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Alveolin proteins in the Toxoplasma inner membrane complex form a highly interconnected structure that maintains parasite shape and replication [1]

['Peter S. Back', 'Molecular Biology Institute', 'University Of California', 'Los Angeles', 'California', 'United States Of America', 'Vignesh Senthilkumar', 'Department Of Microbiology', 'Immunology', 'Molecular Genetics']

Date: 2024-09

Apicomplexan parasites possess several specialized structures to invade their host cells and replicate successfully. One of these is the inner membrane complex (IMC), a peripheral membrane-cytoskeletal system underneath the plasma membrane. It is composed of a series of flattened, membrane-bound vesicles and a cytoskeletal subpellicular network (SPN) comprised of intermediate filament-like proteins called alveolins. While the alveolin proteins are conserved throughout the Apicomplexa and the broader Alveolata, their precise functions and interactions remain poorly understood. Here, we describe the function of one of these alveolin proteins in Toxoplasma, IMC6. Disruption of IMC6 resulted in striking morphological defects that led to aberrant invasion and replication but surprisingly minor effects on motility. Deletion analyses revealed that the alveolin domain alone is largely sufficient to restore localization and partially sufficient for function. As this highlights the importance of the IMC6 alveolin domain, we implemented unnatural amino acid photoreactive crosslinking to the alveolin domain and identified multiple binding interfaces between IMC6 and 2 other cytoskeletal IMC proteins—IMC3 and ILP1. This provides direct evidence of protein–protein interactions in the alveolin domain and supports the long-held hypothesis that the alveolin domain is responsible for filament formation. Collectively, our study features the conserved alveolin proteins as critical components that maintain the parasite’s structural integrity and highlights the alveolin domain as a key mediator of SPN architecture.

Funding: This work was supported by the NIH grants AI123360 to P.J.B., AI139201 and AI137767 to G.E.W., and GM071940 to Z.H.Z. P.S.B. was also supported by the Ruth L. Kirschstein National Research Service Award GM007185 and UCLA Molecular Biology Institute (MBI) Whitcome Fellowship. A.K.S. was supported by the NIAID predoctoral training grant AI055402. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In this study, we report the successful knockout of the T. gondii alveolin (TgIMC6). We determine its critical role in maintaining parasite shape and describe the downstream consequences on parasite motility and host cell invasion. We also characterize the severe replication errors exhibited by Δimc6 parasites. To determine the significance of the alveolin domain, we use deletion analyses to demonstrate that the alveolin domain is largely sufficient to restore IMC6 localization and function. We then implement the recently developed photoreactive crosslinking approach to pinpoint direct interactions and map the binding interfaces between the IMC6 alveolin domain and other cytoskeletal proteins of the SPN. This firmly establishes the alveolin domain as a major region of protein–protein contact.

In apicomplexans, the alveolins are believed to be the major constituents of the subpellicular network (SPN) [ 12 , 13 , 15 – 21 ]. The SPN is a highly interwoven mesh of filaments that underlies the membrane vesicles of the IMC and provides a cytoskeletal foundation for the parasite [ 9 , 18 ]. The alveolins are categorized by the presence of a conserved alveolin domain, a region of the protein containing proline and valine-rich repeats [ 1 ]. It is speculated that the alveolin domain mediates the formation of filaments via protein–protein interactions that ultimately establish the SPN. However, direct experimental evidence is lacking, largely due to the detergent insoluble nature of these proteins that limit the use of standard interaction methods such as co-immunoprecipitation (co-IP). To overcome this barrier, we recently adapted unnatural amino acid (UAA) photocrosslinking to T. gondii, which identifies interacting partners within the native environment of the parasite [ 22 ]. This approach uses the zero-length crosslinker p-azidophenylalanine (Azi), enabling us to map specific binding interfaces that provide structural information regarding the interaction [ 23 ]. We previously used this technique to determine that TgILP1 binds 2 alveolin proteins, IMC3 and IMC6. However, these interactions were mapped to their variable N- and C-terminal regions rather than the core alveolin domains. Thus, the alveolin domains remain unexplored in this family of proteins. Functionally, only some of the T. gondii alveolin proteins have been studied so far. Of these, many were shown to play a role in replication or in providing tensile strength, but none were shown to be important or essential for parasite fitness [ 24 , 25 ]. In contrast, the alveolins that have eluded characterization are typically those with low genome-wide CRISPR screen (GWCS) phenotype scores, suggesting essentiality [ 26 ].

In the Apicomplexa, the alveoli are called the inner membrane complex (IMC) and are situated underneath the plasma membrane as in other alveolates. The IMC has been studied most extensively in T. gondii and Plasmodium spp. and contains 3 main functions. It first serves as a platform for the glideosome, an actin-myosin motor that powers gliding motility and host cell invasion [ 8 ]. Second, it provides a scaffold for daughter cell assembly throughout the replication process [ 9 – 11 ]. Finally, the apical cap region of the IMC houses the regulatory center for cytoskeletal disassembly during the final stages of replication [ 12 – 14 ]. While these functions are generally conserved in other apicomplexans, it is unlikely that they are conserved in other alveolates. Due to their nonparasitic lifestyle, ciliates and dinoflagellates have likely co-opted their respective alveoli to meet the demands of a free-living environment. Thus, determining the precise roles of the alveolin proteins promises to provide insights into these differences.

The superphylum Alveolata contains a remarkably diverse group of protozoans, including the free-living ciliates and dinoflagellates as well as the parasitic apicomplexans. Despite these lifestyle differences, the alveolates are unified by a peripheral system of membranes underlying the plasma membrane called the alveoli and a conserved group of proteins called the alveolins [ 1 ]. These 2 features define the superphylum and highlight the alveoli as critical cellular structures for the survival of these organisms. Of the alveolates, the parasitic apicomplexans have garnered the most attention due to their severe public health and economic burdens [ 2 ]. Notable human pathogens include Toxoplasma gondii (toxoplasmosis), Plasmodium spp. (malaria), and Cryptosporidium spp. (cryptosporidiosis), which together cause an enormous disease burden globally that results in a tremendous number of fatalities [ 3 – 5 ]. Veterinary pathogens include Neospora spp. and Eimeria spp., which cause large numbers of disease in livestock and subsequent economic losses [ 6 , 7 ].

Results

Disrupting IMC6 causes severe defects in vitro and in vivo Of the alveolin proteins with low phenotype scores, IMC6 was assigned the highest score of −3.19 [26,27]. Recent work from our lab demonstrated that genes with similar phenotype scores (ISAP1: −3.49 and IMC29: −3.95) could be disrupted [28,29]. Thus, we wanted to determine if IMC6 could also be knocked out. As previously reported, IMC6 localizes to the IMC of both maternal and daughter parasites with enrichment in the daughter buds (Fig 1A) [18]. We were indeed successful in generating a knockout strain (Δimc6), verified by the absence of protein expression in immunofluorescence assays (IFA) and by recombination at the genomic locus using PCR (Fig 1B and 1C). We then generated a complementation construct with the full-length IMC6 cDNA driven by its endogenous promoter with a C-terminal 1xV5 epitope tag (Fig 1D). Expressing this construct in Δimc6 parasites restored protein localization and expression similar to wild-type levels as determined by IFA and western blot (complemented strain: IMC6c; Fig 1E and 1F). PPT PowerPoint slide

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TIFF original image Download: Fig 1. Disrupting IMC6 causes severe fitness and virulence defects. (A) IFA of WT parasites showing proper IMC6 localization in mature and budding parasites. (B) IFA of Δimc6 parasites showing absence of IMC6. (C) PCR verification of the genomic loci from WT (RHΔhxgprt) and Δimc6 parasites. Diagram illustrates primers used to amplify the IMC6 coding sequence (blue arrows) and the site of recombination for the knockout locus (magenta arrows). (D) Diagram of the full-length complementation construct, which includes the endogenous promoter and a V5 epitope tag. Full-length protein is 444 amino acids, with aa128-290 encompassing the alveolin domain. (E) IFA of IMC6c parasites showing restored localization of IMC6 (anti-V5). (F) Western blot of whole cell lysates depicting the absence (Δimc6) and rescue (IMC6c) of IMC6 expression. The difference in migration between WT and IMC6c is due to the V5 epitope tag in the complemented version. ROP13 was used as a loading control. (G) Quantification of plaque areas, where each point represents a biological replicate with 30–40 plaques measured per replicate. Data are plotted as the mean ± SD, and significance was determined using two-way ANOVA. ****: p < 0.0001. (H) Quantification of plaque efficiency, measured by counting the number of plaques relative to the total number of parasites added. Data are plotted as the mean ± SD, and significance was determined using multiple two-tailed t tests. ****: p < 0.0001. (I) Survival curve of mice (n = 4) injected with the indicated parasite strain. All scale bars are 2 μm. The raw data underlying this figure can be found in S1 Data. IFA, immunofluorescence assay; KO, knockout; WT, wild-type. https://doi.org/10.1371/journal.pbio.3002809.g001 To assess the overall fitness cost of disrupting IMC6, we first performed plaque assays. Measuring both plaque area and plaque-forming efficiency indicated a severe 78.8% reduction in plaque size and a 71.7% reduction in plaque efficiency (Fig 1G and 1H). These defects were fully rescued in the IMC6c parasites. To determine if virulence is also affected, we infected mice with 102 wild-type, 105 Δimc6, or 102 IMC6c parasites (Fig 1I). As expected, the mice injected with wild-type or IMC6c parasites succumbed to the infection after 7 days. In contrast, of the 4 mice injected with a 1,000-fold greater number of knockout parasites, 3 survived the infection. This demonstrates a dramatic decrease in virulence that corroborates the in vitro growth defects and highlights the importance of IMC6 for parasite fitness.

Disruption of IMC6 causes extreme shape defects Upon disrupting IMC6, one of the most striking changes was parasite morphology. Δimc6 parasites appeared grossly misshapen in intracellular vacuoles, stained with the IMC markers IMC-associated protein 1 (ISP1), which labels the apical end, and IMC3, which labels the body (Fig 2A). Extracellular Δimc6 parasites were similarly misshapen, as assessed by phase contrast microscopy (Fig 2B). To quantify the shape defects in a nonbiased manner, we used ImageStream imaging flow cytometry [21]. We measured >20,000 individual extracellular parasites for each strain and evaluated parasite morphology under 2 main categories—aspect ratio and circularity (Fig 2C and 2D). As expected, the aspect ratios of wild-type parasites indicated an elongated morphology with a median of 0.59. In contrast, the aspect ratios of knockout parasites were higher with a median of 0.78, indicating a substantially rounder shape on average. This shape defect was fully restored in the complemented parasites. Similarly for circularity, the wild-type and IMC6c populations contained, on average, less circular cells with medians of 4.7 and 5.1, respectively. In contrast, the circularity values for Δimc6 parasites indicated substantially rounder cells with a median of 7.8, again highlighting the grossly misshapen morphology of the knockout parasites. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Δimc6 parasites are extremely misshapen. (A) Phase contrast and IFA of a representative field of Δimc6 vacuoles. ISP1 stains the apical end, while IMC3 stains the body portion of the IMC. Scale bar is 5 μm. (B) Phase contrast field of WT and Δimc6 extracellular parasites. Scale bar is 5 μm. (C) ImageStream analysis of the aspect ratios for each population. Greater aspect ratios indicate rounder objects. Median aspect ratio for WT is 0.59, Δimc6 is 0.78, and IMC6c is 0.61. (D) ImageStream analysis using the circularity parameter, where greater values indicate rounder objects. Median circularity value for WT is 4.72, Δimc6 is 7.78, and IMC6c is 5.05. The raw data underlying these medians can be found in 10.5281/zenodo.13333981. (E, F) Bivariate plots of the Circularity feature vs. the Elongated ML classifier on the WT (E) and Δimc6 (F) samples. The WT population contains 85.3% elongated and 13.3% circular objects. The Δimc6 population contains 46.6% elongated and 46.5% circular objects. This population shift is highlighted by the density plots. The assigned values indicate confidence given by the Elongated ML classifier, where higher values indicate greater confidence of elongatedness. (G) TEM of detergent-extracted and negatively stained parasites. Insets are zoomed in 3-fold. Scale bar is 1 μm. AC, apical complex; IFA, immunofluorescence assay; IMC, inner membrane complex; ISP1, IMC-associated protein 1; ML, Machine Learning; MT, microtubules; SPN, subpellicular network; TEM, transmission electron microscopy; WT, wild-type. https://doi.org/10.1371/journal.pbio.3002809.g002 We extended this quantification by supplementing the circularity measurement with the Elongated Machine Learning (EloML) classifier. The EloML classifier considers a set of 10 features and assigns a binary classification for each object as either elongated or circular (S1 Fig). Taught on 200 wild-type and 200 knockout parasites, the EloML classifier was designed to create a threshold for elongated/circular objects and to assign a confidence value for each one. Upon analyzing our parasites with this approach, the wild-type strain contained 85.3% elongated and 13.3% circular populations (Fig 2E). In contrast, the Δimc6 strain contained 46.6% elongated and 46.5% circular populations (Fig 2F). This dramatic shift in the population is illustrated by the density scatter plots. Taken together, imaging flow cytometry demonstrates that IMC6 is critical to maintain the parasite’s shape. To determine if the shape defects are caused by structural damage to the SPN filaments, we performed transmission electron microscopy (TEM) on detergent-extracted parasites. We found that the parasite ghosts generally resemble the shapes of whole extracellular parasites—elongated for wild-type and swollen for knockout (Fig 2G, additional images in S2 Fig). Importantly, we found that the SPN and microtubule array are clearly present and generally intact in the knockout parasites, indicating that they are not overly sensitive to detergent extraction. Thus, while subtle differences may exist, the cytoskeleton of Δimc6 parasites appears to remain largely intact despite the severe shape defects.

IMC6 is important for proper parasite replication As the subtle defects in motility and invasion are likely insufficient to produce the severe plaque defects of Δimc6 parasites, we evaluated whether replication is affected since the localization of IMC6 is enriched in daughter buds. We first assessed overall replication by quantifying the number of parasites per vacuole at 24 and 32 hours postinfection (hpi) (Fig 4A and 4B). At 24 hpi, the majority of wild-type vacuoles contained 8 parasites. In contrast, Δimc6 vacuoles were more evenly split between 4 and 8 parasites per vacuole, indicating they are progressing significantly slower compared to wild-type parasites. This defect became more pronounced at 32 hpi, where the majority of wild-type vacuoles contained 16 parasites, while a significantly fewer number of Δimc6 vacuoles contained 16 (Fig 4B). Thus, Δimc6 parasites can progress through endodyogeny, albeit at a considerably slower rate than wild-type parasites. PPT PowerPoint slide

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TIFF original image Download: Fig 4. IMC6 is important for proper endodyogeny. (A, B) Replication assays conducted at 24 (A) and 32 (B) hpi. Each category represents the number of parasites per vacuole. Triplicate experiments were performed by quantifying >300 total vacuoles across at least 15 different fields per replicate. Data are plotted as the mean ± SD, and significance was calculated using two-way ANOVA. (A) shows significance for 4 and 8 parasites per vacuole. (B) shows significance for 4, 8, and 16 parasites per vacuole. **: p = 0.0018. ****: p < 0.0001. (C-H) Representative IFAs depicting various replication defects, including asynchrony (C), multi-daughters (D), disorganization (E), breaks in IMC structure (F), and incomplete separation in mature (G) and budding (H) parasites. All IFAs were conducted approximately 30 hpi. All scale bars are 2 μm. (I) Quantification of abnormal vacuoles as defined by the presence of any one of the replication defects. Triplicate experiments were performed with >200 total vacuoles counted across at least 15 different fields per replicate. Data are plotted as the mean ± SD, and significance was calculated using two-tailed t tests. ****: p < 0.0001. (J) Phase contrast and IFA of a field of extracellular Δimc6 parasites. White arrows highlight incompletely separated parasites. Scale bar is 5 μm. (K) Quantification of the incomplete separation phenotype in extracellular parasites. Triplicates performed by counting >300 individual parasites across at least 15 different fields per replicate. Data are plotted as the mean ± SD, and significance was calculated using two-tailed t tests. ****: p < 0.0001. The raw data underlying this figure can be found in S1 Data. hpi, hours postinfection; IFA, immunofluorescence assay; IMC, inner membrane complex. https://doi.org/10.1371/journal.pbio.3002809.g004 To pinpoint specific replication defects, we first stained Δimc6 parasites with a series of markers to evaluate various parasite organelles. These IFAs indicated that the apicoplast, mitochondria, plant-like vacuole (PLV/VAC), micronemes, and rhoptries localize properly and appear unaffected in the absence of IMC6 (S4 Fig). We then stained parasites with the IMC markers ISP1 and IMC3 to assess the fidelity of endodyogeny. We noticed several errors including asynchronous division, >2 daughter buds per maternal parasite, grossly unorganized vacuoles, large breaks in the IMC structure, and incomplete daughter separation (Fig 4C–4H). Many of the Δimc6 vacuoles exhibited multiple replication defects simultaneously. Thus, rather than quantifying each defect individually, we categorized each vacuole as either standard or abnormal depending on the presence of any one of these replication defects. This revealed an astonishing 92.8 ± 6.1% of abnormal Δimc6 vacuoles compared to 7.4 ± 2.7% of abnormal wild-type vacuoles and 4.7 ± 1.4% of abnormal IMC6c vacuoles (Fig 4I). We were particularly intrigued by the incompletely separated parasites. During the final stages of endodyogeny, the parasites seem to stall very early during cytokinesis, resulting in conjoined bodies (Fig 4G). We even observed conjoined parasites that have begun the next round of division, suggesting that cytokinesis is decoupled from the cell cycle in these parasites (Fig 4H). This defect was also seen in naturally egressed extracellular parasites, with 19.4 ± 3.5% of Δimc6 parasites exhibiting this phenotype (Fig 4J and 4K). To determine if this is linked to basal complex formation, we endogenously tagged BCC1 in both wild-type and knockout parasites [31,32]. First, we assessed properly separated parasites and found that BCC1 localizes to the basal ends of each mature and daughter parasite (S5A and S5B Fig). Moreover, the maternal basal complexes appear to be fully contracted, indicating that both the formation and contraction of the basal complex are unaffected in knockout parasites. We then evaluated the basal complex in incompletely separated parasites, which again showed proper BCC1 localization in mature and daughter cells (S5C Fig). This indicates that the cytokinesis phenotype is not due to basal complex defects. Taken together, the severe defects in replication combined with slower invasion likely explain the drastic growth defects in vitro and virulence defects in vivo.

The alveolin domain is largely sufficient for IMC6 localization and partially sufficient for its function We and others have previously reported that the alveolin domain is largely sufficient for the proper localization of IMC3, IMC6, and IMC8 [18,22]. However, these studies were done in wild-type parasites, so the functional significance of the alveolin domain has not been tested. Thus, we generated IMC6 deletion constructs that truncate the N-terminal and/or C-terminal regions flanking the alveolin domain and expressed them in Δimc6 parasites. We first assessed the localization of each truncated protein and found results consistent with our previous study [22]. The localization of IMC62-290 closely resembles that of the full-length IMC6, while both IMC6128-290 and IMC6128-444 exhibit slightly more cytoplasmic staining (Fig 5A–5C). This demonstrates that the alveolin domain alone is sufficient to restore much of the protein’s localization, although the N-terminal portion of the protein is also important. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Domain analysis of IMC6 correlates parasite form and function. (A) Diagram and IFA of IMC62-290, showing proper localization. (B) Diagram and IFA of IMC6128-290, showing partial IMC localization with additional cytoplasmic staining. (C) Diagram and IFA of IMC6128-444, showing partial IMC localization with additional cytoplasmic staining. (D) Quantification of plaque areas for each deletion construct. The data for WT, Δimc6, and IMC6c were collected in the same experiment as those from Fig 1G and are shown here again to facilitate a direct comparison. Triplicates were performed by measuring 30–40 plaques per replicate, data are plotted as the mean ± SD, and significance was calculated using two-way ANOVA. ****: p < 0.0001. (E) Quantification of plaque efficiency. The data for WT, Δimc6, and IMC6c were collected in the same experiment as those from Fig 1H and are shown here again to facilitate a direct comparison. Data are plotted as the mean ± SD, and significance was calculated using two-tailed t tests. ****: p < 0.0001. (F, G) ImageStream analysis of each population, showing aspect ratio (F) and circularity (G). The data for WT, Δimc6, and IMC6c were collected in the same experiment as those from Fig 2C and 2D and are shown here again for ease of comparison. Median aspect ratio for IMC62-290 is 0.68, IMC6128-290 is 0.73, and IMC6128-444 is 0.73. Median circularity value for IMC62-290 is 6.04, IMC6128-290 is 6.63, and IMC6128-444 is 6.71. All scale bars are 2 μm. The raw data underlying these medians can be found in 10.5281/zenodo.13333981, and the raw data underlying the graphs can be found in S1 Data. IFA, immunofluorescence assay; IMC, inner membrane complex; WT, wild-type. https://doi.org/10.1371/journal.pbio.3002809.g005 To dissect the function of each domain, we performed plaque assays. Measuring both plaque area and plaque efficiency revealed a pattern that mimics the localization data. IMC62-290 fully rescues the plaque size and partially rescues plaque efficiency. On the other hand, IMC6128-290 and IMC6128-444 partially rescue plaque size and does not rescue plaque efficiency at all (Fig 5D and 5E). We then used ImageStream to determine if growth defects are linked to parasite shape. Consistent with the plaque assays, the quantifications for aspect ratio and circularity followed a similar pattern (Fig 5F and 5G). Parasites expressing IMC62-290 most resembled the elongated shape of wild-type parasites, though not fully. In contrast, parasites expressing IMC6128-290 or IMC6128-444 moderately rounded, indicating a partial rescue of shape. Phase contrast images of each complemented strain further support the partial rescue of shape (S6 Fig). Taken together, this domain analysis demonstrates that the alveolin domain alone is largely sufficient for localization and partially sufficient for function, with the N-terminal third of the protein also contributing an important role. We additionally uncover a strong correlation between parasite shape and parasite fitness, with more rounded parasites exhibiting decreased fitness. Thus, IMC6 provides a critical structural role that defines parasite shape, which ultimately impacts parasite motility, invasion, and replication.

IMC3 binds IMC6 at multiple residues across the alveolin domain To identify the crosslinked partners of IMC6, we considered candidates whose localization resembles IMC6 and predicted molecular weight represents the upshifted product. For the larger crosslinked products at approximately 140 and 175 kDa, IMC3 appeared to be the most likely candidate. To determine if IMC3 is a crosslinked partner of IMC6, we chose N162 to carry out preliminary experiments as proof of concept. We first performed a denaturing co-IP experiment, in which the irradiated and crosslinked samples are boiled in 1% SDS before being diluted to standard RIPA buffer conditions for the IP (Fig 8A) [22]. Probing with anti-hemagglutinin (HA) indicated that we successfully purified both uncrosslinked and crosslinked products, migrating at the same molecular weights as the whole cell lysate samples in Fig 7B. Probing with anti-IMC3 demonstrated that IMC3 is indeed the binding partner for IMC6 at this residue. As a complementary approach, we endogenously tagged IMC3 with a spaghetti monster Myc (smMyc) epitope tag in the amber mutant parasite strain, performed photoreactive crosslinking, and compared the upshift sizes between the untagged and smMyc-tagged samples (Fig 8B). We observed a significant size difference between the untagged and tagged versions, confirming that IMC3 binds IMC6 at residue N162. Based on this preliminary evidence, we evaluated the remaining residues using the endogenous tagging approach as this provides a definitive identity of the binding partner and produces more consistent results than co-IPs. PPT PowerPoint slide

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TIFF original image Download: Fig 8. IMC3 binds the IMC6 alveolin domain at multiple sites. (A) Western blot of IMC6-N162* denaturing IP following Azi incorporation and UV treatment. Black arrowhead represents uncrosslinked IMC62-290-3xHA (approximately 45 kDa). Purple arrowhead represents uncrosslinked IMC3 nonspecifically captured in the IP (about 85 kDa). Red arrowhead represents the crosslink-of-interest, seen in both the HA and IMC3 blots (approximately 175 kDa). (B-L) Western blot of whole cell lysates following Azi incorporation and UV treatment. For each residue, both WT and IMC3-smMyc strains were subjected to photoreactive crosslinking and analyzed on the same blot for direct comparison. Black arrowheads represent uncrosslinked IMC62-290-3xHA (about 45 kDa). Blue arrowheads indicate the crosslinked product at approximately 140 kDa, which is not shifted further by the IMC3-smMyc tag. Red arrowheads represent the crosslinks-of-interest, which migrate even higher in the IMC3-smMyc sample (about 175 kDa in the WT vs. approximately 200 kDa in the IMC3-smMyc). Orange arrowheads represent faint crosslinks-of-interest with similarly higher migration in the IMC3-smMyc sample. Every blot was detected with anti-HA. Azi, p-azidophenylalanine; HA, hemagglutinin; IP, immunoprecipitation; smMyc, spaghetti monster Myc; WT, wild-type. https://doi.org/10.1371/journal.pbio.3002809.g008 We thus endogenously tagged IMC3 with smMyc in the other 10 residues of similar-sized crosslinked products, performed photoreactive crosslinking on both the untagged and tagged versions, and analyzed each pair by western blot (Fig 8C–8L). Five of these residues (Y221, E229, E243, E245, and K277) showed an unmistakable size difference due to the smMyc tag, from approximately 175 kDa in the untagged version to about 200 kDa in the tagged version (indicated by red arrowheads). Three other residues (K224, Q234, and K240) showed a similar size difference (orange arrowheads). While these almost certainly bind to IMC3 as well, we denoted them as likely IMC3-binding due to the fainter upshifts of the tagged versions. Nonetheless, this demonstrates that IMC3 binds IMC6 via an array of residues spanning the alveolin domain. We also observed upshifts migrating at about 140 kDa throughout the alveolin domain (blue arrowheads). These, however, did not shift higher in the IMC3smMyc strain, indicating that IMC3 is not the binding partner for this crosslinked product. While the approximately 140 kDa crosslink was the only product for K236 and D249, it was an additional product for all other residues. This suggests that the same residue may have the capacity to interact with both IMC3 and a second, unknown protein or that some background occurs with this shifted product.

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