Septin 7 interacts with centromere-associated protein E and is required for its kinetochore localization.
Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubules and the kinetochore. Septin (SEPT) belongs to a conserved family of polymerizing GTPases localized to the metaphase spindle during mitosis. Previous study showed that SEPT2 depletion results in chromosome mis-segregation correlated with a loss of centromere-associated protein E (CENP-E) from the kinetochores of congressing chromosomes (1). However, it has remained elusive as to whether CENP-E physically interacts with SEPT and how this interaction orchestrates chromosome segregation in mitosis. Here we show that SEPT7 is required for a stable kinetochore localization of CENP-E in HeLa and MDCK cells. SEPT7 stabilizes the kinetochore association of CENP-E by directly interacting with its C-terminal domain. The region of SEPT7 binding to CENP-E was mapped to its C-terminal domain by glutathione S-transferase pull-down and yeast two-hybrid assays. Immunofluorescence study shows that SEPT7 filaments distribute along the mitotic spindle and terminate at the kinetochore marked by CENP-E. Remarkably, suppression of synthesis of SEPT7 by small interfering RNA abrogated the localization of CENP-E to the kinetochore and caused aberrant chromosome segregation. These mitotic defects and kinetochore localization of CENP-E can be successfully rescued by introducing exogenous GFP-SEPT7 into the SEPT7-depleted cells. These SEPT7-suppressed cells display reduced tension at kinetochores of bi-orientated chromosomes and activated mitotic spindle checkpoint marked by Mad2 and BubR1 labelings on these misaligned chromosomes. These findings reveal a key role for the SEPT7-CENP-E interaction in the distribution of CENP-E to the kinetochore and achieving chromosome alignment. We propose that SEPT7 forms a link between kinetochore distribution of CENP-E and the mitotic spindle checkpoint.
Zhu
Mei
M
Division of Cellular Dynamics, Hefei National Laboratory for Physical Sciences at Microscale and the University of Science and Technology of China, Hefei 230027, China.
Wang
Fengsong
F
Yan
Feng
F
Yao
Phil Y
PY
Du
Jian
J
Gao
Xinjiao
X
Wang
Xiwei
X
Wu
Quan
Q
Ward
Tarsha
T
Li
Jingjing
J
Kioko
Steve
S
Hu
Renming
R
Xie
Wei
W
Ding
Xia
X
Yao
Xuebiao
X
D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D020090:Chromosome Segregation / Q000502:physiology*; D002877:Chromosomes, Human / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*; D064246:Mad2 Proteins; D008677:Metaphase / Q000502:physiology; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D058112:Septins; D008941:Spindle Apparatus / Q000235:genetics / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D034741:RNA, Small Interfering; D012097:Repressor Proteins; C068688:centromere protein E; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases; D010744:Phosphoric Monoester Hydrolases; C113158:SEPTIN7 protein, human; D058112:Septins