Regulation of membrane trafficking by signalling on endosomal and lysosomal membranes

Abstract

Endosomal and lysosomal membrane trafficking requires the coordination of multiple signalling events to control cargo sorting and processing, and endosome maturation. The initiation and termination of signalling events in endosomes and lysosomes is not well understood, but several key regulators have been identified, which include small GTPases, phosphoinositides, and Ca2+. Small GTPases act as master regulators and molecular switches in a GTP-dependent manner, initiating signalling cascades to regulate the direction and specificity of endosomal trafficking. Phosphoinositides are membrane-bound lipids that indicate vesicular identities for recruiting specific cytoplasmic proteins to endosomal membranes, thus allowing specificity of membrane fusion, fission, and cargo sorting to occur within and between specific vesicle compartments. In addition, phosphoinositides regulate the function of membrane proteins such as ion channels and transporters in a compartment-specific manner to mediate transport and signalling. Finally, Ca2+, a locally acting second messenger released from intracellular ion channels, may provide precise spatiotemporal regulation of endosomal signalling and trafficking events. Small GTPase signalling can regulate phosphoinositide conversion during endosome maturation, and electrophysiological studies on isolated endosomes have shown that endosomal and lysosomal Ca2+ channels are directly modulated by endosomal lipids. Thus trafficking and maturation of endosomes and lysosomes can be precisely regulated by dynamic changes in GTPases and membrane lipids, as well as Ca2+ signalling. Importantly, impaired phosphoinositide and Ca2+ signalling can cause endosomal and lysosomal trafficking defects at the cellular level, and a spectrum of lysosome storage diseases.

Figure 1. Endosomal trafficking network

A schematic view of the endosomal trafficking network. Vesicular pH and predominant membrane phosphoinositides on different compartments are represented by different colours. During endocytosis, a piece of the plasma membrane is excised and enters the cytosol in the form of a nascent endosome (NE; a). Nascent endosomes fuse with each other (b) and recruit early endosomal proteins to become early endosomes (EE; b). Membrane receptors are sorted and recycled back to the plasma membrane through recycling endosomes (RE; c). Material destined for degradation is passed on to the late endosomes (LE; d), which are also referred to as multi-vesicular bodies (MVB) due to the intraluminal vesicles (ILVs) that contain membrane proteins sorted for degradation. Hydrolytic enzymes are transported to late endosomes through transport vesicles (TV) from Golgi (e). Membrane receptors carrying the enzymes are shuttled back to Golgi through retrograde transport. Late endosomes mature into lysosomes (LY) either through further acidification, or through fusion with existing lysosomes (f). During starvation or when organelles are damaged, lysosomes also accept cargo from autophagosomes (AP) carrying damaged organelles or cytosolic material for degradation (g). The resulting autophagic lysosomes (AL) are usually larger than endocytic lysosomes. Lysosomes can undergo Ca²⁺-dependent exocytosis (h). Lysosomal membrane proteins are recycled from autophagic lysosomes by fission processes that happen on tubular structures (i). The mechanism of recycling of membrane proteins from endocytic lysosomes has yet to be established (j).

Figure 2. A proposed model of the phosphoinositide–Ca²⁺–membrane fusion pathway

A, the initiation of vesicle fusion is mediated by the cooperation of Rab proteins and tethering complexes, which coordinate the assembly of the SNARE complex. B, after the SNARE complex is assembled, the vesicles are in a ready-to-fuse state. C, an increase in the membrane PI(3,5)P₂ concentration activates Ca²⁺ influx into the cytosol, which acts as a trigger for vesicle fusion.

Figure 3. Fluorescently tagged phosphoinositide probes

Cos7 cells are transfected with green fluorescent protein (GFP)-tagged phosphoinositide-binding domains. A, a PI(3)P probe, FYVE^hrsX2-GFP, is concentrated in EEA1-positive vesicles. mCherry-EEA1 is co-transfected to visualize early endosomes. B, the PI(4,5)P₂ probe, PLCδ-PH-GFP, is mainly localized in the plasma membrane at rest. mCherry is co-transfected to define cell morphology. Scale bars represent 20 μm.

Figure 4. A lysosome-targeted genetically encoded Ca²⁺ sensor

GCaMP5 contains a circularly permutated enhanced GFP (EGFP). The N-terminal of the fragment containing residues 149–238 is linked to the M13 peptide, while the C-terminal of the fragment containing residues 1–144 is linked to calmodulin (CaM). The binding of Ca²⁺ ions to calmodulin causes a conformational change and calmodulin then binds to the M13 peptide, bringing the two parts of the permutated EGFP together, reconstituting a functional EGFP. When GCaMP5 is attached to a lysosomal membrane protein (e.g. TRPML1), it works as a sensor for local Ca²⁺ release.