Previously, we demonstrated that functional abrogation of pericentrin disrupts centrosome and spindle organization in several systems (Doxsey et al. 1994
). Based on these observations, we reasoned that an artificial elevation of pericentrin levels would provide additional information on protein function and interaction. To this end, we constructed and expressed an HA-Pc in COS-7 cells, and examined centrosome and spindle composition and function.
As expected, HA-Pc had an electrophoretic mobility of ~220 kD and was found in both Triton X-100 soluble and insoluble fractions ( A). Immunofluorescence analysis demonstrated that the more abundant detergent soluble fraction was distributed throughout the cytoplasm ( D, inset), whereas the detergent insoluble fraction colocalized with γ tubulin at centrosomes ( and ). Centrosome localization of HA-Pc was unaltered when microtubules were depolymerized, suggesting that the protein was an integral component of centrosomes and not simply bound there by microtubules (data not shown).
Figure 1 HA-Pc overexpression has no detectable effect on microtubule nucleation or organization. A, Triton X-100 soluble (lane 1) and insoluble fractions (lane 2) of HA-Pc–expressing COS-7 cells immunoblotted with anti-HA antibodies. Detergent extracted (more ...)
Mitotic Spindles Are Structurally and Functionally Disrupted in Pericentrin-overexpressing Cells
The organization of microtubules in interphase HA-Pc expressing cells was indistinguishable from control cells ( D). Moreover, there was no detectable difference in microtubule nucleation from centrosomes (, E–H). The most dramatic consequence of HA-Pc expression was disruption of mitotic spindle organization ( and ). A significant fraction of mitotic COS-7 cells at all expression levels exhibited spindle defects (75.7 ± 6.1%, n = 423), compared with nontransfected cells (2.5 ± 1.5%, n = 598) and vector DNA transfected cells (3.0 ± 1.0, n = 201). Three categories of spindle defects were observed. Spindles with structural defects were detected in 36.2% of transfected cells and included multipolar, monopolar, and distorted spindles (, D–I, also see , , , and ). Mispositioned spindles were observed in 22.0% of the cells, and were often positioned far from the cell center (, J–L). Spindles with misaligned, missegregated, and mono-oriented chromosomes were commonly observed (42.5%; , also see , , , and ). Spindle defects occurred alone or in combination.
Figure 2 Mitotic spindle organization and positioning is impaired in HA-expressing cells. Immunofluorescence staining of microtubules (or γ tubulin; K) in nontransfected (A–C) and HA-Pc–overexpressing COS-7 cells (D–L). (more ...)
Figure 3 Chromosomes in pericentrin-overexpressing cells are misaligned and missegregated, creating aneuploidy. A pericentrin-overexpressing COS-7 cell (A) with a chromosome that is not aligned on the metaphase plate (B, arrow). Note metaphase DNA overexposed (more ...)
Figure 5 Localization of several centrosome and kinetochore proteins are unaltered in pericentrin-expressing cells. HA-expressing COS-7 cells were immunolabeled for dynactin subunits (dynamitin, A–D; p150glued, E) or proteins involved in spindle (more ...)
Despite the presence of improperly attached chromosomes, HA-Pc cells progressed through mitosis and were frequently observed in later stages of mitosis with missegregated chromosomes ( and ). The percentage of mitotic figures in the population of HA-Pc–expressing cells (3.1 ± 0.9%, n = 3,490) was not significantly different from control cells transfected with other constructs or mock transfected cells (2.9 ± 1.0 to 4.4 ± 2.1%, n = 5,002), and the cell viability and growth rate appeared unchanged. Nuclei exhibited a remarkably wide variation in DNA content. Values ranged from zero to five times those of controls ( and ), demonstrating that the cells were becoming aneuploid. From this analysis, we conclude that pericentrin overexpression causes multiple mitotic spindle defects leading to chromosome missegregation and aneuploidy.
Cytoplasmic Dynein Is Dissociated from Multiple Cellular Sites in HA-Pc–expressing Cells
To test the possibility that cytoplasmic dynein or dynactin contributed to the pericentrin overexpression phenotype, we examined the distribution of these protein complexes in pericentrin-overexpressing cells. The level of cytoplasmic dynein immunoreactivity in mitotic cells was dramatically reduced at spindle poles (8–12-fold; , G–J). The motor appeared to be specifically displaced from spindle poles and not simply masked from antibody access for several reasons. First, diminished dynein staining was detected with two independent antibody preparations raised against the DIC (L5, polyclonal and 74.1, monoclonal). Second, control cells expressing β-galactosidase (, A–C) or untransfected cells (, D–F) had normal levels of dynein at their poles ( J). Third, there was no detectable change in the distribution and abundance of several other centrosome and spindle pole components. The centrosome localization and levels of the dynactin subunits, dynamitin (, A–D) and p150glued, did not appear to be altered, although there was some variability in p150glued levels in prometaphase ( E). There was no apparent change in the level of γ tubulin at individual spindle poles, even in cells with multiple poles (, J–N). This suggests that multipolar spindles have normal centrosomes at their poles, each with the appropriate amount of γ tubulin (see Discussion). The spindle pole protein, NuMA, also appeared to be localized normally to poles of mitotic spindles (, F–I).
Dynein immunofluorescence is reduced at spindle poles in mitotic HA-Pc–expressing cells. HA-Pc–expressing metaphase cells stained with antidynein antibody (74.1) show significantly reduced levels of dynein immunofluorescence at spindle poles (G–I), compared with β-galactosidase–expressing control cells (A–C) or nontransfected control cells (D–F). Quantification of dynein immunofluorescence at spindle poles (J). Open bars, mock (vector) transfected cells; filled bars, pericentrin-transfected cells. Each bar represents an average value obtained from at least 65 cells. The dynein level on kinetochores is reduced in a prometaphase HA-Pc–expressing cell (N–P), compared with a nontransfected control cell (K–M). Horizontal series are of the same cell. Bars, 10 μm (bar in C for A–C, bar in P for D–P). Images in all panels were exposed and processed similarly.
Cytoplasmic dynein was also dramatically reduced at kinetochores (, K–P). In contrast, kinetochore localization and levels of dynactin (, A–D, arrowheads; and data not shown) and the kinesin-related protein, CENP-E (, O–R; Yen et al. 1992
; Lombillo et al. 1995
), both appeared unchanged.
Consistent with defects in the Golgi complex induced by overexpression of the dynamitin subunit of dynactin (Burkhardt et al. 1997
), HA-Pc overexpression caused dispersal of Golgi elements. This was observed by immunostaining with antibodies to the Golgi protein, p58 (Bloom and Brashear 1989
; ; 77 ± 3.3%, n
= 251). In adjacent nontransfected control cells, Golgi complexes had the characteristic tightly focused appearance and were found in the perinuclear region of the cells ( B, arrowheads; 95.6%, n
= 497). Disruption of the Golgi complex was also observed using a green fluorescent protein (GFP)-tagged N-acetylglucosamine transferase in cotransfection experiments with pericentrin (data not shown). Golgi complex dispersal did not appear to result from impaired microtubule integrity, as no detectable changes in the microtubule network were observed (see D).
Figure 6 Golgi complexes are disrupted in pericentrin-overexpressing cells. An HA-Pc COS-7 cell (A) showing dispersal of the Golgi complex as revealed by staining with anti-p58 antibodies (B, center). In adjacent nontransfected cells, the Golgi complexes are well (more ...)
Pericentrin Interacts Directly with Cytoplasmic Dynein through the Light Intermediate Chain
The loss of cytoplasmic dynein from spindle poles and kinetochores, and the abrogation of cellular functions mediated by dynein (spindle positioning, Golgi complex organization) suggested that overexpressed pericentrin sequestered the motor in the cytoplasm. This was tested directly by coimmunoprecipitation assays. Antibodies to both DIC and DHC precipitated HA-Pc ( A, lanes 5 and 6), whereas a control IgG preparation did not ( A, lane 7). Conversely, antibodies to HA, but not to control IgGs, precipitated DIC ( A, lanes 1–3). Under the same conditions, antibodies to dynactin components (dynamitin and p150glued
) did not precipitate detectable amounts of HA-Pc ( A, lanes 8 and 9), although they immunoprecipitated other proteins of the dynactin complex ( A, lanes 11, 12). In cells metabolically labeled with [35
S]methionine, HA-Pc was specifically immunoprecipitated with antibodies to DHC, but not to preimmune sera ( B). Moreover, despite very low levels of endogenous pericentrin in nontransfected control cells (Doxsey et al. 1994
), we were able to specifically detect DHC after immunoprecipitation of pericentrin from lysates prepared from large numbers of cells ( C). These results suggest that overexpressed pericentrin binds to and sequesters dynein in the cytoplasm, and prevents it from associating with its cellular targets.
To determine whether the dynein–pericentrin interaction was direct or indirect, we cotransfected cells with HA-Pc and individual dynein and dynactin subunits, and performed a series of immunoprecipitation and immunoblot analyses. Immunoprecipitation of HA-pericentrin failed to pull down the DHCs and DICs or the dynactin subunits p150Glued and dynamitin ( A). However, a myc-tagged rat cytoplasmic dynein light intermediate chain (Hughes, S., A. Purohit, S. Doxsey, and R. Vallee, manuscript in preparation) and its COOH-terminal fragment N174 clearly coimmunoprecipitated with HA-pericentrin ( A). When cells cotransfected with the LIC N174 fragment and HA-Pc were labeled with [35S]methionine, the only bands specifically immunoprecipitated with anti-HA antibodies were HA-Pc and N174 ( B). Dynactin did not appear to be required for the pericentrin–LIC interaction since overexpression of dynamitin had no effect on the ability of the proteins to coimmunoprecipitate (data not shown). These results provide strong evidence for a direct interaction between HA-Pc and the light intermediate chain of cytoplasmic dynein.