Conservation of the WD-repeat, microtubule-binding protein, EMAP, in sea urchins, humans, and the nematode C. elegans

Abstract

The echinoderm microtubule-associated protein (EMAP) is the most abundant microtubule-binding protein in the first cleavage mitotic apparatus in sea urchin embryos. The first goal of this study was to determine whether there is sufficient EMAP in the egg and embryo to modify microtubule dynamics during the early cleavages divisions and whether EMAP functions at a specific time or place in the embryo. To accomplish this goal, we examined the relative abundance, tissue distribution, and temporal pattern of EMAP expression during embryonic development. The second goal of this study was to identify important functional domains within the EMAP coding sequence. A conserved sequence might reveal a potential microtubule-binding domain. We cloned, sequenced and compared overlapping EMAP cDNAs from two different sea urchin species that diverged approximately 80 million years ago, and compared these with cDNA sequences from a vertebrate and nematode species. From quantitative immunoblots, we determined the EMAP concentration in eggs to be 4 microM. The steady-state levels of EMAP mRNA and protein accumulated during development, and all three germ layers expressed EMAP. During the early stages of development, EMAP and tubulin were both abundant in the ectoderm, mesoderm and endoderm. However, during late gastrulation and the formation of the early pluteus larvae, EMAP was enriched in the mesoderm, while tubulin staining was most abundant in the archenteron. These results indicate that EMAP may have tissue-specific functions in the late stage embryo. To identify conserved functional domains, we compared the predicted amino acid sequence encoded by Strongylocentrotus purpuratus and Lytechinus variegatus EMAP cDNAs, and determined that these two sea urchin EMAPs were 95% conserved and shared an identical domain organization. A parsimonious analysis of these sea urchin protein sequences, as well as human and C. elegans EMAP sequences was used to construct a gene tree. Together these results suggest that EMAP is an important microtubule protein required at all developmental stages of sea urchins, and whose cellular function may be conserved amongst metazoans.

EML proteins in microtubule regulation and human disease

“…However, analysis of endogenous EML localization has, at least until recently, been hampered by the lack of good, commercially available antibodies. Peptidespecific antibodies raised to EML3 and EML4 have though confirmed localization of these proteins to microtubules in cultured cells (Chen et al, 2015;Tegha-Dunghu et al, 2008). Taken together, the expression and localization studies are indicative of a family of cytoplasmic microtubule-binding proteins with widespread expression but which may have particular roles in neuronal tissues.…”

“…Antibodies raised against EMAP detected a similar sized protein by Western blot in microtubule preparations from a variety of echinoderm species, while it decorated microtubules in sea urchin embryos and adult coelomocytes by immunofluorescence microscopy (Suprenant et al, 1993). Expression of recombinant proteins confirmed even association of mammalian EML1, 2, 3 and 4 with the microtubule lattice (Eichenmuller et al, 2002;Houtman et al, 2007;Pollmann et al, 2006;Richards et al, 2015;Tegha-Dunghu et al, 2008). However, analysis of endogenous EML localization has, at least until recently, been hampered by the lack of good, commercially available antibodies.…”

“…Studies on sea urchin EMAP revealed that microtubule binding was conferred through a region towards the N-terminus of the protein (Eichenmuller et al, 2001). Indeed, it was originally suggested that a highly conserved 'HELP' (hydrophobic ELP) motif of approximately 40 residues within this N-terminus was responsible for microtubule binding (Eichenmuller et al, 2002;Suprenant et al, 2000). However, chimeras that specifically fused the HELP motif to EGFP did not localize to microtubules (Pollmann et al, 2006), and structural studies have since revealed that the HELP motif is an integral part of the TAPE domain and contributes to protein folding rather than microtubule binding (Richards et al, 2014).…”

“…Indeed, sea urchin EMAP, as well as mammalian EML1-4, have all been reported to localize to spindle microtubules in mitosis, as well as the microtubule cytoskeleton in interphase (Eichenmuller et al, 2002;Kielar et al, 2014;Pollmann et al, 2006;Suprenant et al, 1993;Tegha-Dunghu et al, 2008). However, we recently found that there may well be important differences in the relative affinity of EMLs for microtubules in interphase and mitosis, with EML3 and EML4 exhibiting reduced localization to microtubules in mitosis as compared to interphase (Montgomery et al, in preparation).…”

“…This protein, termed EMAP for echinoderm MAP, was the major non-tubulin component present in purified mitotic spindle preparations made from unfertilised sea urchin eggs (Suprenant et al, 1993;Suprenant and Marsh, 1987). Orthologues of EMAP were subsequently identified in other echinoderms, such as starfish and sand dollar, and then in more distant eukaryotes, including flies, worms and vertebrates, where the name of ELP or EML (both for EMAP-like proteins) has been adopted (Hueston et al, 2008;Suprenant et al, 2000). The common property of these proteins is their ability to decorate microtubules.…”