EML3 is a nuclear microtubule-binding protein required for the correct alignment of chromosomes in metaphase.
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.
Tegha-Dunghu
Justus
J
Zentrum für Molekulare Biologie der Universität Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.
Neumann
Beate
B
Reber
Simone
S
Krause
Roland
R
Erfle
Holger
H
Walter
Thomas
T
Held
Michael
M
Rogers
Phill
P
Hupfeld
Kerstin
K
Ruppert
Thomas
T
Ellenberg
Jan
J
Gruss
Oliver J
OJ
D000595:Amino Acid Sequence; D002455:Cell Division; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002877:Chromosomes, Human / Q000378:metabolism*; D006801:Humans; D008677:Metaphase; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008938:Mitosis; D008969:Molecular Sequence Data; D011485:Protein Binding; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics; D008941:Spindle Apparatus / Q000378:metabolism* / Q000648:ultrastructure
C530989:EML3 protein, human; D008869:Microtubule-Associated Proteins; D020543:Proteome