Acentral tenet of nerve growth factor (NGF) action that is poorly understood is its ability to mediate cytoplasmic signaling, through its receptor TrkA, that is initiated at the nerve terminal and conveyed to the soma. We identified an NGF-induced protein that we termed Pincher (pinocytic chaperone) that mediates endocytosis and trafficking of NGF and its receptor TrkA. In PC12 cells, overexpression of Pincher dramatically stimulated NGF-induced endocytosis of TrkA, unexpectedly at sites of clathrin-independent macropinocytosis within cell surface ruffles. Subsequently, a system of Pincher-containing tubules mediated the delivery of NGF/TrkA-containing vesicles to cytoplasmic accumulations. These vesicles selectively and persistently mediated TrkA-erk5 mitogen-activated protein kinase signaling. A dominant inhibitory mutant form of Pincher inhibited the NGF-induced endocytosis of TrkA, and selectively blocked TrkA-mediated cytoplasmic signaling of erk5, but not erk1/2, kinases. Our results indicate that Pincher mediates pinocytic endocytosis of functionally specialized NGF/TrkA endosomes with persistent signaling potential.

In vision, balance, and hearing, sensory receptor cells translate sensory stimuli into electrical signals whose amplitude is graded with stimulus intensity. The output synapses of these sensory neurons must provide fast signaling to follow rapidly changing stimuli, while also transmitting graded information covering a wide range of stimulus intensity and sustained for long time periods. To meet these demands, specialized machinery for transmitter release—the synaptic ribbon—has evolved at the synaptic outputs of these neurons. Here we show that acute disruption of synaptic ribbons by photodamage to the ribbon dramatically reduces both sustained and transient components of neurotransmitter release in mouse bipolar cells and salamander cones, without affecting the ultrastructure of the ribbon or its ability to localize synaptic vesicles to the active zone. Our results indicate that ribbons mediate slow as well as fast signaling at sensory synapses, and support an additional role for the synaptic ribbon in priming vesicles for exocytosis at active zones.

Retrograde signaling by neurotrophins is crucial for regulating neuronal phenotype and survival. The mechanism responsible for retrograde signaling has been elusive, because the molecular entities that propagate Trk receptor tyrosine kinase signals from the nerve terminal to the soma have not been defined. Here, we show that the membrane trafficking protein Pincher defines the primary pathway responsible for neurotrophin retrograde signaling in neurons. By both immunofluorescence confocal and immunoelectron microscopy, we find that Pincher mediates the formation of newly identified clathrin-independent macroendosomes for Trk receptors in soma, axons, and dendrites. Trk macroendosomes are derived from plasma membrane ruffles and subsequently processed to multivesicular bodies. Pincher similarly mediates macroendocytosis for NGF (TrkA) and BDNF (TrkB) in both peripheral (sympathetic) and central (hippocampal) neurons. A unique feature of Pincher-Trk endosomes is refractoriness to lysosomal degradation, which ensures persistent signaling through a critical effector of retrograde survival signaling, Erk5 (extracellular signal-regulated kinase 5). Using sympathetic neurons grown in chamber cultures, we find that block of Pincher function, which prevents Trk macroendosome formation, eliminates retrogradely signaled neuronal survival. Pincher is the first distinguishing molecular component of a novel mechanistic pathway for endosomal signaling in neurons.

Why neurotrophins and their Trk receptors promote neuronal differentiation and survival whereas receptor tyrosine kinases for other growth factors, such as EGF, do not, has been a long-standing question in neurobiology. We provide evidence that one difference lies in the selective ability of Trk to generate long-lived signaling endosomes. We show that Trk endocytosis is distinguished from the classical clathrin-based endocytosis of EGF receptor (EGFR). Although Trk and EGFR each stimulate membrane ruffling, only Trk undergoes both selective and specific macroendocytosis at ruffles, which uniquely requires the Rho-GTPase, Rac, and the trafficking protein, Pincher. This process leads to Trk-signaling endosomes, which are immature multivesicular bodies that retain Rab5. In contrast, EGFR endosomes rapidly exchange Rab5 for Rab7, thereby transiting into late-endosomes/lysosomes for degradation. Sustained endosomal signaling by Trk does not reflect intrinsic differences between Trk and EGFR, because each elicits longterm Erk-kinase activation from the cell surface. Thus, a population of stable Trk endosomes, formed by specialized macroendocytosis in neurons, provides a privileged endosome-based system for propagation of signals to the nucleus.EGF receptor ͉ Erk kinase ͉ neurotrophins ͉ Pincher

Receptor-mediated trafficking of cholesterol between lipoproteins and cells is a fundamental biological process at the organismal and cellular levels. In contrast to the well-studied pathway of LDL receptor-mediated endocytosis, little is known about the trafficking of high-density lipoprotein (HDL) cholesterol by the HDL receptor, scavenger receptor BI (SR-BI). SR-BI mediates HDL cholesteryl ester uptake in a process in which HDL lipids are selectively transferred to the cell membrane without the uptake and degradation of the HDL particle. We report here the cell surface locale where the trafficking of HDL cholesterol occurs. Fluorescence confocal microscopy showed SR-BI in patches and small extensions of the cell surface that were distinct from sites of caveolin-1 expression. Electron microscopy showed SR-BI in patches or clusters primarily on microvillar extensions of the plasma membrane. The organization of SR-BI in this manner suggests that this microvillar domain is a way station for cholesterol trafficking between HDL and cells. The types of phospholipids in this domain are unknown, but SR-BI is not strongly associated with classical membrane rafts rich in detergent-resistant saturated phospholipids. We speculate that SR-BI is in a more fluid membrane domain that will favor rapid cholesterol flux between the membrane and HDL.

Target-derived neurotrophins use retrogradely transported Trk-signaling endosomes to promote survival and neuronal phenotype at the soma. Despite their critical role in neurotrophin signaling, the nature and molecular composition of these endosomes remain largely unknown, the result of an inability to specifically identify the retrograde signaling entity. Using EGF-bound nanoparticles and chimeric, EGF-binding TrkB receptors, we elucidate Trk-endosomal events involving their formation, processing, retrograde transport, and somal signaling in sympathetic neurons. By comparing retrograde endosomal signaling by Trk to the related but poorly neuromodulatory EGF-receptor, we find that Trk and EGF-receptor endosomes are formed and processed by distinct mechanisms. Surprisingly, Trk and EGF-receptors are both retrogradely transported to the soma in multivesicular bodies. However, only the Trk-multivesicular bodies rely on Pincher-dependent macroendocytosis and processing. Retrograde signaling through Pincher-generated Trk-multivesicular bodies is distinctively refractory to signal termination by lysosomal processing, resulting in sustained somal signaling and neuronal gene expression.

The cytomatrix at the active zone (CAZ) is a macromolecular complex that facilitates the supply of release-ready synaptic vesicles to support neurotransmitter release at synapses. To reveal the dynamics of this supply process in living synapses, we used super-resolution imaging to track single vesicles at voltage-clamped presynaptic terminals of retinal bipolar neurons, whose CAZ contains a specialized structure—the synaptic ribbon—that supports both fast, transient and slow, sustained modes of transmission. We find that the synaptic ribbon serves a dual function as a conduit for diffusion of synaptic vesicles and a platform for vesicles to fuse distal to the plasma membrane itself, via compound fusion. The combination of these functions allows the ribbon-type CAZ to achieve the continuous transmitter release required by synapses of neurons that carry tonic, graded visual signals in the retina.DOI: http://dx.doi.org/10.7554/eLife.13245.001

No abstract