Phosphoinositide-binding proteins mark, shape and functionally modulate highly-diverged endocytic compartments in the parasitic protist Giardia lamblia.

Phosphorylated derivatives of phosphatidylinositol (PIPs) are key membrane lipid residues involved in clathrin-mediated endocytosis (CME). CME relies on PIP species PI(4,5)P2 to mark endocytic sites at the plasma membrane (PM) associated to clathrin-coated vesicle (CCV) formation. The highly diverged parasitic protist Giardia lamblia presents disordered and static clathrin assemblies at PM invaginations, contacting specialized endocytic organelles called peripheral vacuoles (PVs). The role for clathrin assemblies in fluid phase uptake and their link to internal membranes via PIP-binding adaptors is unknown. Here we provide evidence for a robust link between clathrin assemblies and fluid-phase uptake in G. lamblia mediated by proteins carrying predicted PX, FYVE and NECAP1 PIP-binding modules. We show that chemical and genetic perturbation of PIP-residue binding and turnover elicits novel uptake and organelle-morphology phenotypes. A combination of co-immunoprecipitation and in silico analysis techniques expands the initial PIP-binding network with addition of new members. Our data indicate that, despite the partial conservation of lipid markers and protein cohorts known to play important roles in dynamic endocytic events in well-characterized model systems, the Giardia lineage presents a strikingly divergent clathrin-centered network. This includes several PIP-binding modules, often associated to domains of currently unknown function that shape and modulate fluid-phase uptake at PVs.

Introduction

Phosphorylated derivatives of the minor membrane phospholipid phosphatidylinositols (PIPs) are surface molecules of most eukaryotic endomembrane compartments [1-3]. PIPs play important roles in diverse pathways including signaling cascades, autophagy and membrane remodeling [2, 4–8]. Their diverse functions are reflected in their distinct subcellular distribution. PI(4,5)P2 is highly enriched at the plasma membrane (PM) together with PI(3,4,5)P3 [4, 5]. PtdIns(4)P’s largest pool is at Golgi membranes, with lower density found at the PM. PI(3)P is converted into PI(3,5)P2 on early endosomes during transition to multivesicular bodies and then late endosomes [6, 7]. PI(3)P is also a marker of phagosomes [8] while PI(5)P marks both the PM and endomembranes [9]. At least 14 distinct PIP-binding modules have been identified in eukaryotes, demonstrating a wide range of selective protein-lipid interactions associated with the PM and internal membranes [10].

In addition to their structural functions in membranes, in model eukaryotes PIPs are involved in spatiotemporal organization of membrane remodeling processes such as clathrin-coated vesicle (CCV) formation during clathrin-mediated endocytosis (CME). In particular, PI(4,5)P2 marks sites of endocytosis at the PM and recruits proteins involved in the formation of CCVs [11]. The protein interactomes of mammalian PI(4,5)P2-binding proteins include the early-acting clathrin interacting partners AP2 [12-15], AP180/CALM [16, 17] and epsin [17, 18]. These factors carry specific PIP-binding domains that can discriminate between PIP variants to achieve membrane targeting specificity.

Giardia lamblia (syn. intestinalis, duodenalis) is a widespread parasitic protist that colonizes the upper small intestine of vertebrate hosts. Its life cycle is marked by the alternation of an environmentally-resistant, infectious cyst stage responsible for parasite transmission, and a trophozoite stage proliferating by binary fission. Nutrient uptake of trophozoites in the lumen of the small intestine is almost entirely routed through peripheral vacuoles (PVs). These organelles are positioned just beneath the PM and are contacted by funnel-shaped invaginations of the PM that are likely conduits for uptake of fluid-phase extracellular material [19].

A recent characterization of the PV protein interactome, using the conserved G. lamblia clathrin heavy chain (GlCHC) as affinity handle, confirmed the endocytic nature of these organelles by highlighting the presence of giardial AP2 (GlAP2) subunits, the single dynamin-like protein GlDRP and a putative clathrin light chain Gl4259 (GlCLC; [19]). Notably absent were components for CCV uncoating and disassembly, consistent with a lack of measurable clathrin assembly turnover and in line with observations that CCVs are missing in G. lamblia and clathrin assemblies are static and long-lived. Therefore, G. lamblia presents an unusual endocytic system, characterized by divergent endocytic compartments (PVs) associated to static clathrin assemblies that are not predicted to form ordered arrays or higher-order structures such as CCVs, yet are closely membrane-associated.

Included in the giardial CHC interactome were three proteins with predicted PIP-binding domains: FYVE domain protein Gl16653 and two PX-domain proteins (Gl7723 and Gl16595), the latter part of a six-member protein family (Table 1; [19, 20]). In a previous study, we hypothesized that Gl16653 (GlFYVE), Gl7723 (GlPXD1) and Gl16595 (GlPXD2) act as PIP-binding adaptors to link and maintain static clathrin assemblies at the PM and PV membrane interface in G. lamblia [19]. We further postulated that a perturbation of PIP-binding protein levels and/or function would lead to impaired fluid-phase uptake by affecting PV functionality. To test these hypotheses, we performed an in-depth functional characterization of all previously-identified PIP-binding proteins associated to clathrin at PVs. We assessed their lipid-binding preferences and visualized their subcellular localizations using electron microscopy and both conventional and super resolution light microscopy. By manipulating protein levels and/or function we could elicit novel fluid-phase uptake and PV morphology-related phenotypes, thereby establishing PIPs as a link between the role of clathrin as a membrane remodeling protein and PV-based endocytosis in G. lamblia. Furthermore, we used a combination of co-immunoprecipitation and in silico annotation techniques to expand protein interactomes established previously, thereby discovering a new set of PIP-binding proteins with roles likely reaching beyond the PV compartment. Lastly, we propose an updated working model summarizing the complex networks between PIP-binding proteins and clathrin assemblies at PVs.

Table 1

G. lamblia PIP-binding proteins.

A compilation of all PIP-binding domains identified in the Giardia Genome Database (www.giardiadb.org; GDB) using previously characterized domains [24] as baits for HMM-based homology searches (column 1). Predicted giardial orthologs are present for PIP-binding domains ENTH, PH, FYVE, PX, BAR, FERM and PROPPINs (column 2) and mostly retrieve the correct domains when used as baits for reverse HHpred searches (column 4). Except for Glepsin, GlPXD2 and GlPROP1 and 2, all others are currently annotated on GDB as generically “hypothetical”, i.e. of unknown function (column 6). Each orthologue was assigned a name used throughout this report (column 7). Functional domain predictions using SMART (http://smart.embl-heidelberg.de/; column 8) and subcellular localization data (column 9) either previously reported or acquired in this study (column 10), are also included.

Domain	G. lamblia orthologs (UniProtKB entry, GDB gene_ID, Probability/	Seq.	3D structure homologue (HHPRED *.pdb, Probability/E-value, Seq. id./sim. (%/%)1, name used by HHPRED	3D structure homologue (I-TASSER *.pdb)2	G. lamblia ortholog annotation on GDB	Name used in this study	Domains (online tool SMART)	Localisation	References
(UniProtKB entry)	E-value	id./sim. (%/%)1
ENTH (1h0a/O88339)	A8BIK9	28/56.1	6enr 100/2.4e-32 23/35 Epsin-1	3onk	EH domain binding protein epsin 2	Glepsin	Pfam: ENTH (4.8e-33)	Ventral disc/PVs	Ebneter 2014, Touz 2015
	GL50803_3256
	100/6e-37
PH	A8BII4 GL50803_17195	36/61.3	1tqz 100/4.4e-52 36/61.3 NECAP-1	1tqz	Hypothetical protein	GlNECAP1	Pfam: DUF1681 (5.5e-43)	PVs/Cytosolic	Zumthor 2016
(1tqz/Q9CR95)	(100/3.6e-55)
FYVE	A8BDZ8 GL50803_16653 (98.17/2.1e-9)	39/92.1	3mpx 92.07/0.11 19/33.4 FYVE/RhoGEF and PH-domain containing protein	1joc	Hypothetical protein	GlFYVE	SMART: FYVE (6.38e-18)	PVs	Sinha 2011, Zumthor 2016
(1hyi/Q15075)	A8B288 GL50803_16801 (89.99/0.031)	28/75.6	3t7l 99.08/3.4e-13 34/96.2 Zinc finger FYVE domain-containing protein	1vfy	Hypothetical protein	Gl16801	FYVE/PHD zinc finger (1.6e-05)	PVs	This study
PX	A8B343 GL50803_7723	20/36.4	3lui 97.19/2.5e-5 20/32.1 SNX-17	4oxw	Hypothetical protein	GlPXD1	SMART: PX (7.2e-11)	PVs	Zumthor 2016, Jana 2017
(1h6h/Q15080)	(96.41/0.00011)
	A8B344 GL50803_16595	18/21.1	10cs 97.64/1.0e-6 23/36.2 SNX GRD19	4on3	Liver stage antigen-like protein	GlPXD2	SMART: PX (6.01e-11)	PVs	Zumthor 2016, Jana 2017
	(99.43/3.8e-15)
	A8B341 GL50803_16596	18/23	4ikb 98.81/2.4e-10 22/38 SNX-11	3iq2	Hypothetical protein	GlPXD3	SMART: PX (2.11e-06)	PVs	Jana 2017
	(99.55/1.7e-16)
	A8B322	21/27.9	4pqo 98.18/2.9e-7 14/20.2 SNX-14	3iq2	Hypothetical protein	GlPXD4	SMART: PX (1.62e-02)	PVs	Jana 2017
	GL50803_42357
	(99.51/6e-16)
	A8BIZ8	17/16.3	4ikb 99.59/2.0e-16 20/32.5 SNX-11	2mxc	Hypothetical protein	GlPXD5	SMART: PX (2.03e-08)	PVs	Jana 2017
	GL50803_16548
	(99.56/1.6–16)
	D3KH98	-3	4ikd 99.67/5.0e-18 15/35.5 SNX-11	1xtn	Hypothetical protein	GlPXD6	SMART: PX (1.89e-02)	PVs	Jana 2017
	GL50803_24488
	(-/-)
BAR	A8BZ00	11/2.8	2v0o 96.41/0.047 11/14.7 FCH domain	2v0o	Hypothetical protein	GlBAR1	-	PVs/Cytosolic	Morrison 2007
(2efl/q96ru3)	GL50803_15487
	(95.09/0.055)
	A8BMB7	10/4.5	1uru 95.53/0.25 16/20.6 Amphiphysin	1uru	Hypothetical protein	GlBAR2	-	PVs/Cytosolic	Morrison 2007
	GL50803_14045
	(95.67/0.027)
FERM (1e5w/P26038)	A8BC43	12/11.1	6d2k 100/2.9e-38 10/7.7 FERM,ERHGEF and PH-containing protein	5mv9	Hypothetical protein	GlFERM	-	Cytosolic	This study
	GL50803_115468
	(100/7.5e-37)
PROPPINs (4exv/q6cn23)	A8B6Z3	21/36.3	3vu4 100/1.2e-36 23/38.1 KmHsv2	4exv	WD-40 repeat family protein	GlPROP1	SMART: WD40/WD40 (9.6e-02/1.79e-1)	PVs/Cytosolic	Rout 2016
	GL50803_10822
	(100/7.5e-37)
	A8BTE2	16/25.9	5nnz 100/1.1e-36 14/8.5	4exv	WD-40 repeat family protein	GlPROP2	SMART: WD40/WD40 (2.80e-03/14.8)	PVs/Cytosolic	This study
	GL50803_16957
	(100/7.5e-37)

1Values for identity and similarity refer only to the predicted PIP-binding module. Seq. -Sequence. Id.—Identity. Sim.—Similarity.

2The .pdb identifiers in this column were used as modelling templates for Giardia PIP-binding proteins (S1 Fig).

3Protein GL50803_24488 was found by searching GDB for PXD protein paralogues.

Results

The G. lamblia genome encodes at least seven distinct PIP-binding modules

Given that several types of PIP-binding modules have been identified in eukaryotes, we determined how many endocytosis-associated module types were actually represented in the Giardia genome, in addition to the known G. lamblia epsin, FYVE and PXD variants [19-23]. For this reason, we selected a total of 14 protein types from various organisms known to harbour PIP-binding domains, some of them involved in endocytosis. These are: ANTH (AP180 N-terminal homology), ENTH (epsin N-terminal homology), PH (Pleckstrin homology domain), FYVE (Fab1, YOTB, Vac1 and EEA1), PX (Phox homology), BAR (bin, amphiphysin and Rvs), FERM (4.1, ezrin, radixin, moiesin), PROPPINs (β-propellers that bind PIs), C2 (conserved region-2 of protein kinase C), GOLPH3 (Golgi phosphoprotein 3), PDZ (postsynaptic density 95, disk large, zonula occludens), PTB (phosphotyrosine binding), Tubby modules and the PH-like module of the endocytosis-associated NECAP1 protein [24]. Representatives for each module were used as baits for the HMM-based tool HHpred [25] for protein structure prediction and the detection of remotely related sequences in the G. lamblia predicted proteome (Table 1). Putative Giardia protein homologs (Table 1) were then subjected to the online tools SMART [26, 27] and InterProScan [28] to identify conserved structural domains and sequence motifs within a query sequence (Fig 1A).

Fig 1

Functional domain prediction analysis and subcellular localization of G. lamblia PIP-binding proteins.

(A) Predicted functional domains for all identified PIP-binding proteins including positions of repetitive motifs and putative lipid and Zn -binding residues using HHPRED, HMMER and InterProScan. Ptd–Phosphatidylinositol. (B) Conventional confocal light-microscopy analysis of representative non-transgenic trophozoites labelled with Dextran-OG (first panel) to mark PV lumina and of antibody-labelled trophozoites expressing HA-tagged PIP-binding protein reporters. Except for Glepsin-HA and HA-GlFERM, all tested reporter proteins localize in close proximity to peripheral vacuoles (PVs) at the cell cortex. Epitope-tagged HA-GlPXD5 and GlPROP1-HA additionally show signal distribution throughout the cell. Cells were imaged at maximum width, where nuclei and the bare-zone are at maximum diameter. Epitope-tagged Glepsin-expressing cells were imaged at maximum width of the ventral disk. Scale bar: 1 μm. (C-I) Confocal STED microscopy analysis of trophozoites expressing epitope-tagged PIP-binding reporter proteins for GlPXD1-6, GlFYVE-HA and GlNECAP1-HA (red channel) co-labelled with Dextran-OG as a marker for PV lumina (green channel). As shown in the merged insets, although all reporters are clearly PV-associated, reporters for proteins GlFYVE-HA and HA-GlPXD1 and 2 are proximal to the PM with respect to Dextran-OG, indicating they reside at the PV-PM interface. In contrast, reporters for HA-GlPXD3 and GlNECAP1-HA appear to intercalate PVs. Scale bars: 1 μm for full cell and inset images.

This data mining approach detected high-confidence homologs for hitherto undiscovered G. lamblia proteins containing PH-like, FERM, BAR, FYVE and PROPPINs PIP-binding domains (Table 1, Fig 1A). No homologs could be found for the ANTH, GOLPH3, PDZ, PTB, Tubby and PH PIP-binding module types. Specifically for the C2 PIP-binding module, we could not detect significantly-related Giardia sequences using the query sequence 1CZS [29] although ORF numbers GL50803_16728, GL50803_114201, GL50803_17406 and GL50803_14855 all code for predicted C2-containing phosphoinositide kinases [30, 31].

Protein GL50803_17195 (GlNECAP1) is a predicted NECAP1 homolog containing a PH-like domain. Similarly, a conserved PH-like domain found at the C-terminus of FERM proteins was correlated with high confidence to protein GL50803_115468 (GlFERM). Immunofluorescence assay (IFA) and confocal microscopy imaging of a tagged GlFERM reporter detects a diffused cytosolic subcellular distribution (Fig 1B). In contrast, BAR domain-containing proteins GL50803_15847 and GL50803_14045 (GlBAR1 and 2, respectively) localize in close proximity to PVs, similar to tagged reporters for PROPPINs GL50803_10822 (GlPROP1) and GL50803_16957 (GlPROP2) (Fig 1B).

Since GlPXD1-2, GlFYVE and GlNECAP1 were experimentally shown to be associated to giardial clathrin assemblies [19], we selected these proteins and GlPXD3-6 for more detailed subcellular localization experiments. Stimulated emission-depletion (STED) microscopy in co-labelling experiments with Dextran-OG as a marker for fluid-phase endocytosis unequivocally confirmed accumulation for GlPXD1-4 and 6, GlFYVE and GlNECAP1 epitope-tagged reporters at PVs (Fig 1C–1I). The signal generated by GlPXD5 reporters was insufficient for a conclusive localization using STED microscopy but was shown to localize in close proximity to PVs using conventional confocal microscopy (Fig 1B).

To extend the initial annotation of giardial PIP-binding proteins we performed multiple sequence alignment (MSA) analyses for each giardial PIP-binding module with selected orthologs to delineate lipid-binding motifs and residues critical for PIP recognition (S1 Fig). In silico structural analyses of the lipid-binding domains of giardial proteins and their closest homologs were performed ab initio using the online tool I-TASSER [32-34]. Comparative analysis of structure models generated with I-TASSER clearly demonstrated positional conservation of residues critical for PIP binding (S1 Fig).

Taken together, in silico analysis identifies seven distinct PIP-binding module types encoded in the G. lamblia genome, conserved on both sequence and structural levels. Subcellular localization of epitope-tagged variants by fluorescence microscopy indicates clear association to PVs with the exception of Glepsin [21].

PIP-binding proteins associated with clathrin assemblies present distinct lipid-binding profiles in vitro

PX domains [35] and FYVE [36-38] preferentially bind PI(3)P. Even though PH domains have rather promiscuous binding preferences, a subset of PH domains binds strongly to PtdIns(3,4,5)P3 and PtdIns(4,5)P2, as well as PtdIns(3,4)P2 [39-41]. Based on the presence of conserved residues for lipid-binding in the giardial PXD1-6, FYVE and NECAP1 proteins (S1 Fig), we hypothesized that their lipid-binding preferences would also be conserved [20]. We tested this experimentally by expressing MBP-fused, epitope-tagged GlPXD1-6, GlFYVE and GlNECAP1 lipid-binding domains (S2A and S2B Fig). Recombinant fusion proteins were affinity-purified and used in lipid binding assays either for commercially-available PIP gradients as membrane-supported arrays (1.56–100 pmol/spot) (Fig 2A) or membrane strips spotted with defined amounts (100 pmol/spot) of PIPs (S2C Fig). The negative control for binding consisted of a PIP array probed with purified epitope-tagged MBP alone, whereas the positive control consisted of a PIP array probed with a commercially-available anti-PI(4,5)P2 antibody (Fig 2A).

Fig 2

Lipid-binding properties of selected giardial PIP-binding domains.

(A) Membrane-supported lipid arrays spotted with gradients of different phosphorylated variants of phosphatidylinositol (PtdIns), from 100pmol (A) to 1.56pmol/spot (G), were probed with fixed amounts (2.5 μg) of clathrin assemblies-associated epitope-tagged PIP-binding domains from GlPXD1-6, GlNECAP1 and GlFYVE, followed by immunodetection of the epitope tag. The protein fusion partner MBP (MBP alone) and for antibodies raised against PI(4,5)P2 (anti-PI(4,5)P2) were included as negative and positive controls for binding, respectively. No signal using arrays was obtained for MBP-GlPXD4 and MBP-GlPXD5 however, binding preferences for these fusions were determined using lipid strips (S2A and S2B Fig). (B) Plots of densitometric analyses using FIJI for each MBP-fused PIP-binding domain and each spotted PI/PIP residue based on array data presented in (A). (C) Testing of the binding affinity of the MBP-fused PIP-binding domain from GlNECAP1 on a wider range of lipid residues detects cardiolipin as the preferred substrate.

Quantification of the chemiluminescence signals shows a marked preference of MBP-GlPXD1 for PI(4,5)P2 in PIP gradients (Fig 2B) which was corroborated by experiments using PIP strips (S2C Fig). Under these conditions, GlPXD2, 3, and 6 show unexpectedly promiscuous binding preferences, with GlPXD2 presenting a marked affinity for PI(3)P and PI(4,5)P2, GlPXD3 for PI(3)P and to a lesser extent PI(5)P, and GlPXD6 for PI(3)P, PI(4)P, and PI(5)P (Fig 2B). These data were in line with results from independent PIP strip experiments (S2C and S2D Fig). MBP-GlPXD4 and MBP-GlPXD5 binding preferences could only be probed using PIP strips (S2C Fig), showing in both cases a marked affinity for PI(3,5)P2 and PI(4,5)P2 (S2C and S2D Fig). Binding preferences for MBP-GlFYVE could not be determined, given that no signal was ever obtained on both PIP arrays and strips (Fig 2A, S2C and S2E Fig). Surprisingly, testing of GlNECAP1 consistently detected cardiolipin as the preferred lipid moiety (Fig 2C; S2E Fig), with no detectable preference for PIP residues (S2C Fig). Taken together, our data show clearly distinguishable lipid binding profiles in vitro, with varying degrees of promiscuity for different PIP-binding domains.

Depletion of free PI(3)P, PI(4,5)P2 and PI(3,4,5)P3, but not PI(4)P binding sites in vivo inhibits PV-mediated uptake of a fluid-phase marker

The marked preference of GlPXD1-6 for PIP residues PI(3)P and PI(4,5)P2 raised the question whether their perturbation would elicit loss-of-function phenotypes in fluid phase uptake by Giardia trophozoites. Using a combination of commercially available antibodies, heterologous reporter constructs and chemical treatment, we reduced the bioavailability of PI3P, PI(4,5)P2, and in addition PI(3,4,5)P3 and PI(4)P.

Detection of PI(3)P, PI(4,5)P2, and PI(3,4,5)P3 in chemically fixed trophozoites by immunofluorescence microscopy with primary PIP-targeted antibodies highlights enrichment for all PIP moieties in the cortical region containing PVs (S3 Fig).

Ectopic expression of fluorescent high-affinity reporters for PI(3)P and PI(4)P, namely 2xFYVE-GFP and GFP-P4C [42], respectively, in transgenic G. lamblia trophozoites was used to identify membranes enriched for PI(3)P and PI(4)P deposition (Fig 3A–3D). Live microscopy of cells expressing 2xFYVE-GFP shows distinct reporter accumulation in cortical areas consistent with binding to PV membranes (Fig 3B, green panels), whereas representative cells from line GFP-P4C show a more diffused cytosolic staining pattern, with some accumulation at PVs (Fig 3D, green panels). Fluid-phase uptake of Dextran-R was assessed in cells from both transgenic lines, and compared to wild-type cells using quantification of signal intensity. Wild-type control cells and transgenic cells weakly expressing 2xFYVE-GFP (Fig 3A) incorporated large amounts of Dextran-R (Fig 3E). Conversely, a strong 2xFYVE-GFP signal correlated with low amounts of endocytosed Dextran-R detected at the cell periphery and with noticeably enlarged cells (Fig 3B). In contrast, there was no detectable difference in either Dextran-R uptake efficiency (based on fluorescent signal intensity) or cell width between weak (Fig 3C) and strong expressors (Fig 3D) of the GFP-P4C line. Cell width (Fig 3F) and fluid-phase uptake (Fig 3G) aberrant phenotypes in 2xFYVE-GFP cells were recorded with respect to wild-type control and GFP-P4C cells and tested for significance (p>0.05) on 100 cells/line selected in an unbiased fashion. These data translate into a significant negative correlation between expression of the PI(3)P-binding 2xFYVE-GFP reporter and fluid-phase uptake (Fig 3H) whereas only a slight albeit insignificant correlation was found between Dextran uptake and GFP-P4C expression (Fig 3I). Furthermore, performance of the Mann-Whitney test on our data confirmed the significance of the observed changes in cell width only in 2xFYVE-GFP-expressing cells, compared with non-transgenic cells. Specifically, the null hypothesis for no change in width was rejected for the WB vs 2xFYVE-GFP comparison (p(0.05)-value = 2.22045e-16) and accepted for the WB vs GFP-P4C comparison (p(0.05)-value = 0.182377).

Fig 3

Depletion of free PI(3)P, PI(4,5)P2 and PI(3,4,5)P3 binding sites in G. lamblia trophozoites elicits uptake and morphological phenotypes.

(A-D) Light microscopy-based immunofluorescence analysis of representative transgenic trophozoites expressing Legionella-derived PIP-binding constructs. (A-B) Compared to low 2xFYVE-GFP-expressing cells from the same population, reduction of PI(3)P binding sites in cells highly expressing a regulated encystation-dependent epitope-tagged construct 2xFYVE-GFP (GFP) inhibits uptake of fluid-phase marker Dextran-R. Scale bars: 1 μm. (C-D) Expression levels of PI(4)P-binding epitope-tagged construct GFP-P4C expression (GFP) have no visible impact on Dextran-R signal at PVs of transfected cells. Scale bars: 1 μm. (E) Dextran-R uptake in non-transgenic wild-type cells as negative controls for construct-induced uptake phenotypes. Scale bars: 1 μm (F) Box-plot representing the distribution of cell width (in μm) across at least 100 wild-type, 2xFYVE-GFP- and GFP-P4C- expressing cells selected in an unbiased fashion. A statistically significant (two-sided t-test assuming unequal variances, p<0.05) increase in median cell width with respect to non-transgenic cells is detected for 2xFYVE-GFP- but not GFP-P4C- expressing cells. Asterisks indicate statistical significance. n.s.: not significant. (G) Box-plot representing the distribution of measured Dextran-R signal intensity across at least 100 wild-type, 2xFYVE-GFP- and GFP-P4C- expressing cells selected in an unbiased fashion. A statistically significant (two-sided t-test assuming unequal variances, p<0.05) decrease in Dextran-R signal intensity, normalized to wild-type cells (100%), is detected for 2xFYVE-GFP- but not for GFP-P4C- expressing cells. Asterisks indicate statistical significance. n.s.: not significant. (H) A statistically significant (p<0.5) linear correlation exists between Dextran-R signal (x-axis, intensity_red channel [%]) and 2xFYVE-GFP expression (y-axis, intensity_green channel [%]) measured across 100 cells. (I) The apparent linear correlation between GFP-P4C expression (y-axis, intensity_green channel [%]) and Dextran-R signal (x-axis, intensity_red channel [%]) is not statistically significant (p<0.5). (J) Wide-field microscopy-based immunofluorescence analysis of the impact of neomycin treatment on Dextran-R uptake to deplete PI(4,5)P2 binding sites in non-transgenic wild-type cells. With respect to non-treated cells (WT; left panel), Dextran-R signal at PVs is visibly impacted in non-transgenic neomycin-treated cells (WT_Neo; right panel). Scale bars: 10 μm for full wide-field image, 1 μm for a single cell. (K) Box-plot representing the distribution of measured Dextran-R signal intensity across 100 wild-type cells, either untreated (WT) or treated with neomycin (WT_Neo). Neomycin treatment causes a statistically significant (two-sided t-test assuming unequal variances, p<0.05) decrease in Dextran-R signal. Scale bars: wide-field: 10 μm; single cells: 1 μm. For all images, nuclei are labelled with DAPI (blue). Insets: DIC images.

The cationic antibiotic neomycin binds tightly to the headgroup of phosphoinositides with a marked preference for PI(4,5)P2 and, to a lesser extent, PI(3,4,5)P3 [43, 44]. As a means to perturb PI(4,5)P2 and PI(3,4,5)P3 availability in Giardia trophozoites, we tested its effect on fluid-phase uptake by treating wild-type trophozoites with 7.2 mM neomycin followed by uptake of Dextran-R. Quantitative light microscopy image analysis revealed a significantly lower level of Dextran-R in treated trophozoites (p<0.05) (Fig 3J and 3K) which remained vital and motile in the presence of neomycin up to 15mM for 50 minutes (S1–S4 Videos). Taken together, the data indicate that depletion of free binding sites for PI(3)P, PI(4,5)P2, and PI(3,4,5)P3, but not PI(4)P significantly impacts fluid-phase endocytosis through G. lamblia PVs.

Functional characterization of GlPXD1-4 and 6, GlFYVE and GlNECAP1

Manipulation of PIP residue homeostasis elicited PV-dependent fluid-phase uptake phenotypes. We hypothesized that changing expression levels of giardial PIP-binding proteins previously identified in clathrin interactomes would elicit aberrant uptake phenotypes in Giardia trophozoites. In addition, we explored the functional boundaries of each PIP-binding module by defining their protein interactomes. To test this, we used the previously-generated epitope-tagged reporter lines for full-length GlPXD1-4 and 6, GlFYVE and GlNECAP1 (Fig 1C–1I) for assessing the effects of ectopic expression on fluid-phase uptake phenotypes. Furthermore, we used the same lines as a source of tagged “baits” in antibody-based affinity co-immunoprecipitation (co-IP) and identification of reporter-associated protein complexes (Table 2). Further investigation of GlPXD5 was abandoned at this stage due to its intractably low levels of expression.

Table 2

Overview of interactomes derived from epitope-tagged reporter lines for full-length GlPXD1-4 and 6, GlFYVE and GlNECAP1.

Main putative interaction partners are highlighted for each antibody-based affinity co-IP experiment. The type of interaction detected (reciprocal/one-sided) and its relative strength as indicated by the number of exclusive spectral counts associated to each candidate interactor, are also included.

Bait-ORF number	Partners	Interaction	Data	Annotation and reference(Zumthor et al., 2016 if not indicated)
GlPXD1	GL50803_102108	Reciprocal—strong	S4A Fig	GlCHC
GL50803_7723
	GL50803_4259	Reciprocal—strong	S4A Fig	GlCLC
	GL50803_21423	Reciprocal—strong	S4A Fig	GlAP2-β
	GL50803_17304	Reciprocal—weak	S4A Fig	GlAP2-α
	GL50803_8917	One sided—strong	S4A Fig	GlAP2-μ
	GL50803_14373	One sided—weak	S4A Fig	GlDRP
	GL50803_16595	One sided—weak	S4A Fig	GlPXD2
GlPXD2	GL50803_102108	Reciprocal—strong	S4A Fig	GlCHC
GL50803_16595	GL50803_4259	One sided—weak	S4A Fig	GlCLC
	GL50803_21423	Reciprocal—strong	S4B Fig	GlAP2-β
	GL50803_17304	Reciprocal—strong	S4B Fig	GlAP2-α
	GL50803_8917	One sided—strong	S4B Fig	GlAP2-μ
	GL50803_5328	One sided—strong	S4B Fig	GlAP2-σ
	GL50803_42357	Reciprocal weak	S4B Fig	GlPXD4
	GL50803_17195	One sided—weak	S4B Fig	GlNECAP1 (this study)
GlPXD3	GL50803_102108	Reciprocal—strong	S4C Fig	GlCHC
GL50803_16596	GL50803_21423	Reciprocal—strong	S4C Fig	GlAP2-β
	GL50803_17304	Reciprocal—strong	S4C Fig	GlAP2-α
	GL50803_14373	One sided—weak	S4C Fig	GlDRP
	GL50803_15411	Reciprocal—strong	S4C Fig	Gl15411
	GL50803_9606	One sided—weak	S4C Fig	Gl9605 (this study)
GlPXD4	GL50803_102108	One sided—strong	S4A Fig	GlCHC
GL50803_42357	GL50803_21423	One sided—weak	S4A Fig	GlAP2-β
	GL50803_14373	One sided—strong	S4A Fig	GlDRP
	GL50803_16595	Reciprocal weak	S4A Fig	GlPXD2 (this study)
GlPXD6	GL50803_102108	One sided—strong	S4A Fig	GlCHC
GL50803_24488	GL50803_21423	One sided—strong	S4A Fig	GlAP2-β
	GL50803_14373	One sided—strong	S4A Fig	GlDRP
	GL50803_16596	One sided—weak	S4A Fig	GlPXD3 (this study)
	GL50803_16653	One sided—weak	S4A Fig	GlFYVE (this study)
GlFYVE GL50803_16653	GL50803_102108	Reciprocal—strong	S4D Fig	GlCHC
	GL50803_14373	Reciprocal—strong	S4D Fig	GlDRP
GlNECAP1	GL50803_102108	One sided—strong	S4E Fig	GlCHC
GL50803_17195	GL50803_17304	One sided—strong	S4E Fig	GlAP2-α
	GL50803_21423	One sided—strong	S4E Fig	GlAP2-β
	GL50803_14373	One sided—strong	S4E Fig	GlDRP

The extended interactomes of GlPXD1, GlPXD4 and GlPXD6

Epitope-tagged, full-length GlPXD1 is a validated GlCHC interaction partner; its extended interactome confirms association to all core clathrin assembly components (GlCHC, GlCLC, GlDRP, and GlAP2) (Figs 4 and S4A and S1 Table) [19]. A weaker interaction with GlPXD2 was also found. The GlPXD4 interactome includes GlCHC and GlDRP and, uniquely for the GlPXD protein family, a previously confirmed interaction with GlPXD2 [19] albeit detected at lower stringencies (95_2_95, 2 hits) (Figs 4 and S4A and S4 Table). A putative SNARE protein GL50803_5785, previously identified in the GlTom40 interactome [45], was detected at lower stringencies (95_2_95, 2 hits). Similar to GlPXD1, GlPXD6 showed strong interaction with the β subunit of GlAP2 and GlCHC (Figs 4 and S4A), although the reverse interaction was not detected in the previously-published clathrin-centered interactome [19]. Using lower stringency parameters (95_2_50, 3 hits), revealed interaction with GlFYVE, GlPXD3 and GlDRP (Figs 4 and S4A and S5 Table). The GlPXD6 interactome includes Gl16717, a protein of unknown function predicted to carry a StAR-related lipid-transfer domain (Steroidogenic Acute Regulatory protein, START) domain [46]. Ectopic expression of epitope-tagged GlPXD1, 4 and 6 elicited no discernible PV-related phenotypes.

Fig 4

Overview of core protein interactomes determined from co-IP analyses.

Interactomes for GlFYVE, GlNECAP1 and GlPXD3, 4, and 6 defined by co-IP analysis were integrated with previously published data [19] for core clathrin assembly, GlPXD1, GlPXD2 and GlFYVE interactomes. Solid lines: interactions detected at high stringency. Dashed lines: interactions detected at low stringency. Yellow partners are currently annotated on GDB as “hypothetical protein” i.e. proteins of unknown function.

Ectopic expression of tagged GlPXD2 severely perturbs PV organization

Mining the GlPXD2 protein interactome dataset with high stringency parameters confirmed interactions with GlCHC, GlAP2, and GlPXD4 (Figs 4 and S4B and S2 Table). Furthermore, we identified three predicted SNARE proteins: Gl5785, GL50803_14469 (Gl14469; at lower stringencies 95_2_50, 9 hits), and GL50803_10013 (Gl10013; Figs 4 and S4B) [47]. The SNARE Gl5785 was detected also in the interactomes of GlPXD4 and GlTOM40 [45]. GlNECAP1 was also identified as a GlPXD2 interacting partner, albeit only by applying low stringency parameters (95_2_50, represented by a dashed line, Figs 4 and S4B).

In contrast to ectopic expression of tagged GlPXD1, 4, and 6, expression of an epitope-tagged reporter HA-GlPXD2 elicited a distinct phenotype. In contrast to non-transgenic wild-type cells (Fig 5A) and weakly-expressing HA-GlPXD2 cells (Fig 5B–5D upper panels), gated STED imaging of trophozoites strongly expressing HA-GlPXD2 showed large membranous clusters which also accumulated Dextran-R (Fig 5B) and were bound by both anti-GlCHC (Fig 5C) and anti-PI(3)P (Fig 5D) antibodies.

Fig 5

The impact of GlPXD2 ectopic expression on PV morphology.

(A) STED-microscopy-based immunofluorescence analysis of ventral (upper panel) and dorsal (lower panel) views of representative non-transgenic wild-type cells labelled with Dextran-OG, anti-GlCHC (GlCHC) and anti-PI(3)P antibodies (PI(3)P) detects association of all markers to PVs. Insets: DIC images. Scale bars: 1 μm (B) STED-microscopy based immunofluorescence analysis of transgenic cells either weakly (upper panel, non-phenotypic trophozoite) or strongly (lower panels, phenotypic trophozoite) expressing an epitope-tagged GlPXD2 variant (HA-GlPXD2) and subjected to Dextran-OG uptake detects co-labelled PV-derived peripheral clusters which are also detected with (C) anti-GlCHC (GlCHC) and (D) anti-PI(3)P (PI(3)P) antibodies. Scale bars: single cells: 1 μm; merged insets: 1 μm. (E) tEM experiments detect peripheral PV-associated membranous clusters in cells expressing a tagged GlPXD2 variant (HA-GlPXD2; upper panel and insets) which were not detected in non-transgenic cells (WT; lower panel and insets). Scale bars: single cells: 1 μm; insets: 1 μm.

Transmission electron microscopy (tEM) analysis confirmed the presence of randomly distributed peripheral PV clusters in cells expressing HA-GlPXD2 (Fig 5E; left panel) which were not present in representative wild-type control cells (Fig 5E; right panel).

The GlPXD3 interactome is connected to clathrin assemblies and includes a novel dynamin-like protein

GlDRP, GlCHC, and GlAP2 (α/β subunits) were detected in the GlPXD3 interactome, thereby establishing the association of this PX domain protein with clathrin assembly structures at the PV/PM interface (Figs 4 and S4C and S3 Table). A pseudokinase (Gl15411 [48]) previously identified in GlCHC assemblies was also found in the GlPXD3 interactome (Figs 4 and S4C [19]). Furthermore, the GlPXD3 and Gl15411 interactomes share proteins GL50803_16811 (Gl16811) tentatively annotated as a ZipA protein in GDB, and proteins GL50803_87677 (Gl87677) and GL50803_17060 (Gl17060), annotated as a NEK kinase and an ankyrin-domain carrying protein, respectively (Figs 4 and S4C). Unique interaction partners for GlPXD3 include the SNARE protein Gl7309 [47] and GlNSF (GL50803_114776) [49]. In addition, protein GL50803_103709 carrying a predicted N-terminal BRO domain and protein GL50803_9605 were identified as unique GlPXD3 interaction partners (Figs 4 and S4C). Furthermore, the StAR-related lipid-transfer protein Gl16717, already found in the GlPXD6 interactome was also found to be a low-stringency interaction partner for GlPXD3 and Gl15411, thereby connecting the GlPXD3 and GlPXD6-GlFYVE circuits.

Protein Gl9605, the sixth most abundant hit in the GlPXD3 interactome (S3 Table), and currently annotated as having an unknown function, was localized in close proximity to PVs (Fig 6A) and identified as a highly-diverged dynamin-like protein (Fig 6B).

Fig 6

The extended GlPXD3 interactome includes a novel dynamin-like protein in G. lamblia.

(A) Light-microscopy-based immunofluorescence analysis of representative transgenic trophozoites expressing epitope-tagged reporter variants (green) for protein Gl9605. Cells were imaged at maximum width, where nuclei and the bare-zone are at maximum diameter. Nuclei are labelled with DAPI (blue). Scale bar: 1 μm (B) MSA analysis G1-P-loop, G2 switch 1, G3 switch 2 and G4 regions of the conserved GTPase domains of Gl9605, GlDRP, Campylobacter jejuni DLP1 (Uniprot accession CJ0411) and DLP2 (CJ0412), Nostoc punctiforme BDLP1 (B2IZD3), Bacillus subtilis DynAD1 (P54159), Bacillus cereus DynAD2 (CUB17917), and Escherichia coli LeoA (E3PN25) bacterial dynamin-like proteins (BDLPs), Homo sapiens MFN1 (Q8IWA4), MFN2 (O95140), OPA1 (O60313) and DYN1 (Q05193), and Saccharomyces cerevisiae Fzo1p (P38297). Conserved positions are highlighted in grey. (C) I-TASSER de novo predicted 3D structure for Gl9605 (blue) and its closest known structural homologue, C. jejuni DLP2 (5owvC; green) indicating the GTPAse, neck and trunk regions that characterize BDLPs. A close-up view of the overlapping structures in the GTPase domains of Gl9605 (blue) and C. jejuni DLP2 (5owvC; green) marked with specific residues important for GTP binding and catalytic activity. (D) Quantitative microscopy-based immunofluorescence analysis of Dextran-R signal in cells expressing either a non-mutated full-length epitope-tagged Gl9605 or mutated Gl9605 K73E and S74N variants. In contrast to non-transgenic wild-type controls and Gl9605-HA expressing cells, expression of Gl9605 K73E and S74N variants inhibited Dextran-TxR uptake in a statistically significant fashion (box-plot). Asterisks indicate statistical significance. n.s.: not significant.

In support of this, the predicted GTPase domain in Gl9605 contains signature motifs in the P-loop (G1), switch 1 (G2) and switch 2 (G3) regions [50-52]. Conserved motifs in the G4 region are only partially maintained (Fig 6B). To test residue conservation on a structural level, Gl9605 was subjected to ab initio modelling using I-TASSER and the resulting tertiary structure was superimposed on that of a dynamin-like protein 2 (DLP2 Cj:5ovW) [53], Gl9605’s closest structural homologue (Fig 6C). A structural overlap TM-score of 0.913 suggests an almost perfect structural match, with clear chemical and positional conservation of key residues involved in GTPase activity (Fig 6C). We sought to elicit a dominant-negative phenotype by engineering Gl9605 K73E and S74N mutants [54]. In contrast to either wild-type cells or cells expressing a wild-type epitope-tagged Gl9605 control, expression of Gl9605 K73E and S74N mutant reporters inhibited fluid-phase uptake of Dextran-R in a statistically significant manner (p<0.05; Fig 6D).

Regulated ectopic expression of GlFYVE variants inhibits fluid-phase uptake

GlFYVE is a confirmed interactor of clathrin assemblies [55] through specific association to GlCHC and GlDRP (Figs 4 and S4D and S6 Table). GlFYVE’s extended interactome includes GlPXD6 and GlNECAP1.

To characterize the function of GlFYVE and to test whether a dominant-negative effect on uptake could be elicited, we performed a deletion analysis by generating epitope-tagged C-terminal (pCWP1::NT-GlFYVE-HA) and N-terminal (pCWP1::CT-GlFYVE-HA) truncation constructs. These consist of either the disordered region followed by the FYVE domain (Fig 7A), residues 1–300) or the armadillo repeat-rich (ARM repeats) domains (Fig 7A), residues 301–990), respectively.

Fig 7

Regulated ectopic expression of GlFYVE variants inhibits fluid-phase uptake.

(A) C-terminally epitope-tagged full-length (top; pCWP1::GlFYVE-HA), C-terminal truncated (middle; pCWP1::NT-GlFYVE-HA, residues 1–300) and N-terminal truncated (bottom; pCWP1::CT-GlFYVE-HA, 301–990 residues) constructs for GlFYVE were generated for regulated expression and phenotype testing. (B) Confocal imaging and immunofluorescence analysis of 6hr encysting non-transgenic wild-type cells and cells expressing pCWP1-driven constructs GlFYVE-HA, NT-GlFYVE-HA or CT-GlFYVE-HA (anti-HA) shows statistically significant differences in their ability to take up Dextran-R (two-sided t-test assuming unequal variances, p<0.05). Cells expressing construct pCWP1::NT-GlFYVE-HA present additional structures that are not detected in other lines and do not associate with Dextran-R labelling. Asterisks indicate statistical significance: * p<0.05; ** p<0.005. n.s.: not significant. DIC: differential interference contrast. Scale bars: 1 μm. (C) Confocal imaging and immunofluorescence analysis of 6hr encysting non-transgenic wild-type cells and cells expressing pCWP1-driven constructs GlFYVE-HA, NT-GlFYVE-HA or CT-GlFYVE-HA (anti-HA) using anti-CWP1-TxRed antibody (anti-CWP1) shows that the membrane compartments found in NT-GlFYVE-HA-expressing cells are not related to encystation-specific vesicles. Scale bars: 1 μm. (D) Antibody-based immunofluorescence analysis of GlCHC deposition (anti-CHC) in 6hr encysting non-transgenic wild-type cells and cells expressing pCWP1-driven constructs GlFYVE-HA, NT-GlFYVE-HA or CT-GlFYVE-HA (anti-HA) detects a significant degree of GlCHC association to the CT-GlFYVE-HA variant, with only partial association to NT-GlFYVE-HA and GlFYVE-HA constructs. Scale bars: 1 μm.

Expression of both constructs is regulated by an inducible promoter which is de-repressed during transient induction of encystation, the process during which a flagellated trophozoite differentiates to a cyst [56]. After a short (6h) induction pulse, transfected cells were subjected to Dextran-R uptake. In cells expressing full-length pCWP1::GlFYVE-HA and truncated variants, the amount of Dextran-R accumulated in PVs was significantly (p<0,05) lower (Fig 7B, box plot). Furthermore, IFA analysis of pCWP1::NT-GlFYVE-HA cells revealed the presence of structures which overlapped neither with Dextran-R-labelled PVs (Fig 7B) nor with encystation specific vesicles (ESVs) labeled with the anti-CWP1 antibody (Fig 7C). In contrast, CT-GlFYVE-HA and full length GlFYVE-HA localized predominantly to PVs (Fig 7B–7D). The subcellular localization of GlCHC in these lines and in a wild-type control overlapped with the truncated CT-GlFYVE-HA variant, but only partially with NT-GlFYVE-HA and GlFYVE-HA (Fig 7D).

Ectopic expression of GlNECAP1 significantly impairs fluorescent Dextran uptake

Co-IP using epitope-tagged GlNECAP1 confirmed interaction with clathrin assembly components GlAP2-β, μ and α subunits, GlCHC and GlDRP. Interaction with GlFYVE (S4D Fig) and, at lower stringency also for GlPXD2 (S4B Fig), could be confirmed (Figs 4 and S4E and S7 Table).

Three putative conserved AP2-interacting motifs were identified using multi-sequence alignment; the high affinity WxxF motif at the N-terminus, two residues being invariant throughout evolution, K147 and G149, and AP2-beta linker interacting residues binding sites (Fig 8A) [57].

Fig 8

PV morphology and functionality phenotypes caused by GlNECAP1 ectopic expression.

(A) Multiple sequence alignment analysis of GlNECAP1 and NECAP1 orthologues from Arabidopsis thaliana (Uniprot accession Q84WV7), Trichinella pseudospiralis (A0A0V1JQ20), Caenorhabditis elegans (Q9N489), Echinococcus multilocularis (A0A087VZS0), Ceratitis capitata (W8CD89), Homo sapiens (Q8NC96) and Mus musculus (Q9CR95) identifies conserved motifs and residues for interaction with AP2. GlNECAP1 presents partial conservation, with a WXXF motif (orange) shifted to the N-terminus with respect to other orthologues. (B) Ab initio template-based 3D modelling of G. lamblia and H. sapiens NECAP1 (1tqz) homologues predicts similar structures, with conservation of key residues involved in the interaction between NECAP1 proteins and AP2 complexes (shaded in blue and green). (C) Wild-type non-transgenic control cells (WT) and cells expressing either epitope-tagged GlNECAP1 reporters GlNECAP1-HA, GlNECAP1-APEX2-2HA or the ΔWVIF deletion construct GlNECAP1ΔWVIF-HA (green) were tested for Dextran-R (red) uptake. Dextran-R signal intensity after uptake was significantly (p<0.05) decreased in GlNECAP1-HA- and GlNECAP1-APEX2-2HA- expressing cells when compared to wild-type controls and GlNECAP1ΔWVIF-HA-expressing cells (box-plot). (D) Quantitative tEM analysis of GlNECAP1-APEX2-2HA-expressing cells (upper panels) and wild-type non-transgenic cells (WT; lower panels) shows visibly enlarged PVs in GlNECAP1-APEX2-2HA-expressing cells, with a statistically significant (p<0,05) increase in the median of measured area of peripheral vacuoles (in μm2; box-plot).

De novo 3D modelling confirms overall structural conservation of all key residues in GlNECAP1 compared to mammalian NECAP1 (Fig 8B). Furthermore, the interacting interface of NECAP1 with the β-linker region of AP2 was also identified in the structural model for GlNECAP1 (Fig 8B).

To test whether expression of a GlNECAP1 variant lacking the putative high-affinity motif WVIF could elicit a dominant-negative uptake effect, a deletion construct GlNECAP1ΔWVIF-HA was synthesised (Fig 8A) for ectopic expression. Accumulation of Dextran-R into PVs detected by microscopy was significantly lower (p<0.05) in transgenic cells ectopically expressing GlNECAP1-HA or an APEX- and epitope- tagged variant GlNECAP1-APEX2-2HA compared with wild type controls (Fig 8C, box plot). Conversely, ectopic expression of a deletion construct GlNECAP1ΔWVIF-HA (Fig 8C, GlNECAP1ΔWVIF-HA) had no discernible effect on accumulation of Dextran-R in PVs (Fig 8C, box plot). Ectopic expression of the genetically encoded enzymatic reporter [58, 59] GlNECAP1-APEX2-2HA was associated to significantly enlarged PVs in tEM compared to wild type controls (Fig 8D; S5 Fig).

GlPXD3 associates specifically to PVs as membrane coat

Co-localization studies with Dextran-OG and ectopically expressed HA-GlPXD3 show apparent coating of the entire PV membrane on the cytoplasmic side by the reporter construct (Fig 9A).

Fig 9

GlPXD3 membrane coats as a tool to probe PV size and organization.

(A) A dorsal view of representative cells expressing an epitope-tagged GlPXD3 reporter (red) and co-labelled for Dextran-OG (green). STED confocal imaging followed by signal overlap analysis (scatter plot) shows proximal yet distinct deposition patterns, with GlPXD3 reporters closely associated to Dextran-OG-illuminated PVs. Scale bars: whole cell 1 μm; close-ups 1 μm. (B) 3D STED microscopy (left panel) followed by reconstruction using IMARIS (middle panel) of a representative cell expressing an epitope-tagged GlPXD3 reporter reveals fenestrated GlPXD3-delimited areas distributed under the PM and throughout the whole cell (close-up view of inset in the right panel). Scale bars: whole cell 1 μm; close-ups 1 μm. (C) STED microscopy analysis of PVs in a representative non-transgenic wild-type cell labelled with Dextran-OG. Scale bars: whole cell 1 μm; close-ups 1 μm. (D) Average length of the major and minor principle axes of GlPXD3-delimited fenestrated structures (in red) and Dextran-labelled PV organelles in wild-type non-transgenic cells (in green) measured across at least 100 structures/organelles. (E) STED confocal microscopy analysis of ventral and dorsal views of a representative cell expressing an epitope-tagged GlPXD3 reporter (anti-HA) and co-labelled for GlCHC (anti-CHC) shows how fenestrated GlPXD3-delimited structures are decorated with GlCHC foci. Scatter plots are included for signal overlap analysis. Scale bars: whole cell 1 μm; close-ups 1 μm. (F) Similar to GlCHC, anti-PI(3)P antibodies (anti-PI(3)P) detect foci of PI(3)P accumulation in close proximity to GlPXD3 epitope-tagged reporters (anti-HA) in HA-GlPXD3-expressing cells analyzed with STED microscopy. Scatter plots are included for signal overlap analysis. Scale bars: whole cell 1 μm; close-ups 1 μm.

This provided us with an opportunity to generate measurements of PV organelles in optical sections using 3D STED microscopy followed by reconstruction and rendering with IMARIS. Rendered images show hive-like GlPXD3-labelled structures predominantly in the cortical area of the cell underneath the PM that clearly surround the entire PV membrane (Fig 9B). The major and minor principal axes of these structures measured 437 +/- 93 nm and 271 +/- 60 nm. Consistent with the subcellular localization of this marker on the cytoplasmic side of PV membranes, these values were significantly higher (p≤ 0.05) than those obtained from PVs labeled with Dextran-OG (371 +- 79 nm and 221 +/- 49 nm) (Fig 9C and 9D). Signal overlap of epitope-tagged GlPXD3 with endogenous GlCHC as a marker for the PM-PV interface [19] in fluorescence microscopy is low. The image data indicate that both labels have distinct distributions but may spatially overlap at focal clathrin assemblies in small areas at the PV-PM interface (Fig 9E). Similarly, labelling for both PI(3)P and a reporter GlPXD3 variant showed minimal signal overlap (Fig 9F), despite the strong affinity of the latter for this lipid in in vitro lipid-array binding experiments (Fig 2A and 2B).

Discussion

PIPs and PIP binders in G. lamblia

PIPs are recognized spatiotemporal organizers and decorate the surface of the eukaryotic cell’s plasma and endo–membrane system [1-3]. G. lamblia is no exception; despite its significant reduction in endomembrane complexity, this species maintains a variety of PIP residues, mostly located at the cell periphery. We identified 11 novel proteins, in most cases of unknown function that carry predicted PIP-binding modules and primarily localize in close proximity to PVs.

All hitherto identified PIP-binding proteins in G. lamblia can be loosely grouped in two categories; they are either relatively small proteins (up to 400 amino acid residues) consisting almost entirely of the PIP-binding module (e.g. GlPXD6 and GlNECAP1), or they are large proteins consisting of a single predicted domain for PIP-binding associated to domains of unknown function (e.g. GlPXD2 and GlFYVE). A full functional characterization of the latter is a challenge given the level of genomic sequence divergence in G. lamblia. This makes it currently difficult to determine whether sequences are lineage-specific or so diverged as to be unrecognizable orthologues of previously characterized proteins. Hence, structural annotation of large G. lamblia proteins carrying PIP-binding modules such as GlPXD2 or GlFYVE is limited to the lipid binding domain.

Eight out of 14 identified PIP-binding modules are either directly or indirectly associated to clathrin assemblies. Their PIP binding preferences, as measured using in vitro lipid-binding assays, are clearly distinct despite showing a varying degree of promiscuity, consistent with previously published data [20]. In contrast to previous reports, we could not measure PIP residue binding activity for GlFYVE using in vitro lipid-binding assays [22]. Furthermore, GlNECAP1 showed a distinctive and highly specific binding preference for cardiolipin. This is a surprising finding since cardiolipin is an abundant phospholipid of the inner mitochondrial membrane [60] whose presence in Giardia is controversial [61, 62]. Although GlNECAP1 lacks canonical motifs for cardiolipin binding [63], previous reports on the identification of cardiolipin-binding PH domains [64, 65] lend support to the observation that the PH-like domain in GlNECAP1 could bind cardiolipin, at least in vitro. The evolutionary implications for the presence of cardiolipin in an organism with “bare-bones” mitochondrial remnants i.e. mitosomes, with no maintenance of membrane potential nor ATP synthesis activity [66], provide for an exciting research direction worth pursuing.

An interactome-based model for PIP-binding proteins and clathrin assemblies at PVs

Data derived from APEX-mediated tEM experiments on transgenic trophozoites expressing APEX-tagged clathrin assembly components (GlCHC and GlCLC; [19]) show how larger PVs are associated to more than one PM-derived clathrin-marked invagination (Fig 10A).

Fig 10

A working model for PV-associated nanoenvironments defined by clathrin assemblies and PIP-binding proteins.

(A) Electron microscopy images of G. lamblia cells expressing an APEX2-tagged GlCHC (upper panels) or GlCLC (lower panels) reporter show darker APEX2-derived deposits at the PM-PV interface (arrows). Scale bar: 0.1 μm. (B) IFA analysis of a representative non-transgenic wild-type cell labelled with Dextran-OG and anti-GlCHC antibodies to illuminate PV lumina and the PV-PM interface, respectively. Scale bar: 1 μm. (C) Schematic reconstruction of a surface view (left panel) of the PV system associated to clathrin assemblies (blue) and GlPXD3 coats (red), based on data presented in this report and in [13]. PV membranes and lumina are represented in dark and light green, respectively. Cross-sections at (1) and (2) yield views in the right panel, highlighting foci of clathrin assemblies beneath the PM, above GlPXD3’s coat-like deposition pattern surrounding PVs. (D) An overview of the G. lamblia PIP-binding interactome associated to PVs. All represented PIP-binding proteins were found to contact clathrin assemblies (GlCHC) in either reciprocal (double-headed arrows) or one-way (single-headed arrows) modes of interaction, following filtering of co-IP data either at high (black solid lines) or low (grey dashed lines) stringency.

This is supported by data from IFA and STED microscopy analysis of trophozoites loaded with Dextran-OG and labelled with anti-GlCHC antibodies (Fig 10B). By combining APEX-derived tEM data with STED microscopy data for both Dextran-OG and GlPXD3 labelling, a quantified sub organellar model for PV organization can be built which takes into account organelle size and relative distribution of clathrin assemblies (Fig 10C). In this model, GlPXD3 clearly emerges as a membrane coat that surrounds individual PV organelles (Fig 10C, upper panel) on the cytoplasmic side of clathrin assemblies at the PV-PM interface (Fig 10C, lower panel).

The PV-associated PIP-binding protein interactome appears as a tightly knit molecular network with GlCHC at its center (Figs 4 and 10D). Despite the high level of interconnectivity of distinct PIP-binder interactomes (Fig 4), specific molecular circuits such as the ones defined by the SNARE quartet (S4F Fig), pseudokinase Gl15411 and novel DLP Gl9605 (S4G Fig), as well as StAR-related lipid-transfer protein Gl16717 (S4H Fig), can be recognized. Notably, GlPXD1 and 2 are the only PIP-binders who’s extended interactomes include the G. lamblia putative clathrin light chain (Fig 4 and S4F Fig), arguably GlCHC’s closest binding partner. The GlPXD1 interactome further stands out for enrichment of proteasome-associated components (S1 Table), invoking scenarios concerning clathrin assembly turnover in G. lamblia. Although previous data showed that clathrin assemblies are long-lived stable complexes [19], they would still require remodeling, degradation, and substitution with new components. In the absence of classical components as well as C-terminal motifs on GlCHC for ordered disassembly of clathrin coats, GlPXD1’s proteasome-enriched interactome points to proteasome-mediated degradation of GlCHC assemblies as an alternative process to achieve turnover albeit without recycling of coat components.

In the context of clathrin assembly dynamics, GlNECAP1 once again comes to the forefront. NECAP1 is characterized as an AP2 interacting partner and an important component of CCVs in the assembly phase [57]. Given that CCVs have not been detected in Giardia, this begs the question of the functional role of a NECAP1 cardiolipin-binding orthologue in G. lamblia, which was found to interact with G. lamblia AP2 subunits and GlFYVE. Recent developments in gene knock-out [67] and CRISPR-Cas9-based knock-down [68, 69] methodologies tailored to G. lamblia will be instrumental towards a full functional characterization of GlNECAP1’s function(s).

PIP binding homeostasis and fluid-phase uptake

We initially hypothesized that perturbation of free PIP binding sites would elicit fluid-phase uptake phenotypes by impacting PV functionality. The hypothesis tested positive for PI3P, PI(3,4,5)P3, and PI(4,5)P2. A significant effect on cell width was detected when free PI(3)P binding sites were reduced by ectopic expression of 2xFYVE-GFP (Fig 3B and 3F), linking PIP residues to both endocytic homeostasis and overall maintenance of cell size, possibly in connection to membrane turnover. Complementing these data, ectopic expression of both GlFYVE and GlNECAP1 significantly impacted fluid-phase uptake. Furthermore, ectopic expression of GlNECAP1 induced an enlarged PV phenotype similar to that induced by expression of a predicted GTP-locked GlDRP mutant [70]. As with all uptake phenotypes we elicited and measured using fluorescent dextran as a fluid-phase uptake reporter, it is still unclear whether the defect lies in PV-PM fusion or in the sealing-off of PV lumina.

Ectopic expression of a truncated GlFYVE deprived of its ARM repeats, namely NT-GlFYVE, induced formations of vesicle-like structures of undefined origins. ARM folds are superhelical structures mostly involved in protein-protein interactions [71], suggesting that a loss of these domains may impact GlFYVE function and protein complex formation and may lead to protein aggregation. Importantly, these structures are associated to GlCHC which, in cells expressing the NT-GlFYVE recombinant protein, has lost its almost exclusive PV localization. In line with this hypothesis, the NT-GlFYVE epitope-tagged reporter loses association to PVs. In contrast to the GlFYVE-induced uptake phenotype and despite a severe PV clustering phenotype, HA-GlPXD2-expressing cells still appear to perform fluid-phase uptake comparably to wild-type cells. This suggests that PV morphology can be decoupled from effective PV-mediated uptake. Taken together, these data link PIPs to clathrin assemblies and fluid-phase PV-mediated uptake, providing new insights on clathrin’s hitherto unclear role in Giardia endocytosis.

Beyond clathrin assemblies

Investigation of the molecular milieu within which clathrin-associated PIP-binding proteins operate in G. lamblia revealed two protein sets of special interest. Four predicted SNARE proteins were detected in both the GlPXD2 and GlPXD3 interactomes. Further investigations will be necessary to determine whether the function of this SNARE quartet is indeed fusing PM and PV membranes at contact sites, thereby allowing entry of fluid-phase material into PV organelles.

Another finding of special interest concerns Gl9605, a hitherto unrecognized DLP found in the interactome of GlPXD3 with similarity to bacterial DLPs (BDLPs; S6 Fig). Similar to their eukaryotic counterparts, BDLPs are capable of helical self-assembly and tubulation of lipid bilayers, and were shown to be most closely related to the mitofusins FZO and OPA (S6 Fig) [24, 25], but only distantly related to classical dynamins [26]. BDLPs were also found in the Archaea class Methanomicrobia [72], making the family ubiquitously distributed across all kingdoms. These data show how the DLP/DRP family in G. lamblia has now expanded to include the previously unidentified endocytosis-associated Gl9605 BDLP homologue. GlDRP plays a role in the regulation of PV and encystation-specific vesicle (ESV) size [70]. Although its role in fluid-phase uptake has not been determined, expression of a GTP-locked GlDRP mutant inhibited endocytosis of biotinylated surface proteins [70]. On the other hand, a similar mutational analysis of Gl9605 shows that this DLP variant can elicit a dominant-negative fluid-phase uptake phenotype. Although we did not test Gl9605 involvement in surface protein uptake, the data so far suggest that two distinct DLPs play independent albeit complementary roles in the regulation of PV-mediated fluid-phase uptake and organelle homeostasis.

In this work, we report on the detailed functional characterization of PIP-binding proteins in G. lamblia that associate to clathrin assemblies. Our data reveals a previously unappreciated level of complex interplay between lipid residues and their protein binders in marking and shaping endocytic compartments in this parasite. However, several identified PIP-binding modules appear to associate to PVs independently of clathrin. Their extended interactomes and their involvement in fluid-phase uptake have yet to be investigated but current data point towards a complex network of PIP binders of varying binding preference and affinity, all working in the same subcellular environment, yet, in some cases (GlFERM, GlBAR1 and 2, GlPROP1 and 2, and Gl16801), not directly linked to clathrin assemblies. The only known exception is Glepsin whose localization remains controversial due to conflicting reports [21, 73]. We systematically did not detect Glepsin in any of the interactomes for clathrin-associated PIP binders, in line with its localization at the ventral disk [21]. Altogether, the variety of PIP residues and PIP-binding modules in the G. lamblia cortical area containing endocytic PVs underscores their necessity for correct functioning of membrane traffic even in a protist so clearly marked by reduction in endomembrane complexity.

Materials and methods

Giardia lamblia cell culture, induction of encystation and transfection

G. lamblia WBC6 (ATCC catalog number 50803) trophozoites were cultured and harvested applying standardized protocols [56]. Encystation was induced by the two-step method as previously described [74, 75]. Transgenic cell lines were generated using established protocols by electroporation of linearized or circular pPacV-Integ-based plasmid vectors prepared from E. coli as described in [76]. Transgenic lines were then selected for puromycin resistance (final conc. 50 μg ml -1). After selection, transgenic trophozoites carrying episomal or integrated reporter constructs were further cultured with or without puromycin, respectively.

Construction of expression vectors

Oligonucleotide sequences used for cloning in this work are listed in S8 Table. pPacV-Integ-based [34] expression of epitope tagged reporter constructs was driven using either putative endogenous (pE) or encystation-dependent (pCWP1) promoters. Constructs 2xFYVE-GFP and GFP-P4C [42] were kindly provided by Prof. Dr. H. Hilbi (University of Zurich).

PV labelling using fluid-phase markers

Fluid-phase uptake assay in G. lamblia was performed as described previously [26] using dextran coupled to either Oregon Green 488 (Dextran-OG) (Cat. Nr. D-7171, Thermo Fisher Scientific) or Texas-Red (Dextran-R) (Cat. Nr. D-1863, Thermo Fisher Scientific) fluorophores, both at 1mg/ml final concentration. The same protocol was used following treatment with 7.2mM neomycin (G418, Sigma) in supplemented PBS for 45–50 minutes at 37°C.

Co-immunoprecipitation with limited cross-linking

Co-immunoprecipitation of GlPXD1-6, GlNECAP1, and GlFYVE was done as previously reported [19, 45]. Protein input was standardized to 0.8 mg/ml total protein.

Protein analysis and sample preparation for mass spectrometry (MS)-based protein identification

Protein analysis was performed on 4%/10% polyacrylamide gels under reducing conditions (molecular weight marker Cat. Nr. 26616, Thermo Scientific, Lithuania). Immunoblotting was done as described in [77]. Gels for mass spectrometry (MS) analysis were stained using Instant Blue (Expedeon, Prod. # iSB1L) and destained with ultra-pure water.

Mass Spectrometry, protein identification and data storage

MS-based protein identification of de-stained and diced gel lanes was performed as described in [19]. Free access to raw MS data is provided through the ProteomeXchange Consortium on the PRIDE platform [78]. Accession numbers for datasets derived from bait-specific and corresponding control co-IP MS analyses are the following: PXD013890 for GlPXD1, 3 and 6, PXD013897 for GlFYVE, PXD013896 for GlNECAP1 and PXD013899 for GlPXD2 and 4.

In silico co-immunoprecipitation dataset analysis

Analysis of primary structure and domain architecture of putative components of giardial PIP—binding proteins was performed using the following online tools and databases: SMART for prediction of patterns and functional domains (http://smart.embl-heidelberg.de/), pBLAST for protein homology detection (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins), HHPRED for protein homology detection based on Hidden Markov Model (HMM-HMM) comparison (https://toolkit.tuebingen.mpg.de/#/tools/hhpred), PSORTII for sub-cellular localization prediction (https://psort.hgc.jp/form2.html), TMHMM for transmembrane helix prediction (http://www.cbs.dtu.dk/services/TMHMM/), RCSB for 3D structure of homologues (https://www.rcsb.org/), and the Giardia Genome Database to extract organism-specific information such as protein expression levels, predicted molecular sizes and nucleotide/protein sequences (www.giardiaDB.org). The generated co-IP datasets were filtered using a dedicated control-co-IP dataset generated using non-transgenic wild-type parasites. Filtration of the bait-specific co-IP and control-co-IP datasets was done using Scaffold4 (http://www.proteomesoftware.com/products/) with high stringency parameters (95_2_95, FDR 0%) and low stringency parameters (95_2_50, FDR 0%). Furthermore, exclusive hits for bait-specific datasets were manually curated using the following criteria for inclusion into the interactome model: i) exclusive detection with > 3 spectral counts in bait-specific datasets or ii) an enrichment of peptide counts >3 with respect to the ctrl. co-IP dataset. Data presented in S1–S7 Tables show exclusive and non-exclusive protein hits filtered using both stringency levels.

Immunofluorescence analysis (IFA) and light-microscopy

Samples for immunofluorescence analysis of subcellular distribution of reporter proteins by wide-field and laser scanning confocal microscopy (LSCM) were prepared as described previously [33, 35]. Nuclear DNA was labelled with 4’, 6-diamidino-2-phenylindole (DAPI). Antibodies used are the anti-HA antibody (1:50 or 1:100; Anti HA high affinity 3F10, Cat. Nr. 11867423001, Roche), anti-V5 (1:50 or 1:100; V5 Tag Monoclonal Antibody, Cat. Nr. R960-25, Thermo Fisher Scientific), self-made antibodies raised against GlCHC (dilution 1:1000), anti-rat antibody coupled to fluorochrome in case of wide-field or confocal microscopy (1:200; Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488, Cat. Nr. #A11006, Invitrogen) and for STED microscopy (Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594, Cat. Nr. A11007, Invitrogen). Specific PIP residues were detected using anti-PI(3)P (1:100); Purified anti-PI(3)P IgG, Z-P003 Echelon Biosciencies), anti-PI(4,5)P2 (1:100; Purified anti-PI(4,5)P2 IgM, Z-P003 Echelon Biosciencies) and anti-PI(3,4,5)P3 (1:100; Purified anti-PI(3,4,5)P3 IgM, Z-P045 Echelon Biosciencies) followed by an anti-mouse antibody coupled to fluorochrome in all three cases (Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa flour 594, Cat. Nr. A-11005, Thermo Fischer Scientific or Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa flour 488, Cat. Nr. A-11017, Thermo Fischer Scientific). Cells were imaged at maximum width, with nuclei and the bare-zone at maximum diameter. Deconvolution was performed with Huygens Professional (Scientific Volume Imaging). Three-dimensional reconstructions and signal overlap quantification (Mander’s coefficient) in volume images of reconstructed stacks were performed using IMARIS x64 version 7.7.2 software suite (Bitplane AG) or FIJI [79], respectively.

Live cell microscopy of GFP fusion proteins in transgenic Giardia lamblia

Transgenic trophozoites expressing GFP-fusion proteins and non-transgenic lines were harvested and resuspended in approximately 0.5–1 ml of the medium and transferred to a pre-cooled 24 well culture plate placed directly on ice in an ice bucket. After overnight oxygenation in the dark at 4°C, cells were washed in ice-cold PBS supplemented (S-PBS) with 5mM glucose (Cat. No. 49139, Fluka) and 0.1mM ascorbic acid (Cat. No. 95209, Fluka) at pH 7.1. An aliquot of cells in S-PBS was placed on a microscopy slide and left to recover at 37°C for 3 minutes directly before imaging. For the Dextran uptake assay, Dextran-R was added to the cell suspension to a final concentration of 1mg/ml. Cells were incubated in the dark at 37°C for 20 min and were imaged directly or chemically fixed for further processing by IFA.

Super resolution (gSTED) microscopy

Sample preparation was done as described for wide field microscopy and LSCM. For imaging, samples were mounted in ProLong Gold antifade reagent (Cat. Nr. P36934, Thermo Fisher Scientific). Super resolution microscopy was performed on a LSCM SP8 gSTED 3x Leica (Leica Microsystems) at the Center for Microscopy and Image Analysis, University of Zurich, Switzerland. Nuclear labelling was omitted due to possible interference with the STED laser. Further data processing and three-dimensional reconstructions of image stacks were done as described for LSCM.

Sample preparation for transmission electron microscopy

Transgenic trophozoites expressing GlPXD2 (GL50803_16595) and non-transgenic cells were harvested and analyzed by transmission electron microscopy (tEM) as described previously [70].

DAB staining in APEX2 expressing cells

Transgenic trophozoites expressing GlNECAP1-APEX2-2HA, GlCHC-APEX2-2HA and GlCLC-APEX2-2HA were harvested and washed with PBS followed by fixation in 2.5% EM grade glutaraldehyde in cacodylate buffer (100 mM cacodylate (Cat. Nr. 20838), 2mM CaCl2 (Cat. Nr. 21097, Fluka) in PBS) for 1h at RT. Samples were washed twice before and after quenching for 5 min in 20 mM glycine/cacodylate buffer. For staining, cells were resuspended in 500 μl substrate solution containing 1.4 mM DAB tetrahydrochloride (Cat. Nr. D5637, Sigma) with 0.3 mM H2O2 (Cat. Nr. H1009, Sigma) in cacodylate buffer and incubated for 15 min. The reaction was terminated by washing thrice in cacodylate buffer and prepared as described for tEM.

Chemical fixation of DAB-stained cells

DAB stained cell suspicions were post-fixed with 1% aqueous OsO4 for 1 hour on ice, subsequently rinsed three rimes with pure water and dehydrated in a sequence of ethanol solutions (70% up to 100%), followed by incubation in 100% propylene oxide and embedding in Epon/Araldite (Sigma-Aldrich, Buchs, Switzerland). Samples were polymerized at 60°C for 24h. Thin sections were imaged pre- and post- staining with aqueous uranyl acetate (2%) and Reynolds lead citrate.

Expression and purification of bacterial fusion proteins

For each candidate PIP-binding protein, corresponding nucleotide stretches coding for selected amino acid residues (S9 Table) were modified by including an HA-coding sequence either at the 5’ end or the 3’ end and then subcloned into the pMal-2Cx E. coli expression vector (New England Biolabs). The resulting recombinant variants were expressed as maltose-binding protein (MBP) fusions in E. coli (strain Bl21) and grown in LB medium either at 37°C (MBP-GlPXD1, MBP-GlPXD2, MBP-GlPXD3, MBP-GlPXD6, MBP-GlNECAP1 and MBP-GlFYVE) or 30°C (MBP-GlPXD4 and MBP-GlPXD5) to an OD600 = 0.4. Induction of expression was performed by adding 0.2 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside, Cat. Nr. 15529019, Thermo Fischer Scientific) to the cultures and incubating for a further 4 hours. Cells were harvested at 4°C (4,000 x g) and bacterial pellets were resuspended in 5 ml of cold column buffer with 1x PIC (Protease inhibitor cocktail set I; Cat. Nr. 539131-10VL, Merck) and 200 mM PMSF (Cat. Nr. 329-98-6, Sigma Aldrich). Cells were lysed by sonication and centrifuged (20 min, 9,000 x g, 4°C). Cleared supernatant was incubated with amylose resin slurry (Amylose Resin High Flow, Cat. Nr. E8022L, BioLabs) for 4 hours at 4°C on a turning wheel, washed with column buffer and then transferred to an empty column (BioRad). Unbound protein was washed using until background OD280 reached ~0.06. Protein fractions were eluted using 10mM maltose solution and pooled for overnight dialysis in a dialysis cassette (Slide-a-Lyzer, Cat. Nr. 66380, Thermo Fischer Scientific) against 25mM NH4Ac at 4°C and later lyophilized or snap-frozen. Protein fractions were stored at -80°C.

Protein lipid overlay (PLO) assay

PLO assays were performed using two different types of PIP strips (Cat. Nr. P-6001 and P-6002, Echelon) and PIP arrays (Cat. Nr. P-6100, Echelon). PIP strips or PIP arrays were first floated on ultrapure water for 5 min before incubation in blocking buffer (1xPBS containing 0.1%v/v Tween-20 and 3% fatty-acid free BSA (Sigma A7030)) at RT for 1h. For PLO using lipid strips, E. coli-derived lyophilized were reconstituted in 1x PBS and protein concentration was measured using the Bradford assay. For PLO using PIP arrays, snap-frozen protein samples were used. For both types of PLO assays, the equivalent of 0.5 μg/ml of protein in PBS containing 3% fatty acid free BSA were incubated for 1h at RT with gentle agitation. After washing with 1xPBS containing 0.1% v/v Tween-20, PIP–strips were incubated (1h, RT, agitated) with a monoclonal anti-HA antibody (clone 3F10, monoclonal antibody from Roche) at a dilution of 1:500 in blocking buffer. Subsequently strips were washed and incubated (1h, RT, agitated) with a goat-derived polyclonal anti-rat antibody conjugated to HRP at a dilution of 1:2000 in blocking buffer (Cat. Nr. 3050–05, Southern Biotech). After further washing, strips were developed using a chemiluminescent substrate (WesternBright ECL HRP Substrate, Cat. Nr. K-12045-D50).

Densitometric analysis of lipid strips and arrays

Relative quantification of immunoblotting signal intensity on PIP strips and arrays overlaid with PIP-binding proteins was performed using FIJI [79]. For each strip or array, the spot with the highest pixel number was set as a reference for 100% binding; signals coming from all other spots were normalized against it. The data were visualized as bar charts of relative signal intensity as a measure of lipid-binding preference for each PIP-module.

Identification of Giardia orthologues of known PIP-binding domains

PIP-binding domain representatives were used as bait for in silico searches within the Giardia genome database (GDB) (http://giardiadb.org/) using the online tool HHpred (https://toolkit.tuebingen.mpg.de/) to detect remote giardial homologues using hidden Markov models (HMMs; Table 1) [25]. Outputs were firstly evaluated based on the calculated probability and the corresponding E-value for the prediction, with cut-offs for probability and e-value set to 90 and 1e-10, respectively. Sequence identity and similarity were also considered. To validate the prediction, candidate giardial PIP-binding proteins were then utilized as baits to search PDB databases using HHpred to retrieve orthologous PIP-binding proteins/modules. For additional validation, I-TASSER [32-34] was also used to predict hypothetical structures of putative giardial PIP-binding domains next step validation.

Multiple sequence alignment analysis

Multiple sequence alignment using two or more sequences was performed with the Clustal Omega sequence alignment algorithm [80, 81]. The sequences used to compile the alignments shown in S1 Fig were chosen based on representative members for each PIP-binding domain type [1, 10, 82]. Alignments for Figs 6 and 8 were based on previously characterized G1-G4 GTP binding motifs [53] and NECAP1 proteins [57], respectively.

De novo structural modeling and analysis

Ab-initio prediction of hypothetical 3D models presented in S1 Fig was done using I-TASSER [32-34]. The best model was chosen based on the C-score predicted by the algorithm. A C-score is a measure of confidence for a model based on the significance of threading template alignments and the convergence parameters of the structure assembly simulations. It ranges from -5- to 2, with higher C-scores indicating higher confidence. The final 3D structures were displayed using PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.). The superimposition of Giardia PIP-binding proteins with their closest structural orthologue are based on I-TASSER predictions, with structural similarities expressed by TM-score and RMSDa values. The TM-score is computed based on the C-score and protein length. It ranges from 0 to 1, where 1 indicates a perfect match between two structures. RMSDa is the root mean square deviation between residues that are structurally aligned by TM-align [83]. Specifically for GlBAR1 and 2, the structural overlap analysis was performed by selecting positively-charged residues from previously characterized BAR domains shown to play a role in lipid binding [84]. These were manually superimposed on corresponding residues in the predicted GlBAR1 and 2 structures.

Phylogenetic analysis

Selected sequences of GTPase domains (Table 3) were aligned using Clustal Omega tool. The tree construction was submitted to a PHYLogeny Inference Package (PHYLIP) program [85, 86] using random number generator seed set to 111 and number of bootstrap trials set to 10000. The tree was visualized using the on-line tool iTOL and includes branch lengths as a measure of evolutionary distance [87].

Table 3

GTPase domain sequences selected for the phylogenetic analysis of Gl9605, a novel Giardia BDLP.

For each entry, the origin (species and abbreviation), assigned name (protein), unique identifier (UniProtKB) and amino acid stretch used to reconstruct the tree shown in S6 Fig, are provided.

Species and abbreviation	Protein	UniProtKB Identifier	Residue numbers
Aa—Aquifex aeolicus	Aa FtsZ	O66809	1–240
Bv—Bacillus velezensis	Bv DynA	S6FLE6	1–240
Rn—Rattus norvegicus	Rn MFN1	Q8R4Z9	1–240
Hs—Homo sapiens	Hs MFN1	Q8IWA4	1–240
	Hs Dyn1	Q05193	1–240
	Hs Dyn3	Q9UQ16	1–240
	Hs Dyn2	P50570	1–240
	Hs MFN2	O95140	1–240
	Hs DNM1L	O00429	1–240
	Hs ATL1	Q8WXF7	1–240
	Hs GBP1	P32455	1–240
	Hs GBP5	Q96PP8	1–240
	Hs GBP3	Q9H0R5	1–240
	Hs GBP2	P32456	1–240
	Hs MX1	P20591	1–240
	Hs OPA1	O60313	1–277
	Hs ATL3	Q6DD88	1–240
	Hs MX2	P20592	1–240
	Hs GBP4	Q96PP9	1–240
	Hs ATL2	Q8NHH9	1–246
Np—Nostoc punctiforme	Np BDLP	B2IZD3	1–240
Dd—Dictyostelium discoideum	Dd DynA	Q94464	1–240
Ss—Synechocystis sp.	Ss FtsZ	P73456	1–240
Ec—Escherichia coli	Ec LeoA	Q9RFR9	1–240
	Ec FtsZ	AJF44969.1	1–280
Mm—Mus musculus	Mm ATL1	Q8BH66	1–240
Sc—Saccharomyces cerevisiae	Sc SEY1	Q99287	1–240
	Sc FZO1	P38297	1–240
Ce—Caenorhabditis elegans	Ce FZO1	Q23424	1–240
Gl—Giardia lamblia	Gl DRP	E2RU04	1–320
	Gl 9605	A8BAT2	1–320
Bc—Bacillus cereus	Bc BDLP	CUB17917.1	1–280
A—Anoxybacillus sp.	A BDLP1	KXG09432.1	1–280
Cr -Chlamydomonas reinhardtii	Cr FtsZ	BAB91150.1	1–280
Pa—Pseudomonas aeruginosa	Pa BDLP	AMT98798.1	1–280
At—Agrobacterium tumefaciens	At FtsZ	AAC45821.1	1–280
Sm—Sinorhizobium meliloti	Sm FtsZ	AAC45824.1	1–280
Lysinibacillus saudimassiliensis	Ls BDLP	CEA00228.1	1–280

Multiple sequence alignment and structural prediction analysis of G. lamblia PIP-binding domains.

For all PIP-binding modules except GlNECAP1, the sequence of the lipid-binding domain was aligned to its respective homologous domains. Each domain was structurally modelled using I-TASSER (blue) and superimposed on its closest structural homolog (green). For each structural overlay a TM-score and RMSD value are reported, followed by a blow-up of the calculated location of known and predicted PIP-binding motifs. (A-B) Glepsin, (C-E) GlFYVE and Gl16801, (F-L) GlPXD1-6, (M-O) GlBAR1 and GlBAR2, (P-Q) GlFERM and (R-T) GlPROP1 and GlPROP2. (U) Legend to color code for conserved/similar residues. (V) Closest structural homologues, including their origin and identifiers, for structural overlay analysis of PIP-binding modules in G. lamblia. (W) Selected orthologues, including their origin and identifiers, for each G. lamblia PIP-binding module used in the MSA analysis to highlight conserved/similar residues for lipid-binding.

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Lipid-binding properties of Giardia-lipid binding domains.

Lipid-binding and immuno-detection analysis of G. lamblia PIP-binding domains from proteins GlPXD1-6, GlFYVE and GlNECAP1 using lipid strips. (A) Schematic diagram of the pMAL-p2Cx vector used for heterologous expression of individual PIP-binding domains in E.coli. (B) SDS-PAGE analysis of recombinant epitope-tagged MBP-PIP binding domain fusions normalized to 1μg total protein. Protein ladder sizes are included in the first lane. (C) Immuno-detection of epitope-tagged MBP-fusions for each PIP-binding domain overlaid on lipid strips carrying spotted lipid residues at 100 pmol/spot and visualized by chemiluminescence. (D) Lipid binding preferences for all tested MBP-domain fusions, measured using FIJI and visualized as plots of relative signal intensity for each probed lipid residue. Values were normalized to those of lipid residues presenting strongest signal intensity. (E) Lipid binding preferences for GlNECAP1 and GlFYVE investigated using a different set of spotted lipid residues revealed GlNECAP1’s exclusive affinity for cardiolipin.

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Subcellular distribution of PI(3)P, PI(4,5)P2 and PI(3,4,5)P3 in G. lamblia trophozoites.

3D STED microscopy analysis followed by signal overlap and deconvolution of representative non-transgenic wild-type G. lamblia trophozoites co-labeled with anti-GlCHC (in green) antibody and either (A-B) anti-PI(3)P, (C-D) anti-PI(4,5)P2, or (E-F) anti-PI(3,4,5)P3 antibodies (in red). Dorsal and ventral sides are defined with respect to the ventral disk. Scale bar for (A, C, E): 1 μm. Scale bar for (B, D, F): 2 μm. Scale bar for insets in (B, D, F): 0.5 μm.

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Selected PIP-binding protein sub-interactomes and PV-associated nano-environments.

(A) Curated interactomes for GlPXD1, GlPXD4 and GlPXD6. All three epitope-tagged variants used as affinity handles in co-immunoprecipitation experiments identify GlCHC as a strong interaction partner for GlPXD1, 4 and 6. GlPXD1 and 4 further interact with other known clathrin assembly components such as GlCLC, GlAP2 subunits α, β and μ, and GlDRP. GlPXD2, albeit at low stringency, is the only other PXD protein found in all three interactomes. The GlPXD4 interactome includes a putative SNARE protein (5785; [45] while GlPXD6 as an affinity handle pulled down another PIP residue binder, GlFYVE, known to be associated to clathrin assemblies in G. lamblia [19]. (B) The curated extended interactome for GlPXD2 includes all core clathrin assembly components (GlCHC, GlCLC, all GlAP2 subunits, GlDRP [19]) and includes PIP-binders GlPXD4 and GlNECAP1. Three putative SNAREs Gl5785, Gl10013 and Gl14469 were also detected as bona fide GlPXD2 interaction partners, the latter previously detected in the GlPXD4 interactome. (C) Analysis of the extended GlPXD3 interactome using an epitope-tagged variant as affinity handle reveals robust interactions with clathrin assembly components GlCHC, α and β GlAP2 subunits, and GlDRP. Predicted inactive NEK kinase 15411 [48] is similarly associated to clathrin assemblies [19] and further shares proteins Gl16811, Gl87677 and Gl17060 as interaction partners with GlPXD3. Predicted SNARE protein Gl7309, GlNSF (GL50803_1154776) and proteins Gl103709 and Gl9605 are unique GlPXD3 interaction partners. The GlPXD3 interactome is connected to the GlPXD6 circuit both directly and through Gl16717. (D) The extended interactome analysis of epitope-tagged GlFYVE confirmed tight association to GlCHC, GlDRP and GlPXD6. GlNECAP1 as an alternative PIP-binding module was also detected. (E) A GlNECAP1-centered interactome highlights association to clathrin assembly components and to additional PIP-residue binders GlFYVE and GlPXD2. For all interactomes: solid lines: interactions detected at high stringency; dashed lines: interactions detected at low stringency; yellow protein interaction partners are currently annotated on giardiaDB.org as “hypothetical” i.e. of unknown function. (F-H) Nanoenvironments defined by specific sets of interaction partners including clathrin assemblies, PIP-binding proteins, SNARES and proteins of currently unknown function.

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APEX-mediated electron microscopy analysis of GlNECAP1 subcellular deposition.

(A-F) Representative images of transgenic G. lamblia cells expressing construct pE::GlNECAP1-APEX2-2HA showing enlarged PVs and a diffused APEX-dependent cell staining signal. (G-I) Non-transgenic control cells. Scale bar: 1μm.

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Phylogenetic analysis and tree reconstruction for the predicted GTPase domain of the novel dynamin-like protein Gl9605.

Phylogenetic analysis of predicted GTPase domains from the following prokaryotic and eukaryotic species used to compute the tree shown in the figure, including branch lengths as a measure of evolutionary distance: Aa—Aquifex aeolicus, Bv—Bacillus velezensis, Rn—Rattus norvegicus, Hm—Homo sapiens, Np—Nostoc punctiforme, Dd—Dictyostelium discoideum, Ss—Synechocystis sp., Ec—Escherichia coli, Mm—Mus musculus, Sc—Saccharomyces cerevisiae, Ce—Caenorhabditis elegans, Gl—Giardia lamblia, Bc—Bacillus cereus, Ls—Lysinibacillus saudimassiliensis, A—Anoxybacillus sp., Cr—Chlamydomonas reinhardtii, Pa—Pseudomonas aeruginosa, At—Agrobacterium tumefacies, Sm—Sinorhizobium meliloti.

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Proteins identified in the interactome of GlPXD1.

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Proteins identified in the interactome of GlPXD2.

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Proteins identified in the interactome of GlPXD3.

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Proteins identified in the interactome of GlPXD4.

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Proteins identified in the interactome of GlPXD6.

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Proteins identified in the interactome of GlFYVE.

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Proteins identified in the interactome of GlNECAP1.

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List of oligonucleotide names and sequences for construct synthesis.

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Amino acid sequences of lipid-binding modules used in vitro for protein lipid-overlay assay.

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Motility of non-transgenic WB cells following treatment for 50 minutes at 37°C with 0mM neomycin.

(M4V)

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Motility of non-transgenic WB cells following treatment for 50 minutes at 37°C with 2mM neomycin.

(M4V)

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Motility of non-transgenic WB cells following treatment for 50 minutes at 37°C with 7.2mM neomycin.

(M4V)

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Motility of non-transgenic WB cells following treatment for 50 minutes at 37°C with 15mM neomycin.

(M4V)

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7 Oct 2019

Dear Prof. Hehl,

Thank you very much for submitting your manuscript "Phosphoinositide-binding proteins mark, shape and functionally modulate highly-diverged endocytic compartments in the parasitic protist Giardia lamblia" (PPATHOGENS-D-19-01613) for review by PLOS Pathogens. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the manuscript as it currently stands. These issues must be addressed before we would be willing to consider a revised version of your study. We cannot, of course, promise publication at that time.

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Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: Cernikova et al have addressed a longstanding question about the identity of PIP binding proteins in Giardia and their role in regulating endocytic trafficking. They used sensitive bioinformatics tools to identify candidate PIP binding proteins, modeled these proteins to test whether they had conserved residues involved in PIP and lipid binding, and then tested lipid binding activity with in vitro assays. They characterized the localization of these proteins and their relationship to clathrin and fluid phase endocytic cargo using STED super-resolution imaging. Further they explored the function of these proteins in Giardia using a combination of approaches including saturation of binding sites with over expressed PIP binding proteins, dominant negative mutants and chemical inhibitors. They performed crosslinking and pulldown experiments to identify the interactome of several validated lipid binding proteins and established that many of these proteins are in the same clathrin interaction network. They also performed EM demonstrating that some of the dominant negative proteins caused defects in PV morphology. Overall the paper is a major advance and represents a substantial amount of work. What detracts from the work is a focused concise message and a model for how these perturbations block endocytosis. In my opinion there is simply too much data packed into the main figures which will make tracking key points a challenge for the general readership of Plos Pathogens. While the manuscript initially focuses on PIP binding proteins the proteomics results lead to tangents that I think could be reserved for future studies. Rather than following up on new proteins pairwise co-localization studies of the identified lipid binding proteins would lead to a more comprehensive understanding of how these proteins are organized in relationship to each other.

Reviewer #2: Cernikova et al have studied the importance of PIPs in Giardia endocytosis. It is overall a well-performed study but some of studies have been performed earlier and the outline of the paper can be changed to make it more accessible.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: Major Issues:

1. Figure 2 The presented gradients blots seem inconsistent with the strip blots found in the supplement. For example PXD3 very clearly has a dominant preference for PI(3)P on the strip blot but this is less clear on the gradient blot. The number of replicates is not indicated

2. Another issue is that the methods indicates that the MBP fusions were lyophilized. Some proteins cannot tolerate lyophilization as this treatment could impact folding and activity. So were the proteins tested for activity before lyophilization? Of particular interest are the proteins that showed no binding activity. If activity was only tested after lyophilization, then the authors should repeat the lipid binding assays with proteins tested immediately after purification.

3. The authors indicate that their structure prediction identified key preserved residues. If they would mutate some of these for example in the most divergent of the identified proteins with lipid binding activity, this would help support the accuracy of the structure prediction. This could be performed in strip blots or through localization as one would predict the proteins of interest would no longer localize to PVs or bind the blotted lipids.

4. Figure 3. 2mM neomycin seems like a lot of antibiotic and the duration of treatment was not specified. I understand Giardia should not be sensitive to this antibiotic, but I wonder if the authors checked viability of the cells before the endocytosis assay. Also there are two publications that performed bioinformatics to assess phosphoinositide kinase signaling in Giardia and showed that LY29004, an inhibitor of phosphoinositide kinases, impacts cell growth (Cox 2006 and Hernandez 2007). Rather than trying to saturate PIP sites their existence can be blocked using wortmanin or LY294002. One would predict that this would result in altered protein localization and ESV morphology. Additionally, the authors measured the cell width then did a t-test on the median cell size. They should either do a Mann-Whitney U on the median or they should use a t-test to compare means. (Mann-Whitney U is probably better.)

5. Despite extensive super-resolution and EM imaging the authors do not propose any explanation for how their manipulations block endocytosis. Is it that the PVs are no longer open to uptake dextran or is it that they no longer can close so the dextran diffuses away during fixation?

6. Figure 7. How do the authors know that the NT-FYVE is generating a new compartment versus just aggregating? Also I don’t know what relevance this has to the biology of Giardia it seems like an artifact.

7. I have concerns about using the CWP1 promoter to drive over expression. Encystation is promoted by changes in cholesterol and pH which is accompanied by morphological changes that could impact PV function. The authors should, perhaps for their favorite protein, test whether they get similar phenotypes with the tet promoter system. Also it is surprising that the authors did not attempt to use any reverse genetics tools to assess the function of these proteins. Dominant negative and over expression studies can indicate a role but they can also produce misleading results whereas reverse genetics will cleanly indicate if a protein is required for a process of interest.

Reviewer #2: (No Response)

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Minor Issues:

1. Abstract, Author summary and Intro should start by saying “In model eukaryotes” to make it clear that the biology of Giardia is not being discussed.

2. The authors indicate that they could not identify C2 domain containing proteins yet a GiardiaDB text search of C2 yields four proteins. Some of which are phosphoinositide kinases which would be expected to bind PIPs? See GL50803_16728, GL50803_114201, GL50803_17406, GL50803_14855.

3. Figure 1 Panel B can be moved to the supplement as the relevant proteins are shown again in C-I. This also raises the point that the legend stops at C. For panel A, the current size of the DIC insets are too small to see anything, these could be expanded if the panel is moved into the supplement.

4. Several proteomics tables include cytoplasmic HSP70 but the text indicates that no clathrin uncoating proteins were found. Is cytoplasmic HSP70 not involved in clathrin uncoating?

5. Figure 5. While the fact that snare proteins are part of the PXD2 interactome is intriguing, I think including the localization in the main text detracts from a focused story. I recommend removal of panel B. Additionally I think all the interactome data should be combined into one figure to reduce the total number of figures.

6. I am unaware of any particular genetics nomenclature for Giardia, but the authors use of :: to indicate protein fusions is confusing as it is more commonly used to indicate promoter fusions. In particular I was confused by pEHA::GlPXD2 it took me a long time to figure out this is the endogenous promoter driving an N-terminal HA fusion to PXD2. Perhaps pE::HA-GlPXD2 or pE::HA:GlPXD2 would be easier to understand.

7. Figure 6. I think the identification of another dynamin homolog in Giardia is fascinating, but I think its inclusion distracts from a focused story. especially since there is no discussion of how mechanistically the mutant blocks endocytosis. I think a detailed study of this new dynamin and the more conventional dynamin is totally warranted and could be packaged as a focused study with EM work to examine how these proteins are preventing endocytosis. On the other hand if the authors were to perform EM to examine whether the mutants alter ESV morphology this could help explain the endocytosis defect.

8. For the proteomics analysis the methods do not indicate whether individual protein bands, entire lanes, or a certain number of segments were cut out for mass spec analysis?

9. Figure 8 legend. It isn’t clear which promoter was used to drive expression. The authors seem to use ectopic and overexpression interchangeably. I would consider ectopic to be on a plasmid not integrated into the genome and over expression to be when they use the CWP1 promoter to purposely drive expression beyond normal levels. The work would be easier to follow if this was clarified. Is 8D actually using the endogenous promoter as indicated in the graph?

10. Figure 9F dorsal view appears to be in a different orientation than the ventral view?

11. Figure 10. It would be helpful to include a key for the cartoon. Also the authors switched colors for dextran and HA in the middle of the paper. I recommend they always false color HA red and dextran green to match the model regardless of which fluorophores they used. The labels can simply say HA and Dextran and it will help the reader follow the work. 10D-G. In the interest of streamlining the paper I feel that one simplified overall interactome in the working model would be sufficient. The others could be in supplement.

Reviewer #2: 1. Line 44, 55 proteins in what organism(s). In all eukaryotes?

2. Lines 94 and 95. There are of course also amino acid, sugar and nucleoside transporters in the membrane that are crucial for nutrient uptake.

3. I lack more references to already published studies of Giardia PIP binding proteins in the introduction.

4. Line 124. In silico annotation techniques, what specifically is this?

5. Line 132. Not all PIP interacting proteins are associated with endocytosis.

6. Line 175. It is good to make it clear that the localizations were done in this study.

7. Line 192. It is not clear to me why these particular proteins were selected for further studies.

8. Line 197. Was overexpression with another promoter tested on the PXD5 protein to see a stronger signal? I see a signal in 1B.

9. Line 199. G. muris data is now available at NCBI, are all the proteins conserved there?

10. Line 210. This has already been done in an earlier study by Jana et al, 2017.

11. Line 230. Compare new data with old data from Jana. Add the PIP binding data to Table 1.

12. Line 245. How do the authors now that it is saturation, was a titration curve done?

13. Line 251. The controls could have been a parasite overexpressing another PIP domain containing protein not associated with the PVs.

14. Line 264, Neomycin kills Giardia, can this affect uptake?

15. Line 286. The whole interactome part to line 423 can be condensed and the results presented in one table. The follow up data specific for each protein can complement this

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Reviewer #1: No

Reviewer #2: No

6 Jan 2020

Submitted filename: Cernikova et al_response to reviewers.docx

Click here for additional data file.

14 Jan 2020

Dear Prof. Hehl,

We are pleased to inform that your manuscript, "Phosphoinositide-binding proteins mark, shape and functionally modulate highly-diverged endocytic compartments in the parasitic protist Giardia lamblia", has been editorially accepted for publication at PLOS Pathogens.

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***********************************************************

Reviewer Comments (if any, and for reference):

5 Feb 2020

Dear Prof. Hehl,

We are delighted to inform you that your manuscript, "Phosphoinositide-binding proteins mark, shape and functionally modulate highly-diverged endocytic compartments in the parasitic protist Giardia lamblia," has been formally accepted for publication in PLOS Pathogens.

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Michael Malim

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