The N terminus of the Drosophila Numb protein directs membrane association and actin-dependent asymmetric localization

Abstract

Drosophila Numb is a membrane associated protein of 557 amino acids (aa) that localizes asymmetrically into a cortical crescent in mitotic neural precursor cells and segregates into one of the daughter cells, where it is required for correct cell fate specification. We demonstrate here that asymmetric localization but not membrane localization of Numb in Drosophila embryos is inhibited by latrunculin A, an inhibitor of actin assembly. We also show that deletion of either the first 41 aa or aa 41-118 of Numb eliminates both localization to the cell membrane and asymmetric localization during mitosis, whereas C-terminal deletions or deletions of central portions of Numb do not affect its subcellular localization. Fusion of the first 76 or the first 119 aa of Numb to ␤-galactosidase results in a fusion protein that localizes to the cell membrane, but fails to localize asymmetrically during mitosis. In contrast, a fusion protein containing the first 227 aa of Numb and ␤-galactosidase localizes asymmetrically during mitosis and segregates into the same daughter cell as the endogenous Numb protein, demonstrating that the first 227 aa of the Numb protein are sufficient for asymmetric localization.Asymmetric cell divisions in which a mother cell segregates a determinant into one of its daughter cells to make this cell different from its sister cell contribute to generating different cell types in a multicellular organism (1, 2). In Drosophila, the four cells that form an external sensory (ES) organ-one type of sensory structure found in the peripheral nervous systemarise during development from a single sensory organ precursor (SOP) cell that undergoes a series of asymmetric cell divisions (3-5). During these asymmetric divisions, the protein Numb acts as a segregating determinant. In wild-type animals, the SOP cell divides into a IIA cell and a IIB cell. The IIA cell gives rise to the two outer cells (hair and socket cell) whereas the IIB cell forms the two inner cells (neuron and sheath cell).

A Novel Transmembrane Protein Recruits Numb to the Plasma Membrane during Asymmetric Cell Division

“…Thus, NIP appears both necessary and sufficient for the recruitment and retention of Numb in the plasma membrane. Interestingly, a 76-residue fragment upstream of the PTB domain was localized to the neuroblast membrane when expressed in Drosophila (34). Because this fragment does not interact with NIP in vitro, it is likely that it may interact indirectly with NIP or that other mechanisms of Numb membrane localization exist.…”

“…Numb does not contain a transmembrane segment or a lipid anchor, and therefore, its recruitment to the plasma membrane may be mediated by a lipid and/or a protein component of the membrane. The N-terminal region of Numb, including the PTB domain, was shown to be necessary for the membrane recruitment and asymmetric localization of Numb (34). Although the PTB domain was originally identified as a protein module that interacts with NPXpY-containing sequences, some members of the family, such as the Shc and Dab1 PTB domains, are also capable of binding to acidic lipids (46,47) and are therefore implicated in membrane localization.…”

“…Because Numb lacks a transmembrane segment, its recruitment to the membrane would necessarily involve one of several possible mechanisms: by binding to a membrane lipid(s), by directly or indirectly interacting with a membrane protein, or by engaging a protein transporting machinery (32,33). Asymmetric localization of Numb in neuroblasts requires the PTB domain (34). The pivotal role of the PTB domain is further underscored by the observation that it is indispensable not only for Numb localization during sensory organ precursor and neuroblast division but also for the specification of sibling cell fates (34 -36).…”

Conserved Domains of the Nullo Protein Required for Cell-Surface Localization and Formation of Adherens Junctions

“…Interestingly, mutation of the myristoylation site of Numb also failed to affect membrane localization, although truncation of the first 40 amino acids of the N-terminus caused the protein to accumulate in the cytoplasm (Knoblich et al, 1997). It remains to be seen if Numb contains a bipartite localization motif similar to that seen in Nullo and whether the independent membrane targeting activity of myristoyl groups and basic clusters is a common occurrence in Drosophila.…”

“…Although the majority of these genes have been characterized genetically and developmentally, only a handful have actually been shown to be myristoylated (Neel and Young, 1994;Teng et al, 1994;Rossi et al, 1999), and the in vivo role of the myristoylation site has been studied only in the neural protein Numb (Knoblich et al, 1997). Using a yeast NMT system (Duronio et al, 1990), we demonstrated that Nullo is modified by the addition of a myristoyl group, and this is prevented by the substitution of alanine for glycine in position 2.…”