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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 PtK
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 1500 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 PtK 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 Ptk 2 Materials and methods 2.1 Cell culture Rat kangaroo cells (PtK 2.2 Reagents Cytochalasin J (Sigma) was dissolved as a stock solution of 10 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 24 2.4 Immunocytochemistry Cells were cultured onto glass coverslips as previously described. Cultures were treated with 10 Coverslips were incubated for 45 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 PtK 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 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 PtK 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 PtK Fig. 2 Morphology of the MT cytoskeleton and distribution of Arp1 in PtK1 cells treated with 10 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 PtK 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 PtK 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 PtK 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 PtK 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 PtK 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 PtK 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 Ptk 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. 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Correlation between the effects of 24 different cytochalasins on cellular structures and cellular events and those on actin in vitro. J Cell Biol 1982:92:69-78 Received 13 April 2006; accepted 18 April 2006 doi:10.1016/j.cellbi.2006.04.001 |
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355 Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB) |
Abstract
