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.

Fig. 1. Phosphoinositides and Ca²⁺ channels in endolysosomes

A: An overview of PI(P) synthesis in endolysosomes. PI(3)P is synthesized from PI via a PI 3-kinase (Vps34) present in endosomes, while PI(3,5)P₂ is synthesized from PI(3)P via a PI 5-kinase (PIKfyve/Fab1). PIKfyve is a PI(3)P effector that is associated with late endosomes and lysosomes (LELs), and is positively regulated by Vac14. PI(3,5)P₂ can be dephosphorylated by Fig4 or MTM/MTMRs to generate PI(3)P or PI5P, respectively. B: The distribution of PI(3)P and PI(3,5)P₂ associated with the membranes of early and late endosomes, as well as lysosomes, are represented by yellow and red outlines at the membrane of these vesicle types. respectively [35]. TRPML1–3, TRPM2, and TPC1–2 are putative Ca²⁺ release channels associated with endolysosomal membranes that are also labeled in this diagram [69,75,76]. TRPML1–3 channels are predominantly localized to LELs, while TRPML2 and TRPML3 are also associated with recycling and early endosomes, respectively. TPC2 is mainly localized in the LELs, and TPC1 is mainly associated with early and late endosomes. TRPM2 is expressed in specific cell types, and is associated with LELs as well as the plasma membrane. The concentration of Ca²⁺ stores associated with each vesicle type is also indicated, with luminal [Ca²⁺] estimated to be in the micro- to millimolar ranges.

Fig. 2. Two working models for PI(3,5)P₂-dependent TRPML1 activation in endolysosomes

A: “Activation” model. Physiological levels of PI(3,5)P₂ activate TRPML1 by binding to the poly-basic domain in the N- terminus of TRPML1, leading to the release of Ca²⁺. B: “Sensitization” model. In this model, physiological levels of PI(3,5)P₂ are not sufficient to activate TRPML1. Instead, PI(3,5)P₂ is hypothesized to sensitize TRPML1 by converting the channel from a “reluctant” state to a “willing state” (see ref. [92]). Channel opening/activation and Ca²⁺ release are then induced by unidentified trafficking cues, one of which may be the mechanical force generated during pre-fusion membrane curvature/bending. This model confers multiple layers of regulation on TRPML1 activity. Under stress conditions that are analogous to hyperosmotic shock in yeast, acute stimuli may induce high levels of PI(3,5)P₂ to sufficiently activate TRPML1 as shown in A.

Fig. 3. A proposed model for PI(3,5)P₂- and Ca²⁺-dependent endolysosomal membrane fusion

Microdomain specific recruitment of an array of tethering and priming factors has the potential to bring into close proximity these factors between an endolysosomal membrane (lower membrane illustrated) and a membrane of another vesicular compartment (upper membrane illustrated). A: Recruited proteins that may be involved include Rab GTPases (Rab7 for LEL), lipid kinases (PIKfyve for LEL), and several phosphatases (Fig4 and MTM/MTMRs). Activation of the assembled PI(3,5)P₂-metabolizing complex would ensure a transient and local increase in the level of PI(3,5)P₂. PI(3,5)P₂ effectors would also be subsequently recruited and/or activated. B: The release of Ca²⁺ from the lumen of endolysosomes would transiently elevate the levels of juxtaorganellar Ca²⁺ to activate a putative Ca²⁺ sensor protein, such as ALG-2/Synaptotagmin/CaM, to promote the fusion of the lipid bilayers. Both SNARE complex formation and PI(3,5)P₂ may facilitate Ca²⁺ release. An increase in PI(3,5)P₂ levels could also alter the physical properties of the membranes involved, and regulate the interactions between SNARE complexes and Ca²⁺ sensor proteins.