4. EXOCYTOSIS AND ENDOCYTOSIS
4.3. Stimulated exocytosis and exocytosis-coupled endocytosis in ciliates
4.3.1. Assembly of exocytosis sites
After delivery to the cell surface, an estimated period of several minutes may suffice to acquire exocytotic fusion capacity of trichocysts (Plattner et al., 19(3). When Paramecium cells of the temperature-sensitive mutant, nd9 (Beisson et al., 1980; Froissard et ;L1., 20(1), are transferred from a nonper-missive to a pernonper-missive temperature (28 0 C --+ 18 0 C), almost all sites achieve exocytosis competence, paralleled by assembly of "rosettes " (aggre-gates of intramembraneous particleslintegrated proteins seen in freeze-fi-actures; Section 5.2) within hours (Proissard et al., 2(02). This does not contradict our previous estimation of much shorter times for the individual process (Plattner et al., 1993), as this seeming discrepancy is observed with all steps of the exo-endocytosis cycle due to a. certain degree of asynchrony (Knoll et :1.1.,1991:1; Platmer and HentscheJ, 20eJ6).
Interestingly, NSF gene silencing during the 28 QC --+ 18 QC transfer of l1d9 cells can suppress rosette assembly and acquirement of fusion compe-tence (Proissard et al., 20(2); Section 5. This implies that NSF, in this case, is required to establish SNARE complexes, rather than to disassemble them after exocytosis. The latter has been found with some other systems and then tacitly generalized (Litdeton et a1, 20C~1). In fact, the sequence we described has also been found \vith bovine chromaffin cells (Xu et al., 1(99).
Even more strikingly, recent electrophysiological studies with neuronal cells analyzed under conditions of sufficient tirne resolution concludes an ongo-ing interaction of NSF with SNAREs to maintain them in an assembled state ready for fusion (Ktlller et al., 20(8). Since trichocyst docking sites are newly formed over many cell divisions independently of any exocytosis, this also implies a primary role of NSF for SNARE assembly, rather than disassembly after fusion (although NSF may also support SNARE rear-rangelnent after trichocyst exocytosis).
4.3.2. Dynamics of exocytosis in Paramecium
In Pam111eciu 111 , an appropriate stimulus provokes the immediate release of trichocysts. Such a stimulus can be the contact with a predatory ciliate, such as Dileptus, whose attack') are survived selectively by exocytosis-competent cells, as detected by llannnoto andfvliya.ke (1991). Based on this work, subsequent studies by Knoll et a1. (1991 b) have shovvn that local trichocyst release keeps the predator at a distance, thus allU\~ring the ParameciU111 cell to escape. While the actual chemical stimulus is not known, a mechanical stimulus does not produce this phenomenon. In contrast, it can be perfectly mirn1cked by polyarnines such as aminoethyldextran, AED QJhuncr et al., 1984, 1985b).
Meanwhile, AED has been accepted as a standard secretagogue for Pam111eciu111.
The dynamics of synchronous trichocyst exocytosis and exocytosis-cou-pled endocytosis has been thoroughly analyzed by quantitative quenched flow I cryofL'Cation/freeze-fracture EM analysis (Knoll et a1., 1991a; Phuncr and Hentschel, 2006; Plattner et a1., 19(7). Synchronous trichocyst exocytosis upon stimulation with AED occurs within 80 ms, followed by <v 270 ms for endocytotic membrane re sealing and still longer times for pinching offtricho-cyst "ghosts." Again, the individual fusionlresealing pore has a much shorter life-time than registered for the overall phenomenon in the entire cell popula-tion analyzed. For the individual fusion event, we estimate a time requirement
of
<
1 ms, that is, belovv the methodical time resolution which was available tous (Phttner et al., 1993). With liposomes, this has been substantiated in vitro by fast kinetics analysis (Kasson et a1., 20(6) and in vi1l0, by patch-clamp analysis, with mammalian cells (Breckenridge and Almers, 1987), as summarized by Sorensen, 20(5). The open time of a fusion pore (opening to full width) is about in the range of <v 1 s. It is similar when recorded by electrophysiology with mammalian dense core-vesicle systems (Fang et al., 20(8) as it is for the stimulated exo-endocytosis coupling in Para111eciu111. In the latter case, coupling is accelerated with increasing [Ca2+Jo (Pla.ttner et al., 19(7), as it is in mammalian systems (Henkel and Almers, 1996; Rosenboom and Lindau, 1994), as discussed in Section 7.
What is the molecular background of these processes? No ATP is required for membrane fusion per se during trichocyst exocytosis (Vilmart-Seu\iven et al., 1986). There is currently unanimous agreement on this aspect also in other systems (Sorensen, 20(5) though initially this issue had been hotly debated.
In Pam111eciu111, PtSyx1 is considered the t-type SNARE required for trichocyst exocytosis, as silencing of this gene greatly reduces the stimulated exocytotic response (Kissmehl et al, 20(7). Strikingly, PtSyxl is scattered over the entire "somatic" (nonciliary) cell membrane, whereas one would expect concentration at the sites with rosettes, directly above the trichocyst tips.
(The aspect of potential microdomain arrangement is discussed in Section 5.) The rather ubiquitously distributed PtSNAP-25-LP is also recognized over the
130
entire cell boundary (Schilde et aJ, 20(8). Both, PtSyxl and PtSNAP-25-LP may also mediate any other membrane fusion events occurring at the cell surface.
How about other components for stimulated exo-/endocytosis and other membrane fusion processes? Unfortunately, the v-type SNARE pertinent to trichocyst exocytosis could not be identified unequivocally so far, but PtSyb5 is a realistic candidate (Section 3.3). Another open ~uestion
pertinent to trichocyst exocytosis concerns the nature of the Ca2 -sensor which has to be expected at such sites, as outlined in Section 7. Finally, the relevance of calmodulin occurring at trichocyst exocytosis sites (lVlomayezi et aL,1986; Plattner, 1987) expects elucidation. Calmodulin is known to be mandatory for the assembly of a functional trichocyst exocytosis site (Kerboeuf et al., 1993). In mammalian cells, calmodulin binds to synapto-brevin (Queths et aI, 20C~2) and, thus, can affect the arrangement of SNAREs. By intera.ction with the auxiliary protein, l\Ilunc13, calmodulin also drives priming for neurotransmission (Dirnova et al., 20(9). Ho\vever, from its Ca2+ -binding properties and kinetics, calmodulin is generally considered inappropriate to serve as a Ca2+ -sensor for a rapid exocytotic response. Beyond this, calmodulin is a Ca2+ -sensor for the different forms of endocytosis in nerve terminals (\Vu et al., 20(}9) and, thus could exert the same function in ciliates. In sum, calmodulin may be a multifunctional component of dense core-vesicle docking/ exocytosis sites also in ciliates.
Actin is another modulator of exocytotic fusion pore dynamics, for exam-ple, in pancreatic acinar cells (Larina et al., 2007). Remarkably, actin flanks trichocyst docking sites in Paramecium (Kissrnehl et a1., 2(04) and trichocyst docking is reportedly inhibited by cytochalasin B (Beisson and Rossignol, 1975).
Combining fast freezing technology during stimulated synchronous trichocyst release vvith ElVI analysis (Phuner and HenLschel, 2(06) has shown that membrane hlsion probably occurs within a submillisecond time scale and presents itself as a rv 10 nm large spot ("fusion pore"). All this corresponds to the temporal and spatial resolution achievable by the method used (Knoll et al., 1991a; Section 5). Subsequently, the pore expands and thus alluws access of extracellular Ca2+ to the trichocyst contents. This entails vigorous expansion of the trichocyst matrix (Bilinski et aL, J981) by Ca2+ binding to specific matrix: proteins (Klauke et al.,
1(98), as commented in Section 7.2.
Not only trichocyst rnembranes ("ghosts") are internalized by exo-endocytosis coupling, but also dischargeable intact trichocysts can be caused to dedock and eventually to redock. In nondischarge strains of P. tetraurelia, trichocysts can be detached from the cell surface and brought to redocking under conditions described (Pape and Plattner, 1(90) _ This observation is supplemented by the following experiments. Secretory contents release is blocked in a P. caudaturn mutant (\V:ltanabe and Haga, 1996) because of
defective Ca2+ -binding to secretory matri..-x: proteins (I<1auke et aL, 1(98).
In normal cells, contents release can also be inhibited by exocytosis stimula-tion under condistimula-tions unfavorable to matrix expansion, thus resulting in
"frustrated exocytosis" (Kbuke and Plattner, 20(0). This has been visua-lized by the styrene dye FMl-43 that is spontaneously incorporated into the cell membrane and diffuses into the secretory organelle membrane upon fusion (Henkel et al., 1(96). In this case, trichocyst rnembranes fuse ,vith the cell rnembrane, just as during exocytosis, but without contents release. This
"frustrated exocytosis" is followed by resealing, internalization, and redock-ing of the intact organelles which can be stimulated to perform normal exocytosis. The organelle must have a signal indicating its state (Section 3.3.3) because empty "ghosts" would go the degradation pathway.
All this has specified for the first time membrane detachment as a distinctly regulated step.
We summarize this section on exo- and endocytosis as follo\;vs. The highly efficient machinery of stimulated exocytosis is only partly understood on a molecular level, also in ciliates. Actin is found around docking sites underneath the cell membrane (Section 2.32). PtSyxl is involved in stimulated exocytosis, though it is distributed all over the somatic cell surface. Quite uncertain is the type of v-SNARE, possibly PtSyb5, in the trichocyst membrane (Section 3.3.3). No evidence could be found in Pararnecium for any contribu-tion of the proteolipid part of the H+-ATPase to exocytotic membrane fusion, as elaborated in Section 5. Finally, we observe a specific difference between the detachment ofintact trichocysts and their "ghosts" fonned by exocytosis.