Vps8Dp is concentrated at junctions between CV-related
membranes
In budding yeast, the HOPS complex concentrates at the junctions between
membranes destined for fusion during vacuole maintenance (Wang et al.,
2003).To see whether Vps8Dp in Tetrahymena behaves similarly, we
took advantage of the observation that many cells exposed to hypoosmotic
stress showed fragmented CVC structures. As shown in Figure 5A, such
cells contain multiple Dop1p-labeled vacuole-like compartments,
concentrated in the cell posterior and undergoing frequent fusion to
form larger compartments, in addition to what appears to be the central
contractile bladder. Dop1p itself appears to be irregularly distributed,
often as puncta, around the periphery of the labeled compartments.
Interestingly, the CVC similarly fragments into multiple Dop1p-decorated
large vacuole-like compartments under conditions of hyper osmotic
stress, though with the difference that no contractile central bladder
is visible (Fig. 5B).
Vacuole-like compartments formed under hypoosmotic conditions are also
decorated with Vps8Dp (Fig. 5C). The Dop1p-labeled and Vps8Dp-labeled
vacuoles appear to be the same structures, since there is strong
co-localization of the two proteins at such vacuoles in cells expressing
both Dop1p-mNeon and Vps8Dp-mCherry (Fig. S5). However, a notable
difference between the Dop1p and Vps8Dp distributions is that in many
cases the latter appears strikingly concentrated in regions between
adjoining vacuoles, where the membranes come into close contact (Fig.
5C).
The concentration of Vps8Dp-mNeon between adjoining vacuoles/vesicles
could be seen even more strikingly in some osmotically-stressed samples
in which cells occasionally suffered localized plasma membrane rupture
under the pressure from the microscope cover slip. These breaks allowed
leakage of the large Vps8Dp-labeled vesicles into the surrounding
buffer. In such samples, Vps8Dp could be seen highly concentrated at
junctures between the membranes, consistent with a role in tethering and
fusion (Fig. 5D). Moreover, in some cases we could capture membrane
fusion occurring at Vps8Dp-enriched junctions (Fig. 5D, arrowheads).
Interestingly, we also captured episodes in which two of the leaked
vesicles shifted closer to one another, and Vps8Dp accumulated rapidly
at their meeting site just when they came into contact (Fig. 5E and 5F).
Discussion
Genes encoding CORVET subunits underwent a marked expansion in the
lineage leading to Tetrahymena, leading to the six distinct complexes inT. thermophila called 8A-8F based on their unique Vps8 subunit
(Klinger et al., 2013, Sparvoli et al., 2018, Sparvoli et al., 2020).
These complexes are distinct from one another in their localization and
their subunit makeup (Sparvoli et al., 2020). They are accordingly
likely to be individually specialized for distinct functions. We
confirmed this previously for the 8A complex, which we found to be
essential for the formation of lysosome-related organelles called
mucocysts (Sparvoli et al., 2018).
In this manuscript we report analysis of the Vps8 subunit of the 8D
CORVET. Vps8Dp was previously shown to be strongly associated with the
osmoregulatory CVC (Sparvoli et al., 2020, Cheng et al., 2023). Because
the gene was found to be essential for cell viability and therefore
difficult to analyze via gene knockout (Sparvoli et al., 2018), in this
manuscript we instead used a knockdown approach based on induced
expression of a gene-specific RNA hairpin (Howard-Till and Yao, 2006).
One striking finding was that even relatively minor depletion of Vps8Dp
produced cells that were highly sensitive to osmotic shock (Fig. 1).
More informatively, following hairpin induction the cells showed
time-dependent changes in the morphology and functioning of the CVC,
affecting both the spongiome and bladder.
Within the cohorts of knockdown cells in each experiment there was
significant phenotypic heterogeneity, making it difficult to rigorously
establish the order in which different defects were manifest.
Nonetheless, our results suggest that spongiome structure may be more
sensitive to VPS8D dosage compared with the bladder structure,
since at early timepoints following hairpin induction only the spongiome
was reduced in size relative to the wildtype, a response that became
more pronounced at longer time points (Fig. 2J). Interestingly, this
shrinkage of the spongiome is very similar to the response seen when
cells are briefly exposed to hypoosmotic shock (Cheng et al., 2023). The
bladder size in contrast did not detectibly change, but contractions
grew less frequent (Fig. 2G). Since the bladder fills with water
transported from the spongiome, the slowed contraction rate may reflect
less efficient filling from a partially disabled water-collecting
reticulum. The bladder in knockdown cells at these early time points
also showed reduced labeling by Dop1p, with a corresponding increase in
diffuse Dop1p signal in the cytoplasm (Fig. 2A and 2B). The mechanistic
role of Dop1p is not yet understood, but in its absence cells show only
infrequent contraction of an enormously expanded bladder (Cheng et al.,
2016, Cheng et al., 2023).
At later time points following hairpin induction, neither the bladder
nor spongiome was detectable, indicating that VPS8D is essential
for the maintenance of this organelle (Fig. 3). Proteins that in
wildtype cells localized strongly to the CVC were instead found in
dispersed cytoplasmic puncta. This process may be reversible, based on
our observation that some cells in knockdown cultures recover when
hairpin expression is de-induced. Studying the mechanisms underlying
such recovery may help to shed light on trafficking pathways involved in
CVC biosynthesis, which are poorly understood in any lineage and
particularly in Ciliates. The disappearance of both bladder and
spongiome compartments after prolonged VPS8D knockdown is similar
to a phenotype reported in D. discoideum that is linked to a
protein in the BEACH family, called LvsA (Gerald et al., 2002).
Interestingly, pulldown experiments in the Apicomplexan Toxoplasma
gondii suggest physical interactions between CORVET/HOPS and a
BEACH-domain containing protein, but in the context of secretory
organelle biogenesis (Morlon-Guyot et al., 2018).
The established role of CORVETs, based on studies in yeast and animals,
is in tethering and fusion of membrane compartments (Spang, 2016, van
der Beek et al., 2019). Our imaging data are consistent with such a role
for the Vps8Dp-containing CORVET at the CVC. For our imaging, we
exploited conditions in which the CVC bladder, rather than existing as a
single large vacuole, is replaced by a set of smaller vacuoles that can
still be recognized due to their labeling by Dop1p as well as Vps8Dp. We
found that Dop1p has a relatively uniform distribution at the periphery
of such vacuoles, while in contrast Vps8Dp is concentrated at sites
where neighboring vacuoles come into close contact (Fig. 5). In some
cases, these were seen to be sites of subsequent fusion. A similar
phenomenon has been reported of specific protein and lipid accumulation
at the interface between vacuoles undergoing homotypic fusion in yeast,
termed the vertex ring domain, and it includes the HOPS complex (Wang et
al., 2003, Fratti et al., 2004). Interestingly, we witnessed the same
highly polarized Vps8Dp distribution on small Vps8Dp-decorated vacuoles
that were extruded through breaches in the plasma membrane of perforated
cells. In one case, we witnessed rapid Vps8Dp redistribution at the
perimeters of two initially widely-spaced vacuoles when they came into
contact, leading to accumulation at the junction (Fig. 5E). This may be
explained if the 8D-CORVET complexes on each surface are bound to
integral membrane proteins undergoing rapid 2-dimensional diffusion, but
any complex that simultaneously engages with integral membrane proteins
on an adjacent membrane diffuses more slowly. Based on analysis of the
vertex ring complex in yeast, this partitioning is also likely to
involve lipid subdomains (Fratti et al., 2004).
Our images from osmotically-stressed cells are consistent with a role
for Vps8Dp in homotypic tethering and fusion at the CVC, and therefore
support the paradigm for CORVET and HOPS established in other
eukaryotes. However, no Vps8Dp-labeled vertex ring domains are apparent
in cells grown under non-stress conditions, and moreover there is no
step clearly involving homotypic fusion in the Tetrahymenacontractile cycle (Cheng et al., 2023). In Paramecium,
electrophysiological data support a model in which the junctions between
the bladder and emanating spongiome arms undergo fission and subsequent
fusion with each contractile cycle (Tominaga et al., 1998b). Whether
similar fission/fusion cycles occur in Tetrahymena, where the structure
of the bladder/spongiome junction appears quite different, is unknown
(Elliott and Bak, 1964). The role of Vps8Dp is unlikely to be restricted
to either homotypic fusion between bladder-derived vesicles or
spongiome-bladder fusion, since Vps8Dp localizes to puncta throughout
the spongiome (Cheng et al., 2023). Spongiome tubules show dynamic
extension and branching (Cheng et al., 2023), and one possibility is
that the Vps8Dp-containing CORVET complex facilitates homotypic fusion
between branches in a way that is required to maintain a cohesive
reticulum. The retraction of the spongiome at early time points
following VPS8D knockdown may be consistent with this scenario.
However, another broad possibility is that Vps8Dp has activities
independent of a conventional CORVET complex, as a monomer or a smaller
sub-complex, for which examples exist in other eukaryotes for CORVET and
HOPS (Asensio et al., 2013, Lorincz et al., 2016). In pulldowns, Vps8Dp
is associated with the 5 other expected subunits of CORVET (Sparvoli et
al., 2020). However, some details suggest it may be an outlier among theTetrahymena CORVET complexes. First, it is the most biochemically
differentiated, including unique subunit variants for Vps8, Vps16,
Vps18, and Vps33 (Sparvoli et al., 2020). Secondly, Vps8Dp is roughly
200 amino acids longer than the other Vps8 paralogs in Tetrahymena, with
multiple insertions suggesting the potential for additional interactions
and activities. Lastly, Vps8Dp in pulldowns appeared by silver staining
to be significantly more abundant than the other subunits with which it
associates; significantly, no similar imbalance was present for the Vps8
subunits of the other five CORVETs (Sparvoli et al., 2020). Since the
affinity tag for these pulldowns was attached to Vps8Dp, the greater
relative abundance of Vps8Dp could be explained if that particular
CORVET were comparatively unstable. Alternatively or in addition, it
could reflect a super-stoichiometric pool of Vps8Dp in cells. On this
basis, we speculate that Vps8Dp could potentially be playing distinct
roles at the bladder vs. spongiome, as part of different complexes.