A novel approach to study adhesion mechanisms by isolation of the interacting system

Abstract

For decades most investigations into mechanisms of adhesive interactions have examined whole organisms or single cells. Results using whole organisms are often unclear because it may not be known if a probe used in an experiment is directly affecting the cellular interaction under study or if it is an indirect effect resulting from action on some other structure or pathway. Here we develop a novel approach to isolate the structural components of a cellular interaction by dissecting them out of the organism to study them in a pristine environment away from all confounding factors. We used the adhesion between the archenteron and blastocoel roof of the sea urchin gastrula stage embryo as a model that can be replicated in many other developmental and pathological systems. The isolated components of the cellular interaction and those in the whole organism possessed identical cell surface receptors and adhesive affinities.

Figure 1

Sea urchin gastrulation (Based on Gustafson, 1963; Oppenheimer and Carroll 2004).

Figure 2. Whole fixed embryo dissection.

Figures 2A, B show intact 52-hour sea urchin embryos. 2C, D, E show the microdissected archenteron (2E) and blastocoel roof (2D) pieces from the embryos in A, B. 2F, G show that these two microdissected "pieces’ from C, D and E adhered to each other when they were isolated from intact fixed embryos. The structural integrity of the fixed pieces was better than those obtained from live embryos (not shown), but in both cases, the pieces adhered to each other. Scale: Diameter of sea urchin embryos @ 100μm.

Figure 3

Fixed whole 52-hour Lytechinus pictus embryos and pieces labeled with FITC-Lens culinaris agglutinin. 3A, B, C are fixed whole embryos. 3D, E, F are dissected blastocoel roofs and G, H, I are dissected archenterons from these fixed embryos. All bound FITC-Lens culinaris agglutinin after 30 minutes incubation in 0.045μg/μl FITC-Lens culinaris agglutinin in ASW. The embryos bound to the lectin were washed three times in ASW and photographed. The lectin did not appear to wash off. Scale: Diameter of sea urchin embryos @ 100μm.

Figure 4

Pieces of fixed Lytechinus pictus embryo archenterons bound to FITC Lens culinaris agglutinin and not to FITC Lens culinaris/α-methyl-mannose. 4A is a fluorescence image of an archenteron with FITC-Lens culinaris agglutinin without α-methyl-mannose. 4B is a bright field image of the same archenteron. 4C is a fluorescence image of an archenteron incubated with FITC-Lens culinaris agglutinin with α-methyl-mannose. 4D is a bright field image of the archenteron in 4C. Little or no fluorescence was observed on the embryos incubated with the FITC-Lens culinaris agglutinin with α-methyl-mannose (4C). Scale: Length of archenteron @ 80 μm.