Neurons, Chromaffin Cells and Membrane Fusion - Fusion of Biological Membranes and Related Problems - page 362

and its substrates during the critical period of cerebellar granule cell migra-
tion and synaptogenesis suggesting a regulatory role for the enzyme and its
target substrates in cerebellar ontogenesis.
4. ENDOCYTOSIS OF LDV/SECRETORY VESICLES
The membrane of the secretory vesicles, after transient exocytotic
fusion of the vesicle membrane with the plasma membrane, is rapidly and
selectively retrieved back into the cell. While exocytosis requires Ca ++ and
ATP, the subsequent endocytosis can proceed in the virtual absence of Ca ++
and ATP and is largely unaffected by a variety of nucleotide triphosphates
(including nonhydrolyzable analogues) and cyclic nucleotides. While
retrieval of SSV-constituents is well documented (for a review see Südhof,
1995), data on the internalization of noradrenergic LDVs and secretory
vesicles remain fragmentary. Most of our knowledge concerning recycling
of secretory granules/LDVs comes from studies on chromaffin cells, where
retrieved secretory granule constituents have been shown to recycle via
the trans -Golgi network region (Patzak and Winkler, 1986; Philips, 1987;
Hurtley, 1993). In noradrenergic neurons in culture it is shown that after
stimulation, exocytosed LDV-constituents enter the endocytotic pathway
via clathrin-coated pits and clathrin coated vesicles of a relatively smaller
size then the LDV (Annaert et al., 1997). In the same neuronal cultures it
is demonstrated that LDV-membrane constituents are retrieved into early
endosomes after exocytosis, as can be concluded from their colocalisation
with the endosomal markers Rab5 and HRP in subcellular fractiona-
tion analysis and their colocalisation with Rab5 in confocal microscopy
(Partoens et al., 1998). Rab5 is a general specific marker for early endo-
somes (Chavrier et al., 1990) and its occurrence on synaptic vesicles pro-
vided evidence that SSV-membrane constituents recycle via an endosomal
intermediate (Fischer von Mollard et al., 1994; de Hoop et al., 1994). Rab5
regulates the early steps of the endocytotic pathway, i.e. internalization at
the plasma membrane and homotypic fusion of early endosomes with
exchange of contents (Gorvel et al., 1991; Bucci et al., 1992). Therefore, the
presence of Rab5 on both retrieved LDV- and SSV-membranes in the stim-
ulated SCG neurons indicates their possible intermixing and the formation
of a common sorting endosomal compartment. Interestingly, it was recently
reported that Rabphilin3a, previously characterized as a Rab3a-binding
protein (Shirataki et al., 1993) and regulator of regulated exocytosis (Holz
et al., 1994), interacts with the Rab5-Rabaptin5 system (Ohya et al., 1998).
Rab3a, in its GTP-bound state, inhibited the Rabaptin5-Rabphilin3a inter-
action (Ohya et al., 1998) as well as the promoting effect of Rabphilin3a on