MICRODOMAINS AND MEMBRANE FUSION
8. ADDITIONAL ASPECTS OF VESICLE TRAFFICKING
8.1. Guidance and support by microtubules
In this section, \;ve shall address the auxiliary role of microtubules as a kind of long-range targeting aid, the unsettled role of some additional molecules
\vith potential relevance for vesicle trafficking in ciliates and finally the
considerable restrictions of using allegedly organelle- or molecule-specific drugs as a tool for analyzing vesicle trafficking in ciliates. This problem" is largely exemplified by the effects on the microtubule system.
In rnetazoans, micro tubules are considered relevant for fast and direc-tional movement of vesicles of different kinds, rather than for their precise targeting (Hirschberg et al., J 998). In a variety of higher eukaryotic cells, docking of dense core-secretory vesicles takes place by saltatory movement along microtubule rails (Lacy, 1975) and, hence, with the involvement of motor proteins (Soldati and SchJiwa, 20(6). Only the recruitment, but not the release of dense core-secretory vesicles is accelerated by the presence of an intact microtubule system in pancreatic acinar cells (Schnekenburger et aL, 20(9). Endocytosis, at least from some distance from the cell mem-brane on (Nielsen et aL, 1999), also involves micro tubules (Bananis et al., 2000; lVlatteoni and K.reis, 1987). Microtubules contribute not only to trafficking from the early to the late endosome (Aniento et al., 1993), but also to ph~lgosome formation (Harrison and Grinstein, 2002; Khandani et a1., 20(7) and intra-Golgi traHicking (C:1i et al., 2007).
8.1.1. Microtubules in ciliates
Cytoplasmic micro tubules are arranged in a complex pattern in Paramecium (Adoutte et a1.,1991 ; AlIen, 1988; Alien and Fok, 2000; Fok and AJJen, 1988, 1990), in Tetrahymena (Gaertig, 2(H)0), and in other ciliates. They may contain tubulin with different posttranslational modifications in ciliates (Libus6va and Dr[tber, 20(6) such as Paramecium (Adoutte et al, 1991) and Tetrahymena (Gaertig, 20{)O; Penque et aL, 19(1). Ciliates may represent a useful model for analyzing the fimctional meaning of the numerous posttranslational modifica-tions that are also found in metazoans (\Vestermann and \Veber, 2003).
J\iloreover, some of the numerous paralogs generated in Paramecium after gene duplication acquire specific new functions (Aury cL al., 2(06)" This complex scenario still awaits more detailed analysis.
Microtubules £lanc the oral cavity from where a separate population emerges, called the (post)oral fibers. In this region, at least three types of vesicles associate with micro tubules (Schroeder et aL, 1990). This differentia-tion is suppOlted by the differential endowment with SNAREs (Schi1de et a1., 2(10). Additional micro tubules run perpendicular to the oral cavity (Adoutte et aL, 1991) and still others connect the oral cavity with the CYToproct (Allen and \Vol( 1974), as sunmlarized by .A11en and Fok (2000). This set of micro tubules serves recycling of membrane materials as "discoidal vesicles"
generated ft-om the spent phagosome membrane after contents release, thus supporting the formation of a nascent food vacuole. This is true for Parame-ciurn (Schroeder et aL, 1990) and for TetmhYll1ena (Sugita et at, 20(9).
Another chemically defined microtubule population runs around the contractile vacuole and elongates over the radial canals of the osmoregulatory system in Parameciurn (Adoutte et al., "] 991) and in Tetrahymena (Gaertig,
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20()O). In addition, in Paramecium, single distinct micro tubules have been observed to emanate £1'om the cell periphery, deep into the cytoplasm (Adoutte et al., -19(1), and may serve trichocyst docking (Auflierheide, 1977; PJattn.er et aL 1982).
Among oral (and intracytoplasmic) micro tubules in Tetrahymena there are glycinated and glutamylated forms (Gaertig, 20(0). Different microtu-bule subpopulations made of differently posttranslationally modified tubulin participate in phagosOlne processing in T. thennophila (Wloga et al., 2(08).
Microtubule subpopulations differ in sensitivity to depolymerizing drugs, concomitantly diHerent steps of the digestive cycle in Parameciurn have widely different drug sensitivities (Fok et. al., 198.5). Also their cold sensi-tivity differs (Adoutte et al., 1991).
As mentioned, in P tetraurelia, the different microtubule poptllations connected to the oral cavity are associated vvith vesicle types endo,ved with different proteins. The first population, possibly comprising tvvo or more vesicle t)lpes (also with the additional uncertainty of discrimination from acidosomes), contains SNAREs type PtSyb6 (Schilde et al., 2(06), Pt-SNAP-2.5-LP (Sdlilde et al., 20(}8), PtSyb8, PtSyb9, and PtSybl0 (Schildc et al., 2(rlO) as ,vell as PtSyx3 and PtSyx4 (Kissrnebl et a1, 20(7). Furthermore, these vesicles differ in their H+ -ATPase SUs; among others, they contain SUs type al and a4 (\Vassrner et al., 20(6) and they are associated with the oral frlament system. This is made not only of microtubules, but it also contains PtAct5 and PtAct8 (Sehringetal, 2007b). Some of these vesicles are vigorously catapulted from their origin to the periphery of the respective microtubule population, just as previously described by structural video-analysis (Ishida et a1, 200J). Hmv they are associated \vith microtubules and by vvhich motor proteins they are propelled remains open for the time being.
In Paramecium, discoidal recycling vesicles travel along micro tubules to the cytopharynx. This is less evident for the population derived from partially matured phagosomes (AJlen et al., 19(5). Discoidal vesicles origi-nating from the cytoproct are more clearly connected to a specific set of micro tubules by dynein (Schroeder et a1., 1(90). Microtubule binding also occurs with the small cytopharyngeal vesicles, also probably contributing to phagosome biogenesis, since microtubules also emanate longitudinally and perpendicularly to the oral cavity (Adoutte et. al, 1991) and these small vesicles are seen to travel forcefully and unidirectionally, as described above.
The motor proteins, kinesin and dynein, that drive the anterograde ( - -+ +) and retrograde (+ -+ -) vesicle transport, respectively, along micro tubules (Hiroknva and Takemura, 2005; VaJlee et aL, 2()04) also occur in ciliates.
There is much less information about kinesin and its potential contribution to vesicle transport than about dynein. T. thertnophila e:X"Presses ; v 25 and P. tetra-urelia ; v 26 dynein heavy chains (Wilkes et al., 20(8). In Tetrahymena, DYHl encodes a cytoplasmic form required for phagocytosis (independent of oral ciliary activity) (Lee et aL,1(99). In P. multirnicronucieatum, dynein has been
identified by biochem.ical analysis as a two-headed cytosolic form (Schroeder et al., 1990). In P. bUfsmia, its silencing inhibits the formation/detadunent of a food vacuole, thus indicating a process operating in
+ -)- -
direction. Nishihara et a1 (1999) also report cyclosis inhibition by the bona fide dynein ATPase inhibitor, erythro-9-[3(2-hydroxynonyl)] adenine ("EHNA"). Accordingly, fonnation and/or transport offood vacuoles through the cell may be facilitated by microtubules. In agreem.ent with this, nocodazole, an established rnicrotu-bule depoIymerizing agent in ciliates (Plattner et al., 2(J09), greatly reduces cyclosis in Paramecium (Nishihcu'a et al., 1999).In Para m eci lun , the basal bodies of oral ciliary assemblies called "quad-riculus" and "peniculus" are associated with selective dynein isoforms, as these microtubuJes stain with specific antibodies (Asai et al., 199/1). This is the site of vivid transport of small vesicles (Ishid~l et al., 20(H)~d site also stained with GFP constructs of PtAct, PtSyx, PtSyb, and SNAP-25-LP (specified above). These vesicles are transported along with the formation of food vacuoles (Ishida et al., 20Cil). In Paramecium, the oral cavity micro-tubules and the postoral fibers stained differentially when a battery of antibodies was probed (Adoutte et al., 1991); postoral fibers also contain PtSyb9-positive structures (Schilde et al., 2(10). The precise, posttransla-tionaJJy modified microtubule subtype involved in the different steps men-tioned is not always known as yet.
In higher eukaryotes, microtubules may equally contribute to ordered vesicle trafficking from deep inside the cell to the periphery as their disruption causes disintegration of the Golgi apparatus (Pfeffer, 20(7) and abolition of saltatory transport of secretory vesicles (I.:1cy, 1(75). In Parame-cium, some micro tubules emerge from ciliary basal bodies and hang into the cell interior vertically to the cell surface (Glas-Albrecht et al., 1991; Plattner et al., 1982). During saltatory docking (Aufderheide, J977), these micro-tubules can guide trichocysts to the cell periphery. Interestingly, docking follows an inherent polarity of trichocysts, tip first, from the plus to the minus end of microtubules. (An exception is som.e exocytosis-incompetent mutants, such as ptA2, that do not dock their aberrant trichocysts [Pouphile et al., 1986].) This microtubule-guided polarity in Parameciurn is opposite to that in most higher eukaryotic cells (Soldati a.nd Schhwa, 2006). However, plus to minus docking has later on been observed also in MDKC (renal) cells (Bacallao et al., 1989; B et al., 1990). This is supported by the observation that chromaffin granules, isolated from bovine adrenal medulla and injected into Paramecium cells, travel to the plus end (Glas-Albrecht et aL, 1991) which, in chromaflin cells, would carry them to the cell membrane. When similar experiments were conducted with chromaffin granules injected into sea urchin egg cells, granules were docked at the cell
n~embrane (Schermer et al., 1(92), as to be expected from the minus-to-plus orientation of rnicrotubules emanating from the cytocenter in that system.
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In summary, in ciliates rnicrotubules relevant for exo-endocytosis do not emerge from a cytocenter, but microtubule organizing centers are asso-ciated with ciliary basal bodies_ The interaction of dense core-secretory vesicles with microtubules evidently follows, or imposes, an inherent direc-tionality-another novel finding one can derive from the cited work with ciliates_ Unfortunately, analysis of kinesins has been largely neglected with ciliates_
8.1.2. Additional aspects concerning microtubules in ciliates
In P_ multi rnicronucleatu In, the different steps of the phagosomal cycle, from acidosome fusion to fusion with lysosomes, probably involve rather differ-ent populations of microtubules and microfilamdiffer-ents_ This may be inferred from the diflerent sensitivity of the different steps to disrupting drugs (Fok et al., 1985, J987)_ An intriguing interaction of cytoskeletal elements, microtubules and actin, is observed in P_ tetraurelia cells by immunogold EM localization in different areas of intense vesicle trafl.icking (Kissmehl et aL, 20(4)-in agreement with the effects of some drugs aiming at microtubule and microfilament function (Beisson and Rossignol, 1975)_
Mechanisms and functions of these interactions remain to be elucidated in detaiL
Most recently, Cda12p-containing vesicles relevant for cytokinesis (Section 9 _3) have been documented in Tetrah.ymena to travel along cortical microtubules to their site of integration into the cleavage fu.rrow (Zvveif(~l
et at, 20(9)- This microtubular arrangement is considered equivalent to the cytospindle described in more detail in Paramecium where it assembles just prior to cytokinesis (Delgado et al, 1990; lftode et at, 1989)_
In sum, micro tubules form an unspecific long-range guidance system for vesicle trafficking_ CiJiates contain several regularly arranged subpopula-tions of micro tubules, with different posttranslational modificasubpopula-tions and vary-ing drug sensitivity_ In Paramecium, trichocysts are docked, tip first according to their inherent polarity, along microtubules from the plus- to the minus-end_
(In contrast, almost all cells of higher eukaryotes operate in the opposite direction_) In ciliates, subsets of differently modified micro tubules are relevant for phagosome formation, that is, to handle the multiplicity of vesicles that are seen around the oral cavity and which all contain different membrane protein signatures_