fatigue. Muscles isolated from MIP/MTMR14(-/-) mice produced less contractile force, had markedly prolonged relaxation and showed exacerbated fatigue relative to normal muscles. Further analyses revealed that MIP/MTMR14 deficiency resulted in spontaneous Ca(2+) leakage from the internal store - the sarcoplasmic reticulum. This was attributed to decreased metabolism (dephosphorylation) and the subsequent accumulation of MIP/MTMR14 substrates, especially PtdIns(3,5)P(2) and PtdIns (3,4)P(2). Furthermore, we found that PtdIns(3,5)P(2) and PtdIns(3,4)P(2) bound to, and directly activated, the Ca(2+) release channel (ryanodine receptor 1, RyR1) of the sarcoplasmic reticulum. These studies provide the first evidence that finely controlled PtdInsP levels in muscle cells are essential for maintaining Ca(2+) homeostasis and muscle performance.
TRPM2 is a Ca2+-permeable cation channel that is specifically activated by adenosine diphosphoribose (ADPR). Channel activation in the plasma membrane leads to Ca2+ influx and has been linked to apoptotic mechanisms. The primary agonist, ADPR, is produced both extra- and intracellularly and causes increases in intracellular calcium concentration ([Ca2+]i), but the mechanisms involved are not understood. Using short interfering RNA and a knockout mouse, we report that TRPM2, in addition to its role as a plasma membrane channel, also functions as a Ca2+-release channel activated by intracellular ADPR in a lysosomal compartment. We show that both functions of TRPM2 are critically linked to hydrogen peroxide-induced beta cell death. Additionally, extracellular ADPR production by the ectoenzyme CD38 from its substrates NAD+ (nicotinamide adenine dinucleotide) or cADPR causes IP3-dependent Ca2+ release via P2Y and adenosine receptors. Thus, ADPR and TRPM2 represent multimodal signaling elements regulating Ca2+ mobilization in beta cells through membrane depolarization, Ca2+ influx, and release of Ca2+ from intracellular stores.
PtdIns(3,5)P(2) is one of the seven regulatory PPIn (polyphosphoinositides) that are ubiquitous in eukaryotes. It controls membrane trafficking at multiple points in the endosomal/lysosomal system and consequently regulates the size, shape and acidity of at least one endo-lysosomal compartment. PtdIns(3,5)P(2) appears to exert this control via multiple effector proteins, with each effector specific for a subset of the various PtdIns(3,5)P(2)-dependent processes. Some putative PtdIns(3,5)P(2) effectors have been identified, including Atg18p-related PROPPIN [beta-propeller(s) that bind PPIn] proteins and the epsin-like proteins Ent3p and Ent5p, whereas others remain to be defined. One of the principal functions of PtdIns(3,5)P(2) is to regulate the fission/fragmentation of endo-lysosomal sub-compartments. PtdIns(3,5)P(2) is required for vesicle formation during protein trafficking between endo-lysosomes and also for fragmentation of endo-lysosomes into smaller compartments. In yeast, hyperosmotic stress accelerates the latter process. In the present review we highlight and discuss recent studies that reveal the role of the HOPS-CORVET complex and the vacuolar H(+)-ATPase in the process of endo-lysosome fission, and speculate on connections between these machineries and the Fab1p pathway. We also discuss new evidence linking PtdIns(3,5)P(2) and PtdIns5P to the regulation of exocytosis.
Phosphoinositides are implicated in the regulation of a wide variety of cellular functions. Their importance in cellular and organismal physiology is underscored by the growing number of human diseases linked to perturbation of kinases and phosphatases that catalyze interconversion from one phosphoinositide to another. Many such enzymes are attractive targets for therapeutic interventions. Here, we review diseases linked to inheritable or somatic mutations of these enzymes. Phosphatidylinositol (PtdIns), a membrane phospholipid, can be reversibly phosphorylated at the 3, 4, and 5 positions of the inositol ring to generate seven phosphoinositides [PI3P, PI4P, PI5P, PI( 3 , 4 )P 2 , PI( 4 , 5 )P 2 , PI( 3 , 5 )P2, and PI( 3 , 4 , 5 )P 3 ] (FIGURE 1A ). The importance of this metabolism in cell regulation was first established in the context of studies on stimulus-secretion coupling. It was found that many stimuli that trigger secretion also trigger enhanced turnover of PtdIns and phosphoinositides ( 42 ). Subsequently, it became clear that phospholipase C-dependent hydrolysis of PI( 4 , 5 )P 2 to generate the second messenger molecules diacyl glycerol and Ins( 1 , 4 , 5 )P 3 (IP 3 ) is a mechanism through which many cell surface receptors, including many receptors that stimulate secretion, transduce their signals ( 10 ). Diacyl glycerol binds and regulates protein kinase C and a variety of other effectors, whereas IP 3 triggers calcium release from the endoplasmic reticulum ( 10 , 42 ). In another signal transduction pathway, PI( 4 , 5 )P 2 is cleaved by phospholipase A 2 to generate arachidonic acid, a precursor of many signaling molecules.
Membrane fusion underlies many cellular events, including secretion, exocytosis, endocytosis, organelle reconstitution, transport from endoplasmic reticulum to Golgi and nuclear envelope formation. A large number of investigations into membrane fusion indicate various roles for individual members of the phosphoinositide class of membrane lipids. We first review the phosphoinositides as membrane recognition sites and their regulatory functions in membrane fusion. We then consider how modulation of phosphoinositides and their products may affect the structure and dynamics of natural membranes facilitating fusion. These diverse roles underscore the importance of these phospholipids in the fusion of biological membranes.
Mutations of the lipid phosphatase FIG4 that regulates PI(3,5)P(2) are responsible for the recessive peripheral-nerve disorder CMT4J. We now describe nonsynonymous variants of FIG4 in 2% (9/473) of patients with amyotrophic lateral sclerosis (ALS) and primary lateral sclerosis (PLS). Heterozygosity for a deleterious allele of FIG4 appears to be a risk factor for ALS and PLS, extending the list of known ALS genes and increasing the clinical spectrum of FIG4-related diseases.
The signalling lipid PI(3,5)P(2) is generated on endosomes and regulates retrograde traffic to the trans-Golgi network. Physiological signals regulate rapid, transient changes in PI(3,5)P(2) levels. Mutations that lower PI(3,5)P(2) cause neurodegeneration in human patients and mice. The function of Vac14 in the regulation of PI(3,5)P(2) was uncharacterized previously. Here, we predict that yeast and mammalian Vac14 are composed entirely of HEAT repeats and demonstrate that Vac14 exerts an effect as a scaffold for the PI(3,5)P(2) regulatory complex by direct contact with the known regulators of PI(3,5)P(2): Fig4, Fab1, Vac7 and Atg18. We also report that the mouse mutant ingls (infantile gliosis) results from a missense mutation in Vac14 that prevents the association of Vac14 with Fab1, generating a partial complex. Analysis of ingls and two additional mutants provides insight into the organization of the PI(3,5)P(2) regulatory complex and indicates that Vac14 mediates three distinct mechanisms for the rapid interconversion of PI3P and PI(3,5)P(2). Moreover, these studies show that the association of Fab1 with the complex is essential for viability in the mouse.
Niemann-Pick type C1 (NPC1) disease is a neurodegenerative lysosomal storage disorder caused by mutations in the acidic compartment (which we define as the late endosome and the lysosome) protein, NPC1. The function of NPC1 is unknown, but when it is dysfunctional, sphingosine, glycosphingolipids, sphingomyelin and cholesterol accumulate. We have found that NPC1-mutant cells have a large reduction in the acidic compartment calcium store compared to wild-type cells. Chelating luminal endocytic calcium in normal cells with high-affinity Rhod-dextran induced an NPC disease cellular phenotype. In a drug-induced NPC disease cellular model, sphingosine storage in the acidic compartment led to calcium depletion in these organelles, which then resulted in cholesterol, sphingomyelin and glycosphingolipid storage in these compartments. Sphingosine storage is therefore an initiating factor in NPC1 disease pathogenesis that causes altered calcium homeostasis, leading to the secondary storage of sphingolipids and cholesterol. This unique calcium phenotype represents a new target for therapeutic intervention, as elevation of cytosolic calcium with curcumin normalized NPC1 disease cellular phenotypes and prolonged survival of the NPC1 mouse.
TRPML1 (mucolipin 1, also known as MCOLN1) is predicted to be an intracellular late endosomal and lysosomal ion channel protein that belongs to the mucolipin subfamily of transient receptor potential (TRP) proteins. Mutations in the human TRPML1 gene cause mucolipidosis type IV disease (ML4). ML4 patients have motor impairment, mental retardation, retinal degeneration and iron-deficiency anaemia. Because aberrant iron metabolism may cause neural and retinal degeneration, it may be a primary cause of ML4 phenotypes. In most mammalian cells, release of iron from endosomes and lysosomes after iron uptake by endocytosis of Fe(3+)-bound transferrin receptors, or after lysosomal degradation of ferritin-iron complexes and autophagic ingestion of iron-containing macromolecules, is the chief source of cellular iron. The divalent metal transporter protein DMT1 (also known as SLC11A2) is the only endosomal Fe(2+) transporter known at present and it is highly expressed in erythroid precursors. Genetic studies, however, suggest the existence of a DMT1-independent endosomal and lysosomal Fe(2+) transport protein. By measuring radiolabelled iron uptake, by monitoring the levels of cytosolic and intralysosomal iron and by directly patch-clamping the late endosomal and lysosomal membrane, here we show that TRPML1 functions as a Fe(2+) permeable channel in late endosomes and lysosomes. ML4 mutations are shown to impair the ability of TRPML1 to permeate Fe(2+) at varying degrees, which correlate well with the disease severity. A comparison of TRPML1(-/- )ML4 and control human skin fibroblasts showed a reduction in cytosolic Fe(2+) levels, an increase in intralysosomal Fe(2+) levels and an accumulation of lipofuscin-like molecules in TRPML1(-/-) cells. We propose that TRPML1 mediates a mechanism by which Fe(2+) is released from late endosomes and lysosomes. Our results indicate that impaired iron transport may contribute to both haematological and degenerative symptoms of ML4 patients.
The phosphoinositides (PIs) function as efficient and finely tuned switches that control the assembly-disassembly cycles of complex molecular machineries with key roles in membrane trafficking. This important role of the PIs is mainly due to their versatile nature, which is in turn determined by their fast metabolic interconversions. PIs can be tightly regulated both spatially and temporally through the many PI kinases (PIKs) and phosphatases that are distributed throughout the different intracellular compartments. In spite of the enormous progress made in the past 20 years towards the definition of the molecular details of PI-protein interactions and of the regulatory mechanisms of the individual PIKs and phosphatases, important issues concerning the general principles of the organisation of the PI system and the coordination of the different PI-metabolising enzymes remain to be addressed. The answers should come from applying a systems biology approach to the study of the PI system, through the integration of analyses of the protein interaction data of the PI enzymes and the PI targets with those of the 'phenomes' of the genetic diseases that involve these PI-metabolising enzymes.
Autophagy is the engulfment of cytosol and organelles by double-membrane vesicles termed autophagosomes. Autophagosome formation is known to require phosphatidylinositol 3-phosphate (PI(3)P) and occurs near the endoplasmic reticulum (ER), but the exact mechanisms are unknown. We show that double FYVE domain-containing protein 1, a PI(3)P-binding protein with unusual localization on ER and Golgi membranes, translocates in response to amino acid starvation to a punctate compartment partially colocalized with autophagosomal proteins. Translocation is dependent on Vps34 and beclin function. Other PI(3)P-binding probes targeted to the ER show the same starvation-induced translocation that is dependent on PI(3)P formation and recognition. Live imaging experiments show that this punctate compartment forms near Vps34-containing vesicles, is in dynamic equilibrium with the ER, and provides a membrane platform for accumulation of autophagosomal proteins, expansion of autophagosomal membranes, and emergence of fully formed autophagosomes. This PI(3)P-enriched compartment may be involved in autophagosome biogenesis. Its dynamic relationship with the ER is consistent with the idea that the ER may provide important components for autophagosome formation.
Phosphatidylinositol (PI) 3-kinases have been implicated in several aspects of intracellular membrane trafficking, although a detailed mechanism is yet to be established. In this study we demonstrated that wortmannin, a specific inhibitor of PI 3-kinases, inhibited constitutive endocytosis of horseradish peroxidase and transferrin in BHK-21 and TRVb-1 cells. The IC50 was approximately 40 ng/ml (93 nM). In addition, wortmannin blocked the stimulation of horseradish peroxidase uptake by the small GTPase Rab5 but not the stimulation by the GTPase-defective, constitutively activated Rab5 Gln79-->Leu mutant (Rab5:Q79L), providing further evidence that PI 3-kinase activity is essential for the early endocytic process. To further investigate the mechanism, we examined the effect of wortmannin on early endosome fusion in vitro. Wortmannin decreased endosome fusion by 80% with an IC50 value similar to that in intact cells. Addition of Rab5:Q79L but not wild-type Rab5 reversed the inhibitory effect of wortmannin. Furthermore, addition of a constitutively activated PI 3-kinase but not its inactive counterpart stimulated early endosome fusion in vitro. These results strongly indicate that PI 3-kinase plays an important role in regulation of early endosome fusion, probably via activation of Rab5.
PtdIns(3,5)P2 is identified as the product of an agonist-independent, wortmannin-sensitive pathway in resting mouse cells. Results are presented here to indicate that PtdIns(3,5)P2 is formed by phosphorylation of PtdIns3P at the D-5 position, and they suggest that relatively constant cellular levels of PtdIns3P and PtdIns(3, 5)P2 are maintained by the concerted action of PtdIns3P 5-kinase and PtdIns(3,5)P2 5-phosphatase. These studies imply a novel mechanism for the action of PtdIns-specific phosphoinositide 3-hydroxykinases in mammalian cells.
Using a cell-free content mixing assay containing rat liver endosomes and lysosomes in the presence of pig brain cytosol, we demonstrated that after incubation at 37 degrees C, late endosome-lysosome hybrid organelles were formed, which could be isolated by density gradient centrifugation. ImmunoEM showed that the hybrids contained both an endocytosed marker and a lysosomal enzyme. Formation of the hybrid organelles appeared not to require vesicular transport between late endosomes and lysosomes but occurred as a result of direct fusion. Hybrid organelles with similar properties were isolated directly from rat liver homogenates and thus were not an artifact of cell-free incubations. Direct fusion between late endosomes and lysosomes was an N-ethylmaleimide-sensitive factor-dependent event and was inhibited by GDP-dissociation inhibitor, indicating a requirement for a rab protein. We suggest that in cells, delivery of endocytosed ligands to an organelle where proteolytic digestion occurs is mediated by direct fusion of late endosomes with lysosomes. The consequences of this fusion to the maintenance and function of lysosomes are discussed.
The basic reaction mechanisms for membrane fusion in the trafficking of intracellular membranes and in exocytosis are probably identical. But in contrast to regulated exocytosis, intracellular fusion reactions are referred to as 'constitutive' as no final Ca2+-dependent triggering step has been observed. Although transport from the endoplasmic reticulum to the Golgi apparatus in the cell depends on Ca2+, as does endosome fusion and assembly of the nuclear envelope, it is unclear whether Ca2+ triggers these events. Membrane fusion involves several subreactions: priming, tethering and docking. Proteins that are needed for fusion include p115, SNAPs, NSF, SNAREs and small GTPases, which operate in these early reactions, but the machinery that catalyses the final mixing of biological membranes is still unknown. Here we show that Ca2+ is released from the vacuolar lumen following completion of the docking step. We have identified calmodulin as the putative Ca2+ sensor and as the first component required in the post-docking phase of vacuole fusion. Calmodulin binds tightly to vacuoles upon Ca2+ release. Unlike synaptotagmin or syncollin in exocytosis, calmodulin does not act as a fusion clamp but actively promotes bilayer mixing. Hence, activation of SNAREs is not sufficient to drive bilayer mixing between physiological membranes. We propose that Ca2+ control of the latest phase of membrane fusion may be a conserved feature, relevant not only for exocytosis, but also for intracellular, 'constitutive' fusion reactions. However, the origin of the Ca2+ signal, its receptor and its mode of processing differ.
This chapter presents the hypothesis that the Tyrpanosoma cruzi-induced [Ca2+] transients play a role in the lysosome recruitment and fusion process. A prediction of this hypothesis is that [Ca2+] transients should be capable of mobilizing lysosomes for fusion with the plasma membrane in the absence of trypanosomes. This chapter also discusses recent observations that demonstrate that conventional lysosomes can indeed behave as Ca2+ exocytic vesicles in many cell types. These findings, together with recent observations from other groups highlight the existence of a novel pathway of regulated exocytosis involving lysosomes, which is apparently present in most animal cells. Ca2+ is an ubiquitous second messenger involved in the regulation of many cellular processes including cytoskeletal rearrangements and membrane fusion. In the search for specific second messengers induced by T. cruzi in mammalian cells, it is found that the invasive trypomastigote forms are capable of triggering repetitive intracellular free Ca2+ transients in the host cell.
Recombinant v- and t-SNARE proteins reconstituted into separate lipid bilayer vesicles assemble into SNAREpins-SNARE complexes linking two membranes. This leads to spontaneous fusion of the docked membranes at physiological temperature. Docked unfused intermediates can accumulate at lower temperatures and can fuse when brought to physiological temperature. A supply of unassembled v- and t-SNAREs is needed for these intermediates to form, but not for the fusion that follows. These data imply that SNAREpins are the minimal machinery for cellular membrane fusion.
Recent studies into the mechanisms of action of the Ca(2+)-mobilizing messenger NAADP (nicotinic acid-adenine dinucleotide phosphate) have demonstrated that a novel family of intracellular Ca(2+)-release channels termed TPCs (two-pore channels) are components of the NAADP receptor. TPCs appear to be exclusively localized to the endolysosomal system. These findings confirm previous pharmacological and biochemical studies suggesting that NAADP targets acidic Ca(2+) stores rather than the endoplasmic reticulum, the major site of action of the other two principal Ca(2+)-mobilizing messengers, InsP(3) and cADPR (cADP-ribose). Studies of the messenger roles of NAADP and the function of TPCs highlight the novel role of lysosomes and other organelles of the endocytic pathway as messenger-regulated Ca(2+) stores which also affects the regulation of the endolysosomal system.
The delivery of endocytosed cargo to lysosomes occurs through kissing and direct fusion of late endosomes/MVBs (multivesicular bodies) and lysosomes. Live-cell and electron microscopy experiments together with cell-free assays have allowed us to describe the characteristics of the delivery process and determine the core protein machinery required for fusion. The ESCRT (endosomal sorting complex required for transport) machinery is required for MVB biogenesis. The HOPS (homotypic fusion and vacuole protein sorting) complex is required for endosome-lysosome tethering and a trans-SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) complex including the R-SNARE VAMP7 (vesicle-associated membrane protein 7) mediates endosome-lysosome membrane fusion. Protein-binding partners of VAMP7 including the clathrin adaptors AP-3 (adaptor protein 3) and Hrb (HIV Rev-binding protein) are required for its correct intracellular localization and function. Overall, co-ordination of the activities of ESCRT, HOPS and SNARE complexes are required for efficient delivery of endocytosed macromolecules to lysosomes. Endosome-lysosome fusion results in a hybrid organelle from which lysosomes are re-formed. Defects in fusion and/or lysosome reformation occur in a number of lysosome storage diseases.
Phosphoinositide (PI) lipids are essential components of eukaryotic cell membranes. They are produced by mono-, bis- and trisphosphorylation of the inositol headgroup of phosphatidylinositol (PtdIns) and are concentrated in separate pools of cytosolic membranes. PIs serve as markers of the cell compartments and form unique docking sites for protein effectors. Collectively, seven known PIs, the protein effectors that bind them and enzymes that generate or modify PIs compose a remarkably complex protein-lipid signaling network. A number of cytosolic proteins contain one or several effector modules capable of recognizing individual PIs and recruiting the host proteins to distinct intracellular compartment. The recently determined atomic-resolution structures and membrane-targeting mechanisms of a dozen PI effectors have provided insights into the molecular basis for regulation of endocytic membrane trafficking and signaling. In this review, I highlight the structural aspects of the deciphering of the 'PI code' by the most common PI-recognizing effectors and discuss the mechanistic details of their membrane anchoring.
Although fusion mechanisms are highly conserved in evolution and among organelles of the exocytic and endocytic pathways, yeast vacuole homotypic fusion offers unique technical advantages: excellent genetics, clear organelle cytology, in vitro colorimetric fusion assays, and reconstitution of fusion from all-pure components, including a Rab GTPase, HOPS (homotypic fusion and vacuole protein sorting complex), four SNAREs [soluble N-ethylmaleimide-sensitive factor (NSF) attachment receptors] that snare (bind) each other, SNARE-complex disassembly chaperones, and vacuolar lipids. Vacuole fusion studies offer paradigms of the interdependence of lipids and fusion proteins to assemble a fusion microdomain, distinct lipid functions, SNARE complex proofreading through the synergy between HOPS and the SNARE disassembly chaperones, and the role of each fusion protein in promoting radical bilayer restructuring for fusion without lysis.
PIKfyve is a protein and lipid kinase that plays an important role in membrane trafficking, including TGN to endosome retrograde sorting and in insulin-stimulated translocation of the GLUT4 glucose transporter from intracellular storage vesicles to the plasma membrane. We have previously demonstrated that PIKfyve is phosphorylated in response to insulin in a PI3-kinase and protein kinase B (PKB)-dependent manner. However, it has been implied that this was not due to direct phosphorylation of PIKfyve by PKB, but as a result of an insulin-induced PIKfyve autophosphorylation event. Here we demonstrate that purified PIKfyve is phosphorylated in vitro by a recombinant active PKB on two separate serine residues, S318 and S105, which flank the N-terminal FYVE domain of the protein. Only S318, however, becomes phosphorylated in intact cells stimulated with insulin. We further demonstrate that S318 is phosphorylated in response to hyperosmotic stress in a PI3-kinase- and PKB-independent manner. Importantly, the effects of insulin and sorbitol were not prevented by the presence of an ATP-competitive PIKfyve inhibitor (YM20163) or in a mutant PIKfyve lacking both lipid and protein kinase activity. Our results confirm, therefore, that PIKfyve is directly phosphorylated by PKB on a single serine residue in response to insulin and are not due to autophosphorylation of the enzyme. We further reveal that two stimuli known to promote glucose uptake in cells, both stimulate phosphorylation of PIKfyve on S318 but via distinct signal transduction pathways.
Vacuole homotypic fusion has been reconstituted with all purified components: vacuolar lipids, four soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, Sec17p, Sec18p, the Rab Ypt7p, and the hexameric homotypic fusion and vacuole protein sorting complex (HOPS). HOPS is a Rab-effector with direct affinity for SNAREs (presumably via its Sec1-Munc18 homologous subunit Vps33p) and for certain vacuolar lipids. Each of these pure vacuolar proteins was required for optimal proteoliposome clustering, raising the question of which was most directly involved. We now present model subreactions of clustering and fusion that reveal that HOPS is the direct agent of tethering. The Rab and vacuole lipids contribute to tethering by supporting the membrane association of HOPS. HOPS indirectly facilitates trans-SNARE complex formation by tethering membranes, because the synthetic liposome tethering factor polyethylene glycol can also stimulate trans-SNARE complex formation and fusion. SNAREs further stabilize the associations of HOPS-tethered membranes. HOPS then protects newly formed trans-SNARE complexes from disassembly by Sec17p/Sec18p.
The discovery over five decades ago of the lysosome, as a degradative organelle and its dysfunction in lysosomal storage disorder patients, was both insightful and simple in concept. Here, we review some of the history and pathophysiology of lysosomal storage disorders to show how they have impacted on our knowledge of lysosomal biology. Although a significant amount of information has been accrued on the molecular genetics and biochemistry of lysosomal storage disorders, we still do not fully understand the mechanistic link between the storage material and disease pathogenesis. However, the accumulation of undegraded substrate(s) can disrupt other lysosomal degradation processes, vesicular traffic, and lysosomal biogenesis to evoke the diverse pathophysiology that is evident in this complex set of disorders.
We conducted a high-throughput screen for small molecule activators of the TRPML3 ion channel, which, when mutated, causes deafness and pigmentation defects. Cheminformatics analyses of the 53 identified and confirmed compounds revealed nine different chemical scaffolds and 20 singletons. We found that agonists strongly potentiated TRPML3 activation with low extracytosolic [Na(+)]. This synergism revealed the existence of distinct and cooperative activation mechanisms and a wide dynamic range of TRPML3 activity. Testing compounds on TRPML3-expressing sensory hair cells revealed the absence of activator-responsive channels. Epidermal melanocytes showed only weak or no responses to the compounds. These results suggest that TRPML3 in native cells might be absent from the plasma membrane or that the protein is a subunit of heteromeric channels that are nonresponsive to the activators identified in this screen.
Two-pore channels (TPCs or TPCNs) are novel members of the large superfamily of voltage-gated cation channels with slightly higher sequence homology to the pore-forming subunits of voltage-gated Ca(2+) and Na(+) channels than most other members. Recent studies demonstrate that TPCs locate to endosomes and lysosomes and form Ca(2+) release channels that respond to activation by the Ca(2+) mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). With multiple endolysosomal targeted NAADP receptors now identified, important new insights into the regulation of endolysosomal function in health and disease will therefore be unveiled.
J. Neurochem. (2010) 113 , 313–328.
Abstract
Ion channels are classically understood to regulate the flux of ions across the plasma membrane in response to a variety of environmental and intracellular cues. Ion channels serve a number of functions in intracellular membranes as well. These channels may be temporarily localized to intracellular membranes as a function of their biosynthetic or secretory pathways, i.e., en route to their destination location. Intracellular membrane ion channels may also be located in the endocytic pathways, either being recycled back to the plasma membrane or targeted to the lysosome for degradation. Several channels do participate in intracellular signal transduction; the most well known example is the inositol 1,4,5‐trisphosphate receptor (IP 3 R) in the endoplasmic reticulum. Some organellar intracellular membrane channels are required for the ionic homeostasis of their residing organelles. Several newly‐discovered intracellular membrane Ca ²⁺ channels actually play active roles in membrane trafficking. Transient receptor potential (TRP) proteins are a superfamily (28 members in mammal) of Ca ²⁺ ‐permeable channels with diverse tissue distribution, subcellular localization, and physiological functions. Almost all mammalian TRP channels studied thus far, like their ancestor yeast TRP channel (TRPY1) that localizes to the vacuole compartment, are also (in addition to their plasma membrane localization) found to be localized to intracellular membranes. Accumulated evidence suggests that intracellularly‐localized TRP channels actively participate in regulating membrane traffic, signal transduction, and vesicular ion homeostasis. This review aims to provide a summary of these recent works. The discussion will also be extended to the basic membrane and electrical properties of the TRP‐residing compartments.
The mucolipin family of Transient Receptor Potential (TRPML) proteins is predicted to encode ion channels expressed in intracellular endosomes and lysosomes. Loss-of-function mutations of human TRPML1 cause type IV mucolipidosis (ML4), a childhood neurodegenerative disease. Meanwhile, gain-of-function mutations in the mouse TRPML3 result in the varitint-waddler (Va) phenotype with hearing and pigmentation defects. The broad spectrum phenotypes of ML4 and Va appear to result from certain aspects of endosomal/lysosomal dysfunction. Lysosomes, traditionally believed to be the terminal "recycling center" for biological "garbage", are now known to play indispensable roles in intracellular signal transduction and membrane trafficking. Studies employing animal models and cell lines in which TRPML genes have been genetically disrupted or depleted have uncovered roles of TRPMLs in multiple cellular functions including membrane trafficking, signal transduction, and organellar ion homeostasis. Physiological assays of mammalian cell lines in which TRPMLs are heterologously overexpressed have revealed the channel properties of TRPMLs in mediating cation (Ca(2+)/Fe(2+)) efflux from endosomes and lysosomes in response to unidentified cellular cues. This review aims to summarize these recent advances in the TRPML field and to correlate the channel properties of endolysosomal TRPMLs with their biological functions. We will also discuss the potential cellular mechanisms by which TRPML deficiency leads to neurodegeneration.
Cholesterol, an essential component of cell membranes, plays an important role in the maintenance of cellular homeostasis and transmembrane communication within and between cellular compartments. In the brain that contains the highest levels of cholesterol in the body, cholesterol traffic occurs between nerve cells and between intracellular organelles in neurons to subserve normal brain function. Whereas glial cells produce the largest quantities of cholesterol, neurons also acquire cholesterol synthesized by astrocytes. The intracellular organelle endosomes and lysosomes receive and distribute cholesterol through the endocytic and retrograde transport pathways. However, deregulated cholesterol trafficking appears to be involved in the pathogenesis of Alzheimer's disease (AD), Parkinson's disease (PD) and Niemann-Pick disease type C (NPC) diseases. Under the pathological conditions of these neurodegenerative diseases, aberrant molecular interactions or particular depositions of cholesterol have been observed as critical causes to precipitate neuronal cell death. Here, we review the recent advances in terms of the role of cholesterol in healthy brain and molecular mechanisms of cholesterol involvement in AD, PD and NPC diseases. We discuss the different lines of evidence supporting different models of anomalous intracellular cholesterol trafficking with emphasis on cholesterol interactions with alpha-synuclein, NPC1 and NPC2 in AD, PD and NPC.
Membrane trafficking between organelles by vesiculotubular carriers is fundamental to the existence of eukaryotic cells. Central in ensuring that cargoes are delivered to their correct destinations are the Rab GTPases, a large family of small GTPases that control membrane identity and vesicle budding, uncoating, motility and fusion through the recruitment of effector proteins, such as sorting adaptors, tethering factors, kinases, phosphatases and motors. Crosstalk between multiple Rab GTPases through shared effectors, or through effectors that recruit selective Rab activators, ensures the spatiotemporal regulation of vesicle traffic. Functional impairments of Rab pathways are associated with diseases, such as immunodeficiencies, cancer and neurological disorders.
Intracellular trafficking entails the budding, transport, tethering, and fusion of transport vesicles and other membrane carriers. Here we review recent progress toward a mechanistic understanding of vesicle tethering. The known tethering factors are large complexes important for one or more intracellular trafficking pathways and are capable of interacting directly with many of the other principal components of the cellular trafficking machinery. Our review emphasizes recent developments in the in vitro reconstitution of vesicle tethering and the structural characterization of multisubunit tethering factors. The combination of these and other approaches has led to exciting progress toward understanding how these essential nanomachines work.
The ability to sense mechanical and osmotic stimuli is vital to all organisms from mammals to bacteria. Members of the transient receptor potential (TRP) ion-channel family have attracted intense attention for their involvement in mechanosensation. The yeast homologue TRPY1 can clearly be activated by hypertonic shock in vivo and by stretch force under patch clamp. Like its animal counterparts, TRPY1 is polymodal, being gated by membrane stretch force and by cytoplasmic Ca(2+). Here, we investigated how these two gating principles interact. We found that stretch force can induce some channel activation without cytoplasmic Ca(2+). Tens of micromolar Ca(2+) greatly enhance the observed force-induced activities, with open probabilities following well the Boltzmann distribution, in which the two gating energies are summed as exponents. To map this formalism to structures, we found Ca(2+)-binding proteins such as calmodulin or calcineurin to be unnecessary. However, removing a dense cluster of negative charges in the C-terminal cytoplasmic domain of TRPY1 greatly diminishes the Ca(2+) activation as well as its influence on force activation. We also found a strategic point upstream of this charge cluster, at which insertion of amino acids weakens Ca(2+) activation considerably but leaves the mechanosensitivity nearly intact. These results led to a structure-function model in which Ca(2+) binding to the cytoplasmic domain and stretching of the membrane-embedded domain both generate gating force, reaching the gate in parallel.
Disruption of the Transient Receptor Potential (TRP) mucolipin 1 (TRPML1) channel results in the neurodegenerative disorder mucolipidosis type IV (MLIV), a lysosomal storage disease with severe motor impairments. The mechanisms underlying MLIV are poorly understood and there is no treatment. Here, we report a Drosophila MLIV model, which recapitulates the key disease features, including abnormal intracellular accumulation of macromolecules, motor defects, and neurodegeneration. The basis for the buildup of macromolecules was defective autophagy, which resulted in oxidative stress and impaired synaptic transmission. Late-apoptotic cells accumulated in trpml mutant brains, suggesting diminished cell clearance. The accumulation of late-apoptotic cells and motor deficits were suppressed by expression of trpml(+) in neurons, glia, or hematopoietic cells. We conclude that the neurodegeneration and motor defects result primarily from decreased clearance of apoptotic cells. Since hematopoietic cells in humans are involved in clearance of apoptotic cells, our results raise the possibility that bone marrow transplantation may limit the progression of MLIV.
Inositol phospholipids play multiple roles in cell signalling systems. Two widespread eukaryotic phosphoinositide-based signal transduction mechanisms, phosphoinositidase C-catalysed phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) hydrolysis and 3-OH kinase-catalysed PtdIns(4,5)P2 phosphorylation, make the second messengers inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) sn-1,2-diacylglycerol and PtdIns(3,4,5)P3. In addition, PtdIns(4,5)P2 and PtdIns3P have been implicated in exocytosis and membrane trafficking. We now show that when the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe are hyperosmotically stressed, they rapidly synthesize phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2) by a process that involves activation of a PtdIns3P 5-OH kinase. This PtdIns(3,5)P2 accumulation only occurs in yeasts that have an active vps34-encoded PtdIns 3-OH kinase, showing that this latter kinase makes the PtdIns3P needed for PtdIns(3,5)P2 synthesis and indicating that PtdIns(3,5)P2 may have a role in sorting vesicular proteins. PtdIns(3,5)P2 is also present in mammalian and plant cells: in monkey Cos-7 cells, its labelling is inversely related to the external osmotic pressure. The stimulation of a PtdIns3P 5-OH kinase-catalysed synthesis of PtdIns(3,5)P2, a molecule that might be a new type of phosphoinositide 'second messenger, thus appears to be central to a widespread and previously uncharacterized regulatory pathway.
Mucolipidosis, type IV (ML-IV) is an autosomal recessive storage disease that is characterized by lysosomal accumulation of sphingolipids, phospholipids, and acid mucopolysaccharides. Unlike most other storage diseases, the lysosomal hydrolases participating in the catabolism of the stored molecules appear to be normal. In the present study, we examined the hypothesis that the ML-IV phenotype might arise from abnormal transport along the lysosomal pathway. By using various markers for endocytosis, we found that plasma membrane internalization and recycling were nearly identical in ML-IV and normal fibroblasts. A fluorescent analog of lactosylceramide (LacCer) was used to study plasma membrane lipid internalization and subsequent transport. Lipid internalization at 19 degreesC was similar in both cell types; however, 40-60 min after raising the temperature to 37 degreesC, the fluorescent lipid accumulated in the lysosomes of ML-IV cells but was mainly concentrated at the Golgi complex of normal fibroblasts. Biochemical studies demonstrated that at these time points, hydrolysis of the lipid analog was minimal ( approximately 7%) in both cell types. A fluorescence ratio imaging assay was developed to monitor accumulation of fluorescent LacCer in the lysosomes and showed that the apparent concentration of the lipid increased more rapidly and to a greater extent in ML-IV cells than in normal fibroblasts. By 60 min, LacCer apparently decreased in the lysosomes of normal fibroblasts but not in ML-IV cells, suggesting that lipid efflux from the lysosomes was also impaired. These results demonstrate that there is a defect in ML-IV fibroblasts that affects membrane sorting and/or late steps of endocytosis.
GTPases and lipid kinases regulate membrane traffic along the endocytic pathway by mechanisms that are not completely understood. Fusion between early endosomes requires phosphatidylinositol-3-OH kinase (PI(3)K) activity as well as the small GTPase Rab5. Excess Rab5-GTP complex restores endosome fusion when PI(3)K is inhibited. Here we identify the early-endosomal autoantigen EEA1 which binds the PI(3)K product phosphatidylinositol-3-phosphate, as a new Rab5 effector that is required for endosome fusion. The association of EEA1 with the endosomal membrane requires Rab5-GTP and PI(3)K activity, and excess Rab5-GTP stabilizes the membrane association of EEA1 even when PI(3)K is inhibited. The identification of EEA1 as a direct Rab5 effector provides a molecular link between PI(3)K and Rab5, and its restricted distribution to early endosomes indicates that EEA1 may confer directionality to Rab5-dependent endocytic transport.
A number of specific cellular Ca2+ uptake pathways have been described in many different cell types [1] [2] [3]. The possibility that substantial quantities of Ca2+ could be imported via endocytosis has essentially been ignored, although it has been recognized that endosomes can store Ca2+ [4] [5]. Exocrine cells can release significant amounts of Ca2+ via exocytosis [6], so we have investigated the fate of Ca2+ taken up via endocytosis into endosomes. Ca2+-sensitive and H+-sensitive fluorescent probes were placed in the extracellular solution and subsequently taken up into fibroblasts by endocytosis. Confocal microscopy was used to assess the distribution of fluorescence intensity. Ca2+ taken up by endocytosis was lost from the endosomes within a few minutes, over the same period as endosomal acidification took place. The acidification was inhibited by reducing the extracellular Ca2+ concentration, and Ca2+ loss from the endosomes was blocked by bafilomycin (100 nM), a specific inhibitor of the vacuolar proton ATPase. Quantitative evaluation indicated that endocytosis causes substantial import of Ca2+ because of rapid loss from early endosomes.