EML3 is a nuclear microtubule-binding protein required for the correct alignment of chromosomes in metaphase

Abstract

Assembly of the mitotic spindle requires a global change in the activity and constitution of the microtubule-binding-protein array at mitotic onset. An important subset of mitotic microtubule-binding proteins localises to the nucleus in interphase and essentially contributes to spindle formation and function after nuclear envelope breakdown. Here, we used a proteomic approach to selectively identify proteins of this category and revealed 50 poorly characterised human gene products, among them the echinoderm microtubule-associated-protein-like gene product, EML3. Indirect immunofluorescence showed that EML3 colocalises with spindle microtubules throughout all mitotic stages. In interphase, EML3 colocalised with cytoplasmic microtubules and accumulated in interphase nuclei. Using YFP-fusion constructs of EML3, we located a nuclear localisation signal and confirmed the microtubule-binding domain of EML3. Functional analysis of EML3 using time-lapse fluorescence microscopy and detailed end-point analysis of phenotypes after siRNA knockdown demonstrates an important role for EML3 in correct metaphase chromosome alignment. Our proteomic identification screen combined with sensitive phenotypic analysis therefore provides a reliable platform for the identification and characterisation of proteins important for correct cell division

Microtubule association of EML proteins and the EML4-ALK variant 3 oncoprotein require an N-terminal trimerization domain

“…We confirmed co-localization of YFP-tagged human EML1 to the interphase MT network of transiently transfected HeLa cells, as has been previously observed for endogenous EML proteins ( Figure 3) [5][6][7][8]. Overexpression of a tagged protein might affect its regulation and distribution on microtubules, and we shall not comment on these features of the data.…”

“…The archetypal member of this family was identified as the most abundant MAP in dividing echinoderm eggs and embryos [4]. Further members of the EML family associate with MTs and are essential for the proper formation of both the interphase MT network and the mitotic spindle [5][6][7][8]. EML proteins affect microtubule dynamics, and may act as scaffold proteins to localize mitotic kinases to MTs [5,9,10].…”

Crystal structure of EML1 reveals the basis for Hsp90 dependence of oncogenic EML4-ALK by disruption of an atypical β-propeller domain

“…2A) is a characteristic and conserved component of the EML sequence (15) and has been identified previously as carrying its MT-binding activity (17,18). The present structure shows that the HELP motif is not a distinct domain but rather forms part of the hydrophobic core of the TAPE domain and corresponds to those parts of blades 1 and 14 that are buried at the interface of the two β-propellers (Fig.…”

“…The archetypal EML protein was identified as the most abundant MAP in dividing echinoderm eggs and embryos, where it promotes microtubule dynamics (13,14). Microtubule interactions of EML1-4 have been characterized in six EML proteins present in humans (15)(16)(17)(18). More members of the family have been identified in Drosophila and Caenorhabditis elegans, which each have a single homolog (19).…”

“…For example, it has been assumed that the HELP motif forms an independent domain that perhaps mediates specific interactions with microtubules (MTs) or the plasma membrane (15,17,18,20). The extent to which the folding of the WD40 repeat region is disrupted in EML4-ALK fusions is also unclear because the number, position, and arrangement of β-propellers in EML proteins are unknown.…”