DEV015503 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Tyramine competes for dityrosine crosslinks between endogenous proteins. (A) Tyramine (circles) and its analog phenylethylamine (squares) (Sigma-Aldrich, St. Louis, MO, USA) were tested as competitors of endogenous dityrosine crosslinks within S. purpuratus fertilization envelopes. Effective inhibition was assessed by the ability of SFE9 to migrate into an SDS-PAGE gel (Wessel, 1995). Fifteen micrograms of total protein per sample was subjected to SDS-PAGE on pre-cast 4-20% polyacrylamide Tris-Glycine gels (Life-Therapeutics, Frenchs Forest, New South Wales, Australia). Blots were probed simultaneously with rabbit antisera against SFE9 (Wessel, 1995) and soluble protein YP30 (Wessel et al., 2000b), detected by Cy3-conjugated, affinity-purified goat anti-rabbit IgG (Jackson ImmunoReseach Laboratories, West Grove, PA, USA), and imaged using a Typhoon fluorescent scanner run by proprietary software (Amersham Biosciences, Piscataway, NJ, USA). Quantitation of band intensity was made using ImageQuant software (Amersham Biosciences), and data was plotted as shown. (B) Examples of the fertilization envelope phenotypes from select, lower concentrations of tyramine or phenylethylamine. Images were taken thirty minutes after insemination, using an Orca-ER CCD camera (Hammamatsu, Bridgewater, NJ, USA) attached to a Zeiss Axiovert microscope (Carl Zeiss Corporation, Thornwood, NY, USA) driven by Metamorph software (Universal Imaging Corporation, Downingtown, PA, USA). Inhibitor concentrations associated with an absence of fertilization envelopes (arrowheads) were excluded from the best-fit analysis, as their cortical granules never appear to have exocytosed, preventing the activity of ovoperoxidase. The mechanism that causes this failure is not known. Symbols are as in A.

• Supplemental Figure S2 -

  Fig. S2. Tyramide-conjugates are targeted to and retained in tyramine-softened fertilization envelopes. (A) Softened fertilization envelopes isolated from embryos fertilized in the presence of 5 mM tyramine alone, or with tyramide-Alexa Fluor 594 (1:100 dilution) were analyzed using a TCS SP2 AOBS confocal scanning microscope (Leica Microsystems, Bannockburn, IL, USA). Fluorescence and DIC z-series stacks were imaged simultaneously over a 45-µm focal depth using a 594 nm excitation laser and an emission detector window of 605-615 nm. Two-dimension projections were made from the 10 fluorescence images within each z-stack to visualize each structure. Projections of fluorescence stacks are shown above corresponding DIC images of each purified envelope preparation. Source of detail is boxed. Arrowhead indicates a region of high fluorescence. (B) Tyramide-Alexa Fluor 594 was titrated, in the presence of 5 mM tyramine, to determine the optimum ratio of reagents. Plot shows quantification of total fluorescence intensity (AU, arbitrary units) from the fertilization envelope of each zygote, per dilution of tyramide-fluorophore. At least five individuals were measured per concentration. Standard deviation is shown. (C) Representative images of tyramide fluorophore titration, captured using an Orca-ER CCD camera (Hammamatsu, Bridgewater, NJ, USA) attached to an Axiovert microscope (Carl Zeiss Corporation, Thornwood, NY, USA) and driven by Metamorph software (Universal Imaging Corporation, Downingtown, PA, USA). Alexa Fluor 594 fluorescence is shown above corresponding DIC images per dilution tested. Arrowhead indicates a softened fertilization envelope shed from its embryo. Scale bar: 100 µm.