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doi: 10.1242/10.1242/jcs.00084

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Nowa, a novel protein with minicollagen Cys-rich domains, is involved in nematocyst formation in Hydra

Ulrike Engel1,*, Suat Oezbek2, Ruth Engel2, Barbara Petri3, Friedrich Lottspeich4 and Thomas W. Holstein1,{ddagger}

1 Institute of Zoology, Darmstadt University of Technology, 64287 Darmstadt, Germany
2 Department of Biophysical Chemistry, Biozentrum, University of Basel, 4056 Basel, Switzerland
3 Institute of Zoology, University of Munich, 80333 Munich, Germany
4 Max-Planck-Institute of Biochemistry, 82152 Martinsried, Germany
* Present address: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA



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  		Fig. 1. Localization of mAb H22 antigen in the outer wall of nematocysts. (A) Isolated nematocysts of Hydra. The different capsule types are: d, desmoneme; i, holotrichous isorhiza; s, stenotele. One of the stenoteles has discharged (s*). (A') Immunofluorescence of mAb H22 viewed by confocal microscopy (maximum projection) in the isolated nematocysts shown in A. (B) Mature, isolated nematocyte of Hydra. Note the size of the nematocyst vesicle, which fills almost the whole cell (N, nucleus). (C) EM immunogold labeling of mAb H22 in the wall of a Hydra nematocyst. Gold particles are exclusively found in the outer wall (ow) and not in the inner wall (iw). (D) SDS-PAGE and western analysis of whole Hydra and isolated capsules. The protein of 500,000 isolated capsules was compared with 1/5 Hydra (1/5H) by silver-staining. In a western blot, protein of 1 Hydra (1H) and 500,000 capsules separated by SDS-PAGE were probed with mAb H22 and minicollagen antibody. A high molecular weight protein is detected by mAb H22 in isolated capsules, but not in whole Hydra. Minicollagens detected by minicollagen-1 antibody represent the major proteins of the capsule. Bars, 5 µm (A,B); 100 nm (C).

 


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  		Fig. 2. Characterization and isolation of the mAb H22 antigen. (A-C) 2D electrophoresis of capsule proteins according to pI and molecular weight. The protein of two million capsules was separated and analyzed by Coomassie staining (A), western analysis with mAb H22 (B), and silver-staining (C). (D) Assay of N-glycosylation. Solubilized capsule protein was incubated with N-glycosidase F to remove N-linked sugars. The deglycosylated protein and mock control (without enzyme) were separated by SDS-PAGE and probed with mAb H22 (left) and anti-CTLD antibody (right) in western analysis.

 


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  		Fig. 3. Nowa protein from Hydra vulgaris. (A) Nowa domain organization. CTLD, C-type lectin-like domain (smart00034, E-value of 10-17); SCP, homology to SCP domains (smart00198, E-value of 10-3); SP, signal peptide. The putative cleavage site for an N-terminal propeptide after residues KR is indicated by an arrow. (B) Alignment of CTLDs of Nowa and vertebrate proteins. Sequences most similar to the Nowa CTLD were retrieved by a BLAST-search in SWISS-PROT (sp) and TrEMBL (tr) protein databases. (sp P16112), aggrecan core precursor (human); (sp P55066), neurocan core protein precursor (murine); (tr O62623), cartilage proteoglycan (bovine); (tr Q13018), PLA2 RE, secretory phospholipase A2 receptor precursor (human); (tr Q64449), mannose receptor, C type 2 (murine); (tr Q91B90), C-type lectin from Cyprinus carpio; (tr Q91840), Aggretin ß-chain from Agkistrodon rhodostoma; (tr Q9IAM0), Agkisacutacin ß-chain from Agkistrodon acutus. Shading shows conservation of residues of the same similarity group within the aligned sequences: black, 100%; dark grey, 80%; and light grey, 60% conservation. Amino acid similarity groups: DN, EQ, ST, KR, FWY, ILMV. (C) Alignment of cysteines in Cys-rich domains of Nowa and minicollagens. The C-terminal Cys-rich part of Nowa is an eightfold repetition (REP1-REP8) of six characteristically spaced cysteines, similar to the MCCR domain in the N- and C-terminal part of minicollagens; shown for minicollagen-1 (tr Q00484) and minicollagen-2 (tr Q00485) both from Hydra magnipapillata, and minicollagen-Ad (tr Q16990) from Acropora donei.

 


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  		Fig. 4. Recombinant expression of Nowa CTLD. (A) Nowa CTLD construct used for recombinant expression in E. coli. (B) Purified recombinant Nowa CLTD and capsule proteins (500,000 capsules) analyzed by SDS-PAGE and visualized by silver-staining and western analysis.

 


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  		Fig. 6. Immunolocalization of Nowa protein in dividing nematoblasts. (A) The nematocyte differentiation pathway in Hydra. Nematocytes originate from interstitial stem cells (I-cells), which divide three- to five-times after commitment (nematoblasts) and remain interconnected by cytoplasmic bridges forming nests of 8-32 cells (David and Challoner, 1974Go; David and Gierer, 1974Go). After nematocytes have formed a nematocyst (green), nests brake up into single nematocytes, which migrate to the tentacles. (B-D) Confocal microscopy of mAb H22 (green) in dividing nematoblasts. (B) Nematoblast nest in metaphase (optical section) with nuclei (blue) in metaphase condensation numbered 1-8. One of the mAb-H22-positive capsule primordia is indicated by an arrow. (C) Surface projection of the same nest, the boundary of the nest is indicated by a dotted line. (D) Nematoblast in division with metaphase spindle apparatus visualized by mAb anti-tubulin (red). Projections from different angles show the asymmetrical position of the capsule primordium. (E) Continuous [3H]thymidine labeling of mAb-H22-positive nests. The first labeled nests appeared ~5 hours after onset of labelling; values represent single measurements from two independent experiments. Bars, 5 µm.

 


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  		Fig. 5. In-situ hybridization of Nowa transcript in whole mounts of Hydra. (A) Overview of an animal with a small bud showing expression of Nowa in the body column but not in the tentacles. (B) Body column with Nowa-mRNA-positive cells. (B') Enlargement of B with Nowa-mRNA-positive nematoblast or nematocyte nest indicated by an arrow. An adjacent nest with capsules but no Nowa expression is indicated by an arrow and asterisk. Bars, 100 µm (A); 50 µm (B); 10 µm (B').

 


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  		Fig. 7. Immunolocalization of Nowa protein in the early growth phase of nematocyst morphogenesis. (A,B) Confocal microscopy of mAb H22 (green) and minicollagen antibody (red) in early stages of nematocyte differentiation. Nuclei are stained with DAPI (blue). (A) Early and late stages of capsule development in an optical section. (A') Enlargment of one of the early stage nematocytes with nematocyst primordium. Staining of mAb H22 is restricted to the wall and tubular-vesicular structure at the apex (arrow), while minicollagen is detected in the ER and matrix of the capsule, as depicted in the drawing in B. (C) EM-section of nematocyte with nematocyst primordium. Immunogold mAb H22 labeling is found in the capsule matrix (cm), the outer layer of the capsule wall (cw), the Golgi apparatus (g), as indicated by an arrow, and membrane compartments associated with the primordium but not in the ER (n, nucleus). (D,D') Formation of the inner wall layer by minicollagen. Minicollagen is no longer found in the matrix of the capsule but forms the inner wall (iw) adjacent to the mAb-H22-positive outer wall (ow). (E) Isorhizatype nematocyst with tubular-vesicular structures at the growing apex (arrow) in an optical section. Bars, 5 µm (A,A',B,D); 1 µm (C).

 


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  		Fig. 8. Immunolocalization of Nowa protein in the late growth phase of nematocyst morphogenesis. Confocal microscopy of three nematocysts with an outer tubule. (A) Surface projection of mAb H22 (green) and spinalin antibody staining (red). A' shows mAb H22 staining only to visualize staining of the outer tubule. (B) Schematic representation of a nematocyst with an outer tubule. Bars, 5 µm.

 


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  		Fig. 9. MT scaffold around growing nematocysts in the early (A-C) and late (D-H') growth phase of nematocyst morphogenesis. (A,D) Localization of tubulin in Hydra macerates. (B,C,E,F) Confocal microscopy of nematocyst nests in the body of Hydra whole mounts stained by mAb H22 and mAb anti-tubulin or minicollagen and anti-tubulin (all shown as projections). The putative position of the MTOC is indicated by an arrow. (G-H') EM sections through the outer tubule (ot). (G) Tangential section of the outer tubule (see white box in E). The pair of centrioles (ce) are found at the tip of the tubule. MTs are running parallel to the tubule (yellow arrows). (H) Cross-section of the tubule, the enlargement in H' shows the intimate association of MTs with the membrane around the outer tubule. Bars, 5 µm (A-F); 0.5 µm (G); 2 µm (H); 100 nm (E').

 


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  		Fig. 10. Formation of the capsule wall formed from Nowa (green) and minicollagen (red). (A) Protein sorting and transport as detected by minicollagen antibody and mAb H22. Minicollagen and Nowa synthesized in the ER are transported in separate vesicles to the nematocyst vesicle. Nowa is detected by mAb H22 only modification by glycosylation in the Golgi apparatus and forms the outer wall. MTs (yellow) are organized in a scaffold around the growing part of the nematocyst, the MTOC is localized between the Golgi apparatus and the growing apex of the nematocyst. Minicollagen first accumulates in the capsule matrix and is then sorted to the wall to form the inner layer of the wall (3). By further transport of protein-filled vesicles, the outer tubule forms (4). It is subsequently invaginated into the cyst, and spines (s) are formed in the tubule lumen (5). Finally, minicollagen crosslinkage leads to a compaction of the wall structure (6). (B) Model of nematocyst patterning by the MT cytoskeleton. The growing part of the nematocyst vesicle is shown in a schematic cross-section. MTs form a cage around the vesicle and determine its shape (1). The outer wall formed by Nowa on the membrane (2) is used as template for minicollagen assembly. Soluble minicollagen trimers aggregate on the outer wall to form the inner wall (3-5). Finally, Nowa and minicollagen are crosslinked by disulfide bond isomerization to stabilize the structure (6).

 

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