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Direct role of dynein motor in stable kinetochore-microtubule attachment, orientation, and alignment.

Varma D, Monzo P, Stehman SA, Vallee RB - J. Cell Biol. (2008)

Bottom Line: Further, kinetochore microtubule bundles are severely destabilized at reduced temperatures.Dynein HC RNAi and injection of anti-dynein antibody in MG132-arrested metaphase cells produced similar effects.These results identify a novel function for the dynein motor in stable microtubule attachment and maintenance of kinetochore orientation during metaphase chromosome alignment.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA.

ABSTRACT
Cytoplasmic dynein has been implicated in diverse mitotic functions, several involving its association with kinetochores. Much of the supporting evidence comes from inhibition of dynein regulatory factors. To obtain direct insight into kinetochore dynein function, we expressed a series of dynein tail fragments, which we find displace motor-containing dynein heavy chain (HC) from kinetochores without affecting other subunits, regulatory factors, or microtubule binding proteins. Cells with bipolar mitotic spindles progress to late prometaphase-metaphase at normal rates. However, the dynein tail, dynactin, Mad1, and BubR1 persist at the aligned kinetochores, which is consistent with a role for dynein in self-removal and spindle assembly checkpoint inactivation. Kinetochore pairs also show evidence of misorientation relative to the spindle equator and abnormal oscillatory behavior. Further, kinetochore microtubule bundles are severely destabilized at reduced temperatures. Dynein HC RNAi and injection of anti-dynein antibody in MG132-arrested metaphase cells produced similar effects. These results identify a novel function for the dynein motor in stable microtubule attachment and maintenance of kinetochore orientation during metaphase chromosome alignment.

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Role of dynein in kinetochore microtubule stability and kinetochore orientation. LLCPK1 cells transfected with the dynein tail fragment were incubated at 4°C for 10 min and then examined by confocal microscopy. (A) Dynein tail–expressing cell stained with anti-tubulin, DAPI, and anti-myc (inset) showing few remaining kinetochore microtubule bundles. (B) A control cell stained as in A showing numerous cold-stable kinetochore microtubule bundles. (C) Fraction of cells exhibiting decrease in the number of cold-stable microtubule bundles in tail-expressing versus control untransfected cells after cold treatment (n = 90). P = 0.012; two-tailed t test. (D–I) Analysis of kinetochore orientation in tail-expressing LLCPK1 cells. (D–F and D′–F′) Cells in late prometaphase/metaphase (D, D′, E, and E′, pretreated with MG132 as indicated) or prometaphase (F and F′) transfected with the tail were stained with anti-tubulin, CREST, and anti-myc antibodies (D′–F′) as indicated versus DAPI (not depicted). (G and H) Control LLCPK1 cells expressing GFP. The numbered boxes indicate images magnified at right. Insets show GFP staining. (I) Fraction of aligned kinetochore pairs exhibiting different attachment patterns. P = 0.0025; two-tailed t test. Error bars indicate SD from mean from three independent experiments. Bars, 5 μM.
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fig4: Role of dynein in kinetochore microtubule stability and kinetochore orientation. LLCPK1 cells transfected with the dynein tail fragment were incubated at 4°C for 10 min and then examined by confocal microscopy. (A) Dynein tail–expressing cell stained with anti-tubulin, DAPI, and anti-myc (inset) showing few remaining kinetochore microtubule bundles. (B) A control cell stained as in A showing numerous cold-stable kinetochore microtubule bundles. (C) Fraction of cells exhibiting decrease in the number of cold-stable microtubule bundles in tail-expressing versus control untransfected cells after cold treatment (n = 90). P = 0.012; two-tailed t test. (D–I) Analysis of kinetochore orientation in tail-expressing LLCPK1 cells. (D–F and D′–F′) Cells in late prometaphase/metaphase (D, D′, E, and E′, pretreated with MG132 as indicated) or prometaphase (F and F′) transfected with the tail were stained with anti-tubulin, CREST, and anti-myc antibodies (D′–F′) as indicated versus DAPI (not depicted). (G and H) Control LLCPK1 cells expressing GFP. The numbered boxes indicate images magnified at right. Insets show GFP staining. (I) Fraction of aligned kinetochore pairs exhibiting different attachment patterns. P = 0.0025; two-tailed t test. Error bars indicate SD from mean from three independent experiments. Bars, 5 μM.

Mentions: Effects of dynein tail expression on mitotic stage and kinetochore composition during metaphase. (A) Dynein tail–expressing COS7 cells with bipolar spindles were evaluated for chromosome distribution, revealing an increase in late prometaphase cells versus untransfected controls (n = 300). Error bars indicate SD from mean from three independent experiments. (B) Example of cells used for A stained with anti-myc, anti-tubulin, and CREST (not depicted) antibodies versus DAPI (inset). Numbered boxes indicate images magnified below. (C–E) Control COS7 cell expressing a myc vector stained with anti-p150Glued, anti-Mad1, and anti-BubR1 antibodies in each case versus anti-tubulin and anti-myc (not depicted) versus DAPI (inset), showing loss of these proteins from kinetochores at metaphase. (C′–E′) COS7 cells expressing the tail construct and stained as in C–E. The numbered boxes indicate images magnified at right. Dynein tail, p150Glued, Mad1, and BubR1 are all observed to associate with both kinetochores of aligned chromatid pairs, including those kinetochores associated with microtubules. Note examples of misoriented kinetochore pairs in numbered insets (and see Fig. 4). Note that these proteins are absent at spindle poles in tail-expressing cells, in contrast to controls. (F–H and F′–H′) Analysis of Hec1, MCAK, and Kif2b immunoreactivity at aligned kinetochores. Control and tail-expressing COS7 cells were stained using anti-myc (insets) and anti-Hec1, anti-MCAK, and anti-Kif2b antibodies, respectively versus DAPI (n = 150). Mean kinetochore spacing in Hec1-stained cells was decreased relative to controls (see text), which is consistent with loss of tension at kinetochores. MCAK and Kif2b localize to prometaphase kinetochores (G and H) and were normally redistributed to spindle poles in dynein-inhibited cells (G′ and H′). Bars, 5 μM.


Direct role of dynein motor in stable kinetochore-microtubule attachment, orientation, and alignment.

Varma D, Monzo P, Stehman SA, Vallee RB - J. Cell Biol. (2008)

Role of dynein in kinetochore microtubule stability and kinetochore orientation. LLCPK1 cells transfected with the dynein tail fragment were incubated at 4°C for 10 min and then examined by confocal microscopy. (A) Dynein tail–expressing cell stained with anti-tubulin, DAPI, and anti-myc (inset) showing few remaining kinetochore microtubule bundles. (B) A control cell stained as in A showing numerous cold-stable kinetochore microtubule bundles. (C) Fraction of cells exhibiting decrease in the number of cold-stable microtubule bundles in tail-expressing versus control untransfected cells after cold treatment (n = 90). P = 0.012; two-tailed t test. (D–I) Analysis of kinetochore orientation in tail-expressing LLCPK1 cells. (D–F and D′–F′) Cells in late prometaphase/metaphase (D, D′, E, and E′, pretreated with MG132 as indicated) or prometaphase (F and F′) transfected with the tail were stained with anti-tubulin, CREST, and anti-myc antibodies (D′–F′) as indicated versus DAPI (not depicted). (G and H) Control LLCPK1 cells expressing GFP. The numbered boxes indicate images magnified at right. Insets show GFP staining. (I) Fraction of aligned kinetochore pairs exhibiting different attachment patterns. P = 0.0025; two-tailed t test. Error bars indicate SD from mean from three independent experiments. Bars, 5 μM.
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fig4: Role of dynein in kinetochore microtubule stability and kinetochore orientation. LLCPK1 cells transfected with the dynein tail fragment were incubated at 4°C for 10 min and then examined by confocal microscopy. (A) Dynein tail–expressing cell stained with anti-tubulin, DAPI, and anti-myc (inset) showing few remaining kinetochore microtubule bundles. (B) A control cell stained as in A showing numerous cold-stable kinetochore microtubule bundles. (C) Fraction of cells exhibiting decrease in the number of cold-stable microtubule bundles in tail-expressing versus control untransfected cells after cold treatment (n = 90). P = 0.012; two-tailed t test. (D–I) Analysis of kinetochore orientation in tail-expressing LLCPK1 cells. (D–F and D′–F′) Cells in late prometaphase/metaphase (D, D′, E, and E′, pretreated with MG132 as indicated) or prometaphase (F and F′) transfected with the tail were stained with anti-tubulin, CREST, and anti-myc antibodies (D′–F′) as indicated versus DAPI (not depicted). (G and H) Control LLCPK1 cells expressing GFP. The numbered boxes indicate images magnified at right. Insets show GFP staining. (I) Fraction of aligned kinetochore pairs exhibiting different attachment patterns. P = 0.0025; two-tailed t test. Error bars indicate SD from mean from three independent experiments. Bars, 5 μM.
Mentions: Effects of dynein tail expression on mitotic stage and kinetochore composition during metaphase. (A) Dynein tail–expressing COS7 cells with bipolar spindles were evaluated for chromosome distribution, revealing an increase in late prometaphase cells versus untransfected controls (n = 300). Error bars indicate SD from mean from three independent experiments. (B) Example of cells used for A stained with anti-myc, anti-tubulin, and CREST (not depicted) antibodies versus DAPI (inset). Numbered boxes indicate images magnified below. (C–E) Control COS7 cell expressing a myc vector stained with anti-p150Glued, anti-Mad1, and anti-BubR1 antibodies in each case versus anti-tubulin and anti-myc (not depicted) versus DAPI (inset), showing loss of these proteins from kinetochores at metaphase. (C′–E′) COS7 cells expressing the tail construct and stained as in C–E. The numbered boxes indicate images magnified at right. Dynein tail, p150Glued, Mad1, and BubR1 are all observed to associate with both kinetochores of aligned chromatid pairs, including those kinetochores associated with microtubules. Note examples of misoriented kinetochore pairs in numbered insets (and see Fig. 4). Note that these proteins are absent at spindle poles in tail-expressing cells, in contrast to controls. (F–H and F′–H′) Analysis of Hec1, MCAK, and Kif2b immunoreactivity at aligned kinetochores. Control and tail-expressing COS7 cells were stained using anti-myc (insets) and anti-Hec1, anti-MCAK, and anti-Kif2b antibodies, respectively versus DAPI (n = 150). Mean kinetochore spacing in Hec1-stained cells was decreased relative to controls (see text), which is consistent with loss of tension at kinetochores. MCAK and Kif2b localize to prometaphase kinetochores (G and H) and were normally redistributed to spindle poles in dynein-inhibited cells (G′ and H′). Bars, 5 μM.

Bottom Line: Further, kinetochore microtubule bundles are severely destabilized at reduced temperatures.Dynein HC RNAi and injection of anti-dynein antibody in MG132-arrested metaphase cells produced similar effects.These results identify a novel function for the dynein motor in stable microtubule attachment and maintenance of kinetochore orientation during metaphase chromosome alignment.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA.

ABSTRACT
Cytoplasmic dynein has been implicated in diverse mitotic functions, several involving its association with kinetochores. Much of the supporting evidence comes from inhibition of dynein regulatory factors. To obtain direct insight into kinetochore dynein function, we expressed a series of dynein tail fragments, which we find displace motor-containing dynein heavy chain (HC) from kinetochores without affecting other subunits, regulatory factors, or microtubule binding proteins. Cells with bipolar mitotic spindles progress to late prometaphase-metaphase at normal rates. However, the dynein tail, dynactin, Mad1, and BubR1 persist at the aligned kinetochores, which is consistent with a role for dynein in self-removal and spindle assembly checkpoint inactivation. Kinetochore pairs also show evidence of misorientation relative to the spindle equator and abnormal oscillatory behavior. Further, kinetochore microtubule bundles are severely destabilized at reduced temperatures. Dynein HC RNAi and injection of anti-dynein antibody in MG132-arrested metaphase cells produced similar effects. These results identify a novel function for the dynein motor in stable microtubule attachment and maintenance of kinetochore orientation during metaphase chromosome alignment.

Show MeSH
Related in: MedlinePlus