
==== Front
eLife
Elife
eLife
eLife
2050-084X
eLife Sciences Publications, Ltd

100061
10.7554/eLife.100061
version of record
Research Article
Biochemistry and Chemical Biology
Prominin 1 and Tweety Homology 1 both induce extracellular vesicle formation
Bell Tristan A https://orcid.org/0000-0002-3668-8412
bell@molbio.mgh.harvard.edu
12†
Luce Bridget E 1
Hakim Pusparanee https://orcid.org/0000-0002-9018-8179
1
Ananda Virly Y 1
Dardari Hiba https://orcid.org/0009-0008-8794-6697
1
Nguyen Tran H 1
Monshizadeh Arezu https://orcid.org/0009-0005-4326-7754
1
Chao Luke H https://orcid.org/0000-0002-4849-4148
chao@molbio.mgh.harvard.edu
12
1 https://ror.org/002pd6e78 Department of Molecular Biology, Massachusetts General Hospital Boston United States
2 Department of Genetics, Blavatnik Institute, Harvard Medical School Boston United States
Motaung Thabiso Reviewing Editor https://ror.org/00g0p6g84 University of Pretoria South Africa

Campelo Felix Senior Editor https://ror.org/03g5ew477 Institute of Photonic Sciences Spain

† Generate Biomedicines, Somerville, United States.

13 8 2024
2024
13 e10006127 5 2024
08 7 2024
This manuscript was published as a preprint at bioRxiv.08 11 2023

© 2024, Bell et al
2024
Bell et al
https://creativecommons.org/licenses/by/4.0/ This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Prominin 1 (Prom1) is a five-transmembrane pass integral membrane protein that associates with curved regions of the plasma membrane. Prom1 interacts with membrane cholesterol and actively remodels the plasma membrane. Membrane-bending activity is particularly evident in photoreceptors, where Prom1 loss-of-function mutations cause failure of outer segment homeostasis, leading to cone-rod retinal dystrophy (CRRD). The Tweety Homology (Ttyh) protein family has been proposed to be homologous to Prominin, but it is not known whether Ttyh proteins have an analogous membrane-bending function. Here, we characterize the membrane-bending activity of human Prom1 and Ttyh1 in native bilayer membranes. We find that Prom1 and Ttyh1 both induce formation of extracellular vesicles (EVs) in cultured mammalian cells and that the EVs produced are physically similar. Ttyh1 is more abundant in EV membranes than Prom1 and produces EVs with membranes that are more tubulated than Prom1 EVs. We further show that Prom1 interacts more stably with membrane cholesterol than Ttyh1 and that this may contribute to membrane-bending inhibition in Prom1 EVs. Intriguingly, a loss-of-function mutation in Prom1 associated with CRRD induces particularly stable cholesterol binding. These experiments provide mechanistic insight into Prominin function in CRRD and suggest that Prom and Ttyh belong to a single family of functionally related membrane-bending, EV-generating proteins.

Prominin 1
extracellular vesicles
membrane biophysics
protein conformation
cholesterol-binding protein
Research organism

Human
http://dx.doi.org/10.13039/100005237 Helen Hay Whitney Foundation Bell Tristan A http://dx.doi.org/10.13039/100000057 National Institute of General Medical Sciences R35GM142553 Chao Luke H The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.Author impact statementProminin 1 and Tweety Homology 1, two proteins that regulate membrane shape in neural and neuroepithelial cells, have shared evolutionary history and both cause cells to secrete extracellular vesicles.
==== Body
pmcIntroduction

Mammalian cells interact with the extracellular environment through proteins, lipids, and glycans at the plasma membrane. Organized protrusive structures of the membrane such as microvilli and cilia are hotspots for nutrient absorption, cell cycle regulation, and extracellular signaling (Sharkova et al., 2023; Satir and Christensen, 2007). Membrane protrusions are organized both by interactions with the cytoskeleton and by sorting of proteins and lipids within the membrane bilayer (Satir and Christensen, 2007; Brown and London, 2000; Corbeil et al., 2010). Prominin 1 (Prom1) is a five-transmembrane pass integral membrane protein that interacts with membrane cholesterol at sites of membrane protrusion (Corbeil et al., 2013; Thamm et al., 2019). Prom1 was first characterized as the target of AC133-1, a monoclonal antibody raised against pluripotent human epithelial stem cells (Yin et al., 1997; Miraglia et al., 1997). Since then, Prom1 has been identified in the apical membranes of most epithelial and neuroepithelial cell types, but is only natively recognized by AC133-1 in stem cells and photoreceptors (Corbeil et al., 1998; Kemper et al., 2010b; Mak et al., 2012). In photoreceptors, Prom1 associates with a retinal cadherin (Protocadherin-21, Pcdh21) to promote normal outer segment membrane homeostasis (Yang et al., 2008; Burgoyne et al., 2015; Rattner et al., 2001), and several Prom1 mutations are linked to hereditary cone-rod retinal dystrophy (CRRD) (Yang et al., 2008; Zacchigna et al., 2009; Eidinger et al., 2015; Liang et al., 2019; Maw et al., 2000). Most animal genomes also encode at least one paralogous prominin gene (Prominin 2) that is expressed in non-retinal cell types and localizes to positively curved membrane regions (Fargeas, 2013).

When overexpressed, Prom1 dramatically reorganizes the plasma membrane of cultured mammalian cells into long protrusions (Hori et al., 2019; Röper et al., 2000). Small-molecule inhibitors of actin and tubulin do not impair the tubulation phenotype, indicating that cytoskeletal interactions are not strictly required for membrane bending (Hori et al., 2019; Röper et al., 2000). However, cells treated with cholesterol biosynthesis inhibitors or depletion agents do not exhibit membrane tubulation, suggesting that cholesterol plays a pivotal role in the regulation of Prom1 function (Hori et al., 2019; Röper et al., 2000).

Prom1 also induces release of small extracellular vesicles (EVs, <250 nm in diameter) that bleb from the apical plasma membrane of differentiating epithelial stem cells at organized membrane protrusions such as microvilli and cilia (Marzesco et al., 2005; Wood and Rosenbaum, 2015; Dubreuil et al., 2007). Small Prom1 EVs can be detected in saliva, urine, semen, neural tube fluid, and lacrimal fluid of healthy adults (Marzesco et al., 2005; Hurbain et al., 2022).

Recently, the Tweety Homology (Ttyh) protein family was hypothesized to be a distant homolog of the prominins (Hori et al., 2019). Ttyh proteins share prominins’ five-transmembrane topology but have a more minimal extracellular domain (Campbell et al., 2000). Most animals have three paralogous Ttyh proteins (Ttyh1, Ttyh2, and Ttyh3) (Matthews et al., 2007) that all are predominantly expressed in neural tissues (Halleran et al., 2015; Bae et al., 2019; Han et al., 2019; Li et al., 2021; Sukalskaia et al., 2021; Melvin et al., 2022). Notably, overexpression of Ttyh1 at the plasma membrane in cultured cells induces plasma membrane tubulation that is strikingly similar to that observed with Prom1 (Matthews et al., 2007; Jung et al., 2017; Stefaniuk et al., 2010; Wiernasz et al., 2014).

Defining mechanisms of membrane bending by Prominin-family proteins is foundational for understanding retinal disease and stem cell development. Here, we reconstitute EV formation by Prom1 and Ttyh1 to characterize their functions in a native membrane bilayer system. To our knowledge, we present the first evidence that Ttyh1 induces formation of EVs that are functionally and physically analogous to Prom1 EVs. Ttyh1 EVs are more tubulated and protein-rich than Prom1 EVs. Prom1 forms a more stable interaction with cholesterol than Ttyh1, and when depleted of cholesterol, Prom1 EVs mimic the tubulation observed in Ttyh1 EVs. A CRRD-associated Prom1 mutant (W795R) binds cholesterol more stably than wild-type protein, suggesting that a less dynamic interaction between Prom1 and cholesterol inhibits membrane bending. These findings contribute to a path toward mechanistic understanding of Prominin-family protein function in different tissues, including the role of Prom1 in retinal pathology.

Results

Reconstitution and purification of Prom1 EVs

To understand how Prom1 interacts with and reshapes native membranes, we sought a method to purify Prom1 without disturbing protein–lipid interactions. Because Prom1 induces EV formation in epithelial stem cells (Marzesco et al., 2005), we asked whether Prom1 can also form EVs in cultured cells. C-terminally Strep-tagged Prom1 was detected in immunoblots of the conditioned media (CM) from transfected cells, but not in cells treated with a no-plasmid mock transfection (Figure 1A, B). To validate that the ~120 kDa Strep-reactive band we observe is indeed Prom1-Strep, we immunoblotted with the Prom1-specific antibody AC133-1 (Kemper et al., 2010a) and observed robust staining (Figure 1C). To confirm that EV production is not an artifact of transfection, we introduced Prom1-Strep into Expi293 cells by lentiviral transduction to generate a stable polyclonal overexpression cell line. A Strep-reactive band consistent with mature Prom1 was detectable in the CM from these cells over several rounds of cell passaging (Figure 1—figure supplement 1A). We assessed the protein composition of Prom1 EVs using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and silver nitrate staining. Prom1 was a constituent of our samples alongside several other proteins (Figure 1—figure supplement 1). We cannot conclude whether these proteins specifically interact with Prom1 in EVs, are hitchhikers enclosed within the EVs, or originate from endogenous co-purifying EVs.

Figure 1. Reconstitution and purification of Prom1 EVs.

(A) Extracellular vesicle (EV) expression and purification graphic protocol. (B) Anti-Strep immunoblot of cell pellet, conditioned media (CM), clarified conditioned media (CCM), or size-exclusion chromatography (SEC)-purified EVs from mock-transfected or Prom1-Strep-transfected Expi293 cells. Arrowhead indicates the expected molecular weight of Prominin 1 (Prom1). (C) AC133-1 immunoblot of SEC-purified Prom1 EVs. Arrowhead indicates the expected molecular weight of Prom1. (D) Anti-Strep immunoblot of Prom1 EVs treated with or without PNGase F to remove N-glycan moieties. (E) Cryo-transmission electron microscopy (cryo-TEM) images of purified Prom1 EVs. Inset image is magnified to emphasize membrane bilayer density. Images are lowpass filtered to 5 Å to enhance contrast. (F) Negative-stain transmission electron microscopy (NS-TEM) image of purified Prom1 EVs. (G) Measured diameters of Prom1 EVs from cryo-TEM or NS-TEM images (n = 322 and n = 176 for NS-TEM and cryo-TEM measurements, respectively). (H) Anti-Strep (Prom1) and anti-Flag (Pcdh21) immunoblots of CM and CCM from cells transfected with Prom1-Strep, Pcdh21-Flag, or both. Note that Pcdh21 is only detected in CCM when co-expressed with Prom1. Filled and empty arrows indicate expected molecular weights of glycosylated Prom1-Strep and Pcdh21-Flag, respectively. (I) Comparative fluorescence measurements of Pcdh21-mNeonGreen co-immunoprecipitated with or without Prom1-Strep (n = 3, **p < 0.01 by Student’s two-tailed unpaired t test).

Figure 1—source data 1. Raw source gel and blot images.

Figure 1—source data 2. Labelled source gel and blot images.

Figure 1—figure supplement 1. Prom1 induces formation of extracellular vesicles.

(A) Anti-Strep immunoblot of conditioned media (CM) or clarified conditioned media (CCM) from a stable polyclonal Expi293 cell line expressing lentiviral-transduced Prom1-Strep. Arrowhead indicates the expected position of glycosylated Prom1-Strep. P4, P5, P6, and P7 indicate the passage number of the suspension cell culture. (B) Total protein stain of purified Prom1-Strep EVs. Arrowhead indicates the expected position of glycosylated Prom1-Strep. (C) Dynamic light scattering (solution size) measurement of purified Prom1 EVs.

Figure 1—figure supplement 1—source data 1. Raw source gel and blot images.

Figure 1—figure supplement 1—source data 2. Labelled source gel and blot images.

We next purified the Prom1 EVs using differential centrifugation, ultracentrifugal concentration, and size-exclusion chromatography (SEC) based on isolation methods previously described for endogenous small Prom1 EVs (Figure 1A; Marzesco et al., 2005; Huttner et al., 1983). It is important to note that our purification protocol removes any large EVs and midbody remnants, focusing our downstream experiments on small Prom1 EVs. Using dynamic light scattering (DLS), we measured the solution sizes of the purified Prom1 EVs. The sample was monodisperse with an average diameter of 164 ± 14 nm, considerably larger than previously reported 50–80 nm EVs measured by negative-stain transmission electron microscopy (NS-TEM) in samples purified from biological fluids (Marzesco et al., 2005; Figure 1—figure supplement 1). Upon treatment with PNGase F, an enzyme that cleaves N-glycan groups from proteins, the ~120 kDa Prom1 gel band shifted down to its predicted molecular weight of 102 kDa (Figure 1D).

To verify that the purified particles are truly EVs (secreted particles with intact bilayer membranes), we vitrified purified samples and imaged them using cryo-transmission electron microscopy (cryo-TEM) (Figure 1E). We observed spherical EVs with bilayer membranes (Figure 1E, inset). The diameters of EVs measured from cryo-TEM images were bimodal with an average diameter of 117 ± 58 nm, somewhat lower than our measurements from DLS (Figure 1G). We next used NS-TEM to directly compare our reconstituted Prom1 EVs with previously characterized endogenous Prom1 EVs (Marzesco et al., 2005; Figure 1F). We observed a distribution of largely circular EVs ranging in size from ~50 to ~250 nm in diameter, but more skewed toward smaller diameters than observed in the cryo-TEM data (Figure 1G). In addition, EVs had rough edges and internal depressions in NS-TEM, a characteristic feature of EV fixation and dehydration (Marzesco et al., 2005; Figure 1G). Because solution DLS measurements (164 ± 14 nm) suggest slightly larger EV diameters than our NS-TEM (123 ± 73 nm) or cryo-TEM (117 ± 58 nm) measurements, we speculate that sample fixation/dehydration or vitrification may induce deformation and potentially fission of the reconstituted EVs. This effect may have similarly impacted previous characterization of endogenous Prom1 EVs (Marzesco et al., 2005). We conclude that reconstituted Prom1 EVs have similar morphology to endogenous EVs but may be slightly larger in size.

We next asked whether Prom1 in purified EVs retains known functional behavior of Prom1 from endogenous membranes. Prom1 stably binds the retinal cadherin Pcdh21 in photoreceptors (Yang et al., 2008; Burgoyne et al., 2015; Rattner et al., 2001). To determine if Prom1 can co-traffic with Pcdh21 in our EV samples, we co-transfected Expi293 cells with Strep-tagged Prom1 and Flag-tagged Pcdh21 and looked for the presence of each component in cells, CM, and clarified conditioned media (CCM). Pcdh21 was only observed in CCM samples when co-expressed with Prom1, indicating that Prom1 is necessary to traffic Pcdh21 into this class of EVs (Figure 1H). To establish a direct interaction between Prom1 and Pcdh21, we co-expressed Prom1-Strep with mNeonGreen-tagged Pcdh21 and immunopurified Prom1 from EVs solubilized with 1% n-dodecyl-β-D-maltoside (DDM). Pcdh21-associated mNeonGreen reproducibly co-purified with detergent-solubilized Prom1 compared to a control condition lacking Prom1, indicating that the two proteins physically interact in purified EVs (Figure 1I).

Mutations in the Prom1 transmembrane domain impair EV formation

Prom1 binds cholesterol in native membranes and the Prom1–cholesterol interaction is necessary for remodeling the plasma membrane (Röper et al., 2000), but the mechanism of cholesterol-dependent Prom1 function is unclear. We analyzed the sequence of human Prom1 to identify candidate cholesterol recognition amino acid consensus (CRAC, [L/V]-X1−5-[Y/F]-X1−5-[K/R]) or mirrored CRAC (CARC, [K/R]-X1−5-[Y/F]-X1−5-[L/V]) sequences in the transmembrane helices, as these motifs often predict cholesterol binding in membrane proteins (Fantini and Barrantes, 2013). Human Prom1 contained four CRAC and two CARC motifs, of which none were completely conserved among metazoans and only one (CRAC-3) was modestly evolutionarily conserved (Figure 2—figure supplement 1A, Figure 2—figure supplement 1). We therefore turned to a more comprehensive evolutionary analysis of prominin proteins to identify evolutionarily conserved Prom1 transmembrane residues.

We curated prominin sequences from across eukaryotes, considering a prominin to be a sequence with five predicted transmembrane helices, two large extracellular loops, two small intracellular loops, and homology to annotated metazoan prominin sequences. Putative prominin sequences were identified across metazoans as well as in fungi, excavates, SAR (stramenopiles, alveolates, and rhizarians), and green plants (Figure 2—figure supplement 2, Figure 2—figure supplement 3). Strikingly, Trp-795 was nearly perfectly conserved across metazoa and eukaryotic outgroups, making it by far the most conserved residue (excluding Cys residues positioned to form disulfides in AlphaFold2 models) across the curated prominin sequences (Figure 2A, Figure 2—figure supplement 2). The conserved Trp-795 residue was of particular interest as a missense mutation at this site (W795R) is implicated in hereditary CRRD cases (Boulanger-Scemama et al., 2015).

Figure 2. Mutations in the Prom1 transmembrane domain impair EV formation.

(A) AlphaFold2 model of human Prominin 1 (Prom1; Jumper et al., 2021) with residues color coded by level of conservation across a multiple-sequence alignment of metazoan prominin sequences. (B) Possible network of interactions between Trp-795 and several adjacent aromatic residues in human Prom1. (C) Quantification of anti-Strep immunoblots of cellular and clarified conditioned media pools of Prom1-Strep mutants relative to wild-type Prom1-Strep (n = 3, n.s.p > 0.0045, *p < 0.0045, **p < 0.0009, ***p < 0.00009 by Student’s two-tailed unpaired t test with significance thresholds adjusted by Bonferroni correction). Raw blot images from which measurements are derived are included in Figure 2—figure supplement 4. (D) Mean particle diameter of purified Prom1-Strep EVs measured by dynamic light scattering. Error bars indicate standard deviation (SD) (n = 5, n.s.p > 0.007, *p < 0.007 by Student’s two-tailed unpaired t test with significance thresholds adjusted by Bonferroni correction). (E) Confocal fluorescence microscopy images of HeLa cells stably expressing WT (top) or W795R (bottom) Prom1-StayGold (yellow), stained with wheat germ agglutinin (WGA) (blue). Scale bar is 10 μm. Line scan traces across cell junctions are included in Figure 2—figure supplement 5.

Figure 2—figure supplement 1. Putative cholesterol-binding motifs in the Prom1 transmembrane domain.

(A) Multiple sequence alignment of metazoan Prom1 focused on transmembrane domains 1, 2, 3, and 5, with human CRAC and CARC domains highlighted. Red carats indicate key charged, aromatic, or hydrophobic residues that define the CRAC and CARC domains. Alignment visualized using Jalview (Clamp et al., 2004). (B) Trp-795, CRAC-1, CRAC-2, CRAC-3, CRAC-4, CARC-1, and CARC-2 mutation sites superposed onto an AlphaFold2 model (Jumper et al., 2021) of the transmembrane domain of Prom1.

Figure 2—figure supplement 2. Conservation analysis of metazoan Prom1 sequences.

Multiple-sequence alignment of prominin sequences from metazoa with the five transmembrane segments indicated. Red asterisk indicates perfectly conserved non-cysteine residues. Orange asterisk indicates less-than-perfectly conserved non-cysteine residues of interest. Red carat indicates cysteines predicted to form internal disulfides. Double red carat indicates cysteines that are not predicted to form internal disulfides. Alignment visualized using Jalview (Clamp et al., 2004).

Figure 2—figure supplement 3. Phylogenetic analysis of Prom1 sequences.

Inferred phylogenetic relationships between putative prominin homologs identified across eukaryotes. Node labels indicate aLRT branch supports (Anisimova and Gascuel, 2006). Tree visualized using IcyTree (Vaughan, 2017).

Figure 2—figure supplement 4. Blot images used for EV quantification in Figure 1C.

Anti-Strep immunoblots used for quantification of EV production by Prom1 mutants in Figure 2C. Lanes are labeled “C” for cells and “M” for clarified conditioned media.

Figure 2—figure supplement 4—source data 1. Raw source gel and blot images.

Figure 2—figure supplement 4—source data 2. Labelled source gel and blot images.

Figure 2—figure supplement 5. Line scan traces of Prom1 signal in cell lines.

Line scan traces across cell junctions (n = 6) for plasma membrane (WGA) (blue) or Prom1-mStayGold fluorescence signal for WT (left panels) or W795R (right panels) Prom1 (yellow).

We generated Prom1-Strep variants with mutations to disrupt each CRAC or CARC motif, the disease-associated W795R mutation, and point mutations directed against residues predicted to be in close contact with Trp-795 in Alphafold2 models (Figure 2B, Figure 2—figure supplement 4, Jumper et al., 2021). We then assessed EV formation by quantifying the Prom1-Strep signal secreted in EVs or retained in the cellular membranes. We found that the W795R mutation to the Prom1 TM domain significantly disrupted the proportion of Prom1 secreted in EVs (Figure 2C, Figure 2—figure supplement 4 for raw blot images). This effect arose from both decreased expression of Prom1 and increased retention of protein in cells, though neither of these measurements alone met our stringent significance criteria. To better characterize the morphology of the Prom1 variants, we expressed and purified a subset of mutant Prom1 EVs at larger scale. Each of the mutants produced EVs that are 150–200 nm in diameter and monodisperse by DLS analysis, with no mutant EV sizes deviating significantly from that of WT Prom1 EVs (Figure 2D). Thus, transmembrane domain mutations in Prom1 primarily alter the quantity of EVs produced rather than EV size.

To better understand the mechanism of reduced Prom1 EV formation by the CRRD-associated W795R mutant, we engineered HeLa cells that stably express C-terminally StayGold-tagged WT (Prom1-SG) or W795R Prom1 (Prom1[W795R]-SG) under the EF-1α promoter. Prom1-SG signal colocalized with wheat germ agglutinin (WGA)-stained plasma membrane, but Prom1[W795R]-SG was not present at the plasma membrane and instead concentrated on WGA-positive intracellular features (Figure 2E). Despite the apparent low signal of plasma-membrane localized Prom1-SG, line scan analysis shows correlation between Prom1-SG signal across the cell junctions (Figure 2—figure supplement 5, left). In contrast, there is a marked absence of Prom1[W795R]-SG signal localized on the plasma membrane (Figure 2—figure supplement 5, right). We infer that mStayGold-tagged W795R Prom1 is trafficked to the plasma membrane less efficiently than the mStayGold-tagged wild-type protein.

Prominin homolog Ttyh1 produces EVs

Ttyh proteins are proposed prominin homologs that share the five-transmembrane topology of prominins but have shorter extracellular domains (~120 amino acids in Ttyh vs ~280 amino acids in Prom) (Hori et al., 2019; Figure 3A). Prominin and Ttyh both traffic to the plasma membrane and associate with protrusive membrane structures (Hori et al., 2019; Röper et al., 2000; Matthews et al., 2007; Jung et al., 2017; Stefaniuk et al., 2010; Wiernasz et al., 2014). Given the predicted sequence homology between Prom and Ttyh, we hypothesized that Ttyh may also produce EVs. Evolutionary analysis of metazoan Ttyh proteins does not indicate conserved CRAC or CARC sites, nor does it suggest any conserved transmembrane residue analogous to Trp-795 in metazoan prominins (Figure 3B).

Figure 3. Prominin homolog Ttyh1 produces EVs.

(A) Comparison of a Ttyh1 subunit from cryo-transmission electron microscopy (cryo-TEM) structure 7P5J (Sukalskaia et al., 2021) with an AlphaFold2-predicted Prominin 1 (Prom1) monomer (Jumper et al., 2021). (B) Residue-level conservation among metazoan Tweety Homology (Ttyh) proteins plotted onto a subunit of human Ttyh1. No residue analogous to Prom1 Trp-795 is present in Ttyh. (C) Anti-Strep immunoblot comparing Prom1- and Ttyh1-Strep extracellular vesicles (EVs) throughout different stages of purification. Filled and empty arrowheads indicate the expected positions of Prom1 and Ttyh1, respectively. Doublet and higher bands in Ttyh1 lanes are products of on-gel disulfide crosslinking in concentrated samples. (D) Representative negative-stain transmission electron microscopy (NS-TEM) images of Ttyh1-Strep EVs. White arrowheads indicate possible sites of EV fission. (E) Representative cryo-TEM images of Ttyh1-Strep EVs. Magnified insets show bilayer density at highly curved membrane segments. Images are lowpass filtered to 5 Å to enhance contrast. (F) Comparison of Prom1 or Ttyh1 EV diameter in NS-TEM or cryo-TEM images (n = 322, n = 1357, n = 176, and n = 2224 for Prom1 NS-TEM, Ttyh1 NS-TEM, Prom1 cryo-TEM, and Ttyh1 cryo-TEM measurements, respectively). (G) Comparison of Prom1 or Ttyh1 EV roundness in NS-TEM or cryo-TEM images. Secondary plots only include EVs with roundness ≤0.8 (n = 322, n = 1357, n = 122, and n = 1546 for Prom1 NS-TEM, Ttyh1 NS-TEM, Prom1 cryo-TEM, and Ttyh1 cryo-TEM measurements, respectively). (H) Quantification of Prom1 and Ttyh1 protein levels in EVs from anti-Strep Western blots (n = 3). Immunoblots used for quantification are included in Figure 3—figure supplement 1A. (I) Quantification of total EV membrane area from NS-TEM micrographs (n = 236, ****p < 0.0001 by unpaired Mann–Whitney test). Representative micrographs used for quantification are included in Figure 3—figure supplement 1. (J) Anti-Strep immunoblots of fractions from sucrose gradient equilibrium sedimentation of Prom1-Strep EVs (top) or Ttyh1-Strep EVs (bottom). (K) Anti-Strep immunoblots of fractions from sucrose gradient equilibrium sedimentation of EVs from cells co-expressing Prom1-Strep and Ttyh1-Strep. Filled and empty arrowheads indicate the expected positions of Prom1 and Ttyh1, respectively. (L) Quantification of immunoblots in panels K (top) and L (bottom).

Figure 3—source data 1. Raw source gel and blot images.

Figure 3—source data 2. Labelled source gel and blot images.

Figure 3—figure supplement 1. Characterization of Prom1 and Ttyh1 EVs.

(A) Dynamic light scattering measurement of EV diameter for purified Prom1-Strep, Ttyh1-Strep, or Prom1-Strep + Ttyh1-Strep co-expression EVs. (B) Anti-Strep immunoblots used for relative quantification of Prom1 and Ttyh1 levels in purified EVs. Filled and empty arrowheads indicate the expected positions of Prom1 and Ttyh1, respectively. (C) Representative NS-TEM micrographs and image processing intermediates used for membrane area quantification. (D) Quantification of relative protein-to-membrane ratio for Prom1 and Ttyh1 EVs using data from Figure 3I, J. Error bars indicate S.D. (n = 3, ** p < 0.01 by Student’s two-tailed unpaired t test).

Figure 3—figure supplement 1—source data 1. Raw source gel and blot images.

Figure 3—figure supplement 1—source data 2. Labelled source gel and blot images.

We expressed C-terminally Strep-tagged Ttyh1 in Expi293 cells and purified EVs using the same procedure as for Prom1 EVs (Figure 1A). We detected Ttyh1-Strep in CM, CCM, and SEC-purified EV fractions (Figure 3C). DLS indicated that purified Ttyh1 EVs are monodisperse with an average diameter of 180 ± 10 nm, similar to the average diameter of WT Prom1 EVs (164 ± 14 nm) (Figure 3—figure supplement 1).

We next characterized the morphology of purified Ttyh1 EVs using NS-TEM. We found that Ttyh EVs adopted striking long and bent tubular structures with much higher frequency than Prom1 EVs (6.4% of Ttyh1 EVs vs 0.3% of Prom1 EVs, n = 1357 and n = 322, respectively) (Figure 3D, G). Ttyh1 EVs visualized by NS-TEM were on average smaller than Prom1 EVs (Figure 3F, G), and were similarly smaller than expected from solution DLS measurement (Figure 1—figure supplement 1). Furthermore, we observed that the smallest Ttyh1 EVs were similar in diameter to the short-axis caliper diameter of the tubular EVs (42 ± 6 nm) and we observed cases where tubular EVs appeared to be in the process of dividing into smaller EVs (Figure 3D white arrowheads, Figure 3F). Although the NS-TEM conditions deviate from a solvated physiological state, they do suggest that Ttyh1 supports comparatively greater membrane curvature than Prom1 in these EVs.

We further analyzed Ttyh1 EVs by cryo-TEM to verify that the purified sample indeed contained EVs with intact bilayer membranes (Figure 3E). Like Prom1 EVs, Ttyh1 EVs have a bimodal size distribution with local maxima around 60 and 140 nm (Figure 3F). We also observed a population of EVs exhibiting the tubular phenotype seen in NS-TEM that again substantially exceeded that seen with Prom1 EVs (8.8% of Ttyh1 EVs vs 0.8% of Prom1 EVs, n = 2224 and n = 176, respectively) (Figure 3G). This supports the observation that EV bending is more frequent in Ttyh1 EVs than Prom1 EVs.

Molecular crowding is known to contribute to membrane bending (Derganc and Čopič, 2016). To test if Ttyh1 may promote EV membrane bending through a crowding mechanism, we quantified relative protein-to-membrane ratios for Prom1 and Ttyh1 EVs. From identical amounts of transfected cell media, purified EVs contained 8.8 ± 1.0 fold more Ttyh1 protein than Prom1 (Figure 3H, Figure 3—figure supplement 1). Both Prom1 and Ttyh1 EV membrane surface areas were significantly more abundant than the background level of EVs produced in a mock-transfected control culture, but Ttyh1 produced 5.6 ± 0.8 fold more total EV membrane than Prom1 (Figure 3I, Figure 3—figure supplement 1). From these measurements, we estimate that Ttyh1 is present at ~fivefold higher concentration in EV membranes (Figure 3—figure supplement 1). Thus, molecular-crowding effects may be a factor contributing to the increased membrane bending we observe in Ttyh1 EVs.

To further characterize purified Prom1 and Ttyh1 EVs, we subjected purified EVs to equilibrium sucrose gradient sedimentation to resolve populations by density. We observed a single population of Prom1 EVs (centered on fraction 7) but resolved two distinct populations of Ttyh1 EVs with densities lower (centered on fraction 6) and higher (centered on fraction 9) than the Prom1 EVs (Figure 3J). When Prom1 and Ttyh1 were co-expressed, we observed that the resulting EVs contained more Prom1 than Ttyh1 but followed the bimodal distribution of Ttyh1 EVs (Figure 3K). Immunoblots of the sucrose gradient fractions showed that co-expressed Prom1 and Ttyh1 peak in the same sucrose gradient fractions, suggesting that Prom1 and Ttyh1 co-elute in the same EV populations (Figure 3K, L).

Ttyh1 binds cholesterol less stably than Prom1

Because cholesterol interaction is known to regulate membrane bending by Prom1, we asked whether Prom1 and Ttyh1 bind cholesterol similarly (Hori et al., 2019; Röper et al., 2000). To comparatively measure binding, we developed a cholesterol co-immunopurification assay (hereafter referred to as chol-IP) to quantify interaction of Prom1 with fluorophore-labeled cholesterol (Figure 4A). Prom1-Strep or Ttyh1-Strep were transfected into Expi293 cells, and cells were labeled with a low concentration of fluorescent cholesterol. EVs were purified from the CM, solubilized with nonionic detergent (1% DDM), and immunoprecipitated with Strep resin trace labeled with blue fluorescent protein (mTagBFP2-Strep). We then collected epifluorescence micrographs of the resin particles, computationally segmented and filtered each image, and quantified bound cholesterol using mTagBFP2 as a normalizing control (Figure 4A). This approach permits sensitive quantification of bound lipid while efficiently excluding autofluorescent and refractive artifacts.

Figure 4. Ttyh1 binds cholesterol less stably than Prom1.

(A) Cholesterol co-immunoprecipitation (chol-IP) assay graphic protocol. (B) Comparison of relative cholesterol bound by Prom1-Strep or Ttyh1-Strep (n = 3, **p < 0.01 by Student’s two-tailed unpaired t test). (C) Fluorescent cholesterol co-immunopurified by Prom1-Strep or Ttyh1-Strep at different concentrations of n-dodecyl-β-D-maltoside (DDM) detergent. Error bars indicate standard deviation (SD) (n = 3). (D) BODIPY-cholesterol-binding measurements for WT and mutant variants of Prominin 1 (Prom1). Error bars indicate SD (n = 3, n.s.p > 0.0045, *p < 0.0045 by Student’s two-tailed unpaired t test with significance thresholds adjusted by Bonferroni correction). (E) Red fluorescence signal from anti-Strep immunopurification of DDM-solubilized extracellular vesicles (EVs) from cells expressing Prom1-mScarlet (mSc) or both Prom1-mScarlet and Prom1-Strep (Strep + mSc). Error bars indicate SD (n = 3). (F) Comparison between EV yield (Figure 2C) and chol-IP fluorescent cholesterol binding (D), with notable outliers labeled.

Figure 4—figure supplement 1. Fluorescent cholesterol binding by Prom1 and Ttyh1.

Cholesterol co-immunopurification (Chol-IP) measurement of AlexaFluor647-cholesterol binding in Prom1-Strep or Ttyh1-Strep EVs. Error bars indicate S.D. (n = 3, p = 0.053 by Student’s two-tailed unpaired t test).

We performed chol-IP analysis on Prom1 or Ttyh1 EVs labeled with BODIPY-cholesterol. Surprisingly, fluorescent cholesterol robustly co-purified with Prom1 but not with Ttyh1 (Figure 4B). To verify that this difference is not a result of nonspecific fluorophore–protein interactions, we replicated the observation using a different fluorescent cholesterol analog (AlexaFluor647-cholesterol) (Figure 4—figure supplement 1). We further verified that the cholesterol is specifically bound to Prom1 by quantifying bound cholesterol at different concentrations of solubilizing detergent (Figure 4C). Nonspecifically associated fluorescent cholesterol in Ttyh1 samples is delipidated at 0.04–0.2% DDM, 5- to 25-fold below the assay working concentration, suggesting that the assay informs on specific lipid binding that is resistant to delipidation. Although this method cannot inform on transient or kinetically unstable protein–lipid interactions, we infer that Prom1 and Ttyh1 differ in how stably they interact with membrane cholesterol.

Given the striking difference in cholesterol-binding stability between Prom1 and Ttyh1, we next asked whether Prom1 mutants also disrupt cholesterol interaction stability. We purified BODIPY-cholesterol-labeled EVs produced by WT and mutant forms of Prom1 and subjected equal input concentrations of Prom1 to chol-IP analysis. To our surprise, none of the mutants substantially disrupted cholesterol binding, but the W795R mutant and the CARC-1 mutants bound cholesterol significantly more stably than WT Prom1 (Figure 4D). We chose to focus our efforts on the W795R mutant as it is a naturally occurring single-residue mutation with exceptional evolutionary conservation and a clinically validated disease phenotype. To verify that differences in cholesterol binding between wild-type and W795R Prom1 do not arise from a gross oligomerization defect, we purified EVs from cells co-transfected with Strep- and mScarlet-tagged Prom1 and measured co-purification of Prom1-mScarlet on Strep resin. Prom1 W795R co-purified with ~60% as much mScarlet fluorescence as WT Prom1, indicating that W795R indeed multimerizes, albeit with reduced efficiency (Figure 4E). Comparative analysis of EV Yield (Figure 2C) and cholesterol bound (Figure 4D) indicates that though there may be a weak inverse correlation between cholesterol binding and stable cholesterol binding, W795R, CARC-1, and CARC-2 are outliers that bind cholesterol exceptionally tightly (Figure 4F).

Cholesterol-binding stability inversely correlates with Prom1 EV membrane bending

Because Ttyh1 promotes greater membrane curvature than Prom1 but does not stably bind cholesterol, we hypothesized that cholesterol-binding stability may contribute to negative regulation of membrane bending by Prominin-family proteins. If this is the case, then W795R Prom1, which binds cholesterol more stably than WT protein, should not produce EVs that exhibit the tubular morphology observed in Ttyh1 EVs. After purifying W795R Prom1 EVs, we observed a similar solution size for WT (164 ± 14 nm) and W795R EVs (186 ± 13 nm) (Figure 5—figure supplement 1). We measured the size and shape of W795R EVs by cryo-TEM and observed largely spherical vesicles with some local deformations, similar to what we observed with WT Prom1 EVs (Figure 5A). Generally, WT and W795R Prom1 EVs are both spherical, with W795R having no vesicles that fall into our tubular morphology classification (n = 1211) (Figure 5B). Though both large and small diameter EV populations were observed by cryo-TEM, W795R Prom1 had a larger fraction of small EVs than WT Prom1, indicating possible decreased stability or increased fissile propensity in the W795R Prom1 EVs (Figure 5C).

Figure 5. Cholesterol binding stability inversely correlates with Prom1 EV membrane bending.

(A) Representative cryo-transmission electron microscopy (cryo-TEM) images of W795R Prom1-Strep extracellular vesicles (EVs). Magnified insets show bilayer density. Images are lowpass filtered to 5 Å to enhance contrast. (B) Comparison of WT or W795R EV roundness in cryo-TEM images. Secondary plot only includes EVs with roundness ≤0.8 (n = 176 and n = 1211 for WT and W795R Prominin 1 (Prom1) cryo-TEM measurements, respectively.) (C) Comparison of WT or W795R Prom1 EV diameter in cryo-TEM images (n = 122 and n = 821 for WT and W795R Prom1 cryo-TEM measurements, respectively). (D) Cholesterol content of Prom1- or Ttyh1-purified EV samples after treatment with methyl-beta cyclodextrin (mBCD). Error bars indicate standard deviation (SD) (n = 3). (E) Representative negative-stain transmission electron microscopy (NS-TEM) images of purified mBCD-treated EVs. (F) Quantification of EV diameter (top) and roundness (middle) from NS-TEM images, as well as quantification of tubulated EVs (roundness <0.6) (bottom).

Figure 5—figure supplement 1. Dynamic light scattering distributions for Prom1 WT and W795R variants.

Dynamic light scattering EV diameter measurements of purified WT or W795R Prom1-Strep EVs.

If stable cholesterol binding negatively regulates membrane bending, then depleting cholesterol away from EV membranes could induce a Ttyh1-like morphology in Prom1 EVs. We tested this model directly by purifying WT Prom1 or Ttyh1 EVs and treating them with methyl-beta cyclodextrin (mBCD), a compound that extracts cholesterol from membranes (Röper et al., 2000; Zidovetzki and Levitan, 2007). After re-purifying EV samples away from free mBCD and mBCD–cholesterol complexes, Prom1 EVs treated with mBCD (2.5–10 mM) exhibited a decrease in cholesterol content (Figure 5D). A control set of Ttyh1 EVs treated with 0 or 10 mM mBCD showed a similar change in cholesterol content (Figure 5D). Treated and untreated EVs were then analyzed by NS-TEM to compare EV size and morphology (Figure 5E). We observed a 5.4-fold increase in the fraction of Prom1 EVs that deviated from spherical membrane topology after 10 mM mBCD treatment compared to untreated EVs (3.0% vs 0.6% of EVs, n = 232 and n = 356, respectively) (Figure 5F). Importantly, identical treatment of Ttyh1 EVs was used as a nonspecific control for cholesterol depletion from EVs. Ttyh1 EVs treated with mBCD under identical conditions only showed a 1.4-fold increase in deformed EVs (2.1% at 10 mM mBCD vs 1.5% in mock-treated EVs, n = 433 and n = 535, respectively) (Figure 5F). Thus, cholesterol-depleted Prom1 EVs are more prone to membrane deformation than untreated EVs.

Discussion

Prom1 and Ttyh1 bend membranes and form EVs

We report here that both Prom1 and Ttyh1 can induce EV formation in cultured cells upon overexpression (Figures 1B and 3C). Though we have not examined whether Ttyh1 endogenously induces EV formation, we note that it is sufficient to do so in a recombinant system with similar or higher efficiency than Prom1. Given similar membrane remodeling behavior of Prom1 and Ttyh1 in cell culture (Hori et al., 2019; Stefaniuk et al., 2010), Ttyh1 EVs may form by a similar mechanism to Prom1 EV formation. We find that Prom1 and Ttyh1 form EVs that are of similar size and that the two proteins exist in the same pool of EVs when co-expressed (Figure 3K, L).

Membrane bending by Prom1 is critical for maintaining outer segment membrane homeostasis in photoreceptors. Intriguingly, photoreceptors with impaired expression of the disc rim stabilizing protein Peripherin accumulate EVs 200–280 nm in diameter at the outer segment base (Salinas et al., 2017). We observe formation of Prom1 EVs of relatively similar size in our reconstituted system (Figure 1—figure supplement 1). This may be an example of endogenous regulation of Prom1-induced membrane curvature, with curvature-stabilizing proteins like Peripherin preventing the evaginating membrane from budding into EVs.

Ttyh1 forms EVs with membranes that are more tubulated than Prom1 EV membranes

Ttyh1 produces EVs similar in size and density to Prom1 EVs (Figure 3—figure supplement 1, Figure 3J–L). However, Ttyh1 EVs are dramatically more tubulated that Prom1 EVs (Figures 1E, F, 3D, E, G). Several factors could contribute to this increase in membrane bending. First, Ttyh1 is present in EVs at concentrations approximately fivefold higher than Prom1 (Figure 3H, I). Membrane proteins can induce curvature through molecular crowding when enriched at sufficiently high local concentrations (Derganc and Čopič, 2016). Second, Ttyh1 co-purifies with cholesterol less efficiently than Prom1 does (Figure 4B). A confounding effect in our experiments is that molecular crowding may also alter the efficiency with which cholesterol is extracted from EV membranes by detergent. Although we did not observe a kinetically stable interaction between Ttyh1 and cholesterol, it remains to be conclusively determined whether Ttyh1–cholesterol interactions occur in native membranes. Ideally, molecular-crowding and cholesterol-binding effects could be differentiated by modulating protein-to-lipid ratios in EV membranes. Unfortunately, our native purified EVs do not provide a tractable path to perform these experiments. Future experiments reconstituting Prom1 and Ttyh1 into artificial liposomes with variable cholesterol content will be necessary to fully elucidate the mechanistic differences in membrane bending between Prominin and Ttyh proteins.

Cholesterol-binding stability may contribute to membrane-bending regulation by Prom1

We find here that Prom1 in EVs forms a stable interaction with cholesterol that is resistant to delipidation by nonionic detergent (Figure 4B, C). A disease-associated mutation in a strikingly conserved residue in the fifth transmembrane helix of Prom1, W795R, substantially stabilizes cholesterol binding (Figures 2A and 4D). In AlphaFold2 models, Trp-795 forms a series of base–stacking interactions with Phe residues in neighboring transmembrane helices (Figure 2B; Jumper et al., 2021). We find that conservative mutations in several of these adjacent aromatic residues impair EV formation by Prom1, but do not mimic the stable cholesterol binding of W795R (Figures 2C and 4D, F). Cholesterol is asymmetrically distributed in eukaryotic membranes, with a bias toward the outer leaflet (Lorent et al., 2020). Prom1 Trp-795 is predicted to be positioned toward the outer leaflet, possibly potentiating interactions with cholesterol. We also observe increases in cholesterol-binding stability for mutations to two CARC domains in Prom1 (Figure 4D, F). This effect could arise because these residues interface with cholesterol or because they destabilize the transmembrane domain in a manner that mimics the effect of the W795R mutation. Given the poor evolutionary conservation of the two CARC motifs in human Prom1, we favor the latter explanation.

We further show that the W795R mutation prevents efficient trafficking of mStayGold-tagged Prom1 to the plasma membrane (Figure 2E). W795R Prom1 is expressed but primarily localizes to WGA+ intracellular vesicles. This observation explains the low yield of W795R EVs produced in cell culture (Figure 2C). Several studies have demonstrated that protein trafficking through the endomembrane system is sensitive to cholesterol levels (Ridsdale et al., 2006; Stüven et al., 2003), and our findings may suggest an as-yet undefined role for cholesterol in Prom1 membrane trafficking as well.

Prominin and Ttyh proteins are homologous proteins that both promote EV formation

Prominin and Ttyh proteins are both biologically implicated in membrane bending. Prominins localize to cholesterol-rich domains of the plasma membrane and drive protrusion-shed EV formation in differentiating stem cells (Röper et al., 2000; Marzesco et al., 2005; Dubreuil et al., 2007). Ttyh proteins are associated with dendritic spikes in healthy neurons and with tumor microtube formation in aggressive astrocytoma (Matthews et al., 2007; Jung et al., 2017; Stefaniuk et al., 2010; Wiernasz et al., 2014). Given their sequence homology and transmembrane topology, and their similarities in membrane localization, membrane bending, and EV formation behavior, we suggest that Prom and Ttyh proteins be considered members of a broader prominin family of membrane remodeling proteins. Further work will be required to fully characterize the functional specialization of Prom and Ttyh proteins.

Materials and methods

Expression constructs

All Prom1 expression constructs were generated by site-directed mutagenesis from pCS2-Prom1-YFP (a gift from N. Sasai, Nara Institute of Science and Technology). The base Prom1 construct used is the human S1 isoform (NCBI accession NP_001139319.1) (Fargeas et al., 2007). Prom1-Strep was subcloned into pLV-EF1a vector (a gift from K. Hochedlinger, Massachusetts General Hospital) for lentiviral transduction. Human Pcdh21 isoform 1 (NCBI accession NP_149091.1) was synthesized (GenScript) and cloned into a pCDNA3.1 vector (Thermo Fisher) for mammalian cell transfection. mTagBFP2 was subcloned from pBAD (Addgene #54572, RRID:Addgene_54572, a gift from Michael Davidson) into a pET28a vector with a C-terminal Strep tag for bacterial overexpression. Ttyh1 was expressed from a pLX304 vector after addition of C-terminal Strep and His tags to an existing construct (Addgene #161676, RRID:Addgene_161676, a gift from Mike McManus). To stably express fluorescently tagged Prom1 WT and W795R variants in HeLa cells for live cell imaging, Prom1-StayGold (RRID:Addgene_210821) and Prom1[W795R]-StayGold (RRID:Addgene_210822) sequences were synthesized (GenScript) and cloned into a P2555 vector (a gift from S. Jakobs, Max Planck Institute for Biophysical Chemistry) to yield constructs pAH18 and pAH20, respectively. Sequences for constructs generated in this study can be accessed at https://www.addgene.org/browse/article/28243505/.

Cell line construction

Cell lines were regularly tested for mycoplasma contamination. Lentiviral transduction of Prom1-Strep into Expi293 cells was performed using a modification of published protocols (Elegheert et al., 2018; Kutner et al., 2009). Briefly, lentiviral particles were produced in 293T cells (ATCC #CRL-3216, RRID:CVCL_0063) by transient transfection of pLV-EF1a-Prom1-Strep with VSVG (RRID:Addgene_98286) and Delta 8.9 plasmids (a gift from K. Hochedlinger, Massachusetts General Hospital) using the Lipofectamine 3000 system (Thermo Fisher) and incubated overnight at 37°C, 5% CO2. Expi293 cells (Thermo Fisher #A14528, RRID:CVCL_D615) raised for several passages in suspension culture were seeded onto adherent tissue culture plates in adherent culture media (Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1× penicillin/streptomycin (Gibco)) and incubated overnight at 37°C, 5% CO2 to form an adherent monolayer. Transfected cells were exchanged into fresh adherent culture media. Culture media was harvested after an additional 48 hr, filtered through a 0.45-μm vacuum unit, and concentrated from 70 to 0.3 ml in Phosphate Buffered Saline (PBS) buffer by ultracentrifugation according to established protocols (Kutner et al., 2009). Adherent Expi293 cells were infected with concentrated lentiviral particles at 75% confluency in adherent culture media supplemented with 8 μg/ml polybrene (Sigma-Aldrich), then incubated for 48 hr at 37°C, 5% CO2. Cells were gently washed with PBS, exchanged into adherent culture media, and incubated for 24 hr at 37°C, 5% CO2. Subsequently, the cells were subjected to antibiotic selection by exchange into adherent culture media containing 2 μg/ml blasticidin (Gibco) for 10 days with regular exchange into fresh selective media and passaging to prevent cells from achieving full confluency. Selection was considered complete when the majority of cells died and antibiotic-resistant foci recolonized the culture plate. The cells were then trypsinized (Gibco) and transitioned back to suspension culture in modified suspension culture media (Expi293 media (Gibco) supplemented with 1% fetal bovine serum) at a density of 1.0 × 106 live cells per ml of culture and incubated for 48 hr at 37°C, 8% CO2 with 125 rpm rotation. Once the suspension culture reached a density of 3.0 × 106 live cells per ml of culture, cells were re-passaged in 1.0 × 106 live cells per ml of culture in fresh modified suspension culture media supplemented with 1.5 μg/ml blasticidin, and subsequently re-passaged into this media condition every 2 days.

Stable cell lines were constructed using lentiviral and Adeno-associated virus (AAV) transduction methods according to established protocols (Elegheert et al., 2018; Kutner et al., 2009). For generation of stable HeLa cells expressing WT Prom1-StayGold and Prom1[W795R]-StayGold off the AAVS1 locus, the donor plasmids pAH18 or pAH20 were co-transfected with the nuclease plasmid PX458-AAVS1 (a gift from S. Jakobs, Max Planck Institute for Biophysical Chemistry, RRID:Addgene_113194) using Lipofectamine 3000 (Thermo Fisher). Transfected cells were selected with 10 µg/ml blasticidin (Gibco) starting 48 hr post-transfection for 7 days. Stable clones were expanded for 10 days, and single-cell GRP-positive clones were obtained using a FACS AriaII Cell Sorter (BD Biosciences). After clonal expansion, positive clones were detected and verified by fluorescence imaging.

Prom1 and Ttyh1 EV reconstitution

Prom1 and Ttyh1 EVs were reconstituted by expression in Expi293 suspension cells. Briefly, Expi293 cells grown in serum-free Expi293 media at 37°C, 8% CO2 with 125 rpm rotation to a density of 3.0 × 106 live cells per ml of culture were transiently transfected with an appropriate plasmid at 1 μg of DNA per 1 ml of culture using the Expifectamine transfection kit (Thermo Fisher) according to the manufacturer’s protocol. After 48 hr, cultures were centrifuged for 5 min at 500 × g, the media discarded, and the cells resuspended in the same volume of fresh Expi293 media and returned to incubate for an additional 48 hr. After this final incubation, cultures were centrifuged for 5 min at 1500 × g and the CM transferred to clean 50 ml conical tubes.

EVs labeled with fluorescent cholesterol analogs were generated as described above with the following modifications. Two days after transfection, cells were transferred to 50 ml conical tubes, centrifuged for 5 min at 500 × g, then resuspended in an equal volume of Expi293 media with fluorescent cholesterol added to a final concentration of 4 μM. Cells were transferred back to shaker flasks and incubated for 24 hr before CM was harvested.

EV purification

CM was clarified immediately after harvest by centrifuging for 30 min, 10,000 × g at 4°C; then transferring the supernatant to clean tubes and centrifuging again for 1 hr, 21,100 × g at 4°C. The supernatant was transferred to clean 50 ml conical tubes and stored at 4°C until ready for further purification. Clarified CM was transferred to Seton 7030 tubes and each tube underlaid with a 100-μl cushion of 50% sucrose. Tubes were centrifuged in an SW-41 Ti rotor (Beckman Coulter) for 1 hr, 36,000 rpm at 4°C, then ≥200 μl of volume was harvested from the bottom of each tube. In cases where the total harvested volume exceeded 500 μl, the harvested volume was diluted to 11 ml with sterile-filtered PBS buffer, transferred to a final Seton 7030 tube, underlaid with a 100-μl cushion of 50% sucrose, re-centrifuged as described above, and 500 μl of volume harvested from the bottom of the tube. Concentrated EVs were then purified by SEC into sterile-filtered PBS buffer using qEV2-35 nm gravity columns (Izon) at ambient temperature (0.5 ml load volume, 2.5 ml void volume, 1.2 ml harvest volume). Purified EVs were stored at 4°C for up to 8 weeks, over which time no evidence of sample degradation was observed.

Immunoblots

Protein samples were run on 4–20% or 7.5% Mini-PROTEAN TGX SDS–PAGE gels (Bio-Rad), then transferred to Polyvinylidene fluoride (PVDF) membranes using the TurboBlot semi-dry transfer system (Bio-Rad). Blots were washed briefly three times with 10 ml of PBS with 0.1% (v/v) Tween-20 (PBST) buffer, then incubated for 1–2 hr at room temperature in PBST with blocking agent (5 mg/ml bovine serum albumin (Sigma-Aldrich) for anti-Strep blots, 5% nonfat dry milk for all other blots). Blocking solution was removed and primary antibody solution in PBST with blocking agent (1:2000 rabbit anti-Strep (Abcam #76949, RRID:AB_1524455), 1:4000 mouse anti-Flag (Millipore Sigma #F3165, RRID:AB_259529), 1:2500 mouse AC133-1 (Miltenyi #130-111-756, RRID:AB_2751055)) for 2 hr at ambient temperature or for 12–72 hr at 4°C. The blots were then washed three times for 5–10 min with 10 ml PBST. Secondary antibody solution (1:5000 ECL anti-Mouse (Cytiva #NXA931, RRID:AB_772209) or 1:10,000 ECL anti-Rabbit (Cytiva #NA934, RRID:AB_772206)) in PBST with appropriate blocking agent was then added to the blots and incubated for 1 hr at ambient temperature. Blots were incubated with 5 ml Western Lighting ECL solution (PerkinElmer) for 1 min and imaged using the chemiluminescence setting on an Amersham 680 gel imager (GE Healthcare). Blots were adjusted for brightness and contrast using GIMP (GNU Project) and annotated with Illustrator (Adobe). After blotting and imaging, images were adjusted for brightness and contrast and subjected to digital densitometry with ImageJ (Schneider et al., 2012). Resulting measurements were reported normalized to WT Prom1 on each blot to allow comparisons between blots.

Dynamic light scattering

DLS measurements were performed using an SZ-100 Nano Particle Analyzer (Horiba). EVs diluted in PBS to a volume of 1 ml were transferred to a disposable plastic cuvette (Fisher) and measurements were taken using settings for polydisperse liposomes in aqueous buffer. All measurements were taken at 25°C in multiple technical replicates to control for instrument sampling error.

Silver stain

EV samples were run on 7.5% SDS–PAGE Tris–Glycine gels and stained with Pierce Silver Stain for Mass Spectrometry (Thermo Scientific) according to the manufacturer’s protocol.

Glycosylation assays

PNGase F (New England Biolabs) was used to remove N-glycan moieties from proteins under denaturing conditions according to the manufacturer’s instructions.

Cryo-TEM sample preparation and imaging

Prom1 and Ttyh1 EVs were vitrified on 300-mesh gold Quantifoil R 1.2/1.3 + 2 nm Carbon grids (Electron Microscopy Sciences). Briefly, grids were glow discharged in an EasiGlow device (Pelco) set to 5 mA, 30 s, 0.39 mbar, with a 15 s vacuum hold time. The grids were then treated with 5 μl of purified EVs, incubated for 60 s to allow EVs to adhere to the carbon layer, then blotted with a VitroBot Mark IV (Thermo Scientific) set to 22°C, 5 s blot time, +15 blot force, 100% humidity; and plunge frozen in liquid ethane. Vitrified samples were imaged on a Titan Krios microscope (Thermo Scientific) with a Falcon 4 direct electron detector (Thermo Scientific) using counted detection mode, 105,000 × nominal magnification, 0.83 Å pixel size, with 49-frame fractionated collection, 49.8 e−/Å (Satir and Christensen, 2007) total dose, and defocus ranging from −0.8 to −2.0 μm in 0.1 μm increments. Images were processed and analyzed with CryoSparc v. 4.2.1 (Structura Biotechnology). Vesicles were defined and diameter (all EVs) and roundness (only EVs completely visible on one micrograph) were calculated using custom scripts that extend CryoSparc, made publicly available at GitHub, (copy archived at Bell, 2023).

NS-TEM sample preparation and imaging

Formvar carbon film 400-mesh copper grids (Electron Microscopy Sciences) were glow discharged in an EasiGlow device (Pelco) set to 30 mA, 30 s, 0.39 mbar, with a 15 s vacuum hold time. 5 μl of EV sample was applied to a glow-discharged grid and incubated for 60 s at room temperature. The grid was then blotted manually with filter paper (Whatman #43), briefly washed three times with 20 μl of PBS buffer, blotted, washed two times with deionized water, blotted, washed one time with 1.25% (wt/vol) uranyl formate (Electron Microscopy Sciences), and blotted. The grid was then floated for 10 s on a 20 μl of drop of 1.25% uranyl formate, blotted, and allowed to air dry. Imaging was performed on a Tecnai T12 transmission electron microscope (FEI) equipped with an XR16 detector (AMT) operated at an accelerating voltage of 120 kV, 30,000 × nominal magnification, 4.32 Å pixel size, and −1.5 μm defocus. Pixel size was manually calibrated prior to image acquisition using a dedicated calibration waffle grid (Ted Pella). Data collected for EV membrane area quantification were automated using SerialEM (Mastronarde, 2005). Vesicles were defined and analyzed in CryoSparc (Structura Biotechnology) as described above for cryo-TEM data.

Quantification of EV membrane area from NS-TEM images

NS-TEM images were collected using an automated acquisition script. The images were processed to resolve membrane features from background using a custom image processing script made publicly available at GitHub, (copy archived at Bell, 2024a). EV membrane area was calculated for each micrograph as the fraction of pixels in the imaging area that was occupied by membrane features.

Equilibrium sucrose gradient sedimentation analysis

Linear sucrose gradients were prepared in Seton 7030 tubes using sterile-filtered 5% and 30% (wt/vol) sucrose dissolved in PBS buffer using a Gradient Station IP (BioComp). Freshly prepared gradients were loaded into an SW-41 Ti swinging bucket rotor and 200 μl of the purified EVs were layered atop gradients immediately prior to centrifugation. Samples were centrifuged for 5 hr, 22,000 rpm, 4°C, then fractionated into 13 fractions of 930 μl using the Gradient Station IP. Gradient fractions were analyzed by SDS–PAGE and immunoblotting, then quantified as described above.

Evolutionary analysis of Prom and Ttyh proteins

We identified sequences homologous to human Prom1 using BLAST (Sayers et al., 2022) (RRID:SCR_004870) and InterPro (Paysan-Lafosse et al., 2023) (RRID:SCR_006695). Putative prominin sequences were curated to include only sequences with five predicted transmembrane helices by TMHMM (Sonnhammer et al., 1998) (RRID:SCR_014935) in a 2 + 2 + 1 pattern, containing large extracellular loops (>300 amino acids) and two small intracellular loops (<25 amino acids). This broad search revealed prominin sequences across the eukaryotic tree ranging from metazoans to green plants. Multiple-sequence alignment was performed using MAFFT (Katoh et al., 2002) (RRID:SCR_011811) and phylogenetic relationships inferred using IQ-TREE (RRID:SCR_017254) with MODELFIND for evolutionary model selection (Minh et al., 2020; Kalyaanamoorthy et al., 2017). Branch supports were calculated using the approximate likelihood ratio test (Anisimova and Gascuel, 2006). A smaller tree was also constructed spanning only metazoan sequences, with fungi included as an outgroup for rooting. For direct comparison between Prom and Ttyh sequences, homologs of human Ttyh1 were identified for all species included in the prominin metazoan tree and aligned as described above. Figures showing multiple-sequence alignments were generated using JalView (Clamp et al., 2004) (RRID:SCR_006459). Figures showing trees were generated using IcyTree (Vaughan, 2017).

Conservation of each residue within the Prom and Ttyh metazoan trees was calculated using the Livingstone and Barton algorithm implemented in JalView (Livingstone and Barton, 1993). Conservation scores were then plotted onto the AlphaFold2 structure model (Jumper et al., 2021) (RRID:SCR_025454) of human Prom1 or a subunit of the solved cryo-EM structure of human Ttyh1 (Sukalskaia et al., 2021) using PyMol (Schrödinger, RRID:SCR_000305).

Live cell fluorescence microscopy and analysis

Confluent monoclonal HeLa cells stably expressing WT Prom1-StayGold and Prom1[W795R]-StayGold were harvested, seeded onto 35 mm glass-bottom dishes (MatTek Life Sciences) coated with poly-D-lysine (0.1 mg/ml) and allowed to grow overnight at 37°C under 5% CO2. Cells were stained with WGA conjugated with AlexaFluor 647 (W32466, Thermo Fisher Scientific) at 5 µg/ml for 10 min at 37°C, then washed twice with 1× PBS. Cells were placed in Live Cell Imaging Solution (Invitrogen) prior to imaging using a Nikon A1R HD25 point scanning confocal microscope with GaAsP and PMT detectors, equipped with an Apo TIRF 60×/1.49 NA objective lens and Ti2 Z-drive. Temperature, humidity, and CO2 concentrations were controlled with a Live Cell environmental chamber (Oko-Lab). Image acquisition was performed in NIS-Elements (Nikon Instruments Inc, RRID:SCR_014329) and line-scan analysis was performed using Fiji (Schindelin et al., 2012) (RRID:SCR_002285).

Preparation of fluorescent cholesterol analogs

BODIPY-cholesterol was procured commercially (TopFluor cholesterol, Avanti Polar Lipids) and resuspended at 1 mM in ethanol. AlexaFluor647-cholesterol was synthesized from Alkyne Cholesterol and AZDye 647 Azide Plus (Click Chemistry Tools) by Copper(I)-catalyzed azide–alkyne cycloaddition. Briefly, 1 mM AZDye 647 Azide Plus and 2 mM alkyne cholesterol (each from a 10 mM stock prepared in anhydrous Dimethyl sulfoxide) were combined with 2 mM tetrakis(acetonitrile)copper(I) tetrafluoroborate (from a 40 mM stock prepared in ethanol) in a 350 μl reaction brought up to volume with ethanol. The reaction was incubated at 42°C for 30 min then at 70°C for 2 hr with the reaction vessel open to allow solvent to evaporate. Cholesterol was extracted from the final mixture by diluting the solution to 650 μl with PBS buffer and adding 1 ml methanol and 0.5 ml chloroform. The mixture was centrifuged for 2 min at 14,000 × g and the supernatant transferred to a clean vessel, after which 1 ml of chloroform and 2 ml of glacial acetic acid were added and mixed by vortexing. The solution was then concentrated by evaporation in a SPD1010 SpeedVac instrument (Savant) until dried, then resuspended in 100 μl ethanol. This preparation was considered to be at 5 mM labeled cholesterol (assuming 100% yield) for downstream calculations.

Protein expression and purification

A plasmid encoding mTagBFP2-Strep was transformed into E. coli BL21 (DE3) pLysS, grown at 37°C in LB media to OD600 0.5–0.7, induced with 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (Gold Biotechnology), and harvested after 3 hr of expression. Cells were resuspended in 25 ml per liter of culture of Buffer A (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) NaOH pH 7.5, 500 mM NaCl, 20 mM imidazole, 0.5 mM dithiothreitol (DTT)) supplemented with 10 μM leupeptin (Sigma-Aldrich), 1 μM pepstatin A (Sigma-Aldrich), 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich), 1 mg/ml chicken egg lysozyme (Fisher), and 250 U benzonase nuclease (Sigma-Aldrich); and incubated with stirring for 1 hr at 4°C. Cells were then sonicated for 3 min in an ice/water bath with 5 s on/10 s off pulses. The lysate was then clarified by centrifugation in a JA-25.5 rotor (Beckman Coulter) for 45 min, 15,000 rpm, 4°C. The supernatant was then loaded onto two 5 ml HisTrap columns (Cytiva) plumbed in series equilibrated in Buffer A using a peristaltic pump at 1.5 ml/min flow rate. The column was washed with 100 ml of Buffer A and eluted with 20 ml of Buffer B (25 mM HEPES–NaOH pH 7.5, 500 mM NaCl, 300 mM imidazole, 0.5 mM DTT). The protein was found to be ~95% pure by SDS–PAGE, and the concentration of the eluted material was measured using absorbance signal at 280 nm. The sample was divided into small aliquots, flash frozen in liquid nitrogen and stored at −80°C.

Chol-IP assays

Prior to running chol-IP assays, input EVs were quantified by SDS–PAGE and immunoblotting to ensure equal inputs of Prom1-Strep and/or Ttyh1-Strep in each assay. EVs were mixed 4:1 with buffer CIA (25 mM HEPES–NaOH pH 7.8, 150 mM NaCl, 5 mM CaCl2, 5% DDM (Anatrace)) and incubated for 1 hr at 4°C with end-over-end rotation, protected from ambient light. During incubation, 0.025 μl of StrepTactinXT 4Flow resin (IBA) per condition was equilibrated with buffer CIB (25 mM HEPES–NaOH pH 7.8, 150 mM NaCl, 5 mM CaCl2, 1% DDM) in a single pooled reaction. mTagBFP2-Strep was added to the resin at a ratio of 2 fmol mTagBFP2-Strep per 1 μl of resin, and incubated for 15 min at 4°C with end-over-end rotation, protected from ambient light. After one additional wash with buffer CIB, the resin was then divided equally across low-binding 1.5 ml tubes (USA Scientific) so that each condition being tested plus one negative control condition had equal inputs of Blue Fluorescent Protein (BFP) labeled resin. The DDM-treated EVs were then added to the appropriate resin tube and incubated for 1 hr at 4°C with end-over-end rotation, protected from ambient light. Each condition was then washed twice with buffer CIB for 5 min at 4°C with end-over-end rotation, resuspended in 45 μl of buffer CIB, and stored on ice, protected from light. Each condition was sequentially pipetted with a cut pipette tip onto a glass microscope slide (Fisher) and gently covered with an 18 mm × 18 mm no. 1 glass cover slip (Matsunami) layered on carefully to minimize trapped air bubbles. Montaged images of the resin beads were collected using an Axio Observer TIRF microscope (Zeiss) in epifluorescence mode with a Prime 95B camera (Photometrics) running SlideBook v. 6.0.24 software (3i, RRID:SCR_014423). Custom scripts were then used to identify resin beads in each image using 4′,6-diamidino-2-phenylindole (DAPI channel, BFP) signal to both identify beads and normalize fluorescence signal in the Fluorescein isothiocyanate (FITC channel, BODIPY-cholesterol) or CY5 (AlexaFluor647-cholesterol) channels. Images of all resin particles were manually reviewed to ensure the exclusion of air bubbles, or other non-resin fluorescent artifacts from downstream analysis. Analysis scripts have been made publicly available at GitHub, (copy archived at Bell, 2024b).

Immunopurification assays

Co-immunopurification assays from EVs containing fluorescently labeled Prom1 or Pcdh21 were performed as described above for the chol-IP assay, using montaged fluorescence imaging for sensitive and quantitative measurements. Prom1-mScarlet was imaged using CY3 and Pcdh21-mNeonGreen using FITC filter sets.

Cholesterol depletion with mBCD

mBCD (Sigma-Aldrich) was dissolved in sterile-filtered PBS buffer to a final concentration of 20 mM, and a twofold serial dilution series was prepared. For each condition (0, 2.5, 5, and 10 mM mBCD), purified EVs were mixed 1:1 with the appropriate mBCD dilution and incubated for 2 hr at 37°C. After the reaction was complete, the EVs were immediately re-purified over a qEV2-35 nm column (Izon) as described above. Cholesterol in the purified samples was quantified using Amplex Red reagent (Thermo Fisher) according to the manufacturer’s protocol, with fluorescence measurements taken using a SpectraMax M5 plate reader (Molecular Devices). EV morphology was characterized by preparing NS-TEM grids with mBCD-treated samples and analyzing the resulting images as described above.

Funding Information

This paper was supported by the following grants:

http://dx.doi.org/10.13039/100005237 Helen Hay Whitney Foundation to Tristan A Bell.

http://dx.doi.org/10.13039/100000057 National Institute of General Medical Sciences R35GM142553 to Luke H Chao.

Acknowledgements

We thank D Syau (Harvard Medical School) and members of the Chao lab for helpful feedback, and Z Li, S Sterling, R Walsh, M Mayer, and S Rawson from the Harvard Medical School Cryo-EM Center for support with cryo-TEM data collection. We are grateful to Dr. Richard Bouley at the Microscopy Core of the Program in Membrane Biology (PMB) at Massachusetts General Hospital for providing helpful advice and equipment access for live-cell confocal microscopy. We thank the staff at the Center for Regenerative Medicine (CRM) Flow Cytometry Core Facility for helpful support in cytometry sorting and analysis. This work was supported by National Institutes of Health R35GM142553 (to LHC) and the Helen Hay Whitney Foundation (to TAB). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Additional information

Competing interests

Author contributions

Additional files

MDAR checklist

Supplementary file 1. Prom1 mutants used in this study.

Data availability

Electron microscopy datasets and all biochemical data are deposited in a Zenodo repository. Custom software packages are available at GitHub under a GNU General Public License v3.0: vesicle-quant, vesicle-quantification, and bead-assay. Materials generated in this study will be made available on request.

The following dataset was generated:

Bell TA 2023 Electron Microscopy Data for Quantification of Extracellular Vesicles Zenodo 10.5281/zenodo.10034615

10.7554/eLife.100061.sa0
Editor's evaluation
Motaung Thabiso Reviewing Editor https://ror.org/00g0p6g84 University of Pretoria South Africa

This work is important because it significantly advances our understanding of membrane protein functions by establishing the mechanisms by which Prominin-1 and Tweety Homology-1 induce extracellular vesicle (EV) formation. The evidence supporting these findings is compelling as it features rigorous biochemical assays and thorough experimental validation. The study will therefore be of particular interest to researchers in cell biology and membrane dynamics, particularly to researchers with a keen interest in EV formation and its implications for cellular processes.

10.7554/eLife.100061.sa1
Decision letter
Motaung Thabiso Reviewing Editor https://ror.org/00g0p6g84 University of Pretoria South Africa

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

[Editors' note: this paper was reviewed by Review Commons.]

10.7554/eLife.100061.sa2
Author response
Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Summary

Bell et al. overexpress Prom1 or Ttyh1 and test its effect on EV formation from cell lines. They find that Ttyh1 expression leads to an increase in small EVs as well as tubulated EVs, while Prom1 expression leads to a milder increase in small EVs. EV induction by Prom1 is dependent on cholesterol and the authors show that Prom1 makes the cholesterol in EVs more resistant to detergent. The authors show no connection between Ttyh1 EV induction and cholesterol, although they claim it is important. They also show that a disease mutation in Prom1 decreases Prom1 trafficking to the plasma membrane and increases cholesterol resistance to detergent in EVs. The authors also find that the disease mutation decreases the size of the Prom1-induced EVs.

Major Comments

Results – line 99-106 – The EV isolation protocol would remove large EVs like the Prom1+ midbody remnants. It is important to explicitly specify that this study focused on small EVs.

We agree with the reviewers and appreciate the suggestion to make this distinction. We have clarified the Results text (lines 104-105) to specify that our method specifically reconstitutes and isolates small EVs.

Statistics – The t tests appear to have been performed without correction for multiple comparisons (Figure 2C-D, Figure 4D). Given that >10 comparisons were made, this can alter the biological significance of p<0.05 (1 incorrect in 20 comparisons). Please reanalyze with a more appropriate statistical test for multiple comparisons (i.e. ANOVA) or apply a correction to the t test values (i.e. Bonferroni).

We agree with the reviewers that multiple test correction is appropriate for these figures. We have applied Bonferroni correction to the t-tests in Figures2C, 2D, and 4D by adjusting our significance thresholds (α), and included additional text in the figure legend to indicate how and why the correction was performed.

The DLS data does not appear to give any insight into EV size (unlike the EM data) and could be removed from the whole manuscript (or moved to supplemental). The authors should also remove any conclusions based on the DLS data.

We appreciate the reviewers raising this point and agree that the DLS is less informative than our other measurements of EV size and morphology. We have moved all DLS figure panels where EV size is characterized by another method to the Supplement.

Discussion – line 382-383 "Because Prom1 EVs arise directly from blebbing of the plasma membrane23, this finding suggests that Prom1 and Ttyh1 traffic to similar regions of the plasma membrane."

The authors have not examined where Prom1 or Ttyh1 localize in the plasma membrane and can not draw this conclusion. That both proteins promote plasma membrane budding would only suggest that both proteins localize to the plasma membrane, not subregions of the plasma membrane. However, the authors have not demonstrated that Ttyh1 specifically induces plasma membrane budding. The different size of Ttyh1 EVs could be due to different biogenesis mechanisms (i.e. derived from intracellular organelles instead of the plasma membrane), making this statement an over-interpretation on both parts.

This is a fair point. We have removed this sentence from the Discussion (lines 402-403) as the reviewer requests.

Discussion – line 398-400 "Membrane cholesterol is necessary for Prom1-mediated remodeling20,21 and is present at similar levels in purified Prom1 and Ttyh1 EVs (Figure 5E), indicating that it is undoubtedly important for EV formation by both proteins." & line 415-417 "We find that conservative mutations in several of these adjacent aromatic residues impair EV formation by Prom1, but do not mimic the stable cholesterol binding of W795R (Figures2C, 4D). "

The author's data suggests that cholesterol is not important for Ttyh1 to induce EV formation. The authors show that cholesterol depletion does not alter Ttyh1 EV production. Similarly, they find separable effects on cholesterol binding and EV formation with Prom1 mutants, which suggest that there is more to Prom1-mediated EV formation than cholesterol. That cholesterol is present at similar levels can reflect that overexpression of these proteins does not alter the amount of cholesterol in the EV source membrane (i.e. plasma membrane). Also, wouldn't molecular crowding of a membrane protein be predicted to influence how easy it is to extract lipids?

We thank the reviewer for highlighting this imprecisely phrased sentence. We only meant to indicate that cholesterol is present in both sets of EVs and contributes globally to membrane fluidity. We have removed this sentence from the Discussion (lines 419-421) to avoid over-interpretation or confusion.

The reviewer is also correct to point out that molecular crowding could alter how extractable lipids are from EVs. We have included additional explanatory text in the Discussion (lines 421-426) addressing this point.

Discussion – line 431-433 "Our findings suggest that the dynamic interaction of Prom1 with cholesterol may promote efficient maturation and trafficking of Prom1 between the endomembrane system and the plasma membrane.

The authors did not investigate whether depleting cholesterol improved Prom1(W795R) trafficking to the plasma membrane, making this inference untested. Soften interpretation or test experimentally.

We appreciate the reviewer raising this point. We have altered the text in this paragraph (lines449-459) to soften our interpretation of these results, as suggested by the reviewer.

Minor Comments

Abstract – "the EVs produced are biophysically similar"

The authors don't perform any typical biophysical characterization (beyond size and perhaps density), so do they mean physically similar? Given the Prom1 and Ttyh1 EVs can have different shapes and are significantly different sizes, this statement feels misleading.

We thank the reviewer for pointing out the ambiguity around this word. We agree that “physically similar” is a more precise and accurate term, and have revised all instances of this language in the manuscript.

Intro – line 59-60 – "Large Prom1 EVs (500-700 nm in diameter) appear to form from bulk release of membrane from the cell midbody"

Midbody remnants are well defined (if variously named, i.e. flemmingsome) large EVs derived from the spindle midbody, intercellular bridge, and cytokinetic ring. I'm not sure what the authors are trying to express by "bulk release of membrane". Midbody remnants are also a site of membrane tubulation.

The reviewer is correct to point out that midbody remnant release is a well defined process. We originally included this statement to avoid indicating that we are studying the only known class of Prominin EVs, but now recognize that including this creates more confusion that it alleviates. To improve clarity concurrently with the changes referenced above emphasizing that we are specifically studying small EVs, we have removed this reference to the larger class of EVs from the introduction (lines 61-63).

The effect on total numbers of EVs is buried in the y-axes of the EM graphs, making it difficult to distinguish where a higher n of images was examined vs. where there is an increase in EVs. This is especially hard to interpret given the high difference in n values.

The reviewers raise a valid critique of these figure panels. To improve clarity, we have adjusted the y-axes to represent the fraction of EVs rather than the absolute value of EVs, and listed the n values in figure legends.

Figure 2C – Missing WT error bars

We appreciate the reviewer’s concern for the WT error bars in these figures. The measurements underlying these plots are derived from quantification of Western blots. Because the blots have a limited number of lanes, the WT sample was run as a normalization control on each of several sets of blots. By employing this approach, we could make quantitative comparisons within each blot without needing to make direct comparisons between blots, eliminating confounding variables such as blotting times, positions of blots on rotary shakers, developer incubation time, exposure times, etc. Because WT lanes were used for normalization, each “WT” blot condition has its own set of error bars that was used for t-test comparison with the samples that share a blot. For this purely technical reason, we can represent the data either normalized against WT values or with three separate WT measurements for each plot. In the interest of clarity and transparency, we elected to report the values normalized to WT and to include all raw blot images in Supplementary Figure S4. We understand that we could have made this more transparent, so to clarify this decision for readers, we now explicitly reference the raw blot images in both the Results text (lines 185) and in the Figure 2 legend.

Figure 3H, 5C – Why not show raw numbers on the y-axes of the inset graphs like the main graph? Also, if it is only showing a subset of roundness ranges, then the x-axis should not go to 1 (i.e. axis range 0-0.8 would be clearer). I had a hard time figuring out what these insets were trying to show me, so please think about presenting this data more clearly (and larger).

For clarity, we have moved the inset graphs to separate panels alongside the main panel and implemented the requested changes to the axes (see Figures 3G, 5B).

Discussion – line 377 – "Though we do not claim that Ttyh1 endogenously induces EV formation"

This statement could be misinterpreted to say that you do not think endogenous Ttyh1 regulates EV formation. Rephrase as "although we have not examined whether…"

We thank the reviewer for pointing out this unclear sentence and have applied the requested change (line 397).

Discussion – line 400-402 "Our results do not indicate that Ttyh1 does not bind cholesterol, merely that it does not form an interaction that is sufficiently kinetically stable to be co-immunoprecipitated."

The phrasing here is confusing with multiple "not". It is better to leave things open than to say what you have not shown. Rephrase suggestion: "Although Ttyh1 was not able to form a kinetically stable interaction for co-immunoprecipitation, it remains to be determined whether Ttyh1 is able to bind cholesterol."

We thank the reviewer for their suggestion and have modified the sentence to avoid double-negative phrasing (lines 422-426).

Videos – I'm not sure what the two videos add. It's difficult to convince myself that I see plasma membrane labeling in either video, especially in comparison to the over-exposed WGA staining. Also, why are there ~5 sec of empty movie at the end of each?

We appreciate the reviewer’s feedback and have removed the videos from the manuscript.

Reviewer #1 (Significance (Required)):The data is interesting and well presented, but over interpreted in the discussion.

The data on Ttyh1 expression inducing EVs is novel, but limited to overexpression studies.

This study will be of interest to the EV, membrane curvature, and Prmn1/Tthy1 fields

My expertise is in basic research on membrane trafficking (including EV formation) and lipids

We thank the reviewer for their favorable review and helpful suggestions.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

In this study, authors investigated the role of Prom1 and Ttyh1 proteins on EV formation. They showed that both proteins can induce EV formation, while the mechanisms by which they do it might differ slightly. Ttyh1 binding to cholesterol is not as pronounced as Prom1. Surprisingly, cholesterol binding efficiency inversely correlates with EV formation. Also, EVs induced by Tthy1 and Prom1 are structurally different.

My suggestions to improve the manuscript are below.

- Figure 2E is not very convincing. As the authors mentioned, the signal is too low to have a concrete conclusion. The line scans somehow show that WT is more membrane-localized than mutant, but colocalization of Prom1 and WGA seems very similar in both cases. Is it certain that the addition of fluorophore did not change the trafficking? Does endogenous Prom-1 staining look like this? Also, why is WGA staining brighter in mutant sample, just a usual variation or biologically important?

We understand the reviewer’s concern about low signal, but respectfully disagree that the signal is too low to draw a meaningful conclusion. The only point we conclusively make in Figure 2E is that WT Prom1 is more efficiently trafficked to the plasma membrane than W795R Prom1. We feel that this effect is sufficiently well evidenced by the line scan analysis in Supp. Figure S5, where Prom1 peaks are cleanly visible for WT but not for W795R protein.

We observe somewhat variable WGA staining in our experiments, and the differences we show in this figure panel are representative of typical staining variation. We do not draw any biological conclusions from the level of WGA present, only from its localization. Because both the plasma membrane and late endosomes are WGA+, we suspect that the W795R Prom1 is failing to traffic from endosomes to the plasma membrane. However, given the limitations of our fluorescence assay, we have removed any claim beyond the change plasma membrane trafficking efficiency from discussion of this experiment.

We cannot conclude whether the mStayGold fluorophore alters trafficking of Prom1 to the plasma membrane. In response to the reviewer’s comment, we attempted to use immunofluorescence to measure membrane localization of untagged Prom1 with the AC133-1 antibody. Unfortunately, we were unable to optimize this protocol to achieve sufficient membrane staining for quantification. We have softened our interpretation of Figure 2E in the Results and Discussion (lines 203-204, 450) to acknowledge that the effects we observe are only measured with fluorophore-tagged Prom1.

- I also recommend showing the localization of Ttyh1 on cells.

We appreciate the reviewer’s suggestion here, and it is an experiment we considered. One of the challenges we faced in this assay was quantitatively measuring fluorescent signal along cell-boundary plasma membranes without saturating signal from the very bright WGA+ endosomes. Because Ttyh1 globally expresses at higher levels than Prom1 (see Figures 3C, 3I), direct comparison of membrane-localized Prom1 and Ttyh1 is technically challenging in these cells. However, Ttyh membrane localization has been widely reported in other papers (Matthews et al., J. Neurochem, 2007; Jung et al., J. Neurosci., 2017; Sukalskaia et al., Nat. Commun., 2021; Melvin et al., Comm. Biol., 2022) that we now explicitly mention and cite for reader clarity in both the Introduction and Results (lines 69-71, 224-225).

- A graph directly showing cholesterol binding vs EV formation efficiency would be very useful.

We agree with the reviewer that this would be an interesting and useful addition to the paper. We now include this panel in the revised manuscript as Figure 4F.

- "Prominin and Tweety homology proteins are homologous and functionally analogous" involves speculation and authors should clearly mention this. Revealing that they are both contributing to EV formation does not make them definitely functionally analogous.

We agree with the reviewer that this sentence is indeed ambiguous and somewhat speculative. We have revised the section heading to “Prominin and Tweety homology proteins are homologous proteins that both promote EV formation” (lines 461-462) to indicate the specific analogous function we observe.

Reviewer #2 (Significance (Required)):

Overall, it is a useful addition to the field of cell biology, particularly EV field. EV formation and efficiency are both important topics, and this manuscript might give insights.

We thank the reviewer for their favorable review and helpful suggestions.

No competing interests declared.

Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing.

Conceptualization, Resources, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing.

Conceptualization, Resources, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing.

Resources, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – review and editing.

Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing – review and editing.

Resources, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing – review and editing.

Resources, Data curation, Validation, Investigation, Visualization, Methodology, Writing – review and editing.

Conceptualization, Resources, Supervision, Funding acquisition, Project administration, Writing – review and editing.
==== Refs
References

Anisimova M Gascuel O 2006 Approximate likelihood-ratio test for branches: A fast, accurate, and powerful alternative Systematic Biology 55 539 552 10.1080/10635150600755453 16785212
Bae Y Kim A Cho CH Kim D Jung HG Kim SS Yoo J Park JY Hwang EM 2019 TTYH1 and TTYH2 serve as LRRC8A-independent volume-regulated anion channels in cancer cells Cells 8 562 10.3390/cells8060562 31181821
Bell T 2023 Vesicle-quantification swh:1:rev:032952ba5eadf5584504399696993333b9392534 Software Heritage https://archive.softwareheritage.org/swh:1:dir:f728508e242186efa6be4546dcaac7655dfca9ba;origin=https://github.com/tribell4310/vesicle-quantification;visit=swh:1:snp:55235a357950e508d5f67aaf0def5d3c7d92a878;anchor=swh:1:rev:032952ba5eadf5584504399696993333b9392534
Bell T 2024a Vesicle-quant swh:1:rev:cf1e8e7d37420d36bf4a10400f8e1272b313455d Software Heritage https://archive.softwareheritage.org/swh:1:dir:3a37faa65ac020d433d3ae387e3ec466f725fdcf;origin=https://github.com/tribell4310/vesicle-quant;visit=swh:1:snp:54320f5ceb193b477496dc4dcaa7bc78ce4146e2;anchor=swh:1:rev:cf1e8e7d37420d36bf4a10400f8e1272b313455d
Bell T 2024b Bead-assay swh:1:rev:78ab70b5106eeb20226401e3a1f21304c3128f8a Software Heritage https://archive.softwareheritage.org/swh:1:dir:1a7185d4e0199dbf117c44ff96cb7942fbd9c73d;origin=https://github.com/tribell4310/bead-assay;visit=swh:1:snp:025037dfcfb8207cfd9b66056a326563d6398524;anchor=swh:1:rev:78ab70b5106eeb20226401e3a1f21304c3128f8a
Boulanger-Scemama E El Shamieh S Démontant V Condroyer C Antonio A Michiels C Boyard F Saraiva JP Letexier M Souied E Mohand-Saïd S Sahel JA Zeitz C Audo I 2015 Next-generation sequencing applied to a large French cone and cone-rod dystrophy cohort: mutation spectrum and new genotype-phenotype correlation Orphanet Journal of Rare Diseases 10 85 10.1186/s13023-015-0300-3 26103963
Brown DA London E 2000 Structure and function of sphingolipid- and cholesterol-rich membrane rafts The Journal of Biological Chemistry 275 17221 17224 10.1074/jbc.R000005200 10770957
Burgoyne T Meschede IP Burden JJ Bailly M Seabra MC Futter CE 2015 Rod disc renewal occurs by evagination of the ciliary plasma membrane that makes cadherin-based contacts with the inner segment PNAS 112 15922 15927 10.1073/pnas.1509285113 26668363
Campbell HD Kamei M Claudianos C Woollatt E Sutherland GR Suzuki Y Hida M Sugano S Young IG 2000 Human and mouse homologues of the Drosophila melanogaster tweety (tty) gene: A novel gene family encoding predicted transmembrane proteins Genomics 68 89 92 10.1006/geno.2000.6259 10950931
Clamp M Cuff J Searle SM Barton GJ 2004 The jalview java alignment editor Bioinformatics 20 426 427 10.1093/bioinformatics/btg430 14960472
Corbeil D Röper K Weigmann A Huttner WB 1998 AC133 hematopoietic stem cell antigen: human homologue of mouse kidney prominin or distinct member of a novel protein family? Blood 91 2625 2626 9516170
Corbeil D Marzesco AM Wilsch-Bräuninger M Huttner WB 2010 The intriguing links between prominin-1 (CD133), cholesterol-based membrane microdomains, remodeling of apical plasma membrane protrusions, extracellular membrane particles, and (neuro)epithelial cell differentiation FEBS Letters 584 1659 1664 10.1016/j.febslet.2010.01.050 20122930
Corbeil D Karbanová J Fargeas CA Jászai J 2013 Prominin-1 (CD133): molecular and cellular features across species Corbeil D In Prominin-1 (CD133): New Insights on Stem & Cancer Stem Cell Biology Advances in Experimental Medicine and Biology Springer 3 24 10.1007/978-1-4614-5894-4_1
Derganc J Čopič A 2016 Membrane bending by protein crowding is affected by protein lateral confinement Biochimica et Biophysica Acta - Biomembranes 1858 1152 1159 10.1016/j.bbamem.2016.03.009
Dubreuil V Marzesco AM Corbeil D Huttner WB Wilsch-Bräuninger M 2007 Midbody and primary cilium of neural progenitors release extracellular membrane particles enriched in the stem cell marker prominin-1 The Journal of Cell Biology 176 483 495 10.1083/jcb.200608137 17283184
Eidinger O Leibu R Newman H Rizel L Perlman I Ben-Yosef T 2015 An intronic deletion in the PROM1 gene leads to autosomal recessive cone-rod dystrophy Molecular Vision 21 1295 1306 26702251
Elegheert J Behiels E Bishop B Scott S Woolley RE Griffiths SC Byrne EFX Chang VT Stuart DI Jones EY Siebold C Aricescu AR 2018 Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins Nature Protocols 13 2991 3017 10.1038/s41596-018-0075-9 30455477
Fantini J Barrantes FJ 2013 How cholesterol interacts with membrane proteins: an exploration of cholesterol-binding sites including CRAC, CARC, and tilted domains Frontiers in Physiology 4 31 10.3389/fphys.2013.00031 23450735
Fargeas CA Huttner WB Corbeil D 2007 Nomenclature of prominin-1 (CD133) splice variants - an update Tissue Antigens 69 602 606 10.1111/j.1399-0039.2007.00825.x 17498271
Fargeas CA 2013 Prominin-2 and other relatives of CD133 Corbeil D In Prominin-1 (CD133): New Insights on Stem & Cancer Stem Cell Biology Advances in Experimental Medicine and Biology Springer 25 40 10.1007/978-1-4614-5894-4_2
Halleran AD Sehdev M Rabe BA Huyck RW Williams CC Saha MS 2015 Characterization of tweety gene (ttyh1-3) expression in Xenopus laevis during embryonic development Gene Expression Patterns 17 38 44 10.1016/j.gep.2014.12.002 25541457
Han YE Kwon J Won J An H Jang MW Woo J Lee JS Park MG Yoon BE Lee SE Hwang EM Jung JY Park H Oh SJ Lee CJ 2019 Tweety-homolog (Ttyh) family encodes the pore-forming subunits of the swelling-dependent volume-regulated anion channel (VRACswell) in the brain Experimental Neurobiology 28 183 215 10.5607/en.2019.28.2.183 31138989
Hori A Nishide K Yasukuni Y Haga K Kakuta W Ishikawa Y Hayes MJ Ohnuma SI Kiyonari H Kimura K Kondo T Sasai N 2019 Prominin-1 modulates Rho/ROCK-mediated membrane morphology and calcium-dependent intracellular chloride flux Scientific Reports 9 15911 10.1038/s41598-019-52040-9 31685837
Hurbain I Macé AS Romao M Prince E Sengmanivong L Ruel L Basto R Thérond PP Raposo G D’Angelo G 2022 Microvilli-derived extracellular vesicles carry hedgehog morphogenic signals for Drosophila wing imaginal disc development Current Biology 32 361 373 10.1016/j.cub.2021.11.023 34890558
Huttner WB Schiebler W Greengard P De Camilli P 1983 Synapsin I (protein I), a nerve terminal-specific phosphoprotein. III. Its association with synaptic vesicles studied in a highly purified synaptic vesicle preparation The Journal of Cell Biology 96 1374 1388 10.1083/jcb.96.5.1374 6404912
Jumper J Evans R Pritzel A Green T Figurnov M Ronneberger O Tunyasuvunakool K Bates R Žídek A Potapenko A Bridgland A Meyer C Kohl SAA Ballard AJ Cowie A Romera-Paredes B Nikolov S Jain R Adler J Back T Petersen S Reiman D Clancy E Zielinski M Steinegger M Pacholska M Berghammer T Bodenstein S Silver D Vinyals O Senior AW Kavukcuoglu K Kohli P Hassabis D 2021 Highly accurate protein structure prediction with AlphaFold Nature 596 583 589 10.1038/s41586-021-03819-2 34265844
Jung E Osswald M Blaes J Wiestler B Sahm F Schmenger T Solecki G Deumelandt K Kurz FT Xie R Weil S Heil O Thomé C Gömmel M Syed M Häring P Huber PE Heiland S Platten M von Deimling A Wick W Winkler F 2017 Tweety-homolog 1 drives brain colonization of gliomas The Journal of Neuroscience 37 6837 6850 10.1523/JNEUROSCI.3532-16.2017 28607172
Kalyaanamoorthy S Minh BQ Wong TKF von Haeseler A Jermiin LS 2017 ModelFinder: fast model selection for accurate phylogenetic estimates Nature Methods 14 587 589 10.1038/nmeth.4285 28481363
Katoh K Misawa K Kuma K Miyata T 2002 MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform Nucleic Acids Research 30 3059 3066 10.1093/nar/gkf436 12136088
Kemper K Sprick MR de Bree M Scopelliti A Vermeulen L Hoek M Zeilstra J Pals ST Mehmet H Stassi G Medema JP 2010a The AC133 epitope, but not the CD133 protein, is lost upon cancer stem cell differentiation Cancer Research 70 719 729 10.1158/0008-5472.CAN-09-1820 20068153
Kemper K Tol MJPM Medema JP 2010b Mouse tissues express multiple splice variants of prominin-1 PLOS ONE 5 e12325 10.1371/journal.pone.0012325 20808829
Kutner RH Zhang XY Reiser J 2009 Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors Nature Protocols 4 495 505 10.1038/nprot.2009.22 19300443
Li B Hoel CM Brohawn SG 2021 Structures of tweety homolog proteins TTYH2 and TTYH3 reveal a Ca2+-dependent switch from intra- to inter-membrane dimerization bioRxiv 10.1101/2021.08.15.456437
Liang J She X Chen J Zhai Y Liu Y Zheng K Gong Y Zhu H Luo X Sun X 2019 Identification of novel PROM1 mutations responsible for autosomal recessive maculopathy with rod-cone dystrophy Graefe’s Archive for Clinical and Experimental Ophthalmology = Albrecht von Graefes Archiv Fur Klinische Und Experimentelle Ophthalmologie 257 619 628 10.1007/s00417-018-04206-w 30588538
Livingstone CD Barton GJ 1993 Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation Bioinformatics 9 745 756 10.1093/bioinformatics/9.6.745
Lorent JH Levental KR Ganesan L Rivera-Longsworth G Sezgin E Doktorova M Lyman E Levental I 2020 Plasma membranes are asymmetric in lipid unsaturation, packing and protein shape Nature Chemical Biology 16 644 652 10.1038/s41589-020-0529-6 32367017
Mak AB Nixon AML Kittanakom S Stewart JM Chen GI Curak J Gingras A-C Mazitschek R Neel BG Stagljar I Moffat J 2012 Regulation of CD133 by HDAC6 promotes β-catenin signaling to suppress cancer cell differentiation Cell Reports 2 951 963 10.1016/j.celrep.2012.09.016 23084749
Marzesco AM Janich P Wilsch-Bräuninger M Dubreuil V Langenfeld K Corbeil D Huttner WB 2005 Release of extracellular membrane particles carrying the stem cell marker prominin-1 (CD133) from neural progenitors and other epithelial cells Journal of Cell Science 118 2849 2858 10.1242/jcs.02439 15976444
Mastronarde DN 2005 Automated electron microscope tomography using robust prediction of specimen movements Journal of Structural Biology 152 36 51 10.1016/j.jsb.2005.07.007 16182563
Matthews CA Shaw JE Hooper JA Young IG Crouch MF Campbell HD 2007 Expression and evolution of the mammalian brain gene Ttyh1 Journal of Neurochemistry 100 693 707 10.1111/j.1471-4159.2006.04237.x 17116230
Maw MA Corbeil D Koch J Hellwig A Wilson-Wheeler JC Bridges RJ Kumaramanickavel G John S Nancarrow D Röper K Weigmann A Huttner WB Denton MJ 2000 A frameshift mutation in prominin (mouse)-like 1 causes human retinal degeneration Human Molecular Genetics 9 27 34 10.1093/hmg/9.1.27 10587575
Melvin E Kalaninová Z Shlush E Man P Giladi M Haitin Y 2022 TTYH family members form tetrameric complexes at the cell membrane Communications Biology 5 886 10.1038/s42003-022-03862-3 36042377
Minh BQ Schmidt HA Chernomor O Schrempf D Woodhams MD von Haeseler A Lanfear R 2020 IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era Molecular Biology and Evolution 37 1530 1534 10.1093/molbev/msaa015 32011700
Miraglia S Godfrey W Yin AH Atkins K Warnke R Holden JT Bray RA Waller EK Buck DW 1997 A novel five-transmembrane hematopoietic stem cell antigen: isolation, characterization, and molecular cloning Blood 90 5013 5021 9389721
Paysan-Lafosse T Blum M Chuguransky S Grego T Pinto BL Salazar GA Bileschi ML Bork P Bridge A Colwell L Gough J Haft DH Letunić I Marchler-Bauer A Mi H Natale DA Orengo CA Pandurangan AP Rivoire C Sigrist CJA Sillitoe I Thanki N Thomas PD Tosatto SCE Wu CH Bateman A 2023 InterPro in 2022 Nucleic Acids Research 51 D418 D427 10.1093/nar/gkac993 36350672
Rattner A Smallwood PM Williams J Cooke C Savchenko A Lyubarsky A Pugh EN Nathans J 2001 A photoreceptor-specific cadherin is essential for the structural integrity of the outer segment and for photoreceptor survival Neuron 32 775 786 10.1016/s0896-6273(01)00531-1 11738025
Ridsdale A Denis M Gougeon PY Ngsee JK Presley JF Zha X 2006 Cholesterol is required for efficient endoplasmic reticulum-to-Golgi transport of secretory membrane proteins Molecular Biology of the Cell 17 1593 1605 10.1091/mbc.e05-02-0100 16452637
Röper K Corbeil D Huttner WB 2000 Retention of prominin in microvilli reveals distinct cholesterol-based lipid micro-domains in the apical plasma membrane Nature Cell Biology 2 582 592 10.1038/35023524 10980698
Salinas RY Pearring JN Ding JD Spencer WJ Hao Y Arshavsky VY 2017 Photoreceptor discs form through peripherin-dependent suppression of ciliary ectosome release The Journal of Cell Biology 216 1489 1499 10.1083/jcb.201608081 28381413
Satir P Christensen ST 2007 Overview of structure and function of mammalian cilia Annual Review of Physiology 69 377 400 10.1146/annurev.physiol.69.040705.141236 17009929
Sayers EW Bolton EE Brister JR Canese K Chan J Comeau DC Connor R Funk K Kelly C Kim S Madej T Marchler-Bauer A Lanczycki C Lathrop S Lu Z Thibaud-Nissen F Murphy T Phan L Skripchenko Y Tse T Wang J Williams R Trawick BW Pruitt KD Sherry ST 2022 Database resources of the national center for biotechnology information Nucleic Acids Research 50 D20 D26 10.1093/nar/gkab1112 34850941
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B Tinevez J-Y White DJ Hartenstein V Eliceiri K Tomancak P Cardona A 2012 Fiji: an open-source platform for biological-image analysis Nature Methods 9 676 682 10.1038/nmeth.2019 22743772
Schneider CA Rasband WS Eliceiri KW 2012 NIH Image to ImageJ: 25 years of image analysis Nature Methods 9 671 675 10.1038/nmeth.2089 22930834
Sharkova M Chow E Erickson T Hocking JC 2023 The morphological and functional diversity of apical microvilli Journal of Anatomy 242 327 353 10.1111/joa.13781 36281951
Sonnhammer EL von Heijne G Krogh A 1998 A hidden Markov model for predicting transmembrane helices in protein sequences Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology 6 175 182 9783223
Stefaniuk M Swiech L Dzwonek J Lukasiuk K 2010 Expression of Ttyh1, a member of the Tweety family in neurons in vitro and in vivo and its potential role in brain pathology Journal of Neurochemistry 115 1183 1194 10.1111/j.1471-4159.2010.07023.x 20874767
Stüven E Porat A Shimron F Fass E Kaloyanova D Brügger B Wieland FT Elazar Z Helms JB 2003 Intra-Golgi protein transport depends on a cholesterol balance in the lipid membrane The Journal of Biological Chemistry 278 53112 53122 10.1074/jbc.M300402200 14561753
Sukalskaia A Straub MS Deneka D Sawicka M Dutzler R 2021 Cryo-EM structures of the TTYH family reveal a novel architecture for lipid interactions Nature Communications 12 4893 10.1038/s41467-021-25106-4 34385445
Thamm K Šimaitė D Karbanová J Bermúdez V Reichert D Morgenstern A Bornhäuser M Huttner WB Wilsch-Bräuninger M Corbeil D 2019 Prominin-1 (CD133) modulates the architecture and dynamics of microvilli Traffic 20 39 60 10.1111/tra.12618 30328220
Vaughan TG 2017 IcyTree: rapid browser-based visualization for phylogenetic trees and networks Bioinformatics 33 2392 2394 10.1093/bioinformatics/btx155 28407035
Wiernasz E Kaliszewska A Brutkowski W Bednarczyk J Gorniak M Kaza B Lukasiuk K 2014 Ttyh1 protein is expressed in glia in vitro and shows elevated expression in activated astrocytes following status epilepticus Neurochemical Research 39 2516 2526 10.1007/s11064-014-1455-3 25316497
Wood CR Rosenbaum JL 2015 Ciliary ectosomes: transmissions from the cell’s antenna Trends in Cell Biology 25 276 285 10.1016/j.tcb.2014.12.008 25618328
Yang Z Chen Y Lillo C Chien J Yu Z Michaelides M Klein M Howes KA Li Y Kaminoh Y Chen H Zhao C Chen Y Al-Sheikh YT Karan G Corbeil D Escher P Kamaya S Li C Johnson S Frederick JM Zhao Y Wang C Cameron DJ Huttner WB Schorderet DF Munier FL Moore AT Birch DG Baehr W Hunt DM Williams DS Zhang K 2008 Mutant prominin 1 found in patients with macular degeneration disrupts photoreceptor disk morphogenesis in mice The Journal of Clinical Investigation 118 2908 2916 10.1172/JCI35891 18654668
Yin AH Miraglia S Zanjani ED Almeida-Porada G Ogawa M Leary AG Olweus J Kearney J Buck DW 1997 AC133, a novel marker for human hematopoietic stem and progenitor cells Blood 90 5002 5012 9389720
Zacchigna S Oh H Wilsch-Bräuninger M Missol-Kolka E Jászai J Jansen S Tanimoto N Tonagel F Seeliger M Huttner WB Corbeil D Dewerchin M Vinckier S Moons L Carmeliet P 2009 Loss of the cholesterol-binding protein prominin-1/CD133 causes disk dysmorphogenesis and photoreceptor degeneration The Journal of Neuroscience 29 2297 2308 10.1523/JNEUROSCI.2034-08.2009 19228982
Zidovetzki R Levitan I 2007 Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies Biochimica et Biophysica Acta 1768 1311 1324 10.1016/j.bbamem.2007.03.026 17493580
