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Nuf2 and Hec1 Are Required for Retention of the Checkpoint Proteins Mad1 and Mad2 to Kinetochores
- Source :
- Current Biology. 13:2103-2109
- Publication Year :
- 2003
- Publisher :
- Elsevier BV, 2003.
-
Abstract
- Members of the Ndc80/Nuf2 complex have been shown in several systems to be important in formation of stable kinetochore-microtubule attachments and chromosome alignment in mitosis 1, 2, 3, 4, 5, 6, 7, 8 and 9. In HeLa cells, we have shown that depletion of Nuf2 by RNA interference (RNAi) results in a strong prometaphase block with an active spindle checkpoint, which correlates with low but detectable Mad2 at kinetochores that have no or few stable kinetochore microtubules [5]. Another RNAi study in HeLa cells reported that Hec1 (the human Ndc80 homolog) is required for Mad1 and Mad2 binding to kinetochores and that kinetochore bound Mad2 does not play a role in generating and maintaining the spindle assembly checkpoint [6]. Here, we show that depletion of either Nuf2 or Hec1 by RNAi in HeLa cells results in reduction of both proteins at kinetochores and in the cytoplasm. Mad1 and Mad2 concentrate at kinetochores in late prophase/early prometaphase but become depleted by 5-fold or more over the course of the prometaphase block, which is Mad2 dependent. The reduction of Mad1 and Mad2 is reversible upon spindle depolymerization. Our observations support a model in which Nuf2 and Hec1 function to prevent microtubule-dependent stripping of Mad1 and Mad2 from kinetochores that have not yet formed stable kinetochore-microtubule attachments.
- Subjects :
- Mad2
Mad1
Fluorescent Antibody Technique
Gene Expression
Cell Cycle Proteins
Spindle Apparatus
Biology
General Biochemistry, Genetics and Molecular Biology
03 medical and health sciences
0302 clinical medicine
Mad2 Proteins
Humans
Prometaphase
Kinetochores
Mitosis
Metaphase
030304 developmental biology
0303 health sciences
Agricultural and Biological Sciences(all)
Biochemistry, Genetics and Molecular Biology(all)
Kinetochore
Calcium-Binding Proteins
Nuclear Proteins
Phosphoproteins
Cell biology
Repressor Proteins
Cytoskeletal Proteins
Spindle checkpoint
RNA Interference
General Agricultural and Biological Sciences
030217 neurology & neurosurgery
HeLa Cells
Subjects
Details
- ISSN :
- 09609822
- Volume :
- 13
- Database :
- OpenAIRE
- Journal :
- Current Biology
- Accession number :
- edsair.doi.dedup.....c8886a9172e0ad41f5eb2fe2a4833304