Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1999 Dec 13;147(6):1261-74.
doi: 10.1083/jcb.147.6.1261.

Dynein intermediate chain mediated dynein-dynactin interaction is required for interphase microtubule organization and centrosome replication and separation in Dictyostelium

S Ma  1 , L Triviños-Lagos, R Gräf, R L Chisholm
Affiliations

Dynein intermediate chain mediated dynein-dynactin interaction is required for interphase microtubule organization and centrosome replication and separation in Dictyostelium

S Ma et al. J Cell Biol. .

Abstract

Cytoplasmic dynein intermediate chain (IC) mediates dynein-dynactin interaction in vitro (Karki, S., and E.L. Holzbaur. 1995. J. Biol. Chem. 270:28806-28811; Vaughan, K.T., and R.B. Vallee. 1995. J. Cell Biol. 131:1507-1516). To investigate the physiological role of IC and dynein-dynactin interaction, we expressed IC truncations in wild-type Dictyostelium cells. ICDeltaC associated with dynactin but not with dynein heavy chain, whereas ICDeltaN truncations bound to dynein but bound dynactin poorly. Both mutations resulted in abnormal localization to the Golgi complex, confirming dynein function was disrupted. Striking disorganization of interphase microtubule (MT) networks was observed when mutant expression was induced. In a majority of cells, the MT networks collapsed into large bundles. We also observed cells with multiple cytoplasmic asters and MTs lacking an organizing center. These cells accumulated abnormal DNA content, suggesting a defect in mitosis. Striking defects in centrosome morphology were also observed in IC mutants, mostly larger than normal centrosomes. Ultrastructural analysis of centrosomes in IC mutants showed interphase accumulation of large centrosomes typical of prophase as well as unusually paired centrosomes, suggesting defects in centrosome replication and separation. These results suggest that dynactin-mediated cytoplasmic dynein function is required for the proper organization of interphase MT network as well as centrosome replication and separation in Dictyostelium.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Dictyostelium dynein antibodies. Dictyostelium whole cell extracts (lanes 1, 3, and 5) and purified dynein (lanes 2, 4, 6, and 7) were separated on 7.5% SDS-polyacrylamide gels. Gel strips were stained with Coomassie blue (lanes 1 and 2) and Western blots probed with anti-IC antibodies M4 (lanes 3 and 4), IC144 (lanes 5 and 6), or with anti-HC antibody NW127 (lane 7). Positions of dynein subunits are indicated on the right; molecular mass markers (in kD) are indicated on the left. The 55-kD doublet in lane 2 is contaminating tubulin.
Figure 2
Figure 2
Structure and expression of dynein IC truncation mutants. (A) Diagram of predicted IC domain structure in relation to the IC truncations used in this study. ICΔC consists of amino acids 1–278 of the IC sequence. ICΔN106 deletes the NH2-terminal 106 amino acids, whereas ICΔN47 removes the NH2-terminal 47 amino acids. (B) IC truncation mutants are expressed at high levels in wild-type Dictyostelium. Whole cell lysates (2 × 105 cells) were separated on a 7.5% SDS-PAGE gel and the blot probed with IC144 antibody. Control was wild-type cells transformed with pVEII vector alone. ICΔN47a and ICΔN47b are two independent cell lines that differ in their mutant protein expression level. The lower level of ICΔN47 expression in ICΔN74a allows visualization of both endogenous and mutant IC.
Figure 3
Figure 3
ICΔC binds dynactin but not dynein, whereas ICΔN associates with dynein but associates poorly with dynactin. Protein samples of cell lysates (WC) or immunoprecipitates (IP) from ICΔC cells, ICΔN cells, or vector controls were probed with IC144 antibody. Dynein was immunoprecipitated using the NW127 HC antibody (HC IP) and dynactin was immunoprecipitated using the capping protein β antibody R18 (CP IP). The faint double bands at the lower part of the CP IP lanes in control and ICΔC panels are Ig HCs.
Figure 4
Figure 4
ICΔN but not ICΔC cosediments with 20S dynein in sucrose gradients. Cell lysates were fractionated by centrifugation in 5–20% sucrose density gradient and equal volumes of the fractions separated by 7.5% SDS-PAGE. The positions of wild-type and mutant IC were detected with the IC144 antibody; IC mutants were also confirmed by 9E10 mAb that recognizes the myc tag (data not shown). Dynein HC, detected with NW127 antibody, sedimented in fractions 5–7. Cells analyzed are indicated on the left and the positions of relevant proteins are indicated on the right.
Figure 5
Figure 5
Cells expressing IC mutants are larger and flatter than wild-type cells. Phase contrast images of ICΔC or control cells cultured on coverslips for 3 d with or without induction are presented. Bars, 10 μm.
Figure 6
Figure 6
IC mutants cause Golgi dispersion. Control cells (a and b) or IC truncation mutant expressing cells (c–f) induced for 2 d were double-labeled with a comitin mAb to localize the Golgi complex (a, c, and e) and DAPI to visualize the nucleus (b, d, and f). All three IC truncations produced dispersion of the Golgi complex; ICΔC shown (c and e). c and d show IC mutant cells with normal size, whereas e and f show IC mutants with the larger flattened morphology. Bars, 10 μm.
Figure 7
Figure 7
IC mutants alter MT networks, nuclear morphology, and DNA content. Vector controls (a and b) or cells expressing IC truncations (c–j) were stained with antitubulin mAb (a, c, e, g, and i) and DAPI (b, d, f, h, and j). ICΔC and ICΔN showed a similar range of phenotypes; ICΔC cells shown. d, f, h and j show altered nuclear morphology resulting from IC mutant expression. Mutant morphologies include collapsed MT networks forming bundles (c), unusually large MT network with large MTOC (e), multiple cytoplasmic asters (g), and poorly organized MTs lacking a visible organizing center (i). Bars, 10 μm. k shows a typical FACS® profile of control or ICΔC cells induced for 2 d. The x-axis shows the DNA content and y-axis shows the cell count. The dashed line represents controls and the shaded area represents ICΔC cells. 50,000 cells were analyzed for each sample.
Figure 8
Figure 8
Expression of IC truncations produces centrosome abnormalities. Vector control cells (a and b) or cells expressing IC truncations (c–l) were labeled with centrosome antibodies (red), and DAPI (blue) (a, c, e, g, i, and k). l shows an overlay of centrosome (red) and tubulin (green) staining, with the overlapping area in yellow. b, d, f, h and j show phase-contrast images. Centrosome abnormalities include: large centrosome with a large nucleus (c) or with multiple nuclei (e); dumbbell-shaped centrosomes (g); and multiple centrosomes (i and k). In cells with multiple centrosomes each centrosome organizes MTs (l). Bars, 10 μm.
Figure 9
Figure 9
Time course of MT and centrosome phenotypes. Mutant cells grown under repressed conditions were induced. Samples taken daily were fixed and stained with tubulin and centrosome-specific antibodies. The percentage of ICΔC cells (A) and ICΔN47 cells (C) showing normal MT organization, MT bundling, large centrosomes, multiple MT asters, or MTs without an organizing center on each day are presented. B shows the level of ICΔC mutant protein expression determined by densitometric analysis on Western blots of cell lysates.
Figure 10
Figure 10
IC truncations produced centrosomes with increased core lengths and failure to separate. Wild-type (a) or ICΔC cells (b–h) induced for 2 d were analyzed for centrosome morphology by EM. All cells shown are in interphase. A wild-type centrosome (a) is indicated by the arrowhead. (b–d) ICΔC cells with centrosome cores longer than wild-type. (e–h) ICΔC cells showing paired centrosomes suggesting separation defects. N, nucleus. Bars, 200 nm.
Figure 11
Figure 11
Model for the role of dynein in interphase MT organization and mitotic centrosome separation. Also shown is the mechanism by which IC truncation mutants might disrupt dynein function. N, nucleus; C, cytoplasm; V, membranous vesicles. Arrows indicate the direction of force applied on MTs by dynein. +/− depicts the ends of MTs. See Discussion for details.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Barton N.R., Goldstein L.S. Going mobilemicrotubule motors and chromosome segregation. Proc. Natl. Acad. Sci. USA. 1996;93:1735–1742. - PMC - PubMed
    1. Blusch J., Morandini P., Nellen W. Transcriptional regulation by folateinducible gene expression in Dictyostelium transformants during growth and early development. Nucleic Acids Res. 1992;20:6235–6238. - PMC - PubMed
    1. Boleti H., Karsenti E., Vernos I. Xklp2, a novel Xenopus centrosomal kinesin-like protein required for centrosome separation during mitosis. Cell. 1996;84:49–59. - PubMed
    1. Bomsel M., Parton R., Kuznetsov S.A., Schroer T.A., Gruenberg J. Microtubule- and motor-dependent fusion in vitro between apical and basolateral endocytic vesicles from MDCK cells. Cell. 1990;62:719–731. - PubMed
    1. Bruno K.S., Tinsley J.H., Minke P.F., Plamann M. Genetic interactions among cytoplasmic dynein, dynactin, and nuclear distribution mutants of Neurospora crassa . Proc. Natl. Acad. Sci. USA. 1996;93:4775–4780. - PMC - PubMed

Publication types

MeSH terms