Cell type-specific sorting of neuropeptides: a mechanism to modulate peptide composition of large dense-core vesicles - PubMed (original) (raw)
Cell type-specific sorting of neuropeptides: a mechanism to modulate peptide composition of large dense-core vesicles
J Klumperman et al. J Neurosci. 1996.
Abstract
The CNS of Lymnaea stagnalis contains two populations of egg-laying hormone (ELH)-producing neurons that differ in size and topology. In type I neurons, all peptides located C-terminally from the cleavage site Arg-Ser-Arg-Arg180-183 are sorted into secretory large dense-core vesicles (LDCV), whereas N-terminal-located peptides accumulate in a distinct type of vesicle, the large electrondense granule (LEG). Via immunoelectron microscopy, we now show that the second population of ELH-producing neurons, type II neurons, lack LEG and incorporate all proELH-derived peptides into LDCV. This finding provides the first example of a cell type-specific sorting of neuropeptides into LDCV. Furthermore, we provide evidence that LEG are formed through a differential condensation process in the trans-Golgi network and that these bodies are ultimately degraded. Analysis of the endoprotease composition of the two types of proELH-producing neurons suggests that the formation of LEG, and consequently the retention of N-terminal peptides from the secretory pathway, requires the action of a furin-like protein.
Figures
Fig. 1.
Schematic representation of _Lymnaea_proELH in which all basic cleavage sites are indicated. Endoproteolytic cleavage on these sites results in the formation of 11 peptides. The arrow points to the first cleavage site, Arg-Ser-Arg-Arg180–183, that is used in the Golgi of type I neurons (Li et al., 1994). In this study, we used antibodies against N-terminal peptide and ELH to study the targeting of peptides that are located N- or C-terminally from this site.
Fig. 2.
Ultrathin cryosections of type I neurons double-immunolabeled for N-terminal peptide and ELH. LDCV (arrows) were present in the cell body (A), in the axon endings (B), and in the collaterals (C). LEG (bold arrow in_A_) were only present in the cell body. Note the absence of ELH label in the LEG. G, Golgi. Scale bars, 0.1 μm.
Fig. 3.
Frequency diagram of the ELH/(ELH + N-terminal peptide) ratios, as assessed in type I LDCV of animal 1. From this diagram, it is apparent that all type I LDCV belong to a single population.
Fig. 4.
Ultrathin cryosections of type II neurons double-immunolabeled for ELH (small gold particles) and N-terminal peptide (large gold particles). Type I LDCV (arrowheads) were present in the Golgi (G) region (A, B), in clusters in the cell body (C), and in the axons (D). LEG were absent. Note that the intensity of ELH staining is highest in the axon (D). In_D_, a type II axon is closely opposed to a type I axon. A few type I LDCV are visible (arrows), which are larger and less intensely labeled for N-terminal peptide. N, Nucleus. Scale bars, 0.1 μm.
Fig. 5.
Frequency diagram of the ELH/(ELH + N-terminal peptide) ratios, as assessed in type I and type II LDCV of animal 1. From this diagram, it is apparent that type I and type II LDCV form two subpopulations with only partially overlapping ratios.
Fig. 6.
Electron micrographs of a plastic section (A) and ultrathin cryosections (B_–_D) of the TGN of type I neurons. Within condensing vacuoles at the _trans_-Golgi (B) and at various sites in the TGN (A), condensed protein cores with distinct electron densities are found (bold arrows). The lighter part labels for ELH (A), and the darker portion labels for N-terminal peptide (B). The membranes surrounding these differentially condensed proteins may form coated buds (small arrows in A). Sometimes protein cores with similar (C) or distinct (D) protein contents were segregated within the continuous membrane of the TGN (arrows). Scale bars, 0.1 μm.
Fig. 7.
Type I neurons in which lysosomal acid phosphatase (AP) was visualized by enzyme cytochemistry (dense reaction product) and N-terminal peptide by immunogold labeling. LEG positive for AP (bold arrows) were found near the Golgi (A) and more peripherally (B,C). In some AP-positive LEG, the N-terminal peptide label was decreased (C). LEG without AP reaction product (open arrows) were also seen. G, Golgi. Scale bars, 0.1 μm.
Fig. 8.
Putative model of how differential processing of proELH may account for the differential sorting of N-terminal peptide in type I and type II neurons. In type I neurons, the C-terminal part of proELH is sorted into LDCV, whereas the N-terminal intermediate is targeted to LEG and degraded. In type II neurons, both C-terminal proELH-derived and N-terminal proELH-derived peptides are found in LDCV. A possible explanation for the occurrence of N-terminal peptides in LDCV of type II neurons would be that they are still attached to the C-terminal part at the time of LDCV formation. The finding that the endoprotease LFur2 is solely expressed in type I neurons is in line with this hypothesis.
Fig. 9.
Expression of Lfur1 (A,B), Lfur2 (C, D), and LPC2 (G, H) mRNA, as assessed by radioactive cRNA in situ hybridization (black silver grains). Type I (large arrowheads) and type II (small arrowheads) neurons were identified by ELH immunocytochemistry (brown reaction product).A, B, Lfur1 mRNA was only detected in non-ELH-producing neurons (small arrow).C_–_F, LFur2 mRNA was readily detectable in type I neurons (C). In type II neurons, of which high-magnification views are shown in_D_–F, labeling did not exceed background levels. G, H, LPC2 mRNA could be detected in both type I and type II neurons. Non-ELH-producing neurons that express LPC2 are indicated by small arrows. Scale bars, 25 μm.
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