REVIEW

Tetrahymena thermophila:
A Divergent Perspective on
Membrane Trafc
JOSEPH S. BRIGUGLIO
AND AARON P. TURKEWITZ*
The Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago,
Illinois

ABSTRACT

J. Exp. Zool.
(Mol. Dev. Evol.)
322B:500516,
2014

Tetrahymena thermophila, a member of the Ciliates, represents a class of organisms distantly

related from commonly used model organisms in cell biology, and thus offers an opportunity to
explore potentially novel mechanisms and their evolution. Ciliates, like all eukaryotes, possess a
complex network of organelles that facilitate both macromolecular uptake and secretion. The
underlying endocytic and exocytic pathways are key mediators of a cell's interaction with its
environment, and may therefore show nichespecic adaptations. Our laboratory has taken a
variety of approaches to identify key molecular determinants for membrane trafcking pathways in
Tetrahymena. Studies of Rab GTPases, dynamins, and sortilinfamily receptors substantiate the
widespread conservation of some features but also uncover surprising roles for lineagerestricted
innovation. J. Exp. Zool. (Mol. Dev. Evol.) 322B:500516, 2014. 2014 Wiley Periodicals, Inc.
How to cite this article: Briguglio JS, Turkewitz AP. 2014. Tetrahymena thermophila: A divergent
perspective on membrane trafc. J. Exp. Zool. (Mol. Dev. Evol.) 322B:500516.

A LIMITATION OF MODEL ORGANISMBASED CELL

BIOLOGY
Much of the rich detail we possess about cell biological structures
and pathways comes from studies in a handful of well
domesticated model organisms (Boehm and Bonifacino, 2002;
Kourtis and Tavernarakis, 2008; Westermann, 2010; Kimble, 2011),
which are exploited for their inherent biological characteristics
and using the extensive tools that have been developed by large
communities of researchers (Cherry et al., 2012; Lamesch et al.,
2012; McQuilton et al., 2012; Bult et al., 2013; Thompson et al.,
2013). An important generalization that has emerged from many
studies is that a multitude of cell biological mechanisms and
structures are conserved between model organisms (FerroNovick
and Jahn, '94; Finger, '98; Ashburner et al., 2000; Gnczy, 2008;
Simons and Mlodzik, 2008). One might conclude, but we would
argue incorrectly, that structures or mechanisms that are found
only in a subset of organisms (i.e., lineagerestricted) are of
secondary interest for understanding biology. In addition, there
are strong reasons to question the criteria that are commonly used
by many cell biologists to dene a feature as universal.
Historically, the most heavily employed model organisms have
been animal or fungal (Davis, 2004). As a result, many models in
cell biology are heavily biased, if not exclusively based, on data
from organisms that belong to a single eukaryotic lineage

(Hedges, 2002). This is because animals and fungi both belong

to the Opisthokont clade (Baldauf and Palmer, '93; Wainright
et al., '93), just one of what are currently considered to be ve
major surviving eukaryotic lineages (Parfrey et al., 2006). Plants
belong to a 2nd clade (Archeaplastidia) (Turner et al., '99; Reyes
Prieto et al., 2007); an implication is that a comparison of cell
biological data between Arabidopsis and yeast, or between
Arabidopsis and humans, would be a better strategy for
distinguishing between universal versus lineagespecic cellular
features, than a comparison between yeast and humans. The

Grant sponsor: Chicago Biomedical Consortium; grant sponsor: National

Science Foundation (NSF); grant number: MCB1051985; grant sponsor:
National Institutes of Health Training (General Medicine); grant number:
T32 GM007191.


Correspondence to: Aaron P. Turkewitz, The Department of Molecular
Genetics and Cell Biology, The University of Chicago, 920 E. 58th St.,
Chicago, IL 60637.
Email: [email protected]
Received 3 October 2013; Revised 13 January 2014; Accepted 22
January 2014
DOI: 10.1002/jez.b.22564
Published online 14 March 2014 in Wiley Online Library
(wileyonlinelibrary.com).

2014 WILEY PERIODICALS, INC.

DETERMINANTS OF ENDO/EXOCYTOSIS IN CILIATES

501

remaining three clades (Excavata (Simpson and Patterson, '99),

Ameobozoa (CavalierSmith et al., 2004), and SAR (Burki
et al., 2007)); consist largely of unicellular organisms, both
parasitic and freeliving, which are very poorly represented
among the pantheon of model organisms (Baldauf, 2008). An
exception are some parasites (e.g., Toxoplasma, Trypanosomes)
that have won attention because of their importance as pathogens
(Kissinger et al., 2003; Aslett et al., 2010; Hunter and Sibley, 2012;
Stephens et al., 2012). Addressing this asymmetry (an issue
highlighted eloquently by Dacks and Field) in the sampling of
cellular biodiversity by cell biologists could produce a more
accurate understanding both of conserved features, and the
variation that exists within them, and of features that are limited
to specic lineages. A broader, taxonomyinformed sampling
would therefore provide a more accurate picture of the relative
contributions made by inheritance versus innovation in the
emergence of modern cells. In its most ambitious form, such an
initiative would entail developing a broad array of new model
organisms, an exciting but challenging goal.

CILIATES AS MODEL ORGANISMS

An intermediate approach to the same aims, which does not
require developing new model organisms, is to exploit existing
model organisms falling within underexplored, that is, non
Opisthokont, clades. Ciliates represent an attractive group of
organisms for expanding cell biological studies to divergent
lineages. Ciliates, which belong to the StramenopilesAlveolates
Rhizaria (SAR) lineage, are further classied together with
Dinoagellates and Apicomplexa as Alveolates, which refers to
a subplasma membrane compartment called alveoli (Stelly
et al., '91; Plattner et al., 2012; Kono et al., 2013). Unlike the
parasitic Apicomplexa (Sibley, 2004), many Ciliates are free
swimming protozoa, which as a group show remarkable
morphological diversity (Lynn, 2001). Ciliates fall into 11 branches
(Fig. 1) (Lynn, 2003). Importantly, dened laboratory strains and a
wide range of experimental tools have already been established for
two Ciliates in the Oligohymenophorea branch, Tetrahymena
thermophila and Paramecium tetraurelia, and these can be used
for rigorously pursing cell biological questions (Turkewitz
et al., 2002; Collins and Gorovsky, 2005; Beisson et al., 2010).
Excitingly, tool kits are also being developed for Ciliates belonging
to other branches, such as Oxytricha trifallax (Nowacki
et al., 2008; Fang et al., 2012; Swart et al., 2013) and Stentor
coeruleus.
As with researchers using other model organisms, the aim for
those using Ciliates has traditionally been to exploit features that
offer unique experimental advantages (Gibbons and Rowe, '65),
and the most spectacular successes have come from nuclear
dimorphism. Ciliates, though unicellular, maintain two nuclei: a
diploid germline micronucleus (MIC) together with its derived, and
dramatically modied, polyploid macronucleus (MAC) (Prescott, '94). The mechanisms involved in generating the MAC in

Figure 1. Ciliates comprise 11 subbranches, and fall within the

Stramenopile/Alveolate/Rhizaria (SAR) lineage. These relationships
are illustrated as a cladogram, showing classication but not
evolutionary distances. Ciliates, along with Dinoagellates and
Apicomplexans, are Alveolate members of the SAR group
(StramenopilesAlveolatesRhizaria), one of the ve major
eukaryotic lineages (Lynn, 2003; Parfrey et al., 2006;
Baldauf, 2008). The Ciliates fall into 11 classes (Lynn, 2003).

T. thermophila were exploited to yield Nobel Prizewinning

discoveries, namely telomere structure and telomerase
(Blackburn, 2010) as well as catalytic RNA (Cech, 2004), which
are appreciated as fundamental cellular features. An interesting
variation on this theme is represented by ongoing work that
exploits the fact that the MIC must import a different cohort of
cytoplasmic proteins compared to the MAC, since the MIC is silent
while the MAC is actively transcribed. This line of work aims to
dene how nuclear pores (the conduits for macromolecules in and
out of the nucleus) have been remodeled in Ciliates, to rene their
selectivity (Malone et al., 2008; Iwamoto et al., 2009). Thus nuclear
dimorphism can be harnessed to identify deeply conserved
features but also to understand how conserved features can be
rened through lineagespecic innovations.
Our laboratory is interested in the endomembrane system,
which consists of a network of membranous compartments,
including the plasma membrane, interconnected via vesicle
mediated trafc of lipids and proteins (Bonifacino and
Glick, 2004), and which is a hallmark of eukaryotic cells. Like
any complex cellular feature, the endomembrane system in any
J. Exp. Zool. (Mol. Dev. Evol.)

502
given cell must consist of a combination of broadly conserved
structures and mechanisms together with lineagerestricted
elements that arose more recently (Dacks and Field, 2007). For
many pathways, mechanistic studies in Opisthokonts have led to
identication of key determinants (McMahon and Mills, 2004;
Robinson, 2004; Behnia and Munro, 2005; Stenmark, 2009). Due
to the availability of sequenced genomes from many species, it has
recently become possible to pursue rigorous phylogenetic analysis
of such determinants to ask whether orthologous genes are present
in divergent lineages, which is expected for conserved features
deriving from an ancient eukaryotic ancestor (PereiraLeal and
Seabra, 2001; Jekely, 2003; Koumandou et al., 2007, 2011). The
identication and analysis of lineagerestricted features would
help to ll in our picture of cellular organization (PereiraLeal and
Teichmann, 2005; Kienle et al., 2009). One reason is that lineage
restricted elements may reveal adaptive pressures, particularly
because both exocytosis (secretion) and endocytosis (uptake),
which depend on the endomembrane system, are important in the
interaction of cells with their environments.

ENDOCYTOSIS AND EXOCYTOSIS IN TETRAHYMENA

Morphological studies indicate that Tetrahymena maintain a large
set of distinct membrane trafcking pathways (Frankel, 2000).
These include at least four pathways of endocytosis by which
plasma membrane and/or extracellular material is internalized
into the cytoplasm via vesicles. First, clathrincoated vesicles form
at parasomal sacs, which are small invaginations adjacent to cilia
(Allen, '67; Nilsson and van Deurs, '83; Elde et al., 2005). Because
the plasma membrane overlays the alveoli that are organized as an
extensive sheet of abutting cisternae (Fig. 2A, bottom), the
parasomal sacs offer one of the few known places in these cells
where the plasma membrane can make direct contact with the
cytoplasm (Satir and Wissig, '82). A second dramatic form of
endocytosis is phagocytosis of bacteriasized particles, which
occurs at the base of a specialized anterior oral apparatus lined
with beating cilia that sweep in food particles such as bacteria
(Fig. 2A (top), B) (Nilsson, '79; Frankel, 2000). Endocytosis at other
distinct sites is involved in membrane recovery following
exocytosis (Plattner et al., '93) of secretory granules called
mucocysts, and following fusion (Hausmann and Allen, '76) of old
food vacuoles (defecation) at a dened posterior structure called
the cytoproct (also see Fig. 2B) (Allen and Wolf, '79).
Exocytosis, or the fusion of cytoplasmic vesicles with the
plasma membrane, also occurs via several known routes. The best
studied are the aforementioned mucocysts. Hundreds of mucocysts dock in linear arrays at the plasma membrane where they can
undergo release triggered by an extracellular stimulus
(Tiedtke, '76; Satir, '77; Chilcoat et al., '96). In addition to the
regulated secretion of mucocysts, a pathway of constitutive
release has also been inferred, though not identied at the
morphological level, based on the rapid release of newly
synthesized secretory proteins from nonstimulated cells
J. Exp. Zool. (Mol. Dev. Evol.)

BRIGUGLIO AND TURKEWITZ

(Bowman and Turkewitz, 2001; Madinger et al., 2010). Tetrahymena are additionally equipped to secrete hydrolytic enzymes,
which appears to involve secretory lysosomes (Kiy et al., '93).
Other pathways of membrane trafc can also be inferred from
morphological data, though few details are known. A dramatic
example is the remodeling of the oral apparatus, which is
necessary to allow the exchange of meiotic nuclear products in
conjugating cells (Wolfe, '82). By comparing these pathways with
known pathways in other organisms, one may be able to infer or
demonstrate potentially novel features that arose within Ciliates
and/or the SAR lineage.

WHAT RAB GTPases TELL US ABOUT MEMBRANE

TRAFFIC IN TETRAHYMENA
In broad terms, functionally analogous features of the endomembrane system in different lineages may be related to one another in
several different ways, and we consider three classes. First,
analogous features may be truly homologous, that is, both
maintained in a relatively unchanged state during inheritance
from a common ancestor. Second, such homologous features may
be descended from a common ancestral feature, but have
undergone signicant modications in one or both lineages
being compared. Third, features that serve the same function in
two modern lineages could be evolutionarily unrelated, arising
from different origins and by different mechanisms, but have
undergone functional convergence. Both the 2nd and 3rd classes
involve innovations relative to the ancestral state, which may be
at the level of novel genes or higherorder assemblies. It is
reasonable to expect, given the current sampling bias, that many
features currently considered to be conserved throughout
eukaryotes will show greater variation when examined in
divergent lineages. This in turn can provide insights into
fundamental aspects of cellular evolution, such as physical and
genetic constraints on cellular features. In the discussion below,
we outline studies from our laboratory in Tetrahymena that
address both conservation and innovation, and which provide
illustrations of how the latter have shaped membrane trafc in
these cells.
For studying the evolution of the endomembrane network,
large gene families that serve as compartmental determinants
have provided the most informative tools (Dacks et al., 2009). Rab
GTPases, which represent the most numerous group of small
GTPases in eukaryotes, are encoded by gene families in which
each member is associated with one or a small number of
endomembrane compartments (Stenmark, 2009) The association
of Rabs with their target membranes, and with effectors, is
controlled by GTP binding and hydrolysis. A Rab confers
compartmental identity by recruiting effectors that in turn
control the trafcking of proteins and lipids (i.e., vesicular trafc)
to and from that compartment (Segev, 2001; Grosshans
et al., 2006) In many cases, the number of Rabs expressed in
an organism correlates with the complexity of the organism's

DETERMINANTS OF ENDO/EXOCYTOSIS IN CILIATES

503

Figure 2. Morphology and organization of Tetrahymena thermophila. Figure and legend adapted from Figure 1A and B of Bright et al. (2010)
and Figure 22 of Allen ('67). (A) Top: (Bright et al., 2010). Cartoon of a Tetrahymena cell (length 50 mM). All surface features of Tetrahymena,
with the exception of the oral apparatus (OA) (the site of phagocytic ingestion) and cytoproct (CP) (the site of cellular defecation) occur at
repeated positions and therefore form linear arrays, organized by cytoskeletal ribs that run the length of the cell, known as 1 and 2
meridians. (A) Bottom: (Allen, '67). A reconstruction to portray structures positioned just below the cell surface, as seen from outside of a cell
in which the plasma membrane and alveoli have been stripped away from the front half. The reconstruction more clearly illustrates the
positioning of cilia (C) in longitudinal linear 1 meridians, which can be seen running from top to bottom on either side of the image. Although
not prominent in the reconstruction, alveolae are found just beneath the plasma membrane. Alveolae are a monolayer of cisternae underlying
the plasma membrane, and function at least in part as a compartment for storage of mobilizable calcium. The reconstruction illustrates a
mucocyst (MU) docked at a junction between alveolae. The mucocyst sits on a 2 meridian, positioned between ciliabearing 1 meridians. (B)
Prominent structures involved in membrane trafcking in T. thermophila. Phagocytosis begins at the oral apparatus (OA), resulting in
formation of phagosomes (P) that, after undergoing multiple fusion and ssion events, eventually egest undigested material via exocytic
fusion at the cytoproct (CP). Clathrinmediated endocytosis occurs at parasomal sacs (PS), giving rise to endocytic vesicles (E). These endocytic
vesicles coalesce in tubulovesicular endosomes in the cell posterior. Outbound membrane trafcking, including proteins to be secreted,
involves the endoplasmic reticulum (not shown) and Golgi (G), which are present as single cisterna or short stacks near the cell periphery, close
to mitochondria (MI). Some of the secretory cargo is packaged into mucocysts (MU) which dock and subsequently undergo exocytosis at sites
on 1 and 2 meridians. Another prominent organelle is the waterpumping contractile vacuole (CV). Other structures shown: alveolae (A).
macronucleus (polyploid somatic nucleus, M). Micronucleus (diploid germline nucleus, m). Ciliary basal bodies (BB) and the cilia (C) that grow
from them. Rows of cilia cover the entire cell surface, but only a subset are shown here for clarity. An excellent review of these structures is
provided by Frankel (2000).

endomembrane network (PereiraLeal and Seabra, 2001). Budding yeast, renowned for their relative simplicity, express 12
Rabs, whereas humans express about 63 (Stenmark and
Olkkonen, 2001). Importantly, structures that are homologous
in yeast and humans are associated with orthologous Rabs,
consistent with the idea that the common ancestor of fungi and
animals already possessed both those structures and the
associated Rabs, and both structures and Rabs have been

inherited in a conservative fashion (PereiraLeal and

Seabra, 2001). This corresponds to classes 1 or 2, in the paragraph
above. Given this context, expanding the detailed study of Rabs
to divergent lineages could yield important insights into both the
organization and evolution of the endomembrane network
(Brighouse et al., 2010).
Global analysis of the Rabs in T. thermophila, by our laboratory
as well as by SaitoNakano et al., revealed that this singlecelled
J. Exp. Zool. (Mol. Dev. Evol.)

504
organism expresses roughly the same number of Rabs as humans
(Bright et al., 2010; SaitoNakano et al., 2010). In addition,
Tetrahymena express a set of >20 Rablike proteins (J. Briguglio,
manuscript in preparation), which differ from authentic Rabs in
lacking a predicted addition site for a Cterminal prenyl moiety
that is important for targeting to membranes. The number of Rabs
is extravagant relative to many singlecelled organisms (Stenmark
and Olkkonen, 2001), but at least some other protozoa have Rab
families of comparable size. Trichomonas vaginalis and Entamoeba
histolytica, for example, encode Rab families containing 65 (Lal
et al., 2005) and 95 (SaitoNakano et al., 2005) members,
respectively; these numbers should be taken as estimates since
annotation of Rabs is not straightforward (http://www.rabdb.org/
about/). Do these organisms maintain endomembrane networks of
comparable of greater complexity than in human cells? One layer
of complexity in higher organisms is tissuespecic expression
(Ayala et al., '89; Gurkan et al., 2005; Zhang et al., 2007). Similarly,
singlecelled eukaryotes could show stagespecic expression
(e.g., parasites could express different Rabs in different hosts, while
free living cells could adjust for the vagaries of their environment)
(Quevillon et al., 2003; Lal et al., 2005). Tetrahymena, in particular,
adapt quickly and dramatically to different environmental
conditions, for example changing both shape and swimming
behavior when starved for food (Nelsen and Debault, '78). Further
changes occur if cells of a complementary mating type are present,
eventually leading to mating that involves extensive membrane
reorganization (Wolfe and Grimes, '79). Importantly, a database of
T. thermophila gene expression (http://tfgd.ihb.ac.cn/) allows one
to interrogate the levels for any transcript for a variety of different
conditions (Miao et al., 2009; Xiong et al., 2013). Taking advantage
of this resource, we could conclude that nearly all Tetrahymena
Rabs were expressed, many among the 20% most highly
expressed genes in this organism (Bright et al., 2010). The
concurrent expression of 58 Rabs within a single cell, was
consistent with pathways of membrane trafc comparable in
complexity to those in animal cells.
For practical reason, we focused on Rabs transcribed in
vegetative cells and expressed these as GFP fusions, to assign
them to different endomembrane compartments and pathways.
Because few molecular markers are established in these cells, the
assignments were based on previous ultrastructural studies and
further validation with functional probes (e.g., the styryl dye FM4
64 as an endocytic tracer, and India Ink and uorescent bacteria as
ingestible markers for phagosomes). The same Rabs were analyzed
by phylogenetic criteria to ask, as described above, whether
structures analogous with those in fungi and animals were
associated with the orthologous Rabs, consistent with those
structures being related by common descent (Bright et al., 2010).
Experimental data, initially from animals and fungi, had
identied a core group of highly conserved Rabs corresponding
to eight different pathways of membrane trafc, and inferred to be
present in an ancestral eukaryotic cell (PereiraLeal and Seabra,
J. Exp. Zool. (Mol. Dev. Evol.)

BRIGUGLIO AND TURKEWITZ

2001). Those ndings were subsequently extended by broad
sequencebased phylogenetic surveys, but rarely including
characterization of the proteins (Elias et al., 2012). From the
sequencebased plus localization survey of Tetrahymena Rabs,
several different pictures emerged.
Phylogenetic analysis from our laboratory indicated that, a
minority of the Tetrahymena Rabs (27%) could be robustly
identied as orthologs of Rabs in other organisms (Bright et al.,
2010). This conclusion (but see below for other predictions) suggests
that only a minority of the Tetrahymena Rabs and/or membrane
trafcking pathways correspond to Class 1 described above. The
conserved Tetrahymena Rabs belong to six of the core groups
considered to be conserved throughout eukaryotes, including ER
Golgi trafcking, endocytosis, endocytic recycling, late endocytosis, and retrograde Golgi trafcking. The remaining core groups,
corresponding to Golgirelated and regulated exocytic pathways,
appear to have been lost in Tetrahymena. This is unexpected
because, as previously mentioned, Tetrahymena possess a prominent pathway of regulated exocytosis from mucocysts, so the
absence of any conserved regulated exocytic Rabs suggests a
lineagespecic origin for these organelles (Class 3).
Localizing the conserved Rabs (as GFP fusions) conrmed some
expectations but also provided surprises. For example, all four of
the retrograde Golgi Rabs displayed almost identical localizations,
corresponding with the known distribution of the Golgi in this
organism, so that the expansion of this Rab subfamily may reect
a dosage requirement. There may also be a lineagerestricted
contribution, since one highly divergent Rab also showed Golgi
localization. Other pathways may have an even more substantial
lineagerestricted contribution. For example, nine Rabs were
assigned to endocytic pathways based on their colocalization with
FM464. Of these nine only three had clear orthologs in other
organisms, suggesting that the remaining Rabs arose within the
Tetrahymena lineage. These Rabs displayed similar but not
equivalent localizations (e.g., variable overlap with FM464), so
are likely to be specialized for different endocytic subcompartments. Thus, both the Golgi and endosomes in Tetrahymena
appear to have derived from some combination of Classes 1 and 2
histories. Intriguingly, four other Tetrahymena Rabs, which did not
show any detectable overlap with FM464, nonetheless showed
robust orthology to conserved endocytic Rabs in other organisms;
three of these localized to phagocytic structures (Bright
et al., 2010). This case, in which sequenceconserved Rabs have
apparently acquired functions different from their core group,
serves as cautionary reminder of the potential pitfalls of inferring
function based solely on sequence when interrogating divergent
lineages.
One important consideration, as mentioned previously, is that
sequence analysis itself can be ambiguous. The assignment of the
Tetrahymena Rabs by SaitoNakano et al. (2010) utilized reciprocal
Blast searches, rather than the parsimony scores and treebuilding
approaches used by our group. SaitoNakano et al. identied

DETERMINANTS OF ENDO/EXOCYTOSIS IN CILIATES

orthologs for some Tetrahymena Rabs that, in contrast, appeared
lineagerestricted based on our analysis. These differences
underscore the point that it can be difcult to derive robust
phylogenies in divergent lineages, a problem that may be
mitigated with broader sequencing outside of the Opisthokonts.
If SaitoNakano et al.'s conclusions are correct, the endocytic
pathway in Tetrahymena is more substantially, though still
incompletely, derived from conserved Rabs (Class 1) than inferred
by our laboratory's work. While both surveys indicate that
endocytic pathways in Tetrahymena likely contain innovations
that arose within this lineage, the nature of those innovations
remains unclear. Ultimately, these surveys highlight the benet of
predictions based on multiple analytic approaches, but underline
the need for experimental validation.

IDENTIFYING CONCRETE EXAMPLES OF INNOVATION

Whereas a broad survey of Rabs suggested that a number of
pathways in Tetrahymena include nonconserved features, a
concrete example of molecular innovation, in the form of a novel
dynamin described below, was identied in studies of clathrin
mediated endocyosis (CME) in this organism (Elde et al., 2005).
CME is widespread in eukaryotes and has historically been among
the most intensely studied pathways of membrane trafc
(Rappoport, 2008; Traub, 2009). In addition to the clathrin coat,
CME involves a host of other proteins, including a complex
adapter that links the cargo to be internalized with membrane
invagination machinery. This adapter function is provided by the
AP2 heterotetramer, one of a family of AP complexes whose
members function in a variety of compartments (Fig. 3A, bottom)
(Boehm and Bonifacino, 2002; Robinson, 2004; McMahon and
Boucrot, 2011). In Tetrahymena, CME occurs at parasomal sacs,
located anterior to each ciliary basal body (Fig. 3A, top) (Nilsson
and van Deurs, '83; Elde et al., 2005). Since basal bodies are
regularly spaced along cytoskeletal ribs called meridians (Satir
and Wissig, '82), the Tetrahymena cortex contains a globally
ordered array of endocytic portals (Fig. 3B), which facilitates their
analysis by microscopy.
T. thermophila has a single clathrin (heavy chain) gene as well as
unique genes encoding each of the AP2 subunits (Elde et al.,
2005). Exploiting the known distribution of parasomal sacs, both
clathrin and AP2, as GFP fusions, were shown to localize to these
sites (Fig. 3B, top and middle); in contrast, GFPtagged AP1 and 4
complexes localized to other cellular loci. A functional role for
clathrin at parasomal sacs could be demonstrated by conditionally
expressing a truncated clathrin construct, the hub domain, which
acts as a dominantnegative inhibitor in other organisms (Liu
et al., '98). In Tetrahymena, expression of the hub domain inhibited
endocytic uptake, which was measured by uptake of the styryl dye,
FM143 (Elde et al., 2005). Therefore, the phylogenetic and
functional relationships between T. thermophila clathrin and AP
2, and the homologous proteins in other organisms, reinforced the
conclusion that CME is a deeply conserved pathway in eukaryotes.

505
Another central player in CME is actin. Actin contributes to
several stages in metazoan endocytosis (Merrield et al., 2002;
Robertson et al., 2009) and is also involved in fungi (Kaksonen
et al., 2003; Toshima et al., 2006). Surprisingly, standard
approaches to inhibiting actin function had no effect on FM1
43 uptake at parasomal sacs in T. thermophila (Elde et al., 2005),
though they dramatically inhibited actindependent phagocytosis
at the oral apparatus (Elde et al., 2005; Williams et al., 2006). These
results suggested that actin plays little if any role in CME in
Tetrahymena, though it is also possible that phagocytosis and CME
involve different actin isoforms, and that those involved in CME
are resistant to standard pharmacologic inhibition. Consistent
with this possibility, the related Ciliate P. tetraurelia expresses nine
actin isoforms associated with distinct localizations and functions
(Sehring et al., 2010). Moreover, actin sequences are altogether
relatively divergent in Ciliates (Cupples and Pearlman, '86; Drouin
et al., '95), so the loss of broadly conserved drug binding sites is
plausible, especially since Tetrahymena actins appear to have lost
binding sites for phalloidin (Hirono et al., '89). In addition, some
expression proling data in Tetrahymena suggest that proteins
involved in actin dynamics are coregulated with proteins
involved in endocytosis; for example, the gene most tightly
coregulated with the m subunit of AP2 is a homolog of brin, an
actin crosslinker (Nusblat et al., 2012). In summary, the current
functional data suggest that Tetrahymena CME is relatively
actinindependent, which would be highly novel and imply that
other mechanisms have arisen to compensate for the loss of actin
function in this pathway, but more experiments are needed to test
alternative explanations. Interestingly, there is also evidence of
actinindependent endocytosis during specic life stages in
trypanosomes (GarcaSalcedo et al., 2004), which may involve
mechanisms that arose separately from those in ciliates.
Remarkably, CME in T. thermophila requires a protein that is
related, but in an unexpected fashion, to a key protein in animal
cell CME (Elde et al., 2005). The large GTPase called classical
dynamin, as rst shown in fruit ies (van der Bliek and
Meyerowitz, '91), polymerizes around the neck of endocytic
invaginations to form a collar that is required for releasing the
invagination in the form of a vesicle, that is, vesicle ssion
(Hinshaw and Schmid, '95; Merrield et al., 2005) (Fig. 3A,
bottom). Consequently, blocking dynamin function strongly
inhibits CME (van der Bliek and Meyerowitz, '91; McCluskey
et al., 2013). Classical dynamin also recruits effectors including
actinbinding proteins, which may contribute to its late endocytic
role (Mettlen et al., 2009). Notwithstanding this critical role, it is
not clear whether the role of dynamin in CME is ancestral, since
there is only limited evidence that dynamin homologs participate
in CME in most nonOpisthokont lineages (Elde et al., 2005). One
strong possibility is that classical dynamin was recruited to CME
relatively recently, in evolutionary terms, from an ancestral
protein that served a different function, which may have been
mitochondrial scission (Liu et al., 2012).
J. Exp. Zool. (Mol. Dev. Evol.)

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Figure 3. Continued.

J. Exp. Zool. (Mol. Dev. Evol.)

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507

Figure 4. Endocytic dynamins appear on multiple unrelated branches. An updated maximum likelihood tree of eukaryotic dynaminrelated
proteins (DRPs) from Elde et al. (2005). Whereas mitochondriaassociated dynamins appear to have a single origin, endocytic dynamins are
found on multiple, distantly related branches. The tree was constructed from an alignment of the dynamin N domain sequences from which
gaps were deleted. The bootstrap consensus tree was inferred from 1,000 replicates; only values >55% are shown.
T. thermophila expresses eight dynamin homologs (DRP18),
reecting a signicant expansion in the gene family within this
lineage (Elde et al., 2005). Two of the dynamins localized, as GFP
fusions, to parasomal sacs, and of these DRP1 was studied in
detail (Fig. 3B, bottom panel). ImmunoEM analysis of Drp1p
showed that it localized to the sides of clathrincoated areas
within parasomal sacs, consistent with a role in CME. Gene
knockout resulted in cell death, but such cells could be rescued by
introducing an exogenous copy of DRP1 under the transcriptional control of a cadmiuminducible promoter. Using these
cells, it was possible to show that reducing the expression of
DRP1 led to a decrease in CME, measured as above by FM143
uptake (Elde et al., 2005). These results indicate that, in contrast
to the absence of endocytic dynamins in many nonOpisthokonts, a protein in this family is required for CME in
Tetrahymena. This could potentially suggest an ancient role in

endocytosis for dynamins, which were subsequently lost in many

lineages.
Phylogenetic analysis of the Tetrahymena DRPs revealed a
different and more interesting story. A minority of the
Tetrahymena DRPs were closely related to dynamins in many
other species but most, including DRP1, were found on branches
in which the only other members were Tetrahymena or other
Ciliate genes (Fig. 4). In other words, the endocytic dynamins of
Tetrahymena belong to lineagerestricted branches that arose via
expansions (i.e., gene duplication and retention) that occurred
after Ciliates had branched from other eukaryotes (Elde
et al., 2005). Duplicated gene copies can, by acquiring mutations,
also acquire functions that are different from the ancestral gene
(Kellis et al., 2004; Taylor and Raes, 2004). The hypothesis
suggested by the phylogeny was that a duplicated dynamin in
Tetrahymena was recruited to sites of CME, and over time acquired

3
Figure 3. Proteins involved in clathrinmediated endocytosis in Tetrahymena localize to parasomal sacs. Figure and legend adapted from
Figures 1B, 2A, 3D, 4C, and 5B of Elde et al. (2005). (A) Upper panel: In Tetrahymena, coated pits (CP) are found at parasomal sacs near the
base of cilia, as shown in tangential (left) and cross (right) sections. BB, ciliary basal body; C, cilium; CV, coated vesicle; MU, mucocyst.
Bars 200 nm. (Lower panel) Schematic diagram of clathrinmediated vesicle formation in animals. AP2 (red) serves as an adapter. It can
interact with receptors destined for internalization while also recruiting clathrin (green) to the plasma membrane. Clathrin assembly at those
sites enables membrane invagination. Dynamin (blue) assembles at the neck of a nascent vesicle to promote membrane ssion. (B) Left
column: Cells expressing GFPfusions of clathrin (top), AP2 (middle), and Drp1p (bottom). These fusion proteins localize to linear arrays
corresponding to parasomal sacs. The distribution of parasomal sacs was inferred by immunouorescence using an antibody against centrin,
which is used here as a marker for basal bodies (middle column). Centrin localizes to the basal bodies that lie immediately posterior to
parasomal sacs. Bar 5 mm. The merge between columns 1 and 2 is shown in the 3rd column.
J. Exp. Zool. (Mol. Dev. Evol.)

508
an essential role in that pathway. The same events may have
occurred in the animal lineage.
A testable inference of this hypothesis of parallel recruitment
was that mechanisms responsible for targeting dynamin to
clathrincoated pits are likely to differ between animals and
Ciliates. To investigate this issue, the targeting motif in Drp1p was
identied by expressing chimeras between Drp1p and a 2nd T.
thermophila dynamin that localizes to the nuclear envelope, called
Drp6p. These experiments revealed that a 28residue motif in
Drp1p, when swapped with the corresponding region of Drp6p,
was sufcient to target the otherwise Drp6p protein to parasomal
sacs (Elde et al., 2005). Classical dynamin in animals is targeted to
coated pits via a pleckstrin homology (PH) domain that binds to
specic phosphatidyl inositol head groups (Vallis et al., '99). The
28 amino acid motif in Drp1p is not predicted to form a PH
domain; consistent with this, puried Drp1p showed no binding to
phosphatidyl inositides on blots (unpublished) (note that the
phylogenetic analysis discussed above was based solely on the
most highly conserved domains of dynamins, so was not
inuenced by divergent targeting domains). Taken together, the
results suggest that dynamins were recruited at least twice, in
independent lineages, to play essential roles in endocytosis. CME
in Tetrahymena therefore appears to be based upon a core of
deeply conserved proteins but with at least one innovation that is
surprisingly similar to one that developed in animals. That is not to
say that the precise roles played by dynamins during CME in the
two lineages are identical, and indeed Drp1p lacks the PRD domain
present in classical dynamin that is involved in regulating the
actin cytoskeleton (Elde et al., 2005). It is tempting to speculate
that Drp1p provides some function in Tetrahymena CME that is
provided by actin in other lineages.

INDEPENDENT ORIGINS OF REGULATED PEPTIDE

SECRETION?
A striking potential case of cellular innovation has emerged from
detailed studies of mucocysts in Tetrahymena. Elaborate secretory
organelles are found in many Ciliates (Rosati and Modeo, 2003)
and have been extensively studied in the form of Tetrahymena
mucocysts (Turkewitz et al., 2000; Turkewitz, 2004) and the
corresponding organelles in Paramecium, known as trichocysts
(Vayssi et al., 2000). The historical use of two different names
stemmed from the very distinct morphologies of mucocysts and
trichocysts (Hausmann, '78), but more recent molecular work
clearly indicates that they are homologous organelles (Adoutte
et al., '84; Turkewitz et al., '91; Shih and Nelson, '92; Madeddu
et al., '95; Chilcoat et al., '96; Gautier et al., '96; Cowan et al., 2005).
Mucocysts and trichocysts belong to a very broad class of secretory
organelles more generally termed extrusomes. Extrusomes embody
a mode of secretion in which specialized vesicles rst accumulate
in the cytoplasm. In response to extracellular signals the vesicles
can extrude their contents via membrane fusion, called regulated
exocytosis (Kelly, '85). For the Ciliates, the ability to quickly release
J. Exp. Zool. (Mol. Dev. Evol.)

BRIGUGLIO AND TURKEWITZ

large amounts of stored extrusome contents has enabled
sophisticated strategems (Wessenberg and Antipa, '70; Harumoto, '94; Sugibayashi and Harumoto, 2000) for predation or
defense (Rosati and Modeo, 2003). The term extrusome has also
been used to describe secretory organelles in a variety of other
eukaryotes (Hausmann, '78), but whether these are evolutionary
related to those in Ciliates has not been investigated. From our
perspective, a particularly interesting class of secretory vesicles
with extrusomelike features are the dense core granules in
animals, since there has been extensive molecular and mechanistic
analysis of these organelles (Tooze et al., 2001; Crivellato
et al., 2006; Hou et al., 2009).
A dening feature of dense core secretory granules in animals,
which is shared by Ciliate mucocysts and trichocysts, is an
electrondense protein core (Fig. 5A) (Tokuyasu and Scherbaum, '65; Bannister, '72; Michael et al., '87). In both animals and
Ciliates, the cores are formed by protein assembly that is
controlled by proteolytic processing (Adoutte et al., '84; Turkewitz et al., '91; Steiner et al., '96) Moreover, the core proteins in
Ciliates and animals are similar in their acidity (Chilcoat
et al., '96), their capacity for lowafnity calcium binding
(Turkewitz et al., '91; Verbsky and Turkewitz, '98) and their
tendency to aggregate (Gorr et al., '89; Chanat and Huttner, '91)
within the secretory pathway (Rambourg et al., '88; Rahaman
et al., 2009) The last of these properties is likely to be a key
mechanism involved in protein sorting to dense core granules and
mucocysts/trichocysts, respectively (Arvan and Castle, '98; Jain
et al., 2000; Bowman et al., 2009). Notwithstanding these and
other similarities, there is no evidence that dense core granules
and mucocysts/trichocysts arose from the same ancestral
organelle (Elde et al., 2007). The genes encoding the principal
core proteins in mucocysts, called the GRLs (Chilcoat et al., '96;
Cowan et al., 2005), have no identiable homologs in animals.
The same is true for a 2nd major family of proteins in mucocysts,
called GRTs (Fig. 5B) (Bowman et al., 2005), to which we return
below. It is also clear that Ciliates have no homologs to the best
studied enzymes responsible for proteolytic processing of granule
core proteins in animals, the prohormone convertases, or to key
animal proteins involved in regulated exocytosis itself, like the
calcium sensor synaptotagmin (unpublished) (Elde et al., 2007).
The likelihood is that similar functions are being performed by
unrelated genes in Ciliates, as already suggested by the absence of
a Ciliate Rab in the regulated exocytosis subgroup (Bright
et al., 2010). Consistent with this idea, forward genetic analysis in
Paramecium identied a set of Ciliatespecic genes that are
required for regulated exocytosis (Cohen and Beisson, '80;
Bonnemain et al., '92; Klauke et al., '98; Froissard et al., 2004).
Taken together, the evidence suggest that the regulated secretory
organelles of Ciliates and animals had largely independent
evolutionary histories, but converged on similar features (Class 3)
(Elde et al., 2007). But to understand the origins of secretory
organelles in Ciliates, one needs to understand the mechanisms

DETERMINANTS OF ENDO/EXOCYTOSIS IN CILIATES

509

Figure 5. Expression proling identies potential mucocyst biogenesis genes. Figure and legend adapted from Figures 2A, 3A, and 5C of
Briguglio et al. (2013). (A) Electron micrograph of a mucocyst (MU) docked at the plasma membrane illustrating the characteristic elongated
shape and electron dense core. Other structures shown include plasma membrane (PM), Alveolus (A), and mitochondrion (MI). Bar 200 nM.
(B) Simultaneous visualization of mucocysts, by indirect immunouorescence (IF), using monoclonal antibodies against a GRL family protein
(Grl3p) and a GRT family protein (Grt1p). In a cross section through the cell (see cartoon to right for reference), Grl3p (red) is found throughout
the mucocyst while Grt1p is concentrated at the tip which docks at the plasma membrane. (C) The expression proles derived from the
Tetrahymena Functional Genomics Database illustrate the three peak expression prole that is characteristic of mucocyst genes. The
expression proles of the four Tetrahymena sortilins (SOR14, bottom) are similar to those of genes (GRL1, GRL3, GRT1, and IGR1, top)
encoding mucocyst cargo proteins. Each expression prole is normalized to that gene's maximum expression level. Points on the xaxis
correspond to successive timepoints and represent growing, starved, and mating cultures, including three different culture densities (low (Ll),
medium (Lm), and high (Lh)), 7 samples taken during 24 hr of starvation, and 10 samples subsequently taken during 18 hr following
conjugation (Xiong et al., 2013).

J. Exp. Zool. (Mol. Dev. Evol.)

510
underlying their formation, rather than relying on a catalog of the
contents.
We have recently obtained key insights into mechanisms of
mucocyst biogenesis in Tetrahymena by exploiting expression
proling via the aforementioned TFGD (Xiong et al., 2013). We
initially observed that all known mucocyst genes in Tetrahymena
are coregulated, sharing a threepeak expression prole in
growing, starved, and conjugating cultures (Fig. 5C, top). By
using this prole as a screen, we then found a large number of
putative mucocystassociated genes, some of which had Opisthokont homologs. The latter included genes encoding VPS10
domaincontaining proteins (Briguglio et al., 2013) (Fig. 5C,
bottom), called sortilins in animals (Petersen et al., '97), which
were rst identied in yeast as type 1 transmembrane receptors.
More specically, S. cerevisiae Vps10p in yeast is involved in the
sorting of hydrolytic enzymes into vesicles for transport from the
TGN to the vacuole, which is the lysosome equivalent in fungi
(Marcusson et al., '94; Cereghino et al., '95; Deloche et al., 2001).
Sortilins in animals, which expanded into a gene family
(Hermey, 2009), can serve a similar role in sorting to lysosome
related organelles (Herda et al., 2012). Sortilinfamily proteins
may also play additional roles in mammalian secretory granule
formation, discussed briey at the end.
Four sortilins are encoded in the Tetrahymena genome
(Briguglio et al., 2013). Phylogenetic analysis divided them into
two clades; SOR1 and 3 belong to a clade including nonCiliate
sequences, while SOR2 and 4 belong to a Ciliaterestricted clade
(Fig. 6). To test for potential roles in mucocyst formation, each
gene was individually disrupted. The disruption of either lineage
restricted sortilin (2 or 4) resulted in clear mucocyst phenotypes;
most dramatically, SOR4 knockout cells (Dsor4) failed to extrude
any mucocyst content when stimulated. The Dsor4 cells still
accumulated mucocysts (Fig. 7A), but these were decient in at
least two classes of cargo proteins. The rst was the aforementioned GRT family (Fig. 7B), and the 2nd was an aspartyl cathepsin
(Briguglio et al., 2013) shown to play a key role in proteolytic
processing during mucocyst formation (S. Kumar, ms. in
preparation). These results suggested that, in Tetrahymena, the
absence of a sortilin receptor resulted in a failure to sort normal
mucocyst contents to the proper compartment. Indeed, Sor4p
could be coimmunoprecipitated with Grt proteins, consistent
with the idea that it acts as a sorting receptor for these mucocyst
components. Interestingly, the sorting of the other major class of
mucocyst proteins, the Grls, did not depend on SOR4 function
(Briguglio et al., 2013).
Our recent data, combined with insights from studies in
Paramecium (de Loubresse, '93; Vayssi et al., 2001) and another
Ciliate, Pseudomicrothorax dubius (Peck et al., 2012), suggest that
the two classes of mucocyst proteins are transported by different
mechanisms. The rst depends on the aggregation of core
components, and is responsible for delivery of Grl proteins, while
the second depends on classical receptormediated sorting of Grt
J. Exp. Zool. (Mol. Dev. Evol.)

BRIGUGLIO AND TURKEWITZ

Figure 6. The sortilin family of VPS10 domaincontaining

receptors has expanded in Tetrahymena. Figure 2B and legend
directly from Briguglio et al. (2013). The maximum likelihood tree
illustrates a phylogeny of VPS10 domaincontaining receptors
(sortilins) in Alveolates, the taxonomic group consisting of Ciliates,
Apicomplexans, and Dinoagellates. Two of the T. thermophila
sortilins, marked by black circles, cluster with the sortilins from
other Alveolates. In contrast, T. thermophila SOR2 and SOR4,
marked by maroon diamonds, belong to an expansion of sortilins
restricted to Ciliates. Babesia microti (Bm), Cryptosporidium
hominis (Ch), Cryptosporidium muris (Cm), Ichthyophthirius
multiliis (Im), Neospora caninum (Nc), Paramecium tetraurelia
(Pt), Perkinsus marinus (Pm), Plasmodium berghei (Pb), Plasmodium cynomolgi (Pc), Plasmodium falciparum (Pf), Plasmodium
knowlesi (Pk), Plasmodium vivax (Pv), Plasmodium yoelii yoelii (Py),
Tetrahymena thermophila (Tt), Theileria annulata (Ta), Theileria
orientalis (To), Theileria parva (Tp), and Toxoplasma gondii (Tg).

proteins. Surprisingly, the activity of Sor4p as a mucocyst sorting

receptor suggests that the formation of mucocysts and lysosomes
relies on shared cellular machinery. Lending further support to this
idea, we found that the Tetrahymena AP3 adaptor is highly
coregulated with known mucocystassociated genes, including
SOR4. The AP3 adaptor, related to but different from the AP2

511

DETERMINANTS OF ENDO/EXOCYTOSIS IN CILIATES

Figure 7. Disruption of SOR4 results in the loss of specic

mucocyst cargo proteins and aberrant mucocyst morphology.
Figure and legend adapted from Figures 3B and 5B of Briguglio
et al. (2013). (A) Indirect immunouorescent visualization of the
core protein Grl3p in a cross section of Dsor4 cells reveals that
these mutant cells (right) still produce mucocysts, which can be
seen at the periphery of the cell, but the mucocysts exhibit an
abnormally round morphology compared to the wildtype (left, also
compare insets). Bar 5 mm (B) Indirect immunouorescent
visualization of the tip protein Grt1p in WT cells (left) shows
that Grt1p accumulates in the array of docked mucocysts. Here, an
optical section at the cell surface is shown, illustrated by the red
plane in the cartoon. In contrast, there is only background staining
of Grt1p in Dsor4 cells (right), consistent with a failure to sort
Grt1p to mucocysts in the absence of Sor4p. Bar 5 mm

adaptor discussed above for CME, is required for sortilinmediated

sorting to lysosomerelated organelles (LROs) in Opisthokonts
(Raposo et al., 2007). Does this mean that mucocysts represent a
class of LROs? Consistent with this idea, another sortilin is
essential for LRO formation in the Apicomplexan Toxoplasma
gondii, which like Ciliates is an Alveolate (Sloves et al., 2012).
However, given the evidence that Grl protein sorting to
Tetrahymena mucocysts is Sor4pindependent (Briguglio et al.,
2013), it tempting to speculate that mucocyst formation evolved
from the coalescence of an LROrelated pathway with a second
route of membrane trafcking. In future work, one way to
potentially address this question will be to ask whether Grl and Grt

proteins are being delivered to mucocysts in separate vesicles,

reecting separate origins. This possibility is directly raised by
electron microscopy studies on trichocyst formation in Pseudomicrothorax (Peck et al., 2012).
Discerning the evolutionary links between the mechanisms
involved in formation of secretory granules versus mucocysts will
ultimately require that we develop a more complete mechanistic
picture of both, since many steps are still poorly understood. This
point is underscored by recent experimental data. Receptors in the
sortilin family have been implicated by genetic studies, in both
mice and humans, as contributing to physiological responses that
rely on peptides released from secretory granules (Clee et al.,
2006; Goodarzi et al., 2007). In this context, sortilins may be
acting as signaling receptors, but recent results suggest that these
receptors in some cases could be directly involved in granule
formation and not serving a retrieval function. That is, sortilin
family receptors may be playing a wider role in animal secretory
granule formation than envisioned in current models. Similarly,
AP3 appears to be involved in secretory granule formation in
Drosophila and mammalian cells, but the mechanisms involved
are not known (Grabner et al., 2006; Asensio et al., 2010). These
data, informed by our recent results in Tetrahymena, may indicate
surprising mechanistic similarities between the formation of
lysosomes and secretory organelles with dense cores, in multiple
lineages. Since dense core granules are currently featured as a prime
example of nonlysosomal postTGN trafcking in mammalian
cells, validating this new view would signicantly change our
conception of trafcking in the endomembrane network.

CONCLUSIONS
The endomembrane network is a hallmark of eukaryotic cells.
Signal advances over the past 50 years have generated a profound
albeit still incomplete understanding of mechanisms involved in
establishing and maintaining the compartments within this
network, including the details of both endocytic and exocytic
pathways, with the vast majority of this work done in a
phylogenetically narrow range of model organisms. Our lab and
others have investigated a variety of membrane trafcking
pathways in distantly related organisms, the Ciliates. Taken
together, the results in Ciliates lead to some inferences that would
be unsurprising for biologists in many subelds, but that are
frequently overlooked by mainstream cell biologists. While the
endomembrane network is clearly based on many elements that
were already possessed by an ancient eukaryotic ancestor, the
extent of conservation of those elements in any specic modern
lineage cannot be unambiguously discerned by phylogenetic
analysis nor by functional or morphological characterization, but
rather requires a combination of approaches. This is because
conserved proteins can evolve new functions, and because
functionally analogous features can arise via multiple mechanisms. Developing a more nuanced view of the variation in both
origins and mechanisms underlying cellular features will create
J. Exp. Zool. (Mol. Dev. Evol.)

512
new opportunities for discerning general principles, both physical
and genetic, that underlie cellular organization.

ACKNOWLEDGMENTS
We thank our current colleagues Cassandra Kontur and Santosh
Kumar, as well as previous laboratory members whose work
spawned the substance of this review. That work was supported by
grants from the National Institutes of Health (General Medicine),
the National Science Foundation, and the Chicago Biomedical
Consortium. We also thank colleagues working with other Ciliates,
particularly the Paramecium groups of Helmut Plattner, Jean
Cohen, and Linda Sperling, whose work has been key in
understanding pathways discussed here. APT is a member of
the scientic advisory board of Tetragenetics, Inc.
The research described here on Tetrahymena sortilins was
supported by a Catalyst Grant from the Chicago Biomedical
Consortium and by NSF MCB1051985 to A.P.T. J.S.B. was
supported by a National Institutes of Health Training Grant T32
GM007191 and by the NSF through a Graduate Research
Fellowship. This manuscript is dedicated to Joseph Frankel, a
beacon of Tetrahymena cell biology, on the occasion of his
retirement from the University of Iowa.

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