Human ASPM participates in spindle organisation, spindle orientation and cytokinesis.
BACKGROUND
Mutations in the Abnormal Spindle Microcephaly related gene (ASPM) are the commonest cause of autosomal recessive primary microcephaly (MCPH) a disorder characterised by a small brain and associated mental retardation. ASPM encodes a mitotic spindle pole associated protein. It is suggested that the MCPH phenotype arises from proliferation defects in neural progenitor cells (NPC).


RESULTS
We show that ASPM is a microtubule minus end-associated protein that is recruited in a microtubule-dependent manner to the pericentriolar matrix (PCM) at the spindle poles during mitosis. ASPM siRNA reduces ASPM protein at the spindle poles in cultured U2OS cells and severely perturbs a number of aspects of mitosis, including the orientation of the mitotic spindle, the main determinant of developmental asymmetrical cell division. The majority of ASPM depleted mitotic cells fail to complete cytokinesis. In MCPH patient fibroblasts we show that a pathogenic ASPM splice site mutation results in the expression of a novel variant protein lacking a tripeptide motif, a minimal alteration that correlates with a dramatic decrease in ASPM spindle pole localisation. Moreover, expression of dominant-negative ASPM C-terminal fragments cause severe spindle assembly defects and cytokinesis failure in cultured cells.


CONCLUSIONS
These observations indicate that ASPM participates in spindle organisation, spindle positioning and cytokinesis in all dividing cells and that the extreme C-terminus of the protein is required for ASPM localisation and function. Our data supports the hypothesis that the MCPH phenotype caused by ASPM mutation is a consequence of mitotic aberrations during neurogenesis. We propose the effects of ASPM mutation are tolerated in somatic cells but have profound consequences for the symmetrical division of NPCs, due to the unusual morphology of these cells. This antagonises the early expansion of the progenitor pool that underpins cortical neurogenesis, causing the MCPH phenotype.
Section of Ophthalmology and Neuroscience, Wellcome Trust Brenner Building, Leeds Institute of Molecular Medicine, University of Leeds, St. James's University Hospital, Leeds LS9 7TF, UK.
Julie
J
Higgins
Carol
C
Midgley
Anna-Maria
AM
Bergh
Sandra M
SM
Bell
Jonathan M
JM
Askham
Emma
E
Roberts
Ruth K
RK
Binns
Saghira M
SM
Sharif
Christopher
C
Bennett
David M
DM
Glover
C Geoffrey
CG
Woods
Ewan E
EE
Morrison
Jacquelyn
J
Bond
D002455:Cell Division; D002460:Cell Line; D048749:Cytokinesis; D003599:Cytoskeleton; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008870:Microtubules / Q000378:metabolism; D008938:Mitosis; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D022821:RNA Splice Sites; D034741:RNA, Small Interfering / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism / Q000648:ultrastructure*
C467202:ASPM protein, human; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D022821:RNA Splice Sites; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins