title: Dual mechanism controls asymmetric spindle position in ascidian germ cell precursors.
abstract: Mitotic spindle orientation with respect to cortical polarity cues generates molecularly distinct daughter cells during asymmetric cell division (ACD). However, during ACD it remains unknown how the orientation of the mitotic spindle is regulated by cortical polarity cues until furrowing begins. In ascidians, the cortical centrosome-attracting body (CAB) generates three successive unequal cleavages and the asymmetric segregation of 40 localized postplasmic/PEM RNAs in germ cell precursors from the 8-64 cell stage. By combining fast 4D confocal fluorescence imaging with gene-silencing and classical blastomere isolation experiments, we show that spindle repositioning mechanisms are active from prometaphase until anaphase, when furrowing is initiated in B5.2 cells. We show that the vegetal-most spindle pole/centrosome is attracted towards the CAB during prometaphase, causing the spindle to position asymmetrically near the cortex. Next, during anaphase, the opposite spindle pole/centrosome is attracted towards the border with neighbouring B5.1 blastomeres, causing the spindle to rotate (10 degrees /minute) and migrate (3 microm/minute). Dynamic 4D fluorescence imaging of filamentous actin and plasma membrane shows that precise orientation of the cleavage furrow is determined by this second phase of rotational spindle displacement. Furthermore, in pairs of isolated B5.2 blastomeres, the second phase of rotational spindle displacement was lost. Finally, knockdown of PEM1, a protein localized in the CAB and required for unequal cleavage in B5.2 cells, completely randomizes spindle orientation. Together these data show that two separate mechanisms active during mitosis are responsible for spindle positioning, leading to precise orientation of the cleavage furrow during ACD in the cells that give rise to the germ lineage in ascidians.
authors.affiliation: Developmental Biology Unit UMR 7009, UPMC (University of Paris 06), 75015 Paris, France.
authors.forename: François
authors.initials: F
authors.lastname: Prodon
authors.forename: Janet
authors.initials: J
authors.lastname: Chenevert
authors.forename: Céline
authors.initials: C
authors.lastname: Hébras
authors.forename: Rémi
authors.initials: R
authors.lastname: Dumollard
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Faure
authors.forename: Jose
authors.initials: J
authors.lastname: Gonzalez-Garcia
authors.forename: Hiroki
authors.initials: H
authors.lastname: Nishida
authors.forename: Christian
authors.initials: C
authors.lastname: Sardet
authors.forename: Alex
authors.initials: A
authors.lastname: McDougall
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000705:Anaphase; D000818:Animals; D001757:Blastomeres / Q000166:cytology / Q000378:metabolism*; D002453:Cell Cycle / Q000235:genetics; D002455:Cell Division; D018385:Centrosome / Q000378:metabolism*; D003599:Cytoskeleton / Q000235:genetics / Q000378:metabolism*; D005854:Germ Cells / Q000378:metabolism; D008938:Mitosis; D049468:Prometaphase; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D008941:Spindle Apparatus / Q000235:genetics / Q000378:metabolism*; D014561:Urochordata / Q000166:cytology / Q000378:metabolism*
chemical_list: D000199:Actins; D011506:Proteins