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First published online February 15, 2006
Journal of Experimental Biology 209, 956-964 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02031

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An in vivo study of exocytosis of cement proteins from barnacle Balanus improvisus (D.) cyprid larva

Kristin Ödling¹, Christian Albertsson¹, James T. Russell² and Lena G. E. Mårtensson^1,*

¹ Göteborg University, Department of Zoology, Zoophysiology, Medicinaregatan 18 SE-413 90 Göteborg, Sweden
² Section on Cell Biology and Signal Transduction, NICHD, NIH, Building 49, Room 5A-78, 22 Convent Drive, MSC 4480, Bethesda, MD 20892-4480, USA

View larger version (144K): [in a new window]   Fig. 1. The cement gland, as it appears in a living cyprid under differential interference contrast (DIC) optics. The individual granules can be seen as bumps on the cement gland surface. At the apical end, the cement duct, which widens to form the muscular sac, can be seen. CG, cement gland; CD, cement duct; MS, muscular sac; CE, compound eye.

View larger version (130K): [in a new window]   Fig. 2. Appearance of the different types of secretory granules after stimulation of cyprids with dopamine (1 mmol l–1) for 10 min. Cyprids were aldehyde fixed, sectioned and stained with Toluidine Blue. The different types of granules are labelled (1–4). The cement duct, with dissolved proteins, is seen as an extension away from the cement gland towards the muscular sac.

View larger version (22K): [in a new window]   Fig. 3. The different types of granules differ in their overall size. The surface area of the different types of granules was measured by Easy Image Measurements 2000. Overall, 195 of type 1, 113 of type 2, 63 of type 3 and 51 of type 4 were measured, and the sizes were compared using statistical tests. The different types of granules were vastly different in their size except that type 2 granules were not significantly different from type 3 granules.

View larger version (92K): [in a new window]   Fig. 4. Electron microscopy of cement glands in (A) control, unstimulated cyprids and (B) cyprids stimulated with 1 mmol l–1 dopamine for 10 min. The different types of granules are labelled in B and can be compared with Fig. 2. In control, unstimulated animals, most of the granules are type 1, but all the four types of granules are visible in stimulated cement glands (B). Note also that the different types of granules are within the same cell in B. Scale bar: 3.8 µm in A; 2.5 µm in B.

View larger version (150K): [in a new window]   Fig. 5. Electron microscopy of secretory vesicle types. (A) Unstimulated cement gland where most of the cement granules appear densely packed with secretory material. Granule contents appear to have a distinct organization. Scale bar, 0.6 µm. (B) Type 2 granules appear larger and their contents appear amorphous and lack the organization observed in the dense-core vesicles seen in A. Scale bar, 0.6 µm. (C) Type 3 granules appear similar to type 2, except have a `moth-eaten' appearance with clear spaces or hydration channels due to partial loss of contents. Scale bar, 0.25 µm. (D) Type 4 vesicles appear like vacuoles with a reticulated appearance. Note that the reticulated granules appear within the same cell as the dense-core type 1 granules. Scale bar, 0.4 µm.

View larger version (91K): [in a new window]   Fig. 6. Confocal microscopy of an Acridine Orange-stained living cyprid cement gland. The larva was immobilized on a cover slip using Kwik Sil and imaged. (A) Unstimulated cement gland within the living cyprid. (B) The cyprid was stimulated with dopamine (1 mmol l–1) and imaged 15 min later. Note the brightly stained secretory vesicles in the control gland (A) and their loss and the appearance of large vacuoles after stimulation.

View larger version (118K): [in a new window]   Fig. 7. Visualization of cyprid cement secretion under differential interference contrast (DIC) optics. A cyprid larva was immobilized in agarose and observed in a coverslip chamber using an inverted microscope using DIC optics. The montage shows a series of images separated by 10 s intervals. Images are arranged starting at the top and going left to right. Note the appearance of a vacuole that seems to grow larger with time (arrow). Note the increase in size of the cement sac between frames 1 and 12. See supplemental material for a movie sequence of the original data at http://vivaldi.zool.gu.se/film/Movie-2-sm.mov and http://vivaldi.zool.gu.se/film/Movie-2-sm.avi.

View larger version (23K): [in a new window]   Fig. 8. Dopamine stimulation causes secretory vesicle loss and appearance of vacuoles. Cyprids were fixed at different times during exposure to dopamine (1 mmol l–1) and sectioned. Sections were stained with Toluidine Blue, and the different types of vesicles in the stained sections were counted under the microscope. Note that dense-core granules (type 1) reduce in number over time, with a proportionate increase in the number of vacuoles (type 4) after 10 min of dopamine treatment.

View larger version (51K): [in a new window]   Fig. 9. Light microscopy of dopamine-stimulated cement glands. Cement glands were fixed at various times during dopamine exposure, and sections were cut. Toluidine Blue-stained sections were examined under the microscope. Note the absence of vacuoles in the control gland (A) and the appearance of vacuoles after 4 min exposure to dopamine (B), which increase in number after 10 min exposure (C).