title: Removal of Spindly from microtubule-attached kinetochores controls spindle checkpoint silencing in human cells.
abstract: The spindle checkpoint generates a "wait anaphase" signal at unattached kinetochores to prevent premature anaphase onset. Kinetochore-localized dynein is thought to silence the checkpoint by transporting checkpoint proteins from microtubule-attached kinetochores to spindle poles. Throughout metazoans, dynein recruitment to kinetochores requires the protein Spindly. Here, we identify a conserved motif in Spindly that is essential for kinetochore targeting of dynein. Spindly motif mutants, expressed following depletion of endogenous Spindly, target normally to kinetochores but prevent dynein recruitment. Spindly depletion and Spindly motif mutants, despite their similar effects on kinetochore dynein, have opposite consequences on chromosome alignment and checkpoint silencing. Spindly depletion delays chromosome alignment, but Spindly motif mutants ameliorate this defect, indicating that Spindly has a dynein recruitment-independent role in alignment. In Spindly depletions, the checkpoint is silenced following delayed alignment by a kinetochore dynein-independent mechanism. In contrast, Spindly motif mutants are retained on microtubule-attached kinetochores along with checkpoint proteins, resulting in persistent checkpoint signaling. Thus, dynein-mediated removal of Spindly from microtubule-attached kinetochores, rather than poleward transport per se, is the critical reaction in checkpoint silencing. In the absence of Spindly, a second mechanism silences the checkpoint; this mechanism is likely evolutionarily ancient, as fungi and higher plants lack kinetochore dynein.
authors.affiliation: Ludwig Institute for Cancer Research/Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California 92093, USA.
authors.forename: Reto
authors.initials: R
authors.lastname: Gassmann
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Holland
authors.forename: Dileep
authors.initials: D
authors.lastname: Varma
authors.forename: Xiaohu
authors.initials: X
authors.lastname: Wan
authors.forename: Filiz
authors.initials: F
authors.lastname: Civril
authors.forename: Don W
authors.initials: DW
authors.lastname: Cleveland
authors.forename: Karen
authors.initials: K
authors.lastname: Oegema
authors.forename: Edward D
authors.initials: ED
authors.lastname: Salmon
authors.forename: Arshad
authors.initials: A
authors.lastname: Desai
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002875:Chromosomes / Q000235:genetics; D000072159:Dynactin Complex; D004398:Dyneins / Q000378:metabolism; D020868:Gene Silencing / Q000502:physiology*; D018816:Genes, cdc / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics*; D021381:Protein Transport / Q000502:physiology
chemical_list: D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D000072159:Dynactin Complex; D008869:Microtubule-Associated Proteins; C549402:SPDL1 protein, human; D004398:Dyneins