The native structure of cytoplasmic dynein at work translocating vesicles in Paramecium.

In Paramecium multimicronucleatum, the discoidal vesicles, the acidosomes and the 100-nm carrier vesicles are involved in phagosome formation, phagosome acidification and endosomal processing, respectively. Numerous cross bridges link these vesicles to the kinetic side of the microtubules of a cytopharyngeal microtubular ribbon. Vesicles are translocated along these ribbons in a minus-end direction towards the cytopharynx. A monoclonal antibody specific for the light vanadate-photocleaved fragment of the heavy chain of cytoplasmic dynein was used to show that this dynein is located between the discoidal vesicles and the ribbons as well as on the cytosolic surface of the acidosomes and the 100-nm carrier vesicles. This antibody inhibited the docking of the vesicles to the microtubular ribbons so that the transport of discoidal vesicles and acidosomes were reduced by 60% and 70%, respectively. It had little effect on the dynein's velocity of translocation. These results show that cytoplasmic dynein is the motor for vesicle translocation and its location, between the vesicles and the ribbons, indicates that the cross bridges seen at this location in thin sections and in quick-frozen, deep-etched replicas are apparently the working dyneins. Such a working dynein cross bridge, as preserved by ultra-rapid freezing, is 54 nm long and has two legs arising from a globular head that appears to be firmly bound to its cargo vesicle and each leg consists of ≥3 beaded subunits with the last subunit making contact with the microtubular ribbon.

Calmodulin localization and its effects on endocytic and phagocytic membrane trafficking in Paramecium multimicronucleatum.

In ciliates, calmodulin (CaM), as in other cells, has multiple functions, such as activation of regulatory enzymes and modulating calcium-dependent cellular processes. By immunogold localization, CaM is concentrated at multiple sites in Paramecium. It is seen scattered over the cytosol, but bound to its matrix, and is concentrated at the pores of the contractile vacuole complexes and with at least three microtubular arrays. It was localized peripheral to the nine-doublet microtubules of the ciliary axonemes. The most striking localization was on the akinetic side only of the cytopharyngeal microtubular ribbons opposite the side where the discoidal vesicles, acidosomes and the 100-nm carrier vesicles bind and move. CaM was also present at the periphery of the postoral microtubular bundles along which the early vacuole moves and was associated with the cytoproct microtubules that guide the spent digestive vacuoles to the cytoproct. It was not found on the membranes of, or in the interior of nuclei, mitochondria, phagosomes, and trichocysts, and was only sparsely scattered over the cytosolic sides of discoidal vesicles, acidosomes, lysosomes, and digestive vacuoles. Together the associations with specific microtubular arrays and the effects of trifluoperazine and calmidazolium indicate that CaM is involved (i) in vesicle transport to the cytopharynx area for vacuole formation and subsequent vacuole acidification, (ii) in early vacuole transport along the postoral fiber, and (iii) in transporting the spent vacuole to the cytoproct. Higher CaM concentrations subjacent to the cell's pellicle and close to the decorated tubules of the contractile vacuole complex may support a role for CaM in ion traffic.

Relationship between the membrane potential of the contractile vacuole complex and its osmoregulatory activity in Paramecium multimicronucleatum.

The electric potential of the contractile vacuole (CV) of Paramecium multimicronucleatum was measured in situ using microelectrodes, one placed in the CV and the other (reference electrode) in the cytosol of a living cell. The CV potential in a mechanically compressed cell increased in a stepwise manner to a maximal value (approximately 80 mV) early in the fluid-filling phase. This stepwise change was caused by the consecutive reattachment to the CV of the radial arms, where the electrogenic sites are located. The current generated by a single arm was approximately 1.3x10(-10) A. When cells adapted to a hypotonic solution were exposed to a hypertonic solution, the rate of fluid segregation, R(CVC), in the contractile vacuole complex (CVC) diminished at the same time as immunological labelling for V-ATPase disappeared from the radial arms. When the cells were re-exposed to the previous hypotonic solution, the CV potential, which had presumably dropped to near zero after the cell's exposure to the hypertonic solution, gradually returned to its maximum level. This increase in the CV potential occurred in parallel with the recovery of immunological labelling for V-ATPase in the radial arm and the resumption of R(CVC) or fluid segregation. Concanamycin B, a potent V-ATPase inhibitor, brought about significant decreases in both the CV potential and R(CVC). We confirm that (i) the electrogenic site of the radial arm is situated in the decorated spongiome, and (ii) the V-ATPase in the decorated spongiome is electrogenic and is necessary for fluid segregation in the CVC. The CV potential remained at a constant high level (approximately 80 mV), whereas R(CVC) varied between cells depending on the osmolarity of the adaptation solution. Moreover, the CV potential did not change even though R(CVC) increased when cells adapted to one osmolarity were exposed to a lower osmolarity, implying that R(CVC) is not directly correlated with the number of functional V-ATPase complexes present in the CVC.

The vacuolar-atpase of Paramecium multimicronucleatum: gene structure of the B subunit and the dynamics of the V-ATPase-rich osmoregulatory membranes.

Previous studies have shown that the vacuolar-ATPase (V-ATPase) of the contractile vacuole complexes (CVCs) in Paramecium multimicronucleatum is necessary for fluid segregation and osmoregulation. In the current study, immunofluorescence showed that the development of a new CVC begins with the formation of a new pore around which the collecting canals form. The decorated membranes are then deposited around the newly formed collecting canals. Quick-freeze deep-etch techniques reveal that six 10-nm-wide V-ATPase V, sectors, tightly packed into a 20 x 30-nm rectangle, form two rows of these compacted sectors that helically wrap around the cytosolic side of decorated membrane tubules. During new CVC formation, packing of decorated tubules around mature CVCs was temporarily disrupted so that some of these decorated tubules became transformed into decorated vesicles. Freeze-fracturing of these decorated vesicles revealed a highly pitted E-face and a particulate P-face. The V-ATPase was purified for the first time in any ciliated protozoan and shown to contain, as in other cells, the V1 subunits A to E, and four 14-20 kDa polypeptides. The B subunit was cloned and found to be encoded by one gene containing four short introns. This subunit has 510 amino acid residues with a predicted molecular weight of 56.8 kDa, a value similar to B subunits of other organisms. Except for the N- and C-termini, it has a 75% sequence identity with other B subunits, suggesting that the B subunits in Paramecium, like other species, have been conserved and that the entire surface of this subunit may be important in interacting with other subunits.