A new probe illuminates endo-lysosomal Ca²⁺ measurements: A role for vesicular IP₃ receptors?

Signal transduction pathways are constantly evolving into ever more complex pathways that intersect with one another, and one modality—that of Ca²⁺ signalling—is no exception. Having established ER-mediated Ca²⁺ release and Ca²⁺ entry across the plasma membrane as two dominant sources of Ca²⁺ signals, other internal organelles have gradually been welcomed into the Ca²⁺ network, either as Ca²⁺ sources or as Ca²⁺ sinks (or both). These newer players include mitochondria, peroxisomes, the Golgi, and endo-lysosomes (ELs) (as well as secretory vesicles) (1). In spite of their smaller physical size, these other Ca²⁺-signalling organelles exert potent effects on cell biology and manifestly “punch above their weight”.

Understanding when (and how) different Ca²⁺ sources are recruited and how they control Ca²⁺ fluxes across their membranes is a difficult enough task when in isolation, but the system is rendered all the more complex by the fact that these Ca²⁺ sources intersect or overlap with one another in two ways: (a) they can be active at the same time (so that cytosolic Ca²⁺ signals may be a complex summation of multiple Ca²⁺ stores); (b) Ca²⁺ released from one organelle can be transferred to a different neighboring organelle (particularly at membrane contact sites) to either “fill” the recipient compartment or to trigger yet more Ca²⁺ release from it (via Ca²⁺-induced Ca²⁺ release). Tracking the ins and outs of Ca²⁺ within each compartment requires organelle-specific Ca²⁺ reporters targeted to their respective lumina, so that the dynamic changes in their Ca²⁺ content are optically isolated from their neighbors’ content. For the ER and mitochondria, luminal reporters (fluorescent or luminescent) have been successfully optimized and exploited for decades and have unambiguously illuminated how complex organellar Ca²⁺ release/transfer events occur in (patho)-physiological conditions.

However, ELs have proven the enfants terribles among organelles because of their acidic and proteolytic lumina: low pH can crush a reporter’s chromophore, or the Ca²⁺-binding motif (or both), whereas proteolysis of genetically encoded Ca²⁺ indicators (GECIs) can affect targeting and activity. Given the dynamic interactions of ELs with sundry other compartments, the ability to explicitly isolate endo-lysosomal Ca²⁺ signals from those of other sources has been an imperative since the inception of the field. Although chemical reporters have reported Ca²⁺ fluxes in acidic vesicles (2), GECIs have had less success (with a few exceptions). Therefore, the recent paper led by the Alonso group excitingly showcases a new reporter for endo-lysosomal Ca²⁺.

Calvo et al. (3) targeted a novel Ca²⁺ reporter to an endo-lysosomal sub-compartment by its fusion to the luminal tail of VAMP-7, a marker of late endosomes (and secretory vesicles). Instead of a calmodulin-based, purely fluorescent GECI, they used a bioluminescent reporter, the Ca²⁺-binding aequorin protein fused to a GFP variant, which they named endo-lysosomal GFP-aequorin (ELGA). The new probe satisfied all the fundamental criteria of appropriate Ca²⁺ affinity, selective localization to the endo-lysosomal system, and a relative insensitivity to pH down to 6.0, which is crucial. The endo-lysosomal continuum of vesicles exhibits luminal pH varying from 6.5 to 4.5, so the new probe cannot, unfortunately, record from the most acidic lysosomal compartments, but it does report Ca²⁺ in a less acidic subpopulation. Hereafter, the authors referred to this ELGA-labelled compartment as “ELs,” although their identity is slightly unclear when the markers they used are predominantly late-endosome/lysosome.

That aside, calibrating the ELGA signals in three different cell culture models confirmed that the resting [Ca²⁺] of these ELs was ∼300–400 µM, in line with previous estimates of the lysosomal or ER [Ca²⁺] and ample to act as a bona fide Ca²⁺ store. As would be expected, known agonists of endo-lysosomal Ca²⁺-permeable channels elicit sizeable Ca²⁺ release from the ELGA compartments. A huge and unique advantage of a luminal Ca²⁺ probe is that it can be used in permeabilized cells without the reporter (or Ca²⁺) being diluted into the medium. Consequently, cell-impermeant agonists can be used, and NAADP, a dinucleotide messenger that activates TPC channels, evoked a robust Ca²⁺ release in permeabilized cells. This is remarkably one of the few examples where NAADP responses (notoriously fragile) have been preserved in permeabilized mammalian cells. Similarly, agonists of TRPML1 channels (ML-SA1, or the lipid, PI(3,5)P₂) also evoked Ca²⁺ release from these vesicles but not (crucially) from the ER (as measured in parallel with an analogous ER probe). This reinforces that ELGA responses need not be due to ER signal contamination and that ELGA can report a different, non-ER compartment. This result is key for what follows below.

How mammalian ELs fill with Ca²⁺ is uncertain, and although the H⁺ gradient and/or novel Ca²⁺-transporters have been implicated, inhibition of the V-H⁺-ATPase pump with bafilomycin A1 did not prevent Ca²⁺ refilling as measured by ELGA (suggesting that Ca²⁺/H⁺ exchange is not required). In contrast, the surprise was that endo-lysosomal Ca²⁺ refilling (at least in permeabilized cells) was not only ATP-dependent but also sensitive to inhibitors of the ER Ca²⁺ pumps (SERCA). One explanation could be that the ER transfers its Ca²⁺ to ELs (4) and that the SERCA inhibition is an indirect consequence of depleting the “refilling station”. However, the authors could discount this explanation because inter-organelle Ca²⁺ transfer should be blocked with a fast Ca²⁺ chelator, but it did not affect endo-lysosomal refilling. This raises the possibility that SERCA is also present on acidic vesicles to fill them directly, and the authors showed a small degree of colocalization of SERCA and the endo-lysosomal marker LAMP2. This surprising conclusion is at odds with some lysosomal data in the literature, so perhaps the Ca²⁺ handling of this ELGA-labelled compartment differs from that of the later, more acidic lysosomes.

Another unexpected result was that these ELs appear to be decorated with functional IP₃ receptors (IP₃Rs), the canonical Ca²⁺ channels of the ER. ELGA measurements revealed robust Ca²⁺ release in response to IP₃ that was abolished in IP₃R-null cells. Using super-resolution imaging, endogenous IP₃Rs were suggested to have a small degree of overlap with different endo-lysosomal markers (TRPML1, TPC1, LAMP1, and LysoTracker) and with an ELGA variant. The idea that acidic vesicles contain IP₃Rs has some precedents (particularly in secretory vesicles), but the notion of their being on a sub-compartment of ELs is a daring suggestion; if this holds up, then IP₃-mediated Ca²⁺ release from non-ER (and non-Golgi) stores could represent a new paradigm in Ca²⁺ signalling.

In summary, the new endo-lysosomal ELGA probe is a new genetic Ca²⁺ reporter with modest pH insensitivity that can label and record Ca²⁺ fluxes in a sub-compartment of acidic vesicles. This compartment reassuringly bears many of the hallmarks of classical endo-lysosomal pharmacology and markers (e.g., responding to endo-lysosomal channel activation), but it surprisingly shares some of these features with the ER (i.e., IP₃Rs and SERCA). Although the skeptical may express understandable concerns about the ER contaminating the ELGA signals, the authors made strenuous efforts to discount this, and many data are compelling that this is not the case. How SERCA and IP₃Rs might traffic to ELs is an open question, but an important one to answer to bolster this unusual model. Will acidic-vesicle IP₃ signalling be a new avenue of research?