Worm Breeder's Gazette 15(5): 35 (February 1, 1999)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
| 1 | HHMI, Dept of Cell Biology, Duke University Medical Center, Durham, NC |
| 2 | Dept of Biological Sciences, Illinois State University, Normal, IL |
A cell’s identity, interactions with other cells, and ability to adapt to its environment are heavily dependent on the proteins associated on its plasma membrane. Ankyrin, an intracellular protein that couples several integral membrane proteins to the spectrin cytoskeleton, is believed to be instrumental in organizing many membrane proteins into specialized domains. Recently, the L1 family of neuronal cell adhesion molecules was shown to bind ankyrin (1). These proteins, which include L1, NrCAM, NgCAM, neurofascin, and CHL1, are believed to be involved in neurite outgrowth and axonal fasciculation and targeting during embryonic and postnatal vertebrate development.
In Drosophila, a single L1 family homologue, neuroglian, has been identified and characterized. Loss of function mutations in neuroglian result in animals that arrest as larvae. Interestingly, there are no obvious defects in the development of their nervous system (2).
A homologue of the family has been identified in C. elegans (Yuji Kohara and genome sequencing). The homologue, lad-1 (for L1-like Adhesion) is mapped to chromosome IV closely linked tounc-24. Similar to the other members of the L1 family, it contains extracellularly, six Ig domains and five FNIII domains, a transmembrane domain, and a cytoplasmic tail that contains a highly conserved ankyrin-binding domain. We are interested in determining the role of LAD-1 and studying its interaction with UNC-44, the C. elegans ankyrin. As a first step, we looked at lad-1 expression pattern and performed RNAi.
Immunofluorescence using LAD-1 antibodies indicates lad-1 is expressed and localized to cell membranes in several tissues. Lad-1 is expressed particularly strongly in the nervous system with the nerve ring, and the ventral and dorsal nerve cords showing robust staining. In addition to the nervous system, lad-1 is expressed in body wall muscles, hypodermal cells, and the gonad. Interestingly, lad-1 is expressed in the sarcomeres, as well as the cell membranes of body wall muscles. In the gonad, LAD-1 is localized to the membranes of the somatic cells, as well as the membranes of the syncytial germline and oocytes. In the embryo, LAD-1 is localized to membranes where there is cell-cell contact. As expected for an interacting protein, UNC-44 is similarly localized in the same tissues that LAD-1 is, with the exception of the germline and oocytes. While LAD-1 and UNC-44 appear to co-localize in the same tissues, we will further test their interaction using biochemical means.
To determine the consequences of a reduction of LAD-1, RNAi was carried out, using dsRNA targeted to different parts of lad-1. In all cases, only 10% of the progeny of the injected mother gave any phenotype at all, which includes lethality embryonic and larval), Vab, and Unc. lad-1 RNAi in unc-44 mutants are currently being performed to see if unc-44 loss of function enhances these defects.
To further study the role of LAD-1, we attempted to interfere with LAD-1 function using a dominant negative lad-1 construct: a secreted form of LAD-1, which contains only the first three Ig domains. Presumably, the secreted Ig domains would interfere with endogenous LAD-1 function by interacting either with endogenous LAD-1 and/or with other LAD-1 interacting partners. Transgenic lines carrying this construct gave rise to animals that were both Vab and Unc, with some larval lethality. While these phenotypes are similar to that caused by lad-1 RNAi, 80% of the animals carrying this construct gave rise to these phenotypes instead of 10% as seen with RNAi. Apparently, lad-1 is required for viability and proper morphogenesis. Screens for a genetic lad-1 mutant are currently being carried out, which will be useful in further determining the role of lad-1 in C. elegans.