Function and dynamics of slam in furrow formation in early Drosophila embryo.

The Drosophila embryo undergoes a developmental transition in the blastoderm stage switching from syncytial to cellular development. The cleavage furrow, which encloses nuclei into cells, is a prominent morphological feature of this transition. It is not clear how the pattern of the furrow array is defined and how zygotic genes trigger the formation and invagination of interphase furrows. A key to these questions is provided by the gene slam, which has been previously implicated in controlling furrow invagination. Here we investigate the null phenotype of slam, the dynamics of Slam protein, and its control by the recycling endosome. We find that slam is essential for furrow invagination during cellularisation and together with nullo, for specification of the furrow. During cellularisation, Slam marks first the furrow, which is derived from the metaphase furrow of the previous mitosis. Slightly later, Slam accumulates at new furrows between daughter cells early in interphase. Slam is stably associated with the furrow canal except for the onset of cellularisation as revealed by FRAP experiments. Restriction of Slam to the furrow canal and Slam mobility during cellularisation is controlled by the recycling endosome and centrosomes. We propose a three step model. The retracting metaphase furrow leaves an initial mark. This mark and the border between corresponding daughter nuclei are refined by vesicular transport away from pericentrosomal recycling endosome towards the margins of the somatic buds. Following the onset of zygotic gene expression, Slam and Nullo together stabilise this mark and Slam triggers invagination of the cleavage furrow.

Localization of RhoGEF2 during Drosophila cellularization is developmentally controlled by Slam.

Essential for proper function of small GTPases of the Rho family, which control many aspects of cytoskeletal and membrane dynamics, is their temporal and spatial control by activating GDP exchange factors (GEFs) and deactivating GTPase-activating-proteins (GAPs). The regulatory mechanisms controlling these factors are not well understood, especially during development, when the organization and behaviour of cells change in a stage dependent manner. During Drosophila cellularization Rho signalling and RhoGEF2 are involved in furrow canal formation and the organization of actin and myosin. Here we analyze, how RhoGEF2 is localized at the sites of membrane invagination. We show that the PDZ domain is necessary for localization and function of RhoGEF2 and identify Slam as a factor that is necessary for RhoGEF2 localization. We also demonstrate that Slam can recruit RhoGEF2 to ectopic sites. Furthermore we find that the PDZ domain of RhoGEF2 can form a complex with Slam invivo and that Slam transcripts and protein colocalize at the furrow canal and in basal particles. Based on these findings, we propose that accumulation of slam mRNA and protein at the presumptive invagination site provides a spatial and temporal trigger for RhoGEF2-Rho1 signalling.