Different spindle checkpoint proteins monitor microtubule attachment and tension at kinetochores in Drosophila cells.
The spindle assembly checkpoint detects errors in kinetochore attachment to the spindle including insufficient microtubule occupancy and absence of tension across bi-oriented kinetochore pairs. Here, we analyse how the kinetochore localization of the Drosophila spindle checkpoint proteins Bub1, Mad2, Bub3 and BubR1, behave in response to alterations in microtubule binding or tension. To analyse the behaviour in the absence of tension, we treated S2 cells with low doses of taxol to disrupt microtubule dynamics and tension, but not kinetochore-microtubule occupancy. Under these conditions, we found that Mad2 and Bub1 do not accumulate at metaphase kinetochores whereas BubR1 does. Consistently, in mono-oriented chromosomes, both kinetochores accumulate BubR1 whereas Bub1 and Mad2 only localize at the unattached kinetochore. To study the effect of tension we analysed the kinetochore localization of spindle checkpoint proteins in relation to tension-sensitive kinetochore phosphorylation recognised by the 3F3/2 antibody. Using detergent-extracted S2 cells as a system in which kinetochore phosphorylation can be easily manipulated, we observed that BubR1 and Bub3 accumulation at kinetochores is dependent on the presence of phosphorylated 3F3/2 epitopes. However, Bub1 and Mad2 localize at kinetochores regardless of the 3F3/2 phosphorylation state. Altogether, our results suggest that spindle checkpoint proteins sense distinct aspects of kinetochore interaction with the spindle, with Mad2 and Bub1 monitoring microtubule occupancy while BubR1 and Bub3 monitor tension across attached kinetochores.
Logarinho
Elsa
E
Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.
Bousbaa
Hassan
H
Dias
José Miguel
JM
Lopes
Carla
C
Amorim
Isabel
I
Antunes-Martins
Ana
A
Sunkel
Claudio E
CE
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003078:Colchicine / Q000494:pharmacology; D029721:Drosophila Proteins / Q000378:metabolism; D004331:Drosophila melanogaster / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D018386:Kinetochores / Q000187:drug effects / Q000378:metabolism*; D052998:Microcystins; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D008938:Mitosis / Q000187:drug effects; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D017239:Paclitaxel / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D010802:Phylogeny; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D021381:Protein Transport; D020539:Sequence Analysis, Protein; D008941:Spindle Apparatus / Q000378:metabolism*
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; D052998:Microcystins; D009687:Nuclear Proteins; D010456:Peptides, Cyclic; C078588:microcystin; D011494:Protein Kinases; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases; D017239:Paclitaxel; D003078:Colchicine