Structure and Function of Sea Urchin Integrins

Several years ago we cloned and determining the pattern of expression of the sea urchin integrin subunits. The analysis of the sea urchin genome has revealed that there are 8 alpha integirns and 4 beta integrins (Whittaker et al. 2006). The eggs and embryos of sea urchins are an informative model for integrin function in early development. The alphaB BetaC receptor is expressed on unfertilized eggs, but removed during the cortical reaction (Murray et al. 2000). The subunits are re-expressed immediately after fertilization from stored maternal mRNA. The receptor is on the surface of the cell in contact with the hyaline layer and coincident with its re-expression, the actin cytoskeleton re-organizes to form a thick cortex or radially arrayed actin fibers.

Immunofluorescence of integrin expression. a-e are stages of early development (fertilized egg, 30 min, first cleavage, 4 cell, blastula) prepared with anti-beta C. f is a blastula prepared with anti-alpha B. The alphaBbeta C integrin is expressed after fertilization on the apical surfaces of cells, at blastula betaC combines with alphaP on the basal surfaces of he cells.

Using anti-sense morpholinos we have demonstrated that the re-organization of the actin cortex depends on the expression of the beta C integrin subunit. Without integrins, the actin cortex does not form. Embryos injected with beta C antisense morpholino are able to cleave, but the cleavage pattern is irregular and the embryos are a mass of loosely adherent cells that fail to form a blastula. We have concluded that the alphaBbetaC integrin anchors the actin within the cortex, but not the actin of the contractile ring. We hypothesize that the integrins bind a extracellular ligand in the hyaline layer and from a focal adhesion-like complex (see Zito et al. 2010).

Eggs injected with betaC morpholino fail to form radially arranged f-actin fibers in the cortex.

In experiments in which RNA encoding modified forms of beta C integrin are co-injected with morpholino, we can show that full length betaC is able to rescue this phenotype, but deletion of the cytoplasmic domain fails to rescue. We are continuing these studies making specific mutations to determine the residues necessary for organization of the actin cortex. As well, we are using a proteomics approach to determining the components that associate with the cytoplasmic domain of the integrins.

The apical integrin complex is essential for reorganization of the egg cortex. During cleavage the blastocoel forms and a second integrin complex forms on the basal surface of blastomeres interacting with basal lamina components of the extracellular matrix. The integrin subunits of the apical and basal complexes differ and localization data indicates the apical and basal complexes may contain different scaffolding proteins and different kinases. We propose that there are two independent integrin-based signaling complexes formed during cleavage and blastula formation that may have distinct and essential functions in early development. The sea urchin is an excellent model for studies of these pathways and a number of approaches are available to determine their roles in early development.