CeEMAP-95 Function in the Nematode, C. elegans

University Of Kansas Center For Research Inc, Lawrence KS

Investigators

Abstract

Microtubules are critical structural components of eukaryotic cells. They are responsible for the accurate segregation of chromosomes during mitosis and meiosis, and for the intracellular transport of several membrane-bounded organelles and macromolecular complexes. Failure to complete nuclear division results in chromosome missegregation, resulting in aneuploidy, which can have drastic consequences for the organism. In addition, malfunctions in intracellular transport can lead to serious developmental defects. The regulation of microtubule assembly by microtubule-associated proteins (MAPs) is critical to these fundamental biological processes. This project involves a detailed molecular study of a novel microtubule-associated protein, EMAP, first identified in sea urchins and more recently discovered in higher and lower eukaryotes. The fundamental importance of EMAP is underscored by the high degree of sequence conservation from the nematode (C. elegans) to humans. In addition, the gene for human EMAP-1 maps to the Usher syndrome 1a locus, a disease that results in profound deafness and blindness. Sea urchin EMAP has unique effects on microtubule assembly. Microtubules assembled in the presence of EMAP are slightly longer, but significantly more dynamic, with infrequent rescue events. Because EMAP is an abundant component of the mitotic apparatus, EMAP may regulate microtubule assembly dynamics during the early embryonic cell cycles. EMAP is also unusual in that it co-purifies with microtubules that are associated with a subset of mRNAs, poly(A)-binding proteins and ribosomes. The experiments in this project take advantage of Dr. Suprenant's experience in the biochemical and molecular characterization of EMAP and the ability to manipulate EMAP function in the nematode, C. elegans. These experiments are designed to determine whether the C. elegans EMAP homologue, CeEMAP-95, is a bona fide microtubule-binding protein and to identify CeEMAP-95-dependent processes in the worm. Similar to sea urchin EMAP, worm CeEMAP-95 may be involved in regulating microtubule assembly during embryogenesis or perhaps in the generation of cellular asymmetry through the localization of specific developmental determinants. The early stages of this project will involve the generation of specific antibodies, CeEMAP-95 mutant alleles, and recombinant CeEMAP-95 constructs that will provide essential molecular reagents for the continuing study of this interesting protein family. Also, additional microtubule-associated proteins in the worm will be identified and characterized, with the eventual goal of determining whether these proteins act independently, coordinately or in parallel with worm EMAP. Specifically, the following questions will be addressed: 1, is CeEMAP-95 localized to microtubule-rich cell types in the worm? 2, is CeEMAP-95 a regulator of microtubule assembly dynamics? 3, does the loss of function of the CeEMAP-95 gene affect embryonic cell division or neuronal cell function? and 4, what other essential microtubule-binding proteins are present in C. elegans? The long term goal of this work is to identify the cellular functions that require EMAP and to understand how these processes affect the development of a multicellular organism. This will be done through the combination of biochemistry, cellular and molecular biology, and genetics, with both the biochemical demonstration of EMAP's function in vitro and the genetic identification of its function in vivo.