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Cell Biology International (2006) 30, 631–639 (Printed in Great Britain)
Colocalization studies of Arp1 and p150Glued to spindle microtubules during mitosis: The effect of cytochalasin on the organization of microtubules and motor proteins in PtK1 cells
Rollin W. Robinson and Judith A. Snyder*
Department of Biological Sciences, University of Denver, 2190 E. Iliff Avenue, Denver, CO 80208, USA


Abstract

Motor proteins play a fundamental role in the congression and segregation of chromosomes in mitosis as well as the formation of the mitotic spindle. In particular, the dynein/dynactin complex is involved in the maintenance of the spindle, formation of astral microtubules, chromosome motion, and chromosome segregation. Dynactin is a multisubunit, high molecular weight protein that is responsible for the attachment of cargo to dynein. There are a number of major subunits in dynactin that are presumed to be important during mitosis. Arp1 is thought to be the attachment site for cargo to the complex while p150Glued, a side arm of this complex regulates binding to MTs and the binding of dynactin to dynein. We performed colocalization studies of Arp1 and p150Glued to spindle microtubules. Both Arp1 and p150Glued colocalize with spindle MTs as well as cytoplasmic components. When treated with cytochalasin J, Arp1 concentrates at the centrosomes and is less co-localized with spindle MTs. Cytochalasin J has less of an effect on the colocalization of p150Glued with spindle MTs, suggesting that Arp1 may have a cytochalasin J sensitive site.


Keywords: Microtubules, Motor proteins, p150Glued, Arp1, Chromosomes, Mitosis.

*Corresponding author. Tel.: +11 303 871 3537; fax: +11 303 871 3471.


1 Introduction

Cytoplasmic dynein is a large motor protein with a molecular mass of approximately 1500kDa and is composed of two identical heavy chains and a variety of intermediate and light chains. Similar to kinesin, each dynein heavy chain has a large globular ATPase head, which acts as a force-generating engine (Holzbaur and Vallee, 1994; Allan, 1996; Hirokawa, 1998; reviewed in Goldstein and Yang, 2000; Schroer, 2004). Cytoplasmic dynein works with a linker protein, dynactin, in order to attach to its cargo. Dynactin attaches cargo to one end of the dynactin complex, while activating dynein on the other end of the complex, allowing dynein to process along a MT once the cargo has been attached to dynactin (Vaughan and Vallee, 1995; King et al., 2002, 2003; reviewed in Holleran et al., 1998; Schroer, 1996,2004). Furthermore, the mechanisms for dynein/dynactin binding have been assayed in vitro. This interaction leads to MT organization, transport and association of centrosomal MTs, and that dynein intermediate chains may bind to the endomembrane system (King et al., 2003). Dynactin is responsible for the anchoring of MTs at centrosomes and when dynein/dynactin function is blocked, spindles fail to form normally (Quintyne et al., 1999; King et al., 2003). Dynein is also implicated in recruiting astral MTs to the centrosome (Goshima et al., 2005). Furthermore, dynein/dynactin with the help of NuMA can regulate spindle length by maintaining MT depolymerization activities at the centrosomes (spindle poles) (Gaetz and Kapoor, 2004).

Dynactin has several subunits, Arp1, p150glued, p50, p62, p26, and at least two capping proteins. At each end of the Arp1 subunits, there are caps known as CapZ located at the barbed end (Schafer et al., 1994) and Arp11 located at the pointed end (Eckley et al., 1999; reviewed in Schroer, 2004). Immunofluorescence staining shows that the dynactin complex is located at the kinetochore during prometaphase (Echeverri et al., 1996; Howell et al., 2001; Tai et al., 2002). Antibodies to Arp1 showed staining in vertebrate cells in the centrosomal region of the spindle and along kinetochore fibers (Clark and Meyer, 1992; Holleran, 1996; Holleran et al., 1998). Arp1 is an actin-like mini-filament composed of 8–10 subunits containing at least one actin monomer (Kabsch and Holmes, 1995; Holleran, 1996, reviewed in Schroer, 2004). It is the site where particles and organelles (cargo, including chromosomes) are attached to the complex for transport (Allan, 1996; Ahmad et al., 1998; Burkhardt et al., 1997; Echeverri et al., 1996; Schroer, 1994, 1996; Holzbaur and Vallee, 1994; Asai and Wilkes, 2004; reviewed in Vallee et al., 2004; Schroer, 2004).

Arp1 has a sequence similarity to actin-related proteins highest in the nucleotide-binding cleft, or the actin-fold in conventional actin (Kabsch and Holmes, 1995). Sequences vary more in the regions that are externally exposed on the surface of the peptide. Because it has a 53% homology with actin (Holleran, 1996), it may also have the ability to transfer energy into chromosome motion. p150Glued interacts with dynein intermediate chain (DIC), p50, and Arp1 while remaining tethered to the MT network (Karki and Holzbaur, 1995; Vaughan and Vallee, 1995; Vaughan et al., 2002; reviewed in Schroer, 2004). The dynactin arm co-assembles with this flexible sidearm, p150Glued, and p135 isoforms that are instrumental in their binding to MTs. The distal end of the p150Glued sidearm also contains a pair of MT binding sites (one per subunit). The p150Glued subunit of dynactin may also mediate the binding of dynactin to cytoplasmic dynein, the MT based motor (Karki and Holzbaur, 1995). Transient binding by dynactin may stabilize MT interaction and allow the dynein motor to travel along MTs (King et al., 2003). It has been demonstrated that antibodies to p150Glued or the dynein intermediate chain can block the transport of organelles along MTs (Waterman-Storer et al., 1997).

When p150Glued binds to the intermediate chains of dynein, both are found at the plus ends of MTs, and their association is probably mediated by phosphorylation (Vaughan et al., 2002). Adding another facet, Piehl and Cassimeris (2003) found MTs growing towards the nucleus from the centrosome grew longer, but not faster. Malikov et al. (2004) suggest that cytoplasmic dynein nucleates MTs in the absence of the centrosome, but may also control their maintenance in vivo. Antibodies against EB1 or p150glued injected into sea urchin eggs shows that interactions between these two molecules could suppress anaphase astral elongation (Strickland et al., 2005).

Also of interest to this study are the effects of another dynactin subunit, p50. The p50 subunit is presumed to be located on the Arp1 complex at the base of the p150Glued sidearm. Its function, though not well understood, is thought to be the “glue” that holds the dynactin complex together. The overexpression of p50 increased the mitotic index from 2% to over 9% and mitotic cells showed a prometaphase-like configuration. The spindles were found to be asymmetric in shape and lacked astral MTs (Echeverri et al., 1996). On examination of their results, the mitotic cells looked very comparable to Cytochalasin J (CJ) treated PtK1 spindles (Snyder and Cohen, 1996; Wrench and Snyder, 1997). This may be due to the fact that dynein and dynactin are usually coupled together in the mitotic cell; immunocytochemistry localizes these molecules and their complexes to centrosomes, kMTs and kinetochores. Given this colocalization, they may perform an important role in the assembly of MTs at the centrosome, and in chromosome congression and segregation mediated at the kinetochore (King et al., 2003; Strickland et al., 2005).

The relatively high homology of Arp1 with actin, and the role of dynein/dynactin in chromosome movement, led to screening the effect of cytochalasin on mitosis. Cytochalasins are known to be a microfilament poison and possibly an Arp1 poison. Microfilaments have been colocalized with kinetochore fibers using fluorescent rhodamine phalloidin staining (Czaban and Forer, 1992; LaFountain et al., 1992; Sampson et al., 1996), and MFs have been found colocalized with myosin in the mitotic spindle (Silverman-Gavrila and Forer, 2003; Robinson and Snyder, 2005).

We presume CJ treatment has multiple targets in the mitotic spindle. Cytochalasin J could work to disrupt actin filaments thought to run parallel along the kinetochore fibers (Cooper, 1987; Sampson et al., 1996; Robinson and Snyder, 2003, 2005). Furthermore, the work of Wrench and Snyder (1997) showed that CJ affected kinetochore structure, such that the normal trilaminar structure was reduced to one or two lamina in similar treatments presented here. However, there may be another cytochalasin sensitive molecule, which is not a microfilament but instead of an actin-related protein, dynactin. When Ptk1 cells are treated with CJ, the changes in spindle function and architecture can be accounted for. To better understand the interaction between cytoplasmic dynein and dynactin, we studied the colocalization of Arp1 and p150Glued to MTs and used CJ as an assay to disrupt the two systems. Our study involved not only the colocalization of these proteins, but also focused on its effects on the mitotic process. We demonstrate that both Arp1 and p150Glued colocalize with centrosomes, spindle MTs and cytoplasmic vesicles. Both antigens show sensitivity to CJ treatment, however, the effect of CJ on Arp1 is demonstrably more significant than CJ's effect on p150Glued.

2 Materials and methods

2.1 Cell culture

Rat kangaroo cells (PtK1) were grown in monolayer cultures in Hams’ F-12 medium (Sigma) and 10% fetal bovine serum (Atlanta Biological) in a 5% CO2 incubator at 37°C. For immunofluorescence staining experiments, cells were harvested from stock cultures and subcultured onto 22mm2 glass microscope coverslips at 70–80% confluency after 48h. For experimentation, the monolayer was adhered to the coverslip with a fibrinogen-thrombin clot (Sigma) (Forer, 1982).

2.2 Reagents

Cytochalasin J (Sigma) was dissolved as a stock solution of 10mg/ml in DMSO (Sigma) and stored at −70°C. CJ was used at a final concentration of 10μg/ml by suspension directly into conditioned tissue culture medium immediately prior to use. Tissue culture dishes were treated with CJ for 10min at 37°C. The concentration of DMSO was 0.1%, a concentration demonstrated to have no effect on mitosis in PtK1 cells (Snyder and Cohen, 1996).

2.3 Microscopy

Immunofluorescence microscopy was carried out on a Zeiss Axioplan2 equipped with a 100× Plan-NEOFLUAR objective (Carl Zeiss Inc., Thornwood, NY) with a numerical aperture of 1.3. Micrographs were recorded using a Hamamatsu CCD camera from Hamamatsu Photonics (Japan) with the gain limit set at 4. All measurements were made on cells within 24h of experimentation and images were captured on cells within 3–5s following identification. Images were cropped using Adobe PhotoShop. The assembly of figures was also carried out in Adobe PhotoShop (version 7.0, Adobe Systems, Inc., Mountain View, CA).

2.4 Immunocytochemistry

Cells were cultured onto glass coverslips as previously described. Cultures were treated with 10μg/ml CJ in a 37°C incubator, supplemented with 5% CO2 for 10min, and then fixed. CytoSkelFix™ (http://www.cytoskelfix.com) was used for the preservation of MTs and motor proteins since it preserves all cytoskeletal proteins extremely well. After fixation with CytoSkelFix™ for 4min at −20°C, cells were labeled with anti-tubulin, anti-Arp1, and anti-p150Glued. DAPI (Molecular Probes) was applied to stain DNA-containing structures that helped to identify stages of mitosis. Coverslips were rehydrated for 10min in PBS followed by permeabilization for 1min in a 1% Triton X-100 (Sigma) solution in PBS and washed for 10min in PBS before the primary antibodies were applied. The primary antibodies, monoclonal anti-β-tubulin (Sigma, No. T4026), polyclonal anti-Arp1 (Sigma, No. A5601), and polyclonal anti-p150Glued (Santa Cruz Biotechnology, SC9804) were applied to coverslips containing monolayer cultures and then incubated for 45min at 37°C and rinsed 3× briefly in PBS. The secondary antibodies were Alexa Fluor 488 (GAM) and Alexa Fluor 564 (GAR) (Molecular Probes).

Coverslips were incubated for 45min in the secondary antibodies at 37°C, rinsed briefly 3× in PBS then DAPI was applied for 3min at 37°C and rinsed 3× briefly in PBS and 1× briefly in dH2O, then sealed with Gel/Mount mounting medium (Biomeda Corp.).

2.5 Non-specific binding of secondary antibodies

To rule out non-specific binding of fluorescently labeled primary antibodies primary antibodies to β-tubulin, Arp1, and p150Glued were heat-denatured and cells were stained with the same protocol as described in above. When the heat-denatured antibodies were compared to the native antibodies, the denatured antibodies showed little to no fluorescent staining of mitotic or interphase cells (data not shown). To confirm the possibility of the secondary antibodies producing a fluorescent signal, we omitted the primary antibodies and proceeded as above and obtained a faint, non-specific signal in both mitotic and interphase cells (data not shown).

3 Results

3.1 Arp1/MT cytoskeleton localization in untreated PtK1 cells

3.1.1 Metaphase

The transition from prometaphase to metaphase creates a balance of forces between the two half spindles. The sister chromatids are aligned at the metaphase plate and the spindle MTs take on a barrel, or spindle shape due to compressive forces (Fig. 1A, arrow). Anti-tubulin staining showed astral MTs were evenly dispersed and fanned out in a typical array with the most significant staining within the spindle. Fig. 1B represents the distribution of Arp1 antigen throughout the mitotic spindle and the cytoplasm. Arp1 appeared to have a relatively even distribution throughout the cytoplasm, with a greater intensity of staining within the spindle domain. Closer inspection showed that Arp1 was colocalized to kMTs and nkMTs within the spindle, not just near the kinetochore region. There was also anti-Arp1 staining surrounding the chromatin mass beyond the spindle domain (Fig. 1B, arrow). In the merged image of Fig. 1A,B, there was a higher intensity of staining near both centrosomes evidenced by the bright yellow staining (Fig. 1C, arrow).


Fig. 1

Morphology of the MT cytoskeleton and distribution of Arp1 in PtK1 cells. Green indicates MTs, red indicates Arp1 and blue indicates DAPI. (A) Metaphase. Anti-tubulin stained cell showing barrel shaped spindle, arrows. (B) Distribution of Arp1 throughout cell. Arp1 is colocalized to kMTs and NKMTs in the spindle. (C) Merge of (A) and (B) with colocalization of Arp1 and spindle MTs. There is a higher intensity of staining near both centrosomes. (D) Anaphase. There is intense tubulin staining in both the astral and interzonal region of the spindle. Astral MTs emanate from either spindle pole in fan-like pattern. (E) Arp1 is distributed throughout the cell with staining apparent with the remaining spindle MTs and the cytoplasm. Cleavage furrow is apparent, arrow. (F) Merge of (D) and (E) with less staining of Arp1 to spindle MTs located in the interzone between the two chromosome masses, arrow. (G) Telophase. Tubulin staining is apparent on both astral and interzonal MTs. Contractile ring is obvious in the interzone of cell, arrow. (H) Distribution of Arp1 is evident in both the cytoplasm, the interzone region, but not with the astral MTs. I: Merge of (G) and (H). Colocalization of Arp1 to interzone spindle MTs shows a reduced staining pattern with interzonal MTs when compared to anaphase and metaphase cells, arrows. Bar=5μm.


3.1.2 Anaphase

The separation of sister chromatids initiates anaphase; where anaphase A is chromosome-to-pole motion and anaphase B represents pole–pole separation. Fig. 1D shows a cell in late anaphase where the spindle poles have maximized their separation. The localization of Arp1 appeared to be evenly distributed throughout the cell with the cleavage furrow forming between the two chromosome masses in the midzone of the cell (Fig. 1E, arrow). In the merged image of Arp1 and MTs staining in the midzone region between the two sets of chromosomes showed the most staining (Fig. 1F). However, Arp1 did not appear to be evenly distributed along the spindle MTs as seen in metaphase. The nkMTs located in the midzone between the two centrosomes exhibited reduced staining of Arp1 along these MTs when compared to metaphase spindle MTs (Fig. 1F, arrow).

3.1.3 Telophase

At telophase the interzone MTs become more highly bundled due to the formation of the furrow. A contractile ring was evident between the two sets of chromosomes. Anti-tubulin staining demonstrated MTs between the chromosomes and the forming midbody (Fig. 1G, arrow). Arp1 was no longer evenly distributed throughout the forming daughter cells. There was a higher concentration of Arp1 surrounding one of the chromosome masses (though this could be an artifact due to optical sectioning) (Fig. 1H, arrow). Colocalization of Arp1 and MTs were present along MTs nearest the spindle poles (Fig. 1I) with a lack of Arp1 localization along much of the mid-region of the nkMTs between the two centrosomes (Fig. 1I, arrow). Astral MTs were fanned out and extended to the cortical mesh network.

3.2 Arp1/MT cytoskeleton localization in CJ treated PtK1 cells (10μg/ml)

3.2.1 Metaphase

At metaphase the anti-tubulin treated cells showed the spindle lost its barrel shape and was elongated (Fig. 2A). Many spindle MTs were bundled together, some bundles were buckled, and appeared to be unattached to kinetochores or MTs from the opposite spindle (Fig. 2A, white arrows). The astral MT array had been compromised in such a way that the astral MT pattern was barely evident when compared to untreated cells (Fig.1A). Bundling of astral MTs was apparent, particularly in the cortical region of the cell (Fig. 2A, red arrows). Arp1 antigen distributions were uneven throughout the cell with highest intensity of staining coincident with spindle MTs. One or more chromosomes were detached from the spindle or failed to congress to the metaphase plate. Other chromosomes were found at the periphery of the spindle, remaining attached or associated with MTs (Fig. 2B, arrows). In the merged image anti-tubulin and anti-Arp1 showed faint colocalization with little to no colocalization to astral MTs (when compared to untreated cells). Fig. 2C and F (white arrows) show there was little Arp1 staining in the interzonal region of the spindle, compared to the centrosomal regions. The positioning of the spindle axis was turned 45° from the normal 90° spindle axis (co-incident with the longitudinal axis of the cell in PtK1 cells) towards the cellular cortex (Fig. 2C, red arrows).


Fig. 2

Morphology of the MT cytoskeleton and distribution of Arp1 in PtK1 cells treated with 10μg/ml CJ for 10min. Green indicates MTs, red indicates Arp1 and blue indicates DAPI. (A) Metaphase. Barrel shaped spindle is elongated after CJ treatment and bundling and buckling of spindle MTs are apparent, white arrows. Astral MTs are fewer in number, shorter and bundled together, red arrows. Chromosomes reside at the periphery of the spindle. (B) Distributions of Arp1 are concentrated with spindle MTs. Two chromatids are lagging or detached from the spindle, arrows. (C) Merge of (A) and (B). Strong colocalization of Arp1 and spindle pole MTs is evident by the bright yellow color, red arrows. The white arrows are pointing to the lack of colocalization of Arp1 to spindle MTs. (D) Anaphase. Many nkMTs are fragmented and bundled, with some MTs splaying outside the spindle domain, white arrow. Astral MTs are bundled and do not splay out in a fan pattern, red arrows. (E) Distributions of Arp1 show the highest concentration of staining located near one of the spindle poles, arrow. (F) Merge of (D) and (E). Strong colocalization of Arp1 to the spindle poles and the cortical region between the poles and the plasma membrane is shown (arrow). Telophase. (G) MTs are bundled in the interzone region located between the two chromosome masses, white arrows. Astral MTs are bundled together and lack a fan pattern, red arrows. (H) Contractile ring is apparent by the constriction between the two chromosome masses, arrow. Distributions of Arp1 are concentrated with those MTs that end in the cytoplasm of the cell. (I) Merge of (G) and (H). Lack of localization of Arp1 to spindle MTs located in the forming midbody region, though some colocalization is seen with the remnant MTs proximal to the forming nuclei, arrow. Bar=5μm.


3.2.2 Anaphase

Cells in anaphase showed intense anti-tubulin staining in the interzone region. Some nkMTs failed to interact with their opposing nkMTs (Fig. 2D, white arrows). Some bundling of MTs was evident and many MTs were splayed outside the normal spindle domain. Also, a significant fraction of nkMTs were curved towards the periphery of the cell (Fig. 2D, arrows). The astral MT array no longer exhibited the normal astral array pattern. Astral MTs were fragmented or missing from either spindle pole (Fig. 2D, red arrows). The distribution of Arp1 antigens was localized to one end of the cell (Fig. 2E, white arrow) as observed during metaphase treated cells. A few chromosomes remained at the periphery of the spindle and were possibly detached from their kMTs (Fig. 2E, blue staining). The merged image showed strong colocalization of Arp1 and MTs near the centrosome that contained the highest distribution of Arp1 (Fig. 2F, arrow). There was also a strong anti-Arp1 staining in the cortical region of the cell, and not in the interzone of the spindle.

3.2.3 Telophase

In late telophase chromosomes were identified in each forming daughter cell; the nuclear envelope was partially reformed around the decondensed chromosomes while astral MTs appeared fewer in number and shorter in length (Fig. 2G, white arrows). Many MTs were bundled together in the midzone between the two sets of chromosomes and some appeared to be detached from both spindle poles. The distribution of Arp1 antigens was concentrated, particularly around the chromosome mass and the cell cortex. However, this pattern of distribution was also seen in untreated telophase cells. A contractile ring had formed between the two chromosome masses (Fig. 2H, arrow). Colocalization of Arp1 and MTs were present though reduced fluorescence intensity was observed in the midzone region containing a dense packing of MTs. The midzone MTs did not appear to show arp1 localization (Fig. 2I, white arrow).

3.3 p150Glued/MT cytoskeleton localization in untreated PtK1 cells

3.3.1 Metaphase

Anti-tubulin staining showed a typical arrangement of MTs in a spindle shape at metaphase (Fig. 3A). p150Glued staining showed antigen distribution in what appeared to be associated with membrane-containing organelles such as the ER, Golgi and vesicles. The staining was not as punctate or as global a staining as that seen with Arp1 distributions. There was a slightly higher intensity of staining within the spindle domain (Fig. 3B). The merged image showed the distributions of p150Glued colocalized to spindle MTs, although the colocalization staining intensities were much less than Arp1 stained cells. This may be related to the relative stoichiometry of Arp1 and p150Glued in the cell (Fig. 3C, compare with Fig. 1C).


Fig. 3

Morphology of the MT cytoskeleton and the distribution of p150Glued in PtK1 cells. Green indicates MTs, red indicates p150Glued and blue indicates DAPI. Metaphase. (A) Barrel shaped spindle apparent with astral MTs fanned out from spindle poles, arrows. Chromosomes aligned on the metaphase plate. (B) Distribution of p150Glued throughout the cell. p150Glued is associated with spindle MTs and vesicles in the cytoplasm. (C) Merge of (A) and (B). p150Glued shows light staining with spindle MTs. (D) Anaphase. MTs appear straight and in some cases bundled in the interzonal region. (E) Distributions of p150Glued are seen within the spindle higher intensity of staining around one spindle pole, green arrow. White arrows indicate the cleavage furrow. (F) Merge of (D) and (E). Intense colocalization near one spindle pole, arrow. Both tubulin and p150Glued are associated with spindle MTs G: Telophase. Spindle MTs in the interzone are bundled in the central region of the cell, arrows. (H) p150Glued is localized throughout the cytoplasm, with less staining on MTs forming the midbody. (I) Merge of (G) and (H). Arrow indicates lack of colocalization of p150Glued to spindle MTs in the midzone of the cell. Bar=5μm.


3.3.2 Anaphase

At late anaphase nkMTs in the interzone region of the cell appear straight and in some cases bundled, particularly in the middle of the interzone (Fig. 3D). The cleavage furrow was apparent by the hour-glass shape of the cell (Fig. 3E, white arrows) with distributions of p150Glued colocalized with spindle MTs, and also evident in the cytoplasm (Fig. 3E). p150Glued antigens were more concentrated nearest one spindle pole (Fig. 3E, green arrow). In the merged image there was strong colocalization with spindle MTs, yet the cortical region of the cell showed only p150Glued staining. The strongest colocalization appeared nearer to one spindle pole indicated by the bright yellow staining (Fig. 3F, arrow).

3.3.3 Telophase

By late telophase interzonal MTs were concentrated as cytokinesis tended to bundle MTs (Fig. 3G, arrows). p150Glued antigens were distributed throughout the cell, including the lamellopodia. Concentrations of stain indicated these antigens were localized with vesicular compartments (Fig. 3H). Colocalization of p150Glued and MTs was indicated by the yellow color (Fig. 3I). As observed in the Arp1 untreated telophase cells there was less colocalization to the midzone spindle MTs, yet the highest concentration of p150glued was between the chromosomes and the cortical region of the cell, perhaps due to exclusion of vesicles by interzonal MTs (Fig. 3I, arrow).

3.4 p150Glued /MT cytoskeleton localization in CJ treated PtK1 cells (10μg/ml)

3.4.1 Metaphase

Many of the spindle MTs were fragmented and splayed outside of the spindle domain (Fig. 4A, white arrow). The chromosomes failed to show a tight alignment at the metaphase plate, instead aligned at the periphery of the spindle. Bundling and curvature of astral MTs could be observed emanating from the spindle poles towards the periphery of the cell (Fig. 4A, red arrows). The p150Glued distributions appeared to be more concentrated within the spindle domain (Fig. 4B, green arrows) and dispersed in presumptive vesicles in the cytoplasm. One sister chromatid was located outside of the spindle domain and appeared to be detached from the spindle (Fig. 4B, white arrow). The merged image showed strong colocalization of p150Glued with spindle MTs. The chromosome that was detached from the spindle in Fig. 4B was more apparent in the merged image and showed less antigen staining than those chromosomes still in the spindle domain (Fig. 4C, green arrow). The localization of p150Glued to spindle MTs overlaid the staining of anti-tubulin, yet was particularly concentrated between the chromosomes and poles (Fig. 4C, white arrow).


Fig. 4

Morphology of the MT cytoskeleton and the distribution of p150Glued in PtK1 cells treated with 10μg/ml CJ for 10min. Green indicates MTs, red indicates p150Glued and blue indicates DAPI. (A) Metaphase. The spindle is less barrel shaped, white arrow and chromosomes are located at the periphery of the spindle. Astral MTs are bundled and lack a fan pattern, red arrows. (B) P150Glued distributions are concentrated within the spindle region, green arrows. One chromatid has become detached from the spindle, white arrow. (C) Merge of (A) and (B). Detached chromosome is more obvious with the merged image, red arrow. Lack of colocalization of p150Glued to the spindle MTs in the region near the detached chromatid, white arrow. (D) Anaphase. Spindle MTs are bundled and fragmented in the region between the two spindle poles, white arrow. Red arrows indicate astral MTs that are bundled and lack a fanning pattern. (E) p150Glued are more concentrated in the interzone region and preferentially associated with MTs, green arrow. A chromosome appears to be lagging behind or detached from the spindle, white arrow. (F) Merge of (D) and (E). Very little localization of p150Glued can be seen on astral MTs, red arrow. A lack of colocalization of p150Glued to spindle MTs located nearest to the chromosomes masses, white arrow, contrary to its colocalization observed in untreated cells. (G) Telophase. Astral MTs are highly bundled with fewer MTs emanating out from the centrosome, white arrows. Many MTs are fragmented and curved toward the periphery of the cell, red arrow. (H) Cleavage furrow and the shape of the cell has been dramatically altered. The distributions of p150Glued are colocalized with interzonal MTs and some staining is visible in the cortical region of the cell, white arrow. (I) Merge of (G) and (H). There is decreased colocalization p150Glued in the astral region and the interzonal MTs closest to the chromatin masses, white arrow. p150Glued shows faint staining with the splayed MTs near the cortical region of the cell, red arrow. Bar=5μm.


3.4.2 Anaphase

At anaphase the spindle MTs located in the midzone of the cell appeared to be fragmented with some MTs curved outward toward the periphery of the cell (Fig. 4D, white arrow). The astral MTs were bundled and fewer in number, and appeared compressed by the cell cortex (Fig. 4D, red arrows). Some chromosomes lagged behind during chromosome separation or were detached from the spindle (Fig. 4E, white arrow). The distributions of p150Glued were found throughout the cell with a higher concentration of antigens located between the two masses of chromosomes (Fig. 4E, green arrow). The merged image shows the relative colocalization of p150Glued and spindle MTs mostly in the interzone between the two sets of chromosome masses (Fig. 4F, white arrow). However, there was a lack of localization to the astral MTs and around the chromosome masses (Fig. 4F, red arrow).

3.4.3 Telophase

Astral MTs were present outside the spindle with bundling of the remaining astral MTs near the cortex (Fig. 4G, white arrows). Many of the MTs in the interzone region between the two spindle poles were highly misaligned. MTs were curved or splayed towards the periphery of the cell and were no longer attached or aligned with the opposing half spindle (Fig. 4G, red arrow). The relative distributions of p150Glued were more concentrated in regions containing MTs. Staining of the cleavage furrow was less pronounced when compared to untreated cells (Fig. 4H, arrow). Fig. 4I showed colocalization of p150Glued and spindle MTs within the interzone region between the two chromosome masses. However, in the interzone region where spindle MTs were closest to the poles there was more p150Glued antibody localized to spindle MTs (Fig. 4I, white arrow). The region where spindle MTs were unattached and curved outward toward the periphery of the cell lacked p150Glued localization to these MTs (Fig. 4I, red arrow).

4 Discussion

This study described the colocalization of Arp1 and p150Glued with spindle MTs in PtK1 cells during mitosis in both untreated and CJ treated cells. We discuss their role in mitosis because the dynein/dynactin complex has been documented to be involved in the mitotic spindle checkpoint, sister chromatid separation, spindle positioning, spindle elongation, and chromosome movement (Echeverri et al., 1996; Waterman-Storer et al., 1997; Scholey et al., 2003; King et al., 2003; Piehl and Cassimeris, 2003; Schroer, 2004; Strickland et al., 2005). Arp1 and p150Glued are documented to be the major subunits of dynactin. The largest subunit of dynactin, p150Glued, tethers to a MT while interacting with dynein intermediate chain (DIC), p50 (dynamitin), and Arp1. Because p150Glued has an association with MTs it may account for dynein's strong attachment to MTs when carrying large cargo such as chromosomes.

Both Arp1 and p150Glued colocalize to spindle MTs, with small differences in staining patterns. p150Glued shows stronger staining at the centrosomes than Arp1 and is clearly localized to cytoplasmic vesicles. This may be due to their relative stoichiometry in the dynactin complex, since both molecules are necessary for spindle function.

Cytochalasin J is not a particularly potent cytochalasin (Cooper, 1987; Yarhara et al., 1982) yet it has the most significant effect on PtK1 cells mitotic events when compared with similar concentrations of CB, CD, CE and CH (unpublished data). Cytochalasin J treatment of mitotic PtK1 cells indicates its effects not only the microfilament (MF) system, but also the dynein complex (Robinson and Snyder, 2003). Since dynactin contains Arp1 and directly attaches to p150Glued, it may also be sensitive to CJ treatment, particularly since the “actin fold” has a high homology with the ATP binding site with actin filaments (Kabsch and Holmes, 1995). Interestingly, the fact that overexpression of p50 has the same effects on spindle morphology as does CJ (Echeverri et al., 1996), it seems reasonable to assume that Arp1 may be a target for cytochalasins and that dynactin plays a crucial role in mitosis.

One of the most significant changes reported with CJ treatment and its effects on the changes in localization of the three antigens, tubulin, Arp1 and p150Glued are changes in the number and organization of astral MTs. The dynein/dynactin complex is known to be required for spindle organization, particularly organizing centrosomal MTs, (King et al., 2003) and recruiting some kinetochore proteins to the developing spindle (Howell et al., 2001). Centrosome positioning and astral organization may be controlled by Arp1 and p150Glued since CJ was so effective in reorienting the spindle. Since dynein/dynacintin are involved with linkages to the centrosome and cell cortex, as well as recruiting astral MTs to the centrosome, our work strongly suggests that CJ has an effect on both microfilaments, and more profoundly the dynactin complex. Loss of this attachment, could easily explain reorientation of the spindle with CJ treatment.

Cytochalasin J promotes either detachment or reorganization of chromosomes particularly when cells are treated before metaphase (Wrench and Snyder, 1997). It is not clear if this represents a detachment of microfilaments into the kinetochore, the effect of CJ on the dynein/dynactin complex or both.

We are aware that the actin component of ARP1 is unlikely to treadmill like microfilaments. However, it is also possible that the cytochalasin cleft is exposed and may be sensitive to CJ. Similarly, Mad2 and/or BubR1 (Johnson et al., 2004) should be further investigated, as to their localization in Ptk1, but is beyond the scope of this paper. We have recently tried staining the Ptks with CENP antigens, unfortunately, they are human specific, though our current work with HeLa cells, should give us more information about the role of CENPs in perturbed mitotic cells.

The work presented here substantiates a role of Arp1 and p150Glued of dynactin and its interaction with the kinetochore in chromosome movement. We believe that CJ affects long range transport of chromosomes through interaction with Arp1 and p150Glued, as well as a CJ sensitive actin system. Cytochalasin J disrupts the mitotic spindle from prophase through telophase transitions in such a way that chromosomes become detached from the spindle or lag behind not allowing progression through mitosis. CJ has proved to be an interesting and provocative molecule to understand the relationships between MTs and MFs in the process of mitosis.

Acknowledgements

We thank the Barton L. Weller Endowment for support of this research to JAS.

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Received 13 April 2006; accepted 18 April 2006

doi:10.1016/j.cellbi.2006.04.001


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
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