Introduction
Contractile vacuole complexes (CVCs) are a morphologically diverse set of organelles that are found very broadly throughout eukaryotes, and particularly in eukaryotic protists. Their recognized role is in osmoregulation, a critical issue for single-celled organisms inhabiting freshwater and therefore hyperosmotic environments because constant passive water uptake across the plasma membrane threatens cellular via viability, including maintaining physiological cytoplasmic ion concentrations (Naitoh et al., 1997, Tominaga et al., 1998a, Klauke and Plattner, 2000, Kissmehl et al., 2002, Tani et al., 2002, Reuter et al., 2013). Notwithstanding their heterogeneity, CVCs all appear built on similar organizing principles. They are comprised of one or more large vacuolar compartments that function as water-storing bladders, that are ringed by a network of tubular extensions called the spongiome (Allen and Naitoh, 2002). The existing data are consistent with the general idea that cytoplasmic water crosses the spongiome membranes via aquaporins or similar channels and driven by gradients dependent in part on proton-pumping V+-ATPases. Water in the spongiome lumen diffuses to swell the connected bladder. At some point, the bladder membrane fuses with but is not integrated into the plasma membrane, and the soluble contents are rapidly propelled outside the cell by dramatic contraction of the bladder. This membrane fusion event in the Amoebozoan Dictyostelium discoideum is regulated at least in part by triggered calcium release from the bladder lumen, which acts to downregulate a CVC-specific Rab (Parkinson et al., 2014). More generally, ion mobilization from the CVC lumen and/or excretion during bladder contraction may play homeostatic and/or signalling roles (Ladenburger et al., 2006, Malchow et al., 2008, Ladenburger et al., 2009, Patel and Docampo, 2010, Sivaramakrishnan and Fountain, 2012). Following bladder contraction, the fusion pore to the plasma membrane reseals and the bladder is ready for re-filling. Though functionally similar, the precise structures involved in this cycle and their dynamic interactions may differ in important details between lineages. For example, in the Ciliate Paramecium the reticular spongiome is comprised of two distinct zones, and the contractile cycle includes membrane fission and fusion between the spongiome and bladder (Allen, 2000), neither of which features in the CVC of the KinetoplastidTrypanosoma cruzi (Jimenez et al., 2022).
CVCs are not found in yeasts or other lineages in which cells secrete and assemble external walls that can counterbalance osmotic pressures with physical restraints, nor in multicellular organisms like animals where the tonicity of the extracellular milieu is under organismal control. Since the most commonly employed model organisms belong to these groups, CVCs have been relatively understudied and consequently many central issues remain unsettled. One largely open question is how the CVC is integrated with other pathways of eukaryotic membrane trafficking, including the mechanisms that underlie its maintenance in vegetative cells and inheritance during cell division. A number of proteins whose homologs are involved in membrane trafficking in fungi and animals, have been physically and/or functionally associated with the CVC in diverse eukaryotic lineages. Early work in D. discoideum linked both and the clathrin assembly factor AP180 to CVC function, suggesting a putative connection between endocytic trafficking and CVC maintenance (Wen et al., 2009, Macro et al., 2012). The CVC is nonetheless normally isolated from bulk endocytic trafficking, in bothDictyostelium and the Ciliate Tetrahymena thermophila , based on studies using endocytic tracers (Gabriel et al., 1999, Cheng et al., 2023). Whether the same trafficking pathways maintain the CVC in all lineages remains an open question. A related but more far-reaching question is whether CVCs emerged independently in different lineages, or whether they stem from a single ancestral origin. The fact that all known CVCs rely on vacuolar ATPases might argue for the latter, but vacuolar ATPases function in a very broad range of compartments in the endomembrane network (Stevens and Forgac, 1997).
In addition to clathrin and AP180, other endosomal protein homologs were also shown to be physically and/or functionally associated with the CVC in Dictyostelium, including AP2, epsin, the endosomal synaptobrevin homolog Vamp7B, Rabs 11 and 8, and an amphiphysin (I-BAR) family protein (Harris et al., 2001, Stavrou and O’Halloran, 2006, Wen et al., 2009, Linkner et al., 2014, Manna et al., 2023, Du et al., 2008, Essid et al., 2012, Macro et al., 2012). Homologs for a subset of these, including a Rab11 family member, were subsequently found associated with the CVC inTrypanosoma cruzi (Ulrich et al., 2011). The Ciliates are a third, very distantly related lineage with prominent CVCs, about which relatively little is known about membrane trafficking determinants. Comprehensive localization surveys of SNAREs (in Paramecium tetraaurelia ) and of Rabs (in T. thermophila ) provided only ambiguous evidence addressing whether the CVC is an endosome-related compartment, since in both protein families the most prominent CVC-localized members did not belong to conserved endosomal subgroups but instead either belonged to other identifiable subgroups or were lineage-restricted (Bright et al., 2010, Plattner, 2013, Schonemann et al., 2013). A possible exception came from the observation that severalT. thermophila Rabs belonging to conserved endocytic/endosomal subgroups showed secondary localization to the CVC, but with unknown functional significance (Bright et al., 2010). In contrast, strong evidence of an endosomal connection in ciliates came from analysis ofT. thermophila Dop1p, a member of the DOPEY family of proteins that serve endosome-related roles in other lineages. In T. thermophila , Dop1p localizes strongly to the CVC where it is required for normal bladder discharge (Cheng et al., 2016, Cheng et al., 2023).
In this manuscript, we present analysis of T. thermophila VPS8D, a subunit of one of six T. thermophila CORVET (class Ccor e vacuole/e ndosome-t ethering) complexes. In fungi, animals, and plants, CORVET and the related HOPS (ho motypic fusion and p rotein s orting) complexes are key determinants in endo-lysosomal trafficking (van der Beek et al., 2019). Current data support models in which the hetero-hexameric complexes tether two vesicle membranes by binding Rab GTPases in each of the adjoining membranes, and catalyze fusion by interacting with SNAREs (Baker et al., 2015). While many organisms have one CORVET and one HOPS, in the lineage leading to T. thermophilaHOPS was lost and CORVET expanded to a family of six complexes, each with a unique subunit composition and cellular localization (Sparvoli et al., 2018). One of these complexes, which uniquely contains the subunit Vpd8D, was localized in an initial survey to the CVC, and subsequently shown to be present in a punctate distribution at both the bladder and spongiome, in dynamic equilibrium with a cytosolic pool (Sparvoli et al., 2020, Cheng et al., 2023). Attempts to study the function of Vpd8Dp by gene knockout were unsuccessful, since unlike the Vps8 subunits in other CORVET complexes the VPS8D paralog appeared to be essential (Sparvoli et al., 2018). Here we have therefore taken a different approach, using a gene-specific RNA hairpin to partially deplete theVPS8D transcript. The knockdown cells show a variety of CVC-associated phenotypes. The severity of the defects appears to correlate with the intensity of the knockdown, and at the extreme manifests as complete loss of both the bladder and spongiome. Both our functional and localization data are consistent with the idea that inT. thermophila a ciliate-adapted CORVET complex directs critical membrane tethering and fusion cycles in the CVC.