Dare to be different: asymmetric cell division in Drosophila, C. elegans and vertebrates.

One widespread mechanism for the generation of diverse cell types is the unequal inheritance of cell fate determinants. Several such determinants have been identified in the fruitfly Drosophila melanogaster and the worm Caenorhabditis elegans and the molecular machinery responsible for their asymmetric segregation is beginning to be unraveled. To divide asymmetrically, cells establish an axis of polarity, orient the mitotic spindle along this axis and localize cell fate determinants to one side of the cell. During cytokinesis, determinants are then segregated into one of the two daughter cells where they direct cell fate. Here, we outline the steps and factors that are involved in this process in Drosophila and C. elegans and discuss their potential conservation in vertebrates.

Experimental testing of predicted myristoylation targets involved in asymmetric cell division and calcium-dependent signalling.

Evolutionary conservation of N-terminal N-myristoylation within protein families indicates significant functional impact of this lipid posttranslational modification for function. In the MYRbase study (Maurer-Stroh et al., (2004) Genome Biology 5, R21), protein families with relevance to asymmetric cell division in animals and the group of plant calcium-dependent protein kinases (CPKs) have surfaced with many predicted myristoylated members. Here, we describe experimental in vitro verification of predicted myristoylation and explore its impact on subcellular localization for these targets in vivo. Our results confirm that, indeed, Numb isoform A, Neuralized isoforms C and D from Drosophila melanogaster and two Neuralized-like homologues from Mus musculus have the capability for N-terminal myristoylation in vitro and in vivo (in fly tissue and in mouse 3T3 cells respectively) whereas other isoforms such as Neuralized A and B have not. The latter two cases are an examples of different potential of various isoforms for posttranslational modifications. Additionally, the Arabidopsis thaliana CDPKs CPK6, CPK9 and CPK13 are shown to be substrates for myristoylation in vitro, which also affects their subcellular localization (in Arabidopsis protoplasts and tobacco leaves). At the same time, CPK6 and CPK13 do not appear to be substrates of a NMT1-like enzyme; the reasons for differing substrate specificities of NMT homologues in plants are derived from the evolutionary divergence of their N-myristoyl transferase sequences. As a methodical advance, we describe a fast and very sensitive technique (compared to traditional autoradiography) for in vitro testing of myristoylation based on thin layer chromatography read-out of the incorporated radioactive myristoyl anchor with subsequent Western blotting detection for protein yield determination using the same membrane.

The adaptor protein X11Lalpha/Dmint1 interacts with the PDZ-binding domain of the cell recognition protein Rst in Drosophila.

The Drosophila cell adhesion molecule Rst plays key roles during the development of the embryonic musculature, spacing of ommatidia in the compound eye and of sensory organs on the antenna, as well as in the neuronal wiring of the optic lobe. In rst(CT) mutants lacking the cytoplasmic domain of the Rst protein, cell sorting and apoptosis in the eye are affected, suggesting a requirement of this domain for Rst function. To identify potential interacting proteins, yeast two-hybrid screens were performed using the cytoplasmic domains of Rst and its paralogue Kirre as baits. Among several putative interactors, two paralogous Drosophila PDZ motif proteins related to X11/Mint were identified. X11/Mint family members in C. elegans (LIN-10) and vertebrates are believed to function as adaptor proteins and to regulate the assembly of multi-subunit complexes at the synapse, thereby linking the vesicle cycle to cell adhesion. Using genetic, cell biological, and biochemical approaches, we show that the interaction of Rst with X11Lalpha is of biological significance. The proteins interact, for example, in the context of cell sorting in the pupal retina.

The Par complex directs asymmetric cell division by phosphorylating the cytoskeletal protein Lgl.

To generate different cell types, some cells can segregate protein determinants into one of their two daughter cells during mitosis. In Drosophila neuroblasts, the Par protein complex localizes apically and directs localization of the cell fate determinants Prospero and Numb and the adaptor proteins Miranda and Pon to the basal cell cortex, to ensure their segregation into the basal daughter cell. The Par protein complex has a conserved function in establishing cell polarity but how it directs proteins to the opposite side is unknown. We show here that a principal function of this complex is to phosphorylate the cytoskeletal protein Lethal (2) giant larvae (Lgl; also known as L(2)gl). Phosphorylation by Drosophila atypical protein kinase C (aPKC), a member of the Par protein complex, releases Lgl from its association with membranes and the actin cytoskeleton. Genetic and biochemical experiments show that Lgl phosphorylation prevents the localization of cell fate determinants to the apical cell cortex. Lgl promotes cortical localization of Miranda, and we propose that phosphorylation of Lgl by aPKC at the apical neuroblast cortex restricts Lgl activity and Miranda localization to the opposite, basal side of the cell.