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A dynamic interplay between chitin synthase and the proteins Expansion/Rebuf reveals that chitin polymerisation and translocation are uncoupled in Drosophila [1]

['Ettore De Giorgio', 'Institut De Biologia Molecular De Barcelona', 'Ibmb-Csic', 'Parc Científic De Barcelona', 'Barcelona', 'Panagiotis Giannios', 'Institute For Research In Biomedicine', 'Irb Barcelona', 'The Barcelona Institute Of Science', 'Technology']

Date: 2023-01 

Chitin is a highly abundant polymer in nature and a principal component of apical extracellular matrices in insects. In addition, chitin has proved to be an excellent biomaterial with multiple applications. In spite of its importance, the molecular mechanisms of chitin biosynthesis and chitin structural diversity are not fully elucidated yet. To investigate these issues, we use Drosophila as a model. We previously showed that chitin deposition in ectodermal tissues requires the concomitant activities of the chitin synthase enzyme Kkv and the functionally interchangeable proteins Exp and Reb. Exp/Reb are conserved proteins, but their mechanism of activity during chitin deposition has not been elucidated yet. Here, we carry out a cellular and molecular analysis of chitin deposition, and we show that chitin polymerisation and chitin translocation to the extracellular space are uncoupled. We find that Kkv activity in chitin translocation, but not in polymerisation, requires the activity of Exp/Reb, and in particular of its conserved Nα-MH2 domain. The activity of Kkv in chitin polymerisation and translocation correlate with Kkv subcellular localisation, and in absence of Kkv-mediated extracellular chitin deposition, chitin accumulates intracellularly as membrane-less punctae. Unexpectedly, we find that although Kkv and Exp/Reb display largely complementary patterns at the apical domain, Exp/Reb activity nonetheless regulates the topological distribution of Kkv at the apical membrane. We propose a model in which Exp/Reb regulate the organisation of Kkv complexes at the apical membrane, which, in turn, regulates the function of Kkv in extracellular chitin translocation.

Funding: This work was supported by the Spanish Ministerio de Ciencia e Innovación (FPI Fellowship BES-2016-076723 to EDG and BFU-2015-68098-P and PGC2018-098449-B-I00 grants to ML). PG is a researcher in Prof. Jordi Casanova's lab funded by Spanish Ministerio de Ciencia e Innovación (PGC2018-094254-B-100 grant) and the CERCA Program of the Catalan Government. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In this work, we have investigated the cellular and molecular mechanisms of chitin deposition in Drosophila and the roles of exp/reb and kkv in the process. We have found that the activities of Kkv in chitin polymerisation and chitin translocation are uncoupled, and we propose that chitin translocation, but not chitin polymerisation, requires Exp/Reb activity. Our cellular analysis has revealed that when extracellular chitin deposition is prevented, Kkv-polymerised chitin accumulates in the cytoplasm as membrane-less punctae. In addition, we detected a clear correlation between Kkv function in chitin polymerisation and/or translocation and Kkv subcellular localisation. A molecular analysis of Exp/Reb and Kkv proteins, using a structure–function approach, revealed key functions of different conserved motifs of these proteins in chitin polymerisation and extracellular deposition and in protein subcellular localisation. A detailed analysis of the subcellular localisation of Exp/Reb and Kkv indicates that these proteins display a largely complementary pattern at the apical membrane. However, in spite of this complementary pattern, we find that Exp/Reb regulate the pattern of distribution of Kkv protein at the apical membrane. Based on the current understanding of the activity of glycosyltransferases like CES and on the knowledge of Drosophila CHS activity, we propose a model in which Exp/Reb regulate chitin deposition by modulating the distribution and organisation of Kkv complexes at the apical membrane, which would regulate the capacity of Kkv to translocate and release chitin fibers extracellularly.

In Drosophila, CHS-A is encoded by krotzkopf verkehrt (kkv), which is responsible for chitin deposition in ectodermal tissues [ 11 , 12 ]. But besides kkv, our previous work identified another function exerted by expansion (exp) and rebuf (reb) to be required for chitin deposition. Exp and Reb are two homologous proteins, containing a conserved Nα-MH2 domain, that serve the same function, as the presence of only one of them can promote chitin deposition. In the absence of exp/reb, no chitin is deposited in ectodermal tissues, in spite of the presence of kkv, indicating that this function is absolutely required. But most importantly, we found that kkv and exp/reb compose the minimal genetic network, which is not only required, but also sufficient for chitin deposition. Thus, the concomitant expression of the two activities, kkv+exp/reb, promotes increased chitin deposition in ectodermally derived tissues that normally deposit chitin, like the trachea, and ectopic chitin deposition in ectodermally derived tissues that normally do not deposit chitin, like the salivary glands [ 13 ]. In spite of the capital importance of the exp/reb function in chitin deposition, the mechanism of activity of exp/reb has not been identified yet, nor their putative relation/interactions with kkv.

In insects, chitin is found in ectodermal tissues, where it forms chito-protein cuticles, and in the gut, where it forms a Peritrophic Matrix. Chitin is deposited to the extracellular space by chitin synthases (CHS) enzymes, which belong to the family of β-glycosyltransferases, which also includes cellulose synthases (CES) and hyaluronane synthases (HS). Most insect species encode two CHS types, CHS-A, required for chitin deposition in epidermis, trachea, foregut, and hindgut, and CHS-B, required for chitin deposition in the midgut, as a principal component of the peritrophic matrix [ 1 , 2 , 7 ]. The exact mechanism of chitin deposition is not fully elucidated yet, but it is proposed to occur in consecutive steps: (1) polymerisation by the catalytic domain of CHS; (2) translocation through the CHS of the nascent polymer across the membrane and release into the extracellular space; and (3) spontaneous assembly of translocated polymers to form crystalline microfibrils [ 1 , 2 , 8 – 10 ].

Chitin, a polymer of UDP-N-acetylglucosamine (GlcNAc) monomers, is a principal component of the apical extracellular matrix in arthropods. Chitin has a recognised importance in physiology [ 1 , 2 ] but also as a biomaterial [ 3 ]. Chitin and its deacetylated form, chitosan, are nontoxic and biodegradable biopolymers with numerous applications in many sectors such as biomedicine, biotechnology, water treatment, food, agriculture, veterinary, or cosmetics [ 4 , 5 ]. So far, the main commercial sources of chitin are crab and shrimp shells [ 6 ]. Chitin isolation and purification from these sources require several treatments to remove proteins, calcium carbonate, lipids, and pigments, and no standarised methods exist nowadays [ 6 ]. In addition, these treatments have many industrial drawbacks such as high energy consumption, long handling times, solvent waste, high environmental pollution, and high economical costs, among others [ 4 ]. The synthesis of chitin in vitro can represent a more ecological, efficient, and “green” method as an alternative to the chemical procedures. Thus, it is critical to understand the molecular mechanisms of chitin deposition for a streamlined chitin production for multiple applications.

Results

1. The activities of Kkv in chitin polymerisation and translocation are uncoupled, and Exp/Reb activity is specifically required for chitin translocation Chitin deposition is proposed to occur in 3 consecutive steps: (1) chitin polymerisation by CHS; (2) translocation of the nascent polymer through a CHS chitin-translocating channel across the membrane and release into the extracellular space; and (3) spontaneous assembly of translocated polymers to form crystalline microfibrils. In addition, it has also been proposed that the chitin polymerisation and translocation steps are tightly coupled [1,2,8–10]. However, no experimental data are available on Kkv with respect to this model. We aimed to investigate this model by carrying out a molecular and cellular analysis of the roles of Kkv and Exp/Reb in chitin deposition. We found that Kkv can promote extracellular chitin deposition (which requires chitin translocation) in ectodermal tissues only in combination with Exp/Reb activity. kkv overexpression in the tracheal system rescues the lack of chitin in kkv mutants [13] and shows a comparable pattern to endogenous Kkv (S1A–S1D Fig). kkv overexpression does not affect extracellular chitin deposition (which starts from stage 13 as in the wild type) or tracheal morphogenesis (Figs 1A, 1B and S1E; [13]). However, we detected the presence of intracellular chitin punctae at early stages (before stage 14) (Figs 1A’ and S3), which indicates the ability of kkv to polymerise chitin. These intracellular chitin punctae disappeared from stage 14, when chitin is then deposited extracellularly, and were not detected at later stages (Fig 1B’). This switch from intracellular chitin to extracellular chitin perfectly correlates with the expression of exp/reb [13], suggesting that exp/reb promote extracellular chitin deposition. In agreement with this, in exp reb mutants overexpressing kkv, we found intracellular chitin punctae until late stages and no extracellular chitin deposition (Figs 1C and S3; [13]). We also found intracellular chitin punctae and no extracellular chitin when we overexpressed kkv in salivary glands, which do not express exp and reb (Figs 1D and S3; [13]). In contrast, the concomitant overexpression of kkv and exp/reb anticipates and increases extracellular chitin deposition in the trachea (S1E and S1F Fig), which leads to tracheal morphogenetic defects (S1G, S1H and S3 Figs; [13]). In addition, kkv and exp/reb coexpression in salivary glands promotes chitin deposition in the lumen (S1I and S3 Figs; [13]). No intracellular punctae of chitin were detected under these conditions, suggesting that all chitin synthesised by Kkv is deposited extracellularly by Exp/Reb activity. Thus, we propose that the functions of Kkv in chitin polymerisation and translocation are uncoupled and that Exp/Reb activity is required for chitin translocation and release to the extracellular space. In this context, the presence of intracellular chitin would reflect the activity of Kkv in chitin polymerisation that cannot be further processed and translocated due to the absence of Exp/Reb activity. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Analysis of the role of the Nα-MH2 domain of Exp/Reb. All images show projections of confocal sections, except D, I, L, O, and Q, which show single confocal sections. (A, B) The overexpression of GFP-kkv in the trachea leads to the presence of intracellular chitin vesicles at early stages (pink arrowheads) (A, A’). At later stages, intracellular chitin vesicles are not present, and chitin is deposited extracellularly in the lumen (blue arrowheads and inset) (B, B’). (C, C’) In exp reb mutants, the overexpression of GFP-kkv in the trachea produces intracellular chitin punctae until late stages (pink arrowheads). (D) The overexpression of GFP-kkv in salivary glands produces intracellular chitin vesicles (pink arrowheads). (E) Schematic representation of Exp protein. (F, G) In exp reb mutants, the expression of a wild-type form of exp/reb rescues the lack of extracellular chitin deposition (F, white arrow and inset), while expΔMH2/rebΔMH2 do not (G, white arrow points to absence of CBP). (H, I) The co-overexpression of GFP-kkv and expΔMH2 in control embryo does not produce morphogenetic defects in trachea (H) or extracellular chitin deposition in salivary glands (white arrow) (I); however, intracellular chitin punctae are present (pink arrowheads in H, I, and inset in H). (J) The coexpression of GFP-kkv and expΔMH2 in exp reb mutants produces intracellular chitin particles (pink arrowheads) but does not rescue the lack of extracellular chitin deposition. (K, L) RebΔMH2 localises apically in trachea (K) and in salivary glands (L). (M) MH2-reb is not able to rescue the absence of extracellular chitin deposition in exp reb mutants. (N, O) The simultaneous expression of MH2-reb and GFP-kkv does not produce morphogenetic defects or ectopic chitin deposition in trachea (N) and in salivary glands (white arrow in O), but intracellular chitin vesicles are present (pink arrowhead in O). (P, Q) MH2-Exp protein does not localize apically in trachea (P) or in salivary gland (Q). Scale bars A-C, F-H, J, M, N: 25 μm; D, I, K, L, O-Q: 10 μm. https://doi.org/10.1371/journal.pbio.3001978.g001 We note that our experimental approach (using CBP staining to visualise chitin) could not detect intracellular chitin punctae produced by endogenous kkv in the absence of exp/reb in trachea or salivary glands. We propose that endogenous levels of Kkv are limiting and cannot produce sufficient intracellular chitin that we can detect. Therefore, we used the overexpression of kkv, which behaves as the wild-type protein, to augment its activity.

2. Structure–Function analysis of the roles of Exp/Reb in chitin translocation We aimed to understand how Exp/Reb may regulate Kkv-dependent chitin translocation. The only recognisable domain of Exp/Reb identified was an Nα-MH2 [13,14]. However, in the course of this work, we identified a second domain highly conserved, which we called conserved motif 2 (CM2) (Fig 1E). We investigated the functional requirements of each of these two domains in chitin deposition. We generated different UAS transgenic mutant lines with the aim to evaluate their ability to rescue the lack of exp reb activity and their ability to promote chitin deposition when coexpressed with kkv. In agreement with their interchangeable activities, the results we obtained for exp or reb were comparable, so we will refer to them indistinctly.

2.2. The conserved motif 2 is required for Exp localisation We searched for conserved domains by comparing the amino acid sequences of several Exp homologs. We identified a highly conserved region not previously described in the literature. The conserved motif 2 (from now on CM2) contained a region of 8 aa highly conserved (blue square in Fig 2A) followed by a region of 9 aa less conserved (red square in Fig 2A). To investigate the functional activity of the CM2, we generated UASexpΔCM2 transgenic lines. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Analysis of the role of the CM2 domain of Exp/Reb. (A) Alignment of amino acids (aa) sequences of the isoform B of Exp (aa 356–433) and homologs; the blue square indicates 8 aa highly conserved, the red square includes 9 aa less conserved. (B-D) Show projections of confocal sections and (E-F) show single confocal sections. (B) The overexpression of expΔCM2 in an exp reb mutant background rescues the lack of extracellular chitin deposition. (C, D) The simultaneous expression of expΔCM2 and GFP-kkv produces morphogenetic defects in the trachea (arrowheads in C) and ectopic chitin deposition in the lumen of salivary glands (D). (E, F) Overexpressed Exp localises mainly in the apical region (orange arrowheads) with respect the basal domain (yellow arrowheads), while the apical accumulation of overexpressed ExpΔCM2 is less conspicuous (F). (G) Quantifications of accumulation of Exp and ExpΔCM2 in apical versus basal region. n corresponds to the number of ratios analysed (apical/basal ratio per cell), and brackets indicate the number of embryos used. Ratios were obtained from the apical (orange line in E) and basal (yellow line in E) domains of single cells in trachea and salivary glands. The underlying data for quantifications can be found in the S1 Data. Scale bars B, C: 25 μm; D-F: 10 μm. https://doi.org/10.1371/journal.pbio.3001978.g002 The expression of expΔCM2 in an exp reb mutant background rescued the lack of extracellular chitin deposition (Figs 2B and S3), indicating that the protein is functional. In agreement with this, when coexpressed with kkv in the trachea, it produced morphogenetic defects (Figs 2C and S3), comparable to the overexpression of kkv and exp/reb (S1H and S3 Figs). Similarly, expression of kkv and expΔCM2 in salivary glands produced ectopic chitin deposition in the luminal space (Fig 2D). These results indicated that the CM2 is not required for chitin polymerisation and translocation to the extracellular space. In agreement with no requirements of the CM2 in chitin translocation, no intracellular chitin punctae were detected when coexpressing kkv and expΔCM2 (Figs 2D and S3). To further investigate the role of the CM2, we analysed protein localisation. The endogenous Exp and Reb proteins localise mainly apically at the membrane, although a bit of the protein can be detected intracellularly (S2D Fig; [13]). This pattern of subcellular accumulation was maintained when overexpressing Exp (Fig 2E). In contrast, overexpressed ExpΔCM2 did not show such a distinct apical localisation (Fig 2F). We analysed the ratio of accumulation of the Exp proteins (control and ExpΔCM2) in apical versus basal regions. Quantifications indicated a decreased apical enrichment of ExpΔCM2 compared to full-length Exp (Fig 2H). This result indicated that the CM2 is involved in Exp/Reb localisation. However, we could still detect accumulation of ExpΔCM2 at the apical membrane (Fig 2F), which correlated with its activity in chitin deposition. In summary, we identified a highly conserved domain in Exp proteins that is dispensable for chitin polymerisation and translocation but is required for correct protein localisation, which we propose is important for Exp/Reb activity (see Discussion).

3. Structure–Function analysis of two conserved Kkv domains in chitin deposition Kkv is a large protein with multiple functional domains and several transmembrane domains [9,15,16]. As for other members of the β-glycosyltransferase family, it is proposed that the activity of CHS, like Kkv, depends on oligomerisation and interactions with other proteins [1,2,9]. We investigated two Kkv domains with putative roles in direct or indirect interactions with other proteins or in oligomerisation [11,15], the conserved motif WGTRE (amino acids (aa) 1,076–1,080) and a coiled-coil (CC) domain (aa 1,087–1,107) (Fig 3A). PPT PowerPoint slide

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TIFF original image Download: Fig 3. Analysis of the WGTRE and CC domains of Kkv. (A) Schematic representation of Kkv protein (CD, catalytic domain; WGTRE; CC, coiled-coil domain). (B, C, G, I, J) Show projections of confocal sections and (D-F, H, K-N) show single confocal sections. (B) The overexpression of GFP-kkvΔWGTRE in a kkv mutant background does not rescue the absence of extracellular chitin deposition (white arrow, note the absence of CBP) and the protein accumulates in a generalised pattern. (C-D) The overexpression of GFP-KkvΔWGTRE does not produce intracellular chitin punctae, neither in trachea at early stages (C-C’) nor in salivary glands (D). (E-E”‘) GFP-kkvΔWGTRE colocalise with the ER marker KDEL. (F) GFP-KkvΔWGTRE does not colocalise with the marker FK2. (G) The overexpression of GFP-kkvΔCC in a kkv mutant background rescues the lack of extracellular chitin deposition in the trachea (note the presence of CBP staining). (H, I) The simultaneous expression of reb and GFP-kkvΔCC in salivary glands produces ectopic extracellular chitin (H), and no defects in trachea (I). (J) The overexpression of reb in trachea leads to morphogenetic defects. (K, L) Overexpressed GFP-Kkv localises mainly apically (orange arrowheads) although a bit of the protein can be detected in the basal region (yellow arrowheads). (M, N) Apical accumulation of overexpressed GFP-KkvΔCC is less conspicuous. (O) Quantifications of accumulation of GFP-Kkv and GFP-KkvΔCC in apical versus basal region. n corresponds to the number of ratios analysed (apical/basal ratio per cell), and brackets indicate the number of embryos used. Ratios were obtained from the apical (orange line in K) and basal (yellow line in K) domains of single cells in trachea and salivary glands. The underlying data for quantifications can be found in the S1 Data. Scale bars B, C, G, I, J: 25 μm; D-F, H, K-N: 10 μm. https://doi.org/10.1371/journal.pbio.3001978.g003

3.1. The WGTRE domain is required for Kkv ER-exit The conserved motif WGTRE was proposed to be an essential domain for Kkv activity as a point mutation changing the glycine renders an inactive protein [12]. This domain has been suggested to be involved in oligomerisation or interactions with other factors [11]. We generated a protein lacking this domain, GFP-KkvΔWGTRE. To determine the activity of this protein, we assayed its rescuing capacity in a kkv mutant background. While a wild-type form of kkv can rescue the absence of chitin produced by the absence of kkv [13], GFP-kkvΔWGTRE could not, and trachea was defective (Figs 3B and S3). This indicated that the protein is not functional, confirming that the WGTRE domain is essential for chitin production. Accordingly, expression of GFP-kkvΔWGTRE did not produce chitin vesicles in the trachea at early stages (Figs 3C and S3), or in salivary glands (Fig 3D), as GFP-kkv does (Figs 1A, 1D and S3), indicating absence of chitin polymerisation. To better understand the role of this domain, we analysed the localisation of the GFP-KkvΔWGTRE protein. We found no apical accumulation of this protein, neither in a wild-type background nor in a kkv mutant background (Fig 3B). Instead, we found a generalised pattern in the cytoplasm. Costainings with the ER marker KDEL (Fig 3E) indicated that the GFP-KkvΔWGTRE protein is retained in the ER. ER retention may be due either to a defective folding of the protein or to a specific effect of this domain in Kkv trafficking to the membrane. Proteins with a defective folding are degraded from ER upon ubiquitination [17]. To distinguish between these two possibilities, we used the FK2 antibody that recognises mono- and polyubiquitinated conjugates, but not free ubiquitin [18]. From our results, GFP-KkvΔWGTRE and FK2 do not colocalise (Fig 3F), indicating that the protein is not ubiquinated. The results strongly suggested a role for the WGTRE domain in Kkv trafficking from the ER to the membrane. We also concluded that ER exit is required for chitin polymerisation by Kkv.

3.2. The coiled-coil domain is required for Kkv localisation and full Kkv activity Class A CHS contain a CC domain localised C-terminal to the active centre. Potentially, the CC domain could mediate association to yet unknown partner/s, or be involved in protein oligomerisation, regulating CHS localisation or activity [8,15,16]. We generated a protein lacking the CC region, GFP-KkvΔCC. This mutant protein rescued the lack of chitin in a kkv mutant background (Figs 3G and S3), indicating that it is functional. Accordingly, the concomitant expression of reb and GFP-kkvΔCC in salivary glands resulted in deposition of chitin in the luminal space (Figs 3H and S3). Altogether, these results indicated that GFP-KkvΔCC acts as a functional protein in these contexts. We found, however, a condition in which GFP-kkvΔCC behaved differently from GFP-kkv. The overexpression of reb and full-length GFP-kkv in the trachea produced strong morphogenetic defects (S1H and S3 Figs; [13]). In contrast, the overexpression of reb and GFP-kkvΔCC did not produce this abnormal phenotype, and instead, tubes and chitin deposition appeared normal (Figs 3I and S3). Importantly, the overexpression of reb alone also leads to morphogenetic defects, due to the presence of endogenous kkv (Figs 3J and S3). Because the overexpression of reb and GFP-kkvΔCC (in the presence of endogenous kkv) reverts the tracheal defects of overexpression of reb alone, our results suggest that the GFP-kkvΔCC is interfering with the endogenous wild-type Kkv, which can no longer produce a dominant effect in combination with extra Reb. We analysed the localisation of GFP-KkvΔCC to further determine the roles of the CC domain. We found that the protein can still localise at the apical domain, as GFP-Kkv does (Fig 3K–3N); however, we found that the apical enrichment was not as clear as for GFP-Kkv. Quantification of the ratio of protein accumulation in the apical domain versus the basal domain indicated significant differences with respect the control. We confirmed these observations in salivary glands (Fig 3O). Altogether, these results indicate that the CC domain is dispensable for polymerisation and translocation of chitin but plays a role in protein localisation. In addition, the results suggest that GFP-KkvΔCC can interfere with endogenous Kkv, which may indicate a role in protein oligomerisation (see Discussion).

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