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First published online 27 September 2005
doi: 10.1242/jcs.02593

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Yolk granule tethering: a role in cell resealing and identification of several protein components

Department of Cellular Biology and Anatomy, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912, USA

View larger version (180K): [in a new window]   Fig. 1. Tethering and fusion of yolk granules isolated in IM buffer. Tethered yolk granules (A) before (0 second) and (B-D) at various time points after perfusion of Ca2+ (300 µM) into a slide coverslip chamber. Prior to Ca2+ buffer addition the vesicles are tethered to one another, forming large aggregates. These tend to fuse with one another as soon as Ca2+ reaches them, forming a large `patch' vesicle product whose size reflects that of the tethered aggregate. Bar, 10 µm.

View larger version (17K): [in a new window]   Fig. 2. Long-term time course of Ca2+-triggered granule fusion. Granule light scattering, a semi-quantitative measure of granule fusion, was measured at intervals after the addition of Ca2+ buffer to give the following final concentrations: 0.001 µM (IM buffer), ; 11.9 µM, ; 572 µM, ; 1151 µM, . The decrease in light scatter characteristic of fusion in this system was complete at the two higher Ca2+ concentrations at the earliest interval (3 minutes after Ca2+ buffer addition) measurable. No further change in light scattering was recorded over the remaining measurement interval. Additional Ca2+ buffer was added at the 65-minute time point, resulting in the following approximate final concentration levels: <0.001 µM (IM buffer), ; 572 µM, ; 1450 µM, ; 2680 µM, . The granules were pelleted by centrifugation at the 110-minute time point, concentrating them in the well bottom. No change in absorbance, indicative of further fusion, was observed in either case. Error bars indicate s.d. (n=3).

View larger version (78K): [in a new window]   Fig. 3. Washing of yolk granules in the chaotropic salt, KI, inhibits tethering. (A) Granules washed three times post-isolation in IM buffer. (B) Granules washed three times post-isolation in KI buffer. Tethering is no longer evident. (C) Comparison of KI-washed and control, IM-washed granule tethering responses using a novel, rapid, semi-quantitative assay. Granule samples in 96-well plates are centrifuged to pellet the large tethered aggregates. Granules not tethered, which remain suspended in buffer in the upper half of the contents of each well, are then transferred to a fresh well and the absorbance of this transferred measured. Tethering efficiency is an inverse function of measured absorbance. Hence the absorbance of the KI-washed granules (not tethered, see panel B) is higher than that of IM-washed granules (tethered, see panel A). Error bars indicate s.d. (n=6). Bar, 10 µm.

View larger version (165K): [in a new window]   Fig. 4. Restoration of tethering by cytosol. (A) After washing in KI, granules are not capable of the tethering response. (B) Addition of cytosol (100 µg) restores tethering. (C) Ca2+ addition (300 µM) to the non-tethered population of granules produces small fusion products only. (D) Granules tethered by prior cytosol addition, by contrast, fuse upon Ca2+ addition to form large patch vesicle products. Bar, 10 µm.

View larger version (148K): [in a new window]   Fig. 5. Restoration of tethering by factors present in KI salt wash. (A) KI-washed vesicles (tether incompetent) received IM buffer only or (B) IM buffer conditioned by the addition of protein factors (40 µg desalted into IM buffer) stripped from granules during incubation in KI. Tethering is restored by these factors. (C) Fusion products produced by Ca2+ addition to KI-washed granules. (D) Fusion products produced by Ca2+ addition (300 µM) to granules tethered, as in B by the prior addition of tethering factors (TF) from a KI wash of granules. Note the increased size of the resultant fusion products. Bar, 10 µm.

View larger version (7K): [in a new window]   Fig. 6. Lack of tethering activity in a crude yolk granule lysate. Yolk granules were incubated with IM buffer only (IM), with equal amount (100 µg) anion-exchange-purified tethering factor (HQ) or with a crude granule lysate (LYS) and tethering activity measured. The buffer only and lysate samples did not differ significantly from one another, whereas both were significantly different from the tethering factor sample (P<0.005). Error bars indicate s.d. (n=6).

View larger version (23K): [in a new window]   Fig. 7. Purification of a tethering factor from yolk granules. (A) Typical anion exchange column (HQ, BioRad) elution profile. The redissolved ammonium sulfate precipitate of proteins stripped from granules by a KI wash was loaded onto the column. Tethering activity elutes at the central, major peak only. (B) Coomassie Blue-stained SDS gel comparison of the protein constituents of whole yolk granules (Yolk) and the anion exchange fraction enriched in tethering activity (TF). The major yolk protein (MYP), the predominate species of this organelle, is indicated with an asterisk. The seven protein bands characteristic of this fraction are numbered. (C) Enrichment of tethering activity as a function of each purification step, as measured by the semi-quantitative tethering assay. The three fractions assessed are the KI-wash (), the ammonium sulfate precipitate () and the anion-exchange-enriched fraction (). Error bars indicate s.d. (n=3).

View larger version (26K): [in a new window]   Fig. 8. The tethering factor behaves as a high molecular weight complex. (A) The anion exchange fraction enriched in tethering activity elutes predominantly as a single peak at the void volume of a size exclusion column (Sephacryl S-300). This fraction, and none of the others, has tethering activity. Gel electrophoretic analysis of the peak fraction with tethering activity is also shown in this panel. The complex of proteins purified as described above is present in this fraction. (B) Non-denaturing gel electrophoresis of equal amounts (20 µg) of protein from a KI wash (KI wash) and from the anion-exchange-enriched fraction (TF). A high molecular weight species of 670 kDa is evident in both samples, but more prominent in the highly active anion exchange fraction. PS, protein standard markers.

View larger version (18K): [in a new window]   Fig. 9. Monoclonal antibodies raised against the tethering complex react against the 170 kDa subunit and can immunodeplete tethering activity. (A) Western analysis of monoclonal antibody staining of the 170 kDa component of the tethering factor. PS, position of protein standards. (B) Granules were incubated with anion-exchange-purified tethering factor (TF) or with an equivalent amount of tethering factor (15 µg) after it adsorption to protein G beads to which various (numbered columns) monoclonal antibodies had been bound. The activity depleted by protein G beads alone (in the absence of antibodies) has been subtracted from the antibody samples. One sample, a negative control, received IM buffer containing no tethering factor (No TF). Tethering activity was measured in the semi-quantitative assay. Significant immunodepletion (P<0.05) relative to the TF control is indicated for numerous monoclonal antibodies (identified by number). Error bars indicate s.d. (n=3).

View larger version (25K): [in a new window]   Fig. 10. Immunostaining of starfish eggs with a monoclonal antibody raised against tethering factor. (A) A ubiquitous, 1 µm diameter, extranuclear compartment is labeled by a monoclonal antibody raised against the purified tethering complex. This pattern is highly consistent with yolk granule labeling. (B) Higher magnification reveals that the intracellular compartment staining is peripheral (arrowheads). This is consistent with labeling of a peripheral protein. (C) At a low-gain setting, intense staining of the plasma membrane is emphasized in this confocal micrograph. Bar, 10 µm (A,C); 1 µm (B).