Non-muscle myosin II drives critical steps of nematocyst morphogenesis

Abstract

Nematocysts are generated by secretion of proteins into a post-Golgi compartment. They consist of a capsule that elongates into a long tube, which is coiled inside the capsule matrix and expelled during its nano-second discharge deployed for prey capture. The driving force for discharge is an extreme osmotic pressure of 150 bar. The complex processes of tube elongation and invagination under these biomechanical constraints have so far been elusive. Here, we show that a non-muscle myosin II homolog (HyNMII) is essential for nematocyst formation in Hydra. In early nematocysts, HyNMII assembles to a collar around the neck of the protruding tube. HyNMII then facilitates tube outgrowth by compressing it along the longitudinal axis as evidenced by inhibitor treatment and genetic knockdown. In addition, live imaging of a NOWA::NOWA-GFP transgenic line, which re-defined NOWA as a tube component facilitating invagination, allowed us to analyze the impact of HyNMII on tube maturation.

Keywords: Cell biology; Developmental biology.

Graphical abstract

Figure 1

HyNMII expression pattern and immunohistochemical detection in Hydra whole mounts (A) Schematic drawing of nematocyst morphogenesis. The overview image of an early nematocyte shows that nematocysts are secretory products of the Golgi apparatus. Subsequent stages include tube formation, invagination, and capsule maturation. (I) Early tube protrusion. (II) External tube elongation. (III) Invagination/inversion of the tube that is later adorned with spines. (IV) Mature stenotele nematocyst with invaginated, coiled tube in the capsule matrix. rER: rough ER. (B) HyNMII expression is upregulated in endodermal cells of the tentacles in steady-state polyps and in early buds (B′) as determined by ISH. Scale bars = 200 μm. (C) Expression of HyNMII (Uniprot: T2MG36,Hydra Genome Portal 2.0 (https://research.nhgri.nih.gov/hydra/): t8308aep) in published cell clusters identified according to the presence and absence of marker genes by Siebert et al. (https://singlecell.broadinstitute.org/single_cell/study/SCP260/stem-cell-differentiation-trajectories-in-hydra-resolved-at-single-cell-resolution). Dot size and color represent the counts of mapped transcripts in the respective cell cluster. (D) Identification of cnidarian NMII homologs expressed in the nematocyte cell lineage. Published cnidarian single-cell transcriptome datasets^,,,, were searched to identify non-muscular myosin genes enriched in nematocytes using HyNMII as a query. For species in which the nematocyte cluster was not annotated, the expression of minicollagen genes was used as a confirmation (Xenia minicollagens Xenia Hub (http://genome.ucsc.edu/cgi-bin/hgTracks?hubUrl=http://cmo.carnegiescience.edu/gb/hub.txt&genome=xenSp1): Xesp_000432, Xesp_001209, Xesp_001210, Hydractinia minicollagens: Hydractinia Genome Portal Project (https://research.nhgri.nih.gov/hydractinia): HyS0004.75, HyS0004.369, HyS0004.369, HyS0008.263). (E) Western blot detection of recombinant HyNMII motor domain and full-length HyNMII in Hydra lysate as indicated by arrows. The blot was performed using primary antibody solution without (left panel) or with (right panel) 1 mg/mL of the antigenic peptide used for generating the HyNMII antibody. (F–P) Detection of HyNMII by immunohistochemistry. (F) Overview of a whole Hyda bud stained with HyNMII antibody showing signals in developing nematocyte nests throughout the body column. Scale bar = 200 μm. F′ shows the neutralization of specific signals by pre-absorption of the primary antibody with the antigenic peptide at 1 mg/mL. Scale bar = 100 μm. (G) Double staining with HyNMII (magenta) and capsule wall marker CPP1 (green) antibodies showing nematocyte nests in different maturation stages in the gastric region. Scale bar = 50 μm. (H–P). Chronological arrangement of developing stenotele, isorhiza, and desmoneme nematocyst nests as indicated: onset of external tube formation (H, K, N), tube elongation (I, L, O), invaginated tube (J, M, P). (Q) Schematic representation of developmental stages shown in H–P. See also Figure S1 for ultrastructural detection of HyNMII in different developmental stages. Scale bars = H–P: 10 μm.

Figure 2

Blebbistatin treatment leads to tube disintegration (A–E) Depletion of HyNMII-positive nests in the gastric region of polyps that were continuously treated with BBS, fixed and stained for HyNMII (magenta) and CPP1 (green) at different time points as indicated (see also Figures S2A–S2D, which shows the consecutive depletion of nematocyst morphogenetic stages during BBS treatment). Note the appearance of large HyNMII-positive puncta that increase in number from day 1–5 (B–D). (A′–E′) High-magnification images of nematocyst nests in the gastric region of BBS-treated polyps demonstrate HyNMII dissociation from tubes in the form of dot-like structures. Images in A′–D′ show stenoteles in the stage of tube elongation as in Figure 1I and corresponding to stage II in Figure 1A. E′ shows a stenotele nest with coiled internal tubes as in Figure 1J and corresponding to stage IV in Figure 1A. Arrows in B′ indicate bulges mostly forming at tube tips. Arrows in C′ indicate HyNMII-positive dots, while arrowheads indicate external parts of partly invaginated tubes. Arrows in D′ indicate HyNMII-positive puncta in close vicinity to external tubes. See also Figures S2E–S2H, which summarizes typical tube-associated phenotypes observed upon BBS treatment and S2I-J for localization of HyNMII puncta in tentacles. (F) Quantification of HyNMII-positive nests in the gastric region of BBS-treated polyps (see also Table S1). Data represent mean ± SD from 3 animals compared to control polyps. ∗∗∗∗p value <0.0001, ∗p value <0.05. The data were analyzed using a one-way ANOVA test. (G) Rain cloud plot showing the size distribution of HyNMII-positive dots in BBS-treated animals (see also Table S2). The dots were measured along their longest diameter in the upper body region of 3 different animals at day 5 of BBS treatment. (H–L) Polyps of the Cnnos1::GFP transgenic line continuously treated with BBS for 7 days as in A–E show no significant reduction in GFP-positive i-cells in the gastric region. See Figures S2K and S2L for the effect of longer treatments. Cell nuclei were stained with DAPI. Scale bars = A–E: 100 μm; A′–E’: 25 μm; H–L: 20 μm.

Figure 3

Blebbistatin depletes TGN vesicles at growing nematocyst (A) Schematic drawing of early nematocyst development showing the arrangement of microtubules and TGN vesicles around the tip of the growing tube. (B) Electron micrograph of a section showing the nematocyst tube (T) tip surrounded by stacks of the Golgi apparatus (G, pseudocolorized). (C) Double staining with HyNMII (red) and beta-tubulin antibodies (yellow) shows a crest of microtubules surrounding the external tube tips of growing nematocysts. (D) Double staining with Ncol-1pp (magenta) and the capsule wall marker Cnidoin (CN, green) marks the crown of TGN vesicles at the apical protrusion of the growing nematocyst. Scale bars = 10 μm. (E–I) BBS treatment leads to a rapid depletion of Ncol-1pp-positive TGN vesicles in the body region. Days of treatment are indicated. Scale bars = E, F, H, I: 200 μm; H: 100 μm. (J) Nematocyst nest in the gastric region stained with Ncol-1pp (magenta) and Cnidoin (CN, green). Scale bar = 20 μm. (K and L) High-magnification images from animals in E and H showing depletion of TGN vesicles in a single nematocyst nest. For clarity, the Ncol-1pp-stained images (magenta) were converted to grayscale and shown in inverted contrast. Scale bars = 20 μm. (M) Quantification of Ncol-1pp-positive nests in the gastric region of BBS-treated polyps (see also Table S3). Data represent mean ± S.D. from 3 animals compared to control polyps. ∗∗∗p value <0.0005, ∗∗p value <0.005. ∗p value <0.05. The data were analyzed using a one-way ANOVA test.

Figure 4

NOWA is a late tube component that facilitates invagination (A) Detail of the gastric region illustrating major developmental stages of stenoteles stained with NOWA-CTLD antibody. I, onset of external tube formation with several large NOWA granules inside the nematocyst capsule. II; tube elongation at the beginning of, or close to invagination. III, late invagination stage showing the helical shaft region and the partly coiled tube. NOWA spots are completely dissociated throughout the capsule matrix. IV, mature capsule characterized by condensation of NOWA at the fully coiled tube structure and a virtually clear capsule lumen. The inserts show the reduction of NOWA-CTLD-stained structures in animals electroporated with NOWA siRNAs as compared to siGFP-treated controls. (A′) Stenotele nest at stage I stained with NOWA-CTLD (magenta) and CPP1 (green) antibodies. (A″) Stenotele nest as in A′ in an animal treated with NOWA siRNAs showing a reduction of NOWA aggregates in the capsule matrix. (B) Detail of the gastric region illustrating stenotele developmental stages in NOWA::NOWA-GFP transgenic animals shows similar pattern as in A. (C) In mature capsule of the tentacles, the NOWA-CTLD antibody does not stain the tube within the capsule due to the impermeable capsule wall. (D) In contrast to C, mature capsules in transgenic animals show NOWA-positive internal tubes. The arrowhead marks a NOWA-GFP protein aggregate, which normally is dissolved in mature capsules of wild-type animals. See also Figure S3 and Video S1 for visualization of NOWA protein dispersal during invagination. (E) Tube of discharged nematocyst stained with NOWA-CTLD antibody. (F) NOWA-GFP marks both the internal and external tube in a partly discharged mature nematocyst. Scale bars = A–B: 25 μm; A′–A″, C–F: 5 μm. (G) In a Western blot using isolated nematocysts from wild-type and transgenic NOWA::NOWA-GFP animals, NOWA is detected by the NOWA-CTLD antibody at the expected molecular mass of 88 kDa. A weaker second band at about 115 kDa in the transgenic capsule sample marks the NOWA-GFP fusion protein. (H–J) NOWA-CTLD detection by cryosection immunogold-EM (formaldehyde fixation). (H) In stage I nematocysts (see Figures 1A, 1H, and 4A) with protruding tubes, NOWA-CTLD was confined to 0.5–1 μm wide spots within the capsule which first comprised honeycomb-like patterns (arrows) and more loosely arranged strand-like elements in stage II nematocysts (I). Stage III nematocysts showed NOWA-CTLD along the profiles of the inverted tube (J, arrowheads). The multi-layered structure in the center is the spines (SP) that differentiate within the lumen of the tube shaft region. CW = capsule wall. Scale bars = 200 nm. (K) Double staining with HyNMII (magenta) and CPP1 (green) antibodies of a stenotele (nest on the left side), and isorhiza (K′) nest showing thickened double-layered external tubes (white arrowheads) and fine threads of invaginated tubes in the capsule matrix (yellow arrowheads) in siNOWA-treated animals. Scale bars = K, K’: 25 μm. (L) Percentage of nematocyst nests showing partially invaginated tubes in siNOWA and control electroporated Hydras (see also Table S4). Data represent mean ± SD from 20 (ctrl) and 34 (siNOWA) random areas of 85,434.286 μm² in the gastric regions of 3 animals in each treatment group. ∗∗∗∗p value <0.0001. The data were analyzed using an unpaired t-test. (M) Scheme showing the proposed “zipper” mechanism for tube invagination facilitated by NOWA. NOWA conglomerates that appear as large droplet-like structures in the capsule matrix of stages I and II dissociate during the invagination phase (III–IV) into smaller particles connected by cysteine links. We assume that these pass the tubule wall to interact with the chondroitin matrix on the outer surface of the tube via NOWA’s lectin domains. This process could cross-link the invaginating tube surfaces driving invagination in the form of a carbohydrate-lectin “zipper”.

Figure 5

NOWA indicates compromised tube formation in BBS- and siHyNMII-treated animals (A–F) Double staining of control and BBS-treated (4 days) wild-type Hydras with NOWA-CTLD and CPP1 antibodies. (A and D) Early stage of nematocyst development with tube protrusion (see also stage I, Figure 4A) at the apical nematocyst pole (dotted line) and large NOWA-positive protein spots showing asynchronous development in BBS-treated animal. (B and E) Intermediate stages (see also stage III, Figure 4A) with beginning incorporation of NOWA into the triple helical folds of the shaft structures and tubes after invagination show irregular shafts and missing tubes in BBS-treated animal. Note that the NOWA protein spots are only partly dissolved in (E). (C and F) Late-stage nematocysts before final maturation (see also stage IV, Figure 4A) lack the coiled NOWA-positive tube structures (C) in BBS-treated animals, in which the NOWA protein remains as an amorphous aggregate (F). (G–I) NOWA-CTLD and CPP1 double staining of nematocyst nests in animals electroporated with HyNMII siRNAs. (G) Stage I showing partial loss and asynchronous development of nematocysts. (H) Stage III showing compromised and asynchronous tube formation after invagination. (I) Incomplete tubes and partly disaggregated capsule in stage IV nematocyst nest. See also Figure S4 for more detailed phenotypes observed after HyNMII knockdown. (J) Stage II nematocysts in untreated NOWA::NOWA-GFP polyps visualized by live imaging show NOWA protein aggregates in the capsule matrix and intact elongated external tubes. (K) In BBS-treated Hydras (5 days), stage II nematocysts show disaggregation of the tube structure into vesicular bodies (white arrowhead) similar to the HyNMII-positive puncta observed in Figure 2. (L) Mature nematocysts (stage IV) in untreated NOWA::NOWA-GFP Hydras are marked by GFP-positive coiled tubes. (M) In BBS-treated animals (5 days), late-stage nematocysts exhibit failed tube maturation as evidenced by a lack of GFP-positive tube structures. Scale bars = A–L, L–M: 20 μm. J–K: 10 μm.

Figure 6

Effect of cytochalasin D treatment on nematocyst tube stability (A) Nest of stenoteles in early external tube stage stained with Cnidoin (CN) antibody showing drop-like shapes (box indicates magnified example in A′). (B) Stenotele nest at similar stage as in CytD-treated animal showing reduced capsule body sizes and expanded tube diameters (box indicates magnified example in B′). (C) Stenotele nest in early external tube stage stained with HyNMII (magenta) and CPP1 (green) antibodies showing typical narrowing at the capsule neck with a collar-like HyNMII-positive structure (box indicates magnified example in C′). (D) Stenotele nest at similar stage as in C in CytD-treated animal showing reduced capsule body and enlarged tube shaft region (box indicates magnified example in D′). Scale bars = A, B, C, D: 20 μm: A′, B′, C′, D’: 5 μm. (E) Model of the presumptive role of HyNMII and actin filaments in stabilizing by a compressive force the external nematocyst tube that collapses upon CytD treatment. See also Figure S5 for actin localization at the ultrastructural level and Figure S6 for mathematical modeling and simulations of HyNMII-induced membrane shaping.