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First published online 12 December 2006
doi: 10.1242/jcs.03313

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A broad spectrum of actin paralogs in Paramecium tetraurelia cells display differential localization and function

Department of Biology, University of Konstanz, P.O. Box 5560, 78457 Konstanz, Germany

View larger version (17K): [in this window] [in a new window]   Fig. 1. Schematic Paramecium cell. The plasma membrane (pm) is covered with cilia (ci). Along the oral groove, they beat rhythmically to wash food towards the oral cavity (oc). The phagosomal apparatus consists of several elements: as, acidosomes mediating acidification of a food vacuole (fv) after pinching off; cytopharyngal fibers (cf), post oral fibers (pof), cytoproct (cp, site of exocytotic release of spent phagosomes). Dense-core vesicles (trichocysts, tr), are attached to the plasma membrane. Osmoregulation is executed by two contractile vacuole complexes (a, ampula; cv, contractile vacuole; ds, decorated spongiome; ss, smooth spongiome). The red arrows indicate transport routes during phagosomal processing. Discoidal vesicles (dv) and other recycling vesicles (rv) are responsible for return transport of membranes. The endosomal system, composed of parasomal sacs (ps) and early endosomes (ee), is arrayed in a regular fashion. er, endoplasmatic reticulum; ga, golgi apparatus.

View larger version (87K): [in this window] [in a new window]   Fig. 2. GFP localization of members of the act1 subfamily. (A-C) A confocal time series of a cell transformed with GFP-act1-2 reveals actin comet tails that propel vacuoles (two labeled * and +) through the cytoplasm. (D-F) Confocal z series of a cell transformed with GFP-act1-2. Note the irregular distribution of patches around the surface of some food vacuoles (fv). (G,H) Transformation with GFP-act1-6 resulted in diffuse cytosolic staining without any specific localization (G, median; H, superficial plane). (I-N) In cells transformed with GFP-act1-9, actin comet tails on food vacuoles occurred. Food vacuoles can move in opposite directions through the cytoplasm (see the movement of the two vacuoles labeled with * and + within 150 seconds). See supplementary material Movie 1 for the thin, very dynamic tails that can propel some food vacuoles and small vesicles through the cytoplasm and actin filaments that are catapulted into the cytoplasm from the posterior end of the oral cavity. mn, macronucleus; oc, oral cavity; fv, food vacuoles. Bars, 10 µm.

View larger version (125K): [in this window] [in a new window]   Fig. 3. Application of an anti-GFP antibody to cells transformed with GFP-act3-1 constructs. Confocal images from superficial (A) and median (B) focal planes of a dividing cell. Note labeling of cilia (ci), labeling of the cortical `egg-box' relief in A and diffuse staining of the cytoplasm in B. The cleavage furrow is not labeled. Bars, 10 µm.

View larger version (39K): [in this window] [in a new window]   Fig. 4. Immunolocalization of actin using subfamily-specific antibodies. (A) Western blot analysis of affinity-purified anti-act4-1 antibody (lanes 1-3) and anti-act5-1 antibody (lanes 4-6) against the recombinant act1-1 (L261-G366; lanes 1, 4), act4-1 (M1-A160; lanes 2, 5) and act5-1 peptides (H175-L290; lanes 3, 6), respectively. There is no crossreactivity between the isoforms. The double band in lane 2 is probably due to formation of dimers. (B-E) Localization of act4. (B,C) Median and superficial focus plane showing labeling of the oral cavity (oc), around a nascent food vacuole (fv), of cilia and at the cell cortex. (D,E) Median and superficial plane of a dividing cell. Note labeling of the cleavage furrow (cf) and of the old and new oral cavity (oc). (F) Superficial plane of a cell in an earlier stage of division. Again the cleavage furrow is labeled, as well as the old and the new oral cavities. (G) For comparison, a cell in the same dividing state as F, but labeled with an anti-act5 antibody, reveals no labeling of the cleavage furrow. Bars, 10 µm.

View larger version (53K): [in this window] [in a new window]   Fig. 5. GFP localization of act5-1. GFP-tagged act5-1 occurs predominantly throughout the cytoplasm, although it is also found on individual food vacuoles (fv) (arrowhead, A). The focus plane in B reveals a strong signal on different structures of the oral cavity, e.g. the peniculus (p) and the quadrulus (q). A dynamic fiber system emanating from the oral apparatus, also labeled, is indicated by an arrow. mn, macronucleus; oc, oral cavity; c, crystals. Bars, 10 µm.

View larger version (149K): [in this window] [in a new window]   Fig. 6. Transformation of Paramecium cells with the GFP-act8-1 construct resulted in a weak cytosolic signal, labeling around some food vacuoles (fv; A,C) and at the presumed peniculus (p) and the quadrulus (q) of the oral cavity (A,B; C, detail from A). From the latter, a dynamic fiber system originates (B, see also supplementary material Movie 2). Close to the surface round to elongate structures are labeled (arrow in B) which continue deeper into the cytoplasm and probably represent Golgi fields (see text). The superficial plane in D reveals regularly spaced, labeled, very small dots at the plasma membrane (small bar between two labeled structures measures 2 µm; double arrow represents direction of cell axis), i.e. the sites occupied by `parasomal sacs'. (E,F) In immunogold EM analyses, using anti-GFP gold-antibody, label was enriched at the boundary of the rough ER in association with smooth vesicular, round to elongate membrane-bounded elements (arrowhead in E) or in bona fide Golgi fields (F). mn, macronucleus; oc, oral cavity; cv, contractile vacuole. Bars, 10 µm (A-D); 100 nm (E,F).

View larger version (28K): [in this window] [in a new window]   Fig. 7. Comparison of the division rate of cells silenced in different actin subfamilies. Only silencing of act4 and act9 affects the division rate, all other subfamilies show no significant difference when compared with the control (empty pPD vector). While silencing of act9 caused delayed growth, cells silenced in act4 stop dividing and start dying at 72 hours. Error bars represent s.e.m. *s.e.m.=0.

View larger version (47K): [in this window] [in a new window]   Fig. 8. Morphological changes in cells silenced in act4, act7 or act9. Cells silenced in act4 (A) are not able to divide, thus resulting in a `boomerang' appearance. Cells silenced in act7 become slightly oversized with a pointed anterior end ('dolphin' shape, B). Silencing in act9 results in triangle-shaped cells (C). Bars, 10 µm.

View larger version (15K): [in this window] [in a new window]   Fig. 9. Comparison of the phagocytotic capacity (as defined in Materials and Methods) of cells silenced in different actin subfamilies. Although silencing of members of the act1 subfamily, act5 or act6 does not significantly affect phagocytosis (P<0.05; control: pPD vector), it is reduced to 70% in cells silenced in act7, and to 30% in cells silenced in act2 or act3. With act9, in normally shaped cells phagocytosis is slightly impaired, whereas there is a strong inhibition in triangle-shaped cells (10% phagocytotic activity). In cells silenced in act4, no phagocytosis could be observed (*). Data were collected from five different experiments from at least 20 cells per group. Error bars represent s.e.m.

View larger version (9K): [in this window] [in a new window]   Fig. 10. Silencing of act9 affects the pumping cycle of the contractile vacuoles in misshapen transformants. While the osmoregulatory complex works normally in act9-silenced cells with normal shape, its pumping cycle is strongly prolonged in triangle-shaped cells. Feeding with pPD vector does not cause any defect in the cycle of the contractile vacuole complex. Data were collected in three different experiments from at least ten cells per group. wt, wildtype. Error bars represent s.e.m.

View larger version (23K): [in this window] [in a new window]   Fig. 11. Schematic distribution of actin isoforms in the Paramecium cell, as outlined in Table 1. The trafficking scheme is based on published reviews (Fok and Allen, 1990; Allen and Fok, 2000; Plattner and Kissmehl, 2003). Actin distribution is based mainly on the present data obtained with GFP localization in vivo and with antibody labeling, but also takes into account data from previous work (Tiggemann and Plattner, 1981; Kersken et al., 1986a; Kersken et al., 1986b; Kissmehl et al., 2004). The scheme contains elements of the osmoregulatory system (a, ampula; cv, contractile vacuole; ds, decorated spongiome; ss, smooth spongiome), though consistently unlabeled, of the phagosomal apparatus (as, acidosomes; cf, cytopharyngal fibers; cp, cytoproct; ci, cilia) dv, discoidal vesicles and other recycling vesicles, rv; ee, early endosome; er, endoplasmatic reticulum; fv, food vacuole; ga, golgi apparatus; gh, ghosts (from released trichocysts); oc, oral cavity; pm, plasma membrane; pof, post oral fibers; ps, parasomal sacs; tr, trichocysts; gray background, cytosolic.

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