ZW10 helps recruit dynactin and dynein to the kinetochore - PubMed (original) (raw)

ZW10 helps recruit dynactin and dynein to the kinetochore

D A Starr et al. J Cell Biol. 1998.

Abstract

Mutations in the Drosophila melanogaster zw10 gene, which encodes a conserved, essential kinetochore component, abolish the ability of dynein to localize to kinetochores. Several similarities between the behavior of ZW10 protein and dynein further support a role for ZW10 in the recruitment of dynein to the kinetochore: (a) in response to bipolar tension across the chromosomes, both proteins mostly leave the kinetochore at metaphase, when their association with the spindle becomes apparent; (b) ZW10 and dynein both bind to functional neocentromeres of structurally acentric minichromosomes; and (c) the localization of both ZW10 and dynein to the kinetochore is abolished in cells mutant for the gene rough deal. ZW10's role in the recruitment of dynein to the kinetochore is likely to be reasonably direct, because dynamitin, the p50 subunit of the dynactin complex, interacts with ZW10 in a yeast two-hybrid screen. Since in zw10 mutants no defects in chromosome behavior are observed before anaphase onset, our results suggest that dynein at the kinetochore is essential for neither microtubule capture nor congression to the metaphase plate. Instead, dynein's role at the kinetochore is more likely to be involved in the coordination of chromosome separation and/or poleward movement at anaphase onset.

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Figures

Figure 6

Figure 6

Determination of the interaction domains of dynamitin and HZW10. (A) Various NH2- and COOH-terminal deletions of dynamitin (the p50 subunit of dynactin) were constructed and put into the prey vector and tested for their ability to interact with HZW10 bait in the two-hybrid system. Two-hybrid activity was measured by β-galactosidase activity. +++ was arbitrarily set as the strength of the interaction (intensity of blue) between the entire dynamitin protein and HZW10. The coil-coil (coil) and helix-turn-helix (HTH) domains of dynamitin as identified by Echeverri et al. (1996) are marked. (B) Various parts of the HZW10 protein were cloned into the prey vector and tested in the two- hybrid system with dynamitin as bait. Interaction of the entire HZW10 protein with dynamitin is set as +++.

Figure 1

Figure 1

Dhc localization in Drosophila spermatocytes. Blue, DNA; red, Dhc; green, ZW10 protein. (a–c) A prometaphase I figure illustrating the presence of Dhc at the kinetochores of the bivalents. In addition, the colocalization of Dhc and ZW10 is indicated by the yellow signals in c, representing overlap of the sites of Dhc (seen alone in a) and ZW10 staining (seen alone in b). (d) Another prometaphase I figure with Dhc at the kinetochores of the chromosomes in the bivalents. Arrow, a hemispherical zone of Dhc staining on one of the kinetochores of the small fourth chromosome bivalent. (e and f) Metaphase I figures with decreased Dhc staining at the kinetochores (arrows) and with Dhc staining near the poles (e) or along kinetochore microtubules (f). (g and h) Anaphase I figures with Dhc concentrated near the poles. (i) Telophase I figure with Dhc excluded from the reforming nuclei but present at the centrosomes associated with the daughter nuclei. Bar, 10 μm.

Figure 2

Figure 2

Dhc binds to acentric minichromosomes. Two views of the same field are presented to show the relationship between the distribution of DNA (a, c, e, g, and i) and Dhc (b, d, f, h, and j) in primary spermatocytes containing minichromosomes or their deleted derivatives. In all panels, arrows point to the position of the minichromosome(s). (a and b) Dp1187. Other full-length derivatives of Dp1187 (Dp8-23 and γ238) also bind Dhc (data not shown). (c and d) 31E. This deleted minichromosome contains all centromeric heterochromatin, but none of the subtelomeric DNA in Dp1187. (e and f) J21A. This deleted minichromosome contains all the subtelomeric DNA, but only approximately half the centromeric heterochromatin of Dp1187. As the stocks containing the various minichromosomes have not been continuously selected for maintenance of minichromosome number, and because transmission of smaller minichromosomes such as J21A through the male and female is imperfect, some spermatocytes in some individuals contain more than one copy of the minichromosome. (g and h) 19C. This deleted minichromosome is similar to J21A, but has lost ∼150 kb more centromeric heterochromatin. (i and j) 26C. This deleted minichromosome is only 285 kb in length and contains only the subtelomeric DNA, and thus none of the centromeric heterochromatin, of Dp1187. Details on the physical maps of all these minichromosomes can be found in Murphy and Karpen (1995) and in Williams et al. (1998). Bar, 10 μm.

Figure 3

Figure 3

Dhc localization is tension dependent. Two views of the same field are presented to show the relationship between the distribution of DNA (a, c, and e) and Dhc (b, d, and f) in primary spermatocytes containing two univalents: X^Y and C(4)RM (refer to text). Arrows, positions of these univalents; the univalent with less DNA is C(4)RM, the univalent with more DNA in the same panel is X^Y. The remaining DNA staining is due to the large autosomal bivalents that are present in the same spermatocytes. (a and b) Prometaphase I. All kinetochores, including those on the bivalents and those on the univalents, stain with equal intensity. Note that there is only a single spot of Dhc localization associated with the univalents but two spots associated with each bivalent, consistent with the number of kinetochores they contain. (c–f) Metaphase I. Univalents have a much brighter intensity of Dhc staining. Arrowheads, bivalent chromosomes situated on the metaphase plate. Bar, 10 μm.

Figure 4

Figure 4

Dhc fails to localize to kinetochores in zw10 or rod mutant spermatocytes. Two views of the same field are presented to show the relationship between the distribution of DNA (a, c, e, g, i, and k) and Dhc (b, d, f, h, j, and l) in zw10S1/Y mutant testes (a–h) or in rod X−5 mutant testes (i–l). (a–d; i and j) Prometaphase I; (e and f) Metaphase I; (k and l) Anaphase I; (g and h) Metaphase II. Arrows, residual Dhc staining near the poles. Kinetochore localization of Dhc is absent at all stages. Bar, 10 μm.

Figure 5

Figure 5

Dhc fails to localize to the kinetochore in zw10 or rod mutant larval brain neuroblasts. Two views of the same field are presented to show the relationship between the distribution of DNA (a, c, e, g, i, and k) and Dhc (b, d, f, h, j, and l) in neuroblasts from brain preparations treated with colchicine and swelled in hypotonic solution as described in Materials and Methods. (a–f) Wild-type (Oregon-R). (g–j) zw10S1/Y. (k and l) rod X −5. Arrows, clear Dhc staining at the kinetochore regions in wild-type neuroblasts. Dhc in zw10 and rod mutant brains is absent at the kinetochore. Bar, 10 μm.

Figure 7

Figure 7

Colocalization of dynamitin and HZW10. (a–d) A chromosome spread from a HeLa cell arrested in mitosis by nocodazole. (e–h) A HeLa cell in prometaphase. (i–l) A HeLa cell in metaphase. The panels in the first column of each row show staining with anti-dynamitin (a, e, and i); in the second column, staining with anti HZW10 (b, f, and j); and in the third column staining with Hoechst 33258 to visualize DNA (c, g, and k). In the column at the right (d, h, and l), dynamitin staining is shown in red, and HZW10 staining in green; the overlap between these signals is yellow. Bars, 5 μm.

Figure 8

Figure 8

Model for the ZW10/ROD-dependent targeting of dynein to the kinetochore. In this model, a complex containing ZW10 and ROD proteins, as well as potential unknown additional components (?), is associated with the fibrous corona of the prometaphase kinetochore. Direct interactions between ZW10 and the p50 subunit of the dynactin complex then bring dynactin to the kinetochore. Dynactin in turn recruits cytoplasmic dynein to the kinetochore, providing one possible contact between the kinetochore and microtubules. Our results suggest that this contact is sensitive to bipolar tension exerted across the chromosome. As described in the Introduction, additional kinetochore/microtubule interactions are undoubtedly mediated by other microtubule motors such as CENP-E (data not shown). This figure is modified from Vallee et al. (1995).

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