Electrical properties and fusion dynamics ofin vitromembrane vesicles derived from separate parts of the contractile vacuole complex ofParamecium multimicronucleatum

Abstract

SUMMARY The contractile vacuole complex of Paramecium multimicronucleatumtransforms into membrane-bound vesicles on excision from the cell. The I–V relationship was linear in a voltage range of–80 to +80 mV in all vesicles, despite being derived from different parts of the contractile vacuole complex. No voltage-gated unit currents were observed in membrane patches from the vesicles. Vesicles derived from the radial arm showed a membrane potential of >10 mV, positive with reference to the cytosol, while those derived from the contractile vacuole showed a residual (<5 mV) membrane potential. The electrogenic V-ATPases in the decorated spongiome are responsible for the positive potential, and Cl– leakage channels are responsible for the residual potential. The specific resistance of the vesicle membrane (∼6 kΩcm2) increased, while the membrane potential shifted in a negative direction when the vesicle rounded. An increase in the membrane tension (to∼5×10–3 N m–1) is assumed to reduce the Cl– leakage conductance. It is concluded that neither voltage- nor mechano-sensitive ion channels are involved in the control of the fluid segregation and membrane dynamics that govern fluid discharge cycles in the contractile vacuole complex. The membrane vesicles shrank when the external osmolarity was increased,and swelled when the osmolarity was decreased, implying that the contractile vacuole complex membrane is water permeable. The water permeability of the membrane was 4–20×10–7 μm s–1Pa–1. The vesicles containing radial arm membrane swelled after initially shrinking when exposed to higher external osmolarity, implying that the V-ATPases energize osmolyte transport mechanisms that remain functional in the vesicle membrane. The vesicles showed an abrupt (<30 ms),slight, slackening after rounding to the maximum extent. Similar slackening was also observed in the contractile vacuoles in situ before the opening of the contractile vacuole pore. A slight membrane slackening seems to be an indispensable requirement for the contractile vacuole membrane to fuse with the plasma membrane at the pore. The contractile vacuole complex-derived membrane vesicle is a useful tool for understanding not only the biological significance of the contractile vacuole complex but also the molecular mechanisms of V-ATPase activity.

Contractile Vacuole Complex—Its Expanding Protein Inventory

“…The tension measured on the Paramecium CV (Sugino et al, 2005) is compatible with that required for activation of such channels (Kung et al, 2010). Mechanosensitive channels are likely to occur in the Paramecium CVC for the following reasons, although this remains hypothetic for the time being.…”

“…Immuno LM Mercanti et al (2006) P2X cation channels Immuno LM Fountain et al (2007) Nramp/Slc11 GFP Peracino et al (2013) LvsA Functional effects LM Jung et al (2009) MyoJ (myoV type) Immuno LM Jung et al (2009) Continued membrane to membrane interactions also outside the pore and the CV/ ampullae connections. Their occurrence reflects the fusion capacity in vitro of vesicles derived from the Paramecium CVC (Sugino et al, 2005). In vivo fusion may serve for permanently ongoing biosynthesis by ves icle delivery.…”

The contractile vacuole complex of protists – New cues to function and biogenesis

“…However, NSF also occurs in other regions of the CVC except the decorated spongiome (Kissmehl et al, 2002). First of all, the presence of SNAREs indicates that fusion processes in the organelle cannot be merely based on physical forces, as previously suggested (Sugino et al, 2005), particularly since silencing of CVC-resident SNAREs greatly affects organelle function (Schönemann et al, 2013). ''Overt'' membrane fusion is visible at the pore and can be detected also at CV/ampullae connections by membrane capacitance measurements (Sugino et al, 2005).…”

“…This would enable the de-/re-coupling of parts of the spongiome, with a useful feedback to the water/ ion sequestration activity, depending on the physiological requirements. In fact, the fusion capacity of fragments of isolated CVCs has been documented in vitro (Sugino et al, 2005). Due to their high free energy, tubular protrusions are expected to be very liable to fusion, as shown by old (Blumenthal, 1987;Ohki, 1988) and very recent analyses (Shilagardi et al, 2013).…”

Novel vacuoles in Toxoplasma

“…To our knowledge, the aquaporins that have been localized in fungi reside in plasma membranes or ER, not in vacuoles, but several aquaporins exist that have not been localized. j. Physiological studies imply the presence of aquaporins on the contractile vacuoles of ciliates (Sugino et al, 2005) but to our knowledge no aquaporins have been directly localized in ciliates. k. The Plasmodium food vacuole regulates intracellular calcium (Biagini et al, 2003), but the vacuole does not persist into extracellular forms, so cannot affect osmoregulation or ion homeostasis in these forms.…”