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• W2000267454 abstract "Multifunctional structural proteins belonging to the 4.1 family are components of nuclei, spindles, and centrosomes in vertebrate cells. Here we report that 4.1 is critical for spindle assembly and the formation of centrosome-nucleated and motor-dependent self-organized microtubule asters in metaphase-arrested Xenopus egg extracts. Immunodepletion of 4.1 disrupted microtubule arrays and mislocalized the spindle pole protein NuMA. Remarkably, assembly was completely rescued by supplementation with a recombinant 4.1R isoform. We identified two 4.1 domains critical for its function in microtubule polymerization and organization utilizing dominant negative peptides. The 4.1 spectrin-actin binding domain or NuMA binding C-terminal domain peptides caused morphologically disorganized structures. Control peptides with low homology or variant spectrin-actin binding domain peptides that were incapable of binding actin had no deleterious effects. Unexpectedly, the addition of C-terminal domain peptides with reduced NuMA binding caused severe microtubule destabilization in extracts, dramatically inhibiting aster and spindle assembly and also depolymerizing preformed structures. However, the mutant C-terminal peptides did not directly inhibit or destabilize microtubule polymerization from pure tubulin in a microtubule pelleting assay. Our data showing that 4.1 is a crucial factor for assembly and maintenance of mitotic spindles and self-organized and centrosome-nucleated microtubule asters indicates that 4.1 is involved in regulating both microtubule dynamics and organization. These investigations underscore an important functional context for protein 4.1 in microtubule morphogenesis and highlight a previously unappreciated role for 4.1 in cell division. Multifunctional structural proteins belonging to the 4.1 family are components of nuclei, spindles, and centrosomes in vertebrate cells. Here we report that 4.1 is critical for spindle assembly and the formation of centrosome-nucleated and motor-dependent self-organized microtubule asters in metaphase-arrested Xenopus egg extracts. Immunodepletion of 4.1 disrupted microtubule arrays and mislocalized the spindle pole protein NuMA. Remarkably, assembly was completely rescued by supplementation with a recombinant 4.1R isoform. We identified two 4.1 domains critical for its function in microtubule polymerization and organization utilizing dominant negative peptides. The 4.1 spectrin-actin binding domain or NuMA binding C-terminal domain peptides caused morphologically disorganized structures. Control peptides with low homology or variant spectrin-actin binding domain peptides that were incapable of binding actin had no deleterious effects. Unexpectedly, the addition of C-terminal domain peptides with reduced NuMA binding caused severe microtubule destabilization in extracts, dramatically inhibiting aster and spindle assembly and also depolymerizing preformed structures. However, the mutant C-terminal peptides did not directly inhibit or destabilize microtubule polymerization from pure tubulin in a microtubule pelleting assay. Our data showing that 4.1 is a crucial factor for assembly and maintenance of mitotic spindles and self-organized and centrosome-nucleated microtubule asters indicates that 4.1 is involved in regulating both microtubule dynamics and organization. These investigations underscore an important functional context for protein 4.1 in microtubule morphogenesis and highlight a previously unappreciated role for 4.1 in cell division. Protein 4.1, formerly characterized solely as a crucial membrane skeletal protein in mature red cells, is now also recognized to be an important multifunctional structural protein family in nucleated cells. Although protein 4.1 can be plasma membrane-associated in nucleated cells, it also is detected at diverse and interesting subcellular locations during the cell cycle. Protein 4.1 isoforms localize within the nucleus and at centrosomes during interphase, at spindle poles during mitosis, in perichromatin at anaphase, and in the midbody at telophase (1Krauss S.W. Chasis J.A. Rogers C. Mohandas N. Krockmalnic G. Penman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7297-7302Crossref PubMed Scopus (52) Google Scholar, 2Krauss S.W. Larabell C.A. Lockett S. Gascard P. Mohandas N. Chasis J.A. J. Cell Biol. 1997; 137: 275-289Crossref PubMed Scopus (96) Google Scholar, 3De Carcer G. Lallena M.J. Correas I. Biochem. J. 1995; 312: 871-877Crossref PubMed Scopus (69) Google Scholar). Whereas mature red cells express only 80-kDa 4.1, the complex subcellular localization patterns of 4.1 in mammalian cells likely results from expression of several 4.1 isoforms, post-translational modifications and expression of multiple 4.1-related genes (4.1R, G, B, and N) (4Conboy J.G. Chan J. Mohandas N. Kan Y.W. Proc. Natl. Acad. Sci., U. S. A. 1988; 85: 9062-9065Crossref PubMed Scopus (96) Google Scholar, 5Chasis J.A. Coulombel L. Conboy J. McGee S. Andrews K. Kan Y. Mohandas N. J. Clin. Investig. 1993; 91: 329-338Crossref PubMed Scopus (75) Google Scholar, 6Horne W.C. Prinz W.C. Tang E.K. Biochim. Biophys. Acta. 1990; 1055: 87-92Crossref PubMed Scopus (22) Google Scholar, 7Huang J.P. Tang C.J. Kou G.H. Marchesi V.T. Benz Jr., E.J. Tang T.K. J. Biol. Chem. 1993; 268: 3758-3766Abstract Full Text PDF PubMed Google Scholar, 8Subrahmanyam G. Bertics P.J. Anderson R.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5222-5226Crossref PubMed Scopus (55) Google Scholar, 9Walensky L.D. Gascard P. Fields M.E. Blackshaw S. Conboy J.G. Mohandas N. Snyder S.H. J. Cell Biol. 1998; 141: 143-153Crossref PubMed Scopus (107) Google Scholar, 10Walensky L.D. Blackshaw S. Liao D. Watkins C.C. Weier H.U. Parra M. Huganir R.L. Conboy J.G. Mohandas N. Snyder S.H. J. Neurosci. 1999; 19: 6457-6467Crossref PubMed Google Scholar, 11Parra M. Walensky L. Chan N. Snyder S. Mohandas N. Conboy J. Cell. 1998; 9: 265aGoogle Scholar, 12Parra M. Gascard P. Walensky L.D. Snyder S.H. Mohandas N. Conboy J.G. Genomics. 1998; 49: 298-306Crossref PubMed Scopus (97) Google Scholar, 13Parra M. Gascard P. Walensky L.D. Gimm J.A. Blackshaw S. Chan N. Takakuwa Y. Berger T. Lee G. Chasis J.A. Snyder S.H. Mohandas N. Conboy J.G. J. Biol. Chem. 2000; 275: 3247-3255Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 14Gascard P. Lee G. Coulombel L. Auffray I. Lum M. Parra M. Conboy J.G. Mohandas N. Chasis J.A. Blood. 1998; 92: 4404-4414Crossref PubMed Google Scholar). Beyond the characterization of its localization, the current challenge is to decipher functions of 4.1 in various subcellular structures. Although the list remains incomplete, a number of protein 4.1 binding partners have been identified to interact with a specific 4.1 domain in red cells and/or nucleated cells, providing some clues as to potential 4.1 functions (see Fig. 1). Prototypical protein 4.1 (R, red cell) contains several functional domains. An N-terminal extension present only in some isoforms in nucleated cells has been found to interact with the centrosomal protein CPAP (centrosome protein-4.1 associated protein) (15Hung L.Y. Tang C.J. Tang T.K. Mol. Cell. Biol. 2000; 20: 7813-7825Crossref PubMed Scopus (140) Google Scholar). The FERM (4.1/erzin, radixin/moesin) domain interacts with plasma membrane-binding proteins and was recently discovered to also contain a microtubule binding site (16Perez-Ferreiro C.M. Luque C.M. Correas I. J. Biol. Chem. 2001; 276: 44785-44791Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The spectrin-actin binding domain (SABD 1The abbreviations used are: SABD, spectrin-actin binding domain; NuMA, nuclear mitotic apparatus protein; PIPES, 1,4-piperazinediethanesulfonic acid; R, red cell.1The abbreviations used are: SABD, spectrin-actin binding domain; NuMA, nuclear mitotic apparatus protein; PIPES, 1,4-piperazinediethanesulfonic acid; R, red cell.) is capable of forming ternary complexes with spectrin and actin. The C-terminal domain is of particular interest, because it has been found to interact with NuMA (nuclear mitotic apparatus protein) (17Mattagajasingh S.N. Huang S.C. Hartenstein J.S. Snyder M. Marchesi V.T. Benz Jr., E.J. J. Cell Biol. 1999; 145: 29-43Crossref PubMed Scopus (112) Google Scholar). These observations suggest that 4.1 plays diverse roles within the cytoskeleton. Recently we showed (18Krauss S.W. Chen C. Penman S. Heald R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10752-10757Crossref PubMed Scopus (90) Google Scholar, 19Krauss S.W. Heald R. Lee G. Nunomura W. Gimm J.A. Mohandas N. Chasis J.A. J. Biol. Chem. 2002; 277: 44339-44346Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) that 4.1 is essential for the assembly of functional nuclei in interphasic Xenopus egg extracts and requires its capacity to bind actin, which is found closely associated with 4.1 on nuclear filaments in mammalian cells. This latter observation was recently confirmed and extended in a study (20Kiseleva E. Drummond S.P. Goldberg M.W. Rutherford S.A. Allen T.D. Wilson K.L. J. Cell Sci. 2004; (in press)PubMed Google Scholar) that characterized an extensive system of nuclear pore-linked filaments in Xenopus oocytes that contain actin and 4.1 epitopes. Here we provide evidence that 4.1 is also essential during mitosis in Xenopus egg extracts for proper polymerization and organization of the microtubule cytoskeleton. During interphase, centrosomes nucleate and organize a radial array of microtubules. We characterized protein 4.1 previously as an integral centrosome component, resisting vigorous salt/detergent extraction and present at centrosomes independent of microtubules (1Krauss S.W. Chasis J.A. Rogers C. Mohandas N. Krockmalnic G. Penman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7297-7302Crossref PubMed Scopus (52) Google Scholar). By immunofluorescence and cell whole mount electron microscopy 4.1 epitopes localized on centrioles, in the pericentriolar matrix, and on the fibers connecting the centriolar pair (1Krauss S.W. Chasis J.A. Rogers C. Mohandas N. Krockmalnic G. Penman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7297-7302Crossref PubMed Scopus (52) Google Scholar). During mitosis duplicated centrosomes become the poles of the mitotic spindle apparatus, contributing to the organization of microtubules into a bipolar array with their minus ends focused at the poles and their plus ends interacting with chromosomes and overlapping within the center of the spindle. Ultimately responsible for accurate chromosome segregation, spindle function is also dependent on a variety of microtubule-based motor proteins including dynein and kinesin-related proteins that cross-link and sort microtubules according to their structural polarity, and mediate chromosome interactions within the spindle. Proper spindle pole organization is known to depend on the function of NuMA, which interacts with dynein and contributes to microtubule minus-end cross-linking to maintain spindle pole structure (as reviewed in Ref. 21Merdes A. Cleveland D.W. J. Cell Biol. 1997; 138: 953-956Crossref PubMed Scopus (146) Google Scholar) (22Compton D.A. Szilak I. Cleveland D.W. J. Cell Biol. 1992; 116: 1395-1408Crossref PubMed Scopus (184) Google Scholar, 23Yang C.H. Lambie E.J. Snyder M. J. Cell Biol. 1992; 116: 1303-1317Crossref PubMed Scopus (190) Google Scholar, 24Gaglio T. Saredi A. Compton D.A. J. Cell Biol. 1995; 131: 693-708Crossref PubMed Scopus (217) Google Scholar, 25Merdes A. Heald R. Samejima K. Earnshaw W.C. Cleveland D.W. J. Cell Biol. 2000; 149: 851-862Crossref PubMed Scopus (258) Google Scholar, 26Merdes A. Ramyar K. Vechio J.D. Cleveland D.W. Cell. 1996; 87: 447-458Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). Taken together, the observations that protein 4.1 binds to NuMA and localizes to centrosomes and mitotic spindle poles raise the intriguing hypothesis that protein 4.1 is involved in cell division. In this report we used Xenopus egg extracts, a powerful system for in vitro dissection of mitotic microtubule assembly, organization, and function. Xenopus eggs are laid arrested in the metaphase of meiosis II by cytostatic factor, until fertilization triggers a calcium wave promoting entry into the first mitotic cell cycle. Extracts prepared from unfertilized eggs maintain the cytostatic factor metaphase arrest. Mitotic microtubule asters analogous to spindle poles can be assembled in cytostatic factor extracts through the addition of purified centrosomes that directly nucleate microtubules, or by the addition of microtubule stabilizing agents such as Me2SO or taxol that induce microtubule polymerization. In the absence of centrosomes, asters are progressively organized into a focused array of minus ends by motor proteins including dynein and are similarly dependent on the activity of NuMA (24Gaglio T. Saredi A. Compton D.A. J. Cell Biol. 1995; 131: 693-708Crossref PubMed Scopus (217) Google Scholar, 27Gaglio T. Saredi A. Bingham J.B. Hasbani M.J. Gill S.R. Schroer T.A. Compton D.A. J. Cell Biol. 1996; 135: 399-414Crossref PubMed Scopus (257) Google Scholar). Complete mitotic spindle assembly can be induced by adding demembranated Xenopus sperm nuclei, as the centriole-containing basal body of the flagellum remains tightly attached to the sperm and becomes competent to nucleate microtubules in the extract, defining the spindle poles. Thus, the Xenopus system can be used to probe both centrosome-nucleated and motor-dependent organization of microtubules into asters, as well as the more complex events of mitotic spindle assembly. Taking advantage of the open nature of this cell-free system, we present data establishing that protein 4.1 is essential for assembly of these microtubule-based structures by demonstrating that 4.1 depletion of extracts produces aberrant structures. Proper assembly can be restored by the addition of recombinant 4.1. Furthermore we have identified two 4.1 domains critical for spindle, centrosome, and microtubule aster assembly in assays utilizing dominant negative peptides corresponding to 4.1 domains. Our results indicate that 4.1 is involved in regulating both microtubule dynamics and organization and underscore an important functional context for protein 4.1 in cell division. Materials—Expression vectors for His6 fusion peptides were either pMW172 (the gift of Dr. M. Way, European Molecular Biology Laboratory, Heidelberg, Germany) or pET 28 (Novagen). The antibody against NuMA was a very generous gift of Dr. A. Merdes (U. Edinburgh, Scotland). IgGs against 4.1R SABD and 4.1R C-terminal domain were described (2Krauss S.W. Larabell C.A. Lockett S. Gascard P. Mohandas N. Chasis J.A. J. Cell Biol. 1997; 137: 275-289Crossref PubMed Scopus (96) Google Scholar). Fluorescent secondary antibodies were from Molecular Probes. Bovine brain tubulin was prepared according to Ashford et al. (28Ashford A. Andersen S. Hyman A. Celis J. Cell Biology: A Laboratory Handbook. 2. Academic Press, San Diego1998: 206-212Google Scholar). Xenopus Extracts and Assembly Reactions—10,000 × g cytoplasmic Xenopus egg extracts and demembranated sperm nuclei were prepared as described (29Murray A. Methods Cell Biol. 1991; 36: 581-605Crossref PubMed Scopus (802) Google Scholar). For spindle assembly, demembranated Xenopus sperm were added to 20 μl of egg extract on ice with 0.2 mg/ml Texas Red-labeled tubulin, reactions incubated at 20 °C for 30-45 min., diluted with BRB80 (80 mm PIPES, 2 mm MgCl2, 1 mm EGTA, pH 6.8) containing 30% glycerol and 1% Triton X-100 and spun through BRB80 cushions with 40% glycerol onto coverslips (30Sawin K.E. Mitchison T.J. J. Cell Biol. 1991; 112: 941-954Crossref PubMed Scopus (147) Google Scholar). Self-organized microtubule asters were assembled by addition of Me2SO (final concentration 5%) to the egg extract and incubation for 15 min at 20 °C (31Sawin K.E. Mitchison T.J. Mol. Biol. Cell. 1994; 5: 217-226Crossref PubMed Scopus (56) Google Scholar). Centrosome asters were assembled for 15 min at 20 °C after the addition of 1 μl of KE37 centrosomes at 2 × 108/ml, prepared according to Moudjou and Bornens (32Moudjou M. Bornens M. Celis J. Cell Biology: A Laboratory Handbook. 2. Academic Press, London, England1998: 111-119Google Scholar), to 20 μl of extract that had been centrifuged for 30 min at 60,000 rpm in a TLA 100.3 rotor. Centrosome or self-organized aster reactions were diluted with BRB80 containing 15% glycerol and 1% Triton X-100 and spun onto coverslips through a cushion of BRB80 containing 30% glycerol. Indirect Immunofluorescence—In vitro assembled structures on coverslips were fixed in -20 °C MeOH and probed by immunofluorescence as described (33Heald R. Tournebize R. Habermann A. Karsenti E. Hyman A. J. Cell Biol. 1997; 138: 615-628Crossref PubMed Scopus (291) Google Scholar). The concentrations of primary antibodies were: SABD IgG, 5 μg/ml; C-terminal domain IgG, 10 μg/ml; anti-NuMA, 1:50 dilution. Secondary antibodies were used at a 1:100 dilution. Samples probed with equal amounts of control non-immune IgG or without primary antibody or sera showed no fluorescent patterns. Images were captured using a Nikon Eclipse 2000 microscope equipped with a CCD camera and processed using Adobe PhotoShop. Under the imaging conditions used, the limits of resolution of overlap between two fluorophores (e.g. superimposition of red and green signals to generate yellow coloration) was estimated to be ∼300 nm. Expression and Purification of His6-tagged Proteins—Protein 4.1-related peptides were expressed and purified as described (19Krauss S.W. Heald R. Lee G. Nunomura W. Gimm J.A. Mohandas N. Chasis J.A. J. Biol. Chem. 2002; 277: 44339-44346Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Assays with His6 peptides (1-8 μg) were preincubated on ice for 10 min then incubated at 20 °C for assembly of spindles, centrosome asters, or self-organized asters as indicated. Although a range of 1-8 μg was tested for each peptide, the data presented are from experiments using 8 μg of the indicated peptide. Microtubule Co-pelleting Assay—Solutions of 4.1 peptides and of 35 μm tubulin and 1 mm GTP in BRB80 were precleared by centrifugation in a TL-100 rotor for 15 min at 40,000 rpm at 4 °C. Reactions containing 1mm GTP, 35 μm tubulin, 35 μm peptide were mixed on ice, Me2SO was added (final concentration 5%), the reaction was incubated for 30 min at 37 °C, layered onto 40%/BRB80 sucrose cushions, and microtubules were pelleted at 40,000 rpm for 20 min in a TL-100 rotor. Equivalent amounts of supernatant and pellet were analyzed by Western blotting of a 15% SDS-polyacrylamide gel. By this assay, inhibitors of microtubule formation show tubulin remaining in the supernatant (34Wignall S. Gray N. Chang Y.-T. Juarez L. Jacob R. Burlingame A. Schultz P. Heald R. Chem. Biol. 2004; 11: 135-146Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Immunodepletion and Rescue—For 4.1 depletion from Xenopus extracts, protein G-coupled magnetic beads (Dynal) from 100 μl of slurry were mixed with 15 μg of 4.1R SABD, C-terminal domain IgGs or non-immune rabbit IgG for 1 h at 4 °C, the beads were washed twice with 0.1 m sodium phosphate buffer, pH 7.0 (57.7% Na2HPO4 and 42.3% NaH2PO4, v/v), and three times with XB buffer (29Murray A. Methods Cell Biol. 1991; 36: 581-605Crossref PubMed Scopus (802) Google Scholar) then divided into three aliquots. Extract (100 μl) was successively depleted three times by rotation with IgG-coupled beads at 4 °C for 1 h, beads were collected magnetically, and extract was used for the assembly of spindles, centrosome asters, or self-organized asters. Extract depletion was estimated by densitometry of Western blots using an Alpha Imager 2200 and software. In rescue experiments, 1-9 μg of purified bacterially expressed 80-kDa 4.1R was added to 20-μl reactions and incubated on ice for 10 min prior to the initiation of assembly. Reactions in three independent experiments were sampled during 15-45-min incubation periods for the assembly of spindles, centrosomes, or asters. The experiment presented was performed in parallel using the same depleted extract as described (19Krauss S.W. Heald R. Lee G. Nunomura W. Gimm J.A. Mohandas N. Chasis J.A. J. Biol. Chem. 2002; 277: 44339-44346Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). 4.1 Localizes to Mitotic Spindles, Centrosome Asters, and Self-organized Asters Reconstituted in Xenopus Egg Extracts—Previous reports (1Krauss S.W. Chasis J.A. Rogers C. Mohandas N. Krockmalnic G. Penman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7297-7302Crossref PubMed Scopus (52) Google Scholar, 2Krauss S.W. Larabell C.A. Lockett S. Gascard P. Mohandas N. Chasis J.A. J. Cell Biol. 1997; 137: 275-289Crossref PubMed Scopus (96) Google Scholar, 16Perez-Ferreiro C.M. Luque C.M. Correas I. J. Biol. Chem. 2001; 276: 44785-44791Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 17Mattagajasingh S.N. Huang S.C. Hartenstein J.S. Snyder M. Marchesi V.T. Benz Jr., E.J. J. Cell Biol. 1999; 145: 29-43Crossref PubMed Scopus (112) Google Scholar) using a variety of mammalian cells established that 4.1 is localized to centrosomes and mitotic spindle poles. As the Xenopus laevis 4.1 sequence has many highly homologous regions relative to mammalian family members, including the SABD and C-terminal domains (Fig. 1) (19Krauss S.W. Heald R. Lee G. Nunomura W. Gimm J.A. Mohandas N. Chasis J.A. J. Biol. Chem. 2002; 277: 44339-44346Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), we anticipated a similar 4.1 localization in Xenopus. Furthermore, 4.1 function was shown to be conserved between the frog and mammals in studies reconstituting properties of 4.1-deficient human erythrocyte membranes using a recombinant Xenopus 4.1 domain (35Winardi R. Discher D. Kelley C. Zon L. Mays K. Mohandas N. Conboy J.G. Blood. 1995; 86: 4315-4322Crossref PubMed Google Scholar). We first verified that 4.1 epitopes could be detected in centrosomes and mitotic spindles of cultured Xenopus fibroblasts (data not shown). Next we incubated cytostatic factor-arrested Xenopus egg extracts either with demembranated Xenopus sperm to assemble mitotic spindles or with centrosomes from KE37 cells to reconstitute centrosome-based microtubule arrays (referred to as centrosome asters). We also induced the motor-dependent assembly of stabilized microtubules into asters by adding 5% Me2SO to the egg extract (referred to as self-organized asters). This allowed us to examine both centrosome-nucleated and motor-organized structures. In each reaction, microtubules were stained by the addition of trace amounts of rhodamine-labeled tubulin. When each of these structures was probed by immunofluorescence microscopy using affinity-purified antibodies against the 4.1R SABD or C-terminal domain or against the 80-kDa 4.1R, strong 4.1 signals were concentrated at the minus ends of microtubules focused at the spindle poles and also in the center of both centrosome and self-assembled asters (Fig. 2). Immunodepletion of 4.1 Compromises Assembly of Microtubule-based Structures and Can Be Rescued by Supplementation with Recombinant 4.1R—To test whether the protein 4.1 itself is essential for the assembly of spindles, centrosome asters, and self-organized asters, we depleted 4.1 from Xenopus extracts using 4.1 domain-specific affinity-purified IgGs bound to protein G magnetic beads. Previously (19Krauss S.W. Heald R. Lee G. Nunomura W. Gimm J.A. Mohandas N. Chasis J.A. J. Biol. Chem. 2002; 277: 44339-44346Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) we showed by Western blotting that Xenopus extracts contain protein bands from ∼47 to 110 kDa detected by SABD and C-terminal domain IgGs, which were reduced by 50-100% after three rounds of antibody depletion. Using 4.1-immunodepleted extracts, we evaluated the morphology of spindles, centrosomes, and self-organized microtubule asters, and as an additional indicator of structural integrity, we localized the 4.1 binding partner/spindle pole protein NuMA by immunofluorescence. In controls, NuMA largely localized with 4.1 epitopes in a tight focus at the spindle poles, and in the centers of centrosome and self-organized microtubule asters (Figs. 2 and 3). Normal spindles, centrosome asters, and self-organized asters assembled in egg extracts mock-depleted with nonspecific IgG and protein G beads (Fig. 3A). In contrast, there was a dramatic morphological disruption of all microtubule structures assembled in extracts depleted with either 4.1 domain-specific SABD or C-terminal domain IgGs. In spindles assembled in either depleted extract, chromosomes were not aligned equidistant from the poles but were looped out of the spindle midzone (Fig. 3A). Spindles formed in SABD-depleted extracts most often were multipolar, whereas those from C-terminal domain-depleted extracts generally had large unfocused poles. Centrosome asters were disorganized microtubule arrays without an obvious focal center revealed by NuMA staining (Fig. 3B). In SABD-depleted extracts, centrosomes asters often contained multiple small NuMA foci radiating several bundles of microtubules. Similarly, self-organized asters assembled in either the C-terminal domain or SABD-depleted extracts had disorganized microtubules, and NuMA was mislocalized (Fig. 3C). Therefore, depleting SABD- and C-terminal domain-containing 4.1 proteins disrupted spindle, centrosome aster, and self-organized microtubule aster assembly. The aberrant reconstitution of microtubule structures observed in depleted extracts could result either from loss of 4.1 function itself or from loss of an essential 4.1 protein binding partner co-depleted in the reaction. To address this issue we added back-purified recombinant 80-kDa 4.1R to depleted extracts. Strikingly, spindle, centrosome, and aster reconstitution was completely restored by supplementation with recombinant 4.1R in extracts depleted with either SABD or C-terminal domain IgGs, producing structures with morphology and NuMA distribution comparable with controls (Fig. 3, A′, B′, and C′). The rescue of assembly by recombinant 4.1R shows directly that protein 4.1 is essential for the assembly of mitotic spindles, centrosome, and self-organized microtubule asters. Furthermore, this result indicates that a single isoform containing both SABD and C-terminal domains is sufficient to mediate all of the functions of 4.1 necessary for its role in organizing mitotic microtubule arrays in Xenopus egg extracts. Dominant Negative 4.1 Peptides Distort Assembly of Spindles, Centrosomes, and Microtubule Asters in Vitro—Having established that 4.1 is required for proper formation of microtubule structures, we next wanted to test the functions of specific 4.1 domains in spindle, centrosome, and microtubule aster assembly. To this end we added to in vitro assembly reactions bacterially expressed peptides with amino acid sequences corresponding to 4.1 domains. We reasoned that the peptides might act competitively to disrupt 4.1 complexes or to sequester important 4.1 binding partners during the assembly process. We analyzed peptide effects both on morphology and localization of the 4.1 binding partner NuMA as another measure of functional disruption because in controls NuMA localized in a tight focus at spindle poles, in the pericentriolar area of centrosomes and at the centers of self-organized asters (Fig. 3). Initially we analyzed the effects of peptides related to 4.1 SABD and C-terminal domains. We focused on these domains because they (a) have important defined functions, (b) are highly conserved between frog and mammals, (c) were present in recombinant 4.1R used to rescue extracts immunodepleted by SABD and C-terminal domain IgGs, and (d) were demonstrated to profoundly distort nuclear assembly in vitro in egg extracts (19Krauss S.W. Heald R. Lee G. Nunomura W. Gimm J.A. Mohandas N. Chasis J.A. J. Biol. Chem. 2002; 277: 44339-44346Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). For our experiments, we expressed His6-tagged peptides encoded by either exons 16-17 (amino acids 644-705) in the 4.1R spectrin-actin binding domain (SABD) or by exons 20-21 (amino acids 800-858) of the 4.1R C-terminal domain (Fig. 4). As controls for the SABD peptide, we used a variant 4.1N SABD peptide with low amino acid sequence homology to the 4.1R 16-17 peptide and a 4.1R 16-17ΔNF peptide with a deletion of two amino acids within its actin binding domain rendering it unable to bind actin but retaining spectrin binding. As a control for the 4.1R C-terminal domain peptide we used a C-terminal domain peptide in which three valines were changed to alanines, an alteration previously shown to reduce its binding affinity to NuMA Tail I fragment by ∼60-fold (19Krauss S.W. Heald R. Lee G. Nunomura W. Gimm J.A. Mohandas N. Chasis J.A. J. Biol. Chem. 2002; 277: 44339-44346Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Addition of the 4.1R 16-17 SABD peptide produced spindles with less focused microtubules at the poles and more dispersed NuMA in the peripolar area. Often there were multipolar structures. In these spindles, chromatin appeared less condensed at the metaphase plate (Fig. 5A, top row). Centrosome asters, while retaining a small central focus of NuMA staining, had only a few bundled microtubules radiating outward (Fig. 5A, middle row). Self-organized asters also appeared to contain bundled microtubules and had a very diffuse central area of NuMA with additional NuMA epitopes distributed along microtubule bundles (Fig. 5A, bottom row). However, an equal concentration of a deletion mutant in the 4.1R SABD peptide-(4.1R 16-17ΔNF) added to extracts did not affect the assembly of spindles, centrosomes, or asters with respect to the morphol" @default.
• W2000267454 created "2016-06-24" @default.
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• W2000267454 creator A5034746599 @default.
• W2000267454 creator A5044277046 @default.
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• W2000267454 date "2004-06-01" @default.
• W2000267454 modified "2023-10-17" @default.
• W2000267454 title "Two Protein 4.1 Domains Essential for Mitotic Spindle and Aster Microtubule Dynamics and Organization in Vitro" @default.
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