Research Article

A Mitotic Lamin B Matrix Induced by RanGTP Required for Spindle Assembly

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Science  31 Mar 2006:
Vol. 311, Issue 5769, pp. 1887-1893
DOI: 10.1126/science.1122771


Mitotic spindle morphogenesis is a series of highly coordinated movements that lead to chromosome segregation and cytokinesis. We report that the intermediate filament protein lamin B, a component of the interphase nuclear lamina, functions in spindle assembly. Lamin B assembled into a matrix-like network in mitosis through a process that depended on the presence of the guanosine triphosphate–bound form of the small guanosine triphosphatase Ran. Depletion of lamin B resulted in defects in spindle assembly. Dominant negative mutant lamin B proteins that disrupt lamin B assembly in interphase nuclei also disrupted spindle assembly in mitosis. Furthermore, lamin B was essential for the formation of the mitotic matrix that tethers a number of spindle assembly factors. We propose that lamin B is a structural component of the long-sought-after spindle matrix that promotes microtubule assembly and organization in mitosis.

Mitotic spindle assembly and chromosome segregation are dynamic processes requiring the coordinated activities of microtubules (MT), MT-based motors, MT-binding proteins, and chromosomes (1). It was proposed decades ago that a static scaffold (spindle matrix) might tether spindle assembly factors (SAF) and support the assembly and force production of spindle microtubules (2, 3). However, the molecular nature of the spindle matrix remains elusive.

Much progress in understanding spindle morphogenesis and chromosome segregation has come from genetic screens that identified mutated genes causing cell cycle arrests, mitosis-specific defects, or both, in vivo. Such genetic analysis has led to the identification and characterization of SAFs that have specific mitotic functions. However, genetic analyses could overlook mitotic regulators that also have important functions in interphase. For example, the mitotic role of the guanosine triphosphatase (GTPase) Ran, a protein with well-established function in interphase nuclear trafficking (4), was overlooked by genetic studies. Genetic studies could not distinguish between a direct role for Ran in mitosis or an indirect one due to a failure in interphase nuclear trafficking (57). It is now understood that RanGTPase uses similar principles to regulate nuclear functions in interphase and spindle assembly in mitosis (811).

Such dual functions of the Ran system in interphase and mitosis could have more profound implications. From an evolutionary perspective, RanGTPase appears to have branched off relatively early from the related GTPases (12, 13). This may have coincided with the co-evolution of the mitotic spindle apparatus and the interphase nucleus in eukaryotes (13). We reasoned that such coevolving relationships could also lead to sharing of components that regulate both the interphase nucleus and the mitotic spindle apparatus and that such multifunctional components might be missed in genetic screens.

In addition to chromatin, the interphase nucleus contains the nuclear membrane with associated nuclear pore complexes and the nuclear lamina. The major components of the nuclear lamina are the type V intermediate filament proteins called lamins. The lamins are grouped into A and B types on the basis of their biochemical properties (14). Lamin B proteins are ubiquitously expressed in metazoans and are essential for cell viability (15). The A-type lamins are not essential for cell viability, are developmentally regulated, and are expressed primarily in differentiated cells (16). Lamins are important for nuclear functions, including nuclear envelope assembly (17, 18), nuclear size and shape, DNA replication (19), and RNA polymerase II–driven gene expression (20, 21). They also provide mechanical support for the maintenance of the structural integrity of the nucleus and anchorage and organization sites for interphase chromatin (16).

Consistent with the idea that the interphase nuclear components may be used to regulate mitosis, specific nucleoporins are localized to kinetochores, where they regulate the interaction between MTs and kinetochores in mitosis (2224). Moreover, down-regulation of the single lamin gene in Caenorhabditis elegans leads to cell division defects and eventual cell death (25). Because a fraction of lamin B associates with spindles in mitosis in mammalian cells (19, 2628), we used in vivo and in vitro assays to study the mitotic function of lamin B. We show that lamin B is required for spindle assembly and propose that lamin B is a structural component of a spindle-associated matrix that tethers various spindle assembly factors.

Association of lamin B with mitotic spindles. We tested whether the major lamin B isoform in Xenopus eggs, lamin B3 (LB3) (29), was associated with mitotic spindles assembled in extracts made from the cytostatic-factor-arrested Xenopus eggs (M-phase egg extracts). Mitotic spindles were assembled in extracts to which we added Xenopus sperm chromatin, RanGTP (RanL43E and RanQ69L both have point mutations in the effector domain that mimic the GTP-bound state) (30, 31), or RanGTP plus magnetic beads coated with protein kinase Aurora A (AurA beads) (32). AurA beads function as microtubule organizing centers (MTOC) in the presence of RanGTP to stimulate assembly of both MT asters and spindles, whereas beads coated with the catalytically inactive AurA (AurA-AA) fail to do so (33). Immunofluorescence using antibodies to LB3 (34, 35) showed that LB3 was associated with spindles and the peripheral region surrounding the spindle (Fig. 1, A to C) (32). Little LB3 staining could be detected in areas devoid of MT structures. Immunofluorescence staining of HeLa cells showed that a fraction of the human lamin B, LB1 and LB2 (36), was associated with spindles in mitosis (Fig. 1D). Moreover, a fraction of an LB1–green fluorescence fusion protein expressed in HeLa cells was also associated with mitotic spindles (32) (Fig. 1D).

Fig. 1.

Association of lamin B with mitotic spindles assembled in Xenopus egg extracts and HeLa cells. (A) Xenopus lamin B detected by either monoclonal or polyclonal (full-length LB3 protein as antigen) LB3 antibody (green). The monoclonal antibody also recognizes the sperm-specific LB4. MTs were labeled with rhodamin tubulin (red). DNA was labeled with 4′,6′-diamidino-2-phenylindole (DAPI, blue). (B and C) Spindles induced by AurA beads and RanGTP (B) or RanGTP alone (C). LB3 was detected with monoclonal (B) or polyclonal antibodies [antigens are either a peptide corresponding to LB3 (B) or full-length LB3 protein (C), green]. (D) Mitotic spindles as detected by antibodies to LB1 or LB2 or green fluorescent protein (GFP)–tagged LB1 in HeLa cells. Spindle MTs were detected with tubulin antibody (red). Scales: white bars, 10 μm; magnetic beads, 2.8 μm.

Requirement of lamin B for spindle assembly. To determine whether LB has a role in spindle assembly, we reduced expression of either LB1 or LB2 in HeLa cells by small interfering RNA (siRNA) (32) (Fig. 2A). Depletion of either isoform caused an increase in spindle defects (Fig. 2, A to C). Typical mitotic defects included unfocused spindle poles (as judged by localization of γ-tubulin) or poor spindle morphology with mild to severe lack of chromosome congression (Fig. 2C). Consistent with the spindle defects, live imaging revealed that LB siRNA-treated cells spent a longer time in prometaphase and metaphase as compared with controls (Fig. 2D and fig. S1). Thus, spindle assembly and function appear to require an appropriate amount of both LB1 and LB2.

Fig. 2.

Requirement of lamin B for proper spindle assembly and function in mitosis. (A) Immunoblotting to detect LB1, LB2, or tubulin in HeLa cells treated with control or LB siRNAs. (B) Quantification of spindle defects in control or LB siRNA-treated cells. At least 100 mitotic cells were analyzed for each siRNA treatment. Shown are representative quantifications of at least six independent experiments with two different siRNA sequences. (C) Examples of normal and defective spindles (unfocused spindle poles or abnormal spindle lacking chromosome congression) in (B) stained with antibodies to γ-tubulin (green) and α-tubulin (red). Defective spindles are from HeLa cells treated with LB siRNAs. (D) Effect of depletion of LB on the timing of chromosome alignment and segregation. Control or LB siRNA-treated HeLa cells were imaged. The elapsed time from chromosome congression (the appearance of chromosomal bar) to chromosome separation (splitting of the bar into two) (see fig. S1) in 50 to 100 mitotic cells was analyzed for each siRNA treatment. (E) Immunodepletion and add-back of LB3. Rabbit polyclonal or mouse monoclonal antibody to LB3 was used for immunodepletion. 6His-LB3 was added back to the LB3-depleted egg extracts to a final concentration of 0.2 μM. Rabbit or mouse nonimmunized immunoglobulin G was used as a control. (F) Quantification of MT structures in 50 random fields. (G) Examples of different MT structures (red) immunostained with Eg5 antibodies (green). DNA was stained with DAPI (blue). The defective spindle, aster, or half-spindle shown is from LB3-depleted egg extracts. Scale bars, 10 μm.

Because LB has an important function in the interphase nucleus, the spindle defects observed above could be an indirect effect of perturbing interphase nuclear functions. To determine whether LB has a direct role in spindle assembly, we used M-phase egg extracts. Xenopus LB3 was immunodepleted from the M-phase extracts with polyclonal or monoclonal antibodies (32) (Fig. 2E). Depleting LB3 by either antibody resulted in severe disruption of spindle assembly, whereas adding back the bacterially expressed and purified 6His-LB3 partially rescued spindle assembly (Fig. 2, F and G) (32). Therefore, LB appears to have a mitosis-specific function in spindle assembly.

Assembly of lamin B–containing matrix on mitotic spindles induced by RanGTP. Purified soluble LB3 neither bound to MTs assembled from purified tubulin nor promoted MT assembly in vitro (32) (fig. S2). As an intermediate filament protein, LB might regulate SAFs as a polymer or as a soluble protein to promote spindle assembly. We therefore made spindles in egg extracts with either sperm chromatin or AurA beads plus RanGTP, or AurA beads plus 5% dimethylsulfoxide (DMSO), which stimulates assembly of MT asters but not spindles (Fig. 3, A and B) (30, 37). In some experiments, the in vitro–assembled spindles and asters were treated with nocodazole to depolymerize MTs (32). Asters were present in extracts containing AurA beads plus DMSO, but these asters were not associated with AurA beads (Fig. 3B). After depolymerization of MTs with nocodazole, the LB3 antibody revealed a fibrillar-granular matrix surrounding more than 90% of sperm chromatin or AurA beads with RanGTP (Fig. 3, A and B, and fig. S3A). In the reaction containing AurA beads and DMSO, few LB3 matrices were found after the addition of nocodazole, and these matrices did not associate with the AurA-beads (Fig. 3B). The fibrillar-granular appearance (fig. S3A) suggests that the matrix might contain membranes. Indeed, the lipid dye (CMDiI) stained the spindles (fig. S3B) and the matrix (fig. S3C), and Triton X100 treatment completely disrupted the matrix (fig. S3D).

Fig. 3.

Requirement of RanGTP for assembly of LB3 matrices. (A) Association of LB3 matrices with sperm chromatin. Spindle assembly was induced with Xenopus sperm chromatin, and spindle MTs were subsequently depolymerized using nocodazole. MTs (red), LB3 matrix (green), and chromatin (blue) were labeled with rhodamine-tubulin, LB3 monoclonal antibody, and DAPI, respectively. (B) Association of LB3 matrices with AurA beads in the presence of RanGTP. MT assembly was induced with AurA beads and RanGTP or AurA beads and DMSO, and MTs were subsequently depolymerized with nocodazole. The percentages of AurA beads that were associated with MTs (red) or LB3 matrix (green) under different conditions were quantified. (C) Association of LB3 matrices with spindle MTs. Spindle assembly was induced with AurA beads and RanGTP. The presence of MTs, LB3, or TPX2 was examined at the indicated time after nocodazole addition. (D and E) Requirement of RanGTP but not MT polymerization for assembly of LB3 matrices around AurA beads (D) or sperm chromatin (E). AurA beads (D) were incubated with egg extracts with or without RanGTP in the presence or absence of nocodazole. Images show AurA beads (red) and LB3 matrix (green). Results were quantified as above. Sperm chromatin (E) was incubated with M-phase egg extracts in the presence or absence of nocodazole or RanT24N for 5 min. MT (red), LB3 (green), and chromatin (blue). Scales: white bars, 10 μm; magnetic beads, 2.8 μm.

We also assembled spindles on AurA beads with RanGTP, initiated MT depolymerization with nocodazole, and then examined LB3 matrices at various time points. LB3 matrices appeared at all stages of MT disassembly (Fig. 3C). However, the SAF TPX2 (38), which has a similar dynamic behavior to that of tubulin (39), disappeared as MTs were disassembled in these reactions (Fig. 3C). Thus, LB3 appears to associate with MTs as part of a matrix structure in mitosis.

To study whether MT polymerization is necessary for the formation of the LB3 matrix, we assembled LB3 matrix in the absence of MT polymerization. As a control, spindle MTs assembled in the absence of nocodazole were subsequently depolymerized with nocodazole. LB3 matrices assembled around AurA beads in the presence of RanGTP whether MTs were allowed to polymerize or not (Fig. 3D). However, if MTs were allowed to polymerize, a higher percentage of AurA beads was associated with the matrix. Moreover, the matrix assembled with MTs usually surrounded the AurA beads completely, whereas the matrix assembled in the absence of MTs usually partially surrounded the beads. In the absence of RanGTP, little LB3 matrix was formed (Fig. 3D). Therefore, RanGTP, but not MTs, appears to be required for the assembly of LB3 matrix around the AurA beads.

We also examined the requirement of RanGTP and MTs for the assembly of LB3 matrices around sperm chromatin. We inhibited RanGTP production on the sperm chromatin with RanT24N (a dominant negative Ran mutant). More than 90% of the sperm chromatin was associated with an LB3 matrix assembled during a 5-min incubation (Fig. 3E). MTs and LB3 matrix formed quickly, but the two structures did not always associate with one another, and often the formation of the LB3 matrix appeared to precede that of MTs (Fig. 3E and fig. S4). When MT assembly was inhibited by nocodazole, the matrix was still assembled around more than half of the sperm, but these matrices were smaller than those assembled in the presence of MTs (Fig. 3E). RanT24N almost completely inhibited the assembly of LB3 matrix around the sperm chromatin (Fig. 3E).

Retention of SAFs by the lamin B matrix after MT disassembly. Two SAFs, NuMA and Eg5, are proposed to be either components of the putative spindles matrix or tethered to the spindle matrix (40, 41). Poly(ADP-ribose) (PAR) also appears to be part of a static scaffold for proper spindle assembly (39). To test whether the LB3 matrix is part of the putative spindle matrix that tethers SAFs, we used sperm chromatin or AurA beads with RanGTP to induce spindle assembly. After MT disassembly, a number of SAFs [PAR, NuMA, Eg5, and XMAP215 (a SAF that promotes MT assembly)] remained associated with a matrix (Fig. 4A). Both LB3-containing matrices and SAF-containing matrices exhibited similar associations with AurA beads, and the majority of the matrices contained one or two beads (Fig. 4B). Many matrices that associated with two beads resembled spindles in size and shape (Fig. 4A). However, few TPX2-containing matrices remained after MT disassembly. Double immunofluorescence analyses of matrices assembled in the absence of MTs revealed the presence of both LB3 and SAFs in the same matrices (Fig. 4C). Matrices assembled in the absence of MTs were mostly associated with single AurA beads (Fig. 4C).

Fig. 4.

Requirement of LB3 for the assembly of LB3 matrices that contain SAFs. (A) Similarity of LB3 matrices and SAF matrices. Spindle assembly was induced with AurA beads and RanGTP. After MT depolymerization, the remaining structures were immunostained with antibodies to LB3, PAR, NuMA, Eg5, XMAP215, or TPX2. Rhodamine tubulin was used to label MTs. (B) Quantification of LB3 or SAF matrices in 50 random fields from (A) that were associated with 0, 1, 2, or more than 2 beads. (C) Presence of SAF in LB3 matrices. LB3 matrices were assembled with AurA beads and RanGTP in the absence of MT assembly and double immunostained for SAFs (XMAP215, Eg5, or NuMA in green) and LB3 (red). The graph shows the quantification of LB3, NuMA, PAR, XMAP215, Eg5, and TPX2 positive matrices associated with 0, 1, 2, or more than 2 AurA beads in 50 random fields. (D) Requirement of LB3 for the assembly of matrices containing Eg5 and NuMA. Egg extracts were first immunodepleted of LB3, Eg5, or XMAP215 with their respective antibodies and then incubated with sperm chromatin. After depolymerization of MTs, the sperm chromatin was stained with DAPI (blue) and antibodies to LB3, Eg5, or NuMA (green). Rhodamine-tubulin was used to label MTs (red). The percentage of sperm chromatin with associated matrices that contain LB3, Eg5, or NuMA were quantified. When either Eg5 or XMAP215 was depleted from the egg extracts, associations of NuMA, Eg5 (XMAP215 depletion), or LB3 with sperm chromatin as matrices were similar. However, when LB3 was depleted, neither LB3 nor NuMA and Eg5 associated with sperm chromatin as matrices. Shown is a typical graph quantifying the association of LB3 matrix with sperm chromatin. Scales: white bars, 10 μm; magnetic beads, 2.8 μm.

To determine whether LB3 is a structural component of the observed matrices containing SAFs, we immunodepleted LB3 from M-phase extracts and used sperm or AurA beads with RanGTP to stimulate matrix assembly. Depletion of LB3 inhibited the assembly of matrix structures containing LB3, NuMA, or Eg5 (Fig. 4D), as well as XMAP215 and PAR (42). However, depletion of either Eg5 or XMAP215 still allowed assembly of LB3 matrices that contained other SAFs (Fig. 4D). Therefore, lamin B appeared to be required for the assembly of a spindle-associated matrix that contains a number of SAFs. The LB3 matrix induced by RanGTP could be the long-sought-after spindle matrix that tethers SAFs to support spindle assembly.

The assembly of the LB3 matrix required RanGTP but not MTs (Fig. 3). RanGTP stimulates spindle assembly in Xenopus egg extracts by causing the release of bound SAFs from importin α and β (810). The C-terminal domain of LB3 contains a nuclear localization signal (NLS) (36) that bound to bacterially expressed importin α in vitro (fig. S5A). In M-phase extracts, LB3 interacted with importin α and β, and this interaction was disrupted in the presence of RanGTP (fig. S5B). To determine a potential role for importin α and β in the maintenance of the LB3 matrix, spindles assembled in the egg extracts were diluted by a factor of 100 in extract buffer in the presence of nocodazole and purified importin α and β. Because RanGTP concentration is reduced by dilution, it would not be sufficient to sequester the added importin α and β. We found that importin α and β severely disrupted LB3 matrices containing SAFs (fig. S5C) (32). The addition of more RanGTP along with importins prevented the disruption (fig. S5C). This suggests that one function of RanGTP in stimulating the assembly of the LB3 matrix in mitosis might be to displace LB3 from importin α and β.

Stimulation of MT assembly by the isolated LB3 matrix. The presence of SAFs in the LB3 matrix could promote polymerization and organization of MTs during spindle assembly. We developed a procedure to biochemically enrich the matrix. Because AurA beads remained associated with spindles and matrices, we could recover the beads from the egg extracts with a magnet (32). Structures retrieved from M-phase extracts were treated with nocodazole to depolymerize MTs, and the remaining matrices were washed (32) (Fig. 5A). The matrices could alternatively be released from the AurA beads by repeated pipetting, and the released matrices and AurA beads were separately analyzed. Immunoblotting showed that the released matrices contained LB3, NuMA, PAR, Eg5, and XMAP215 but lacked detectable TPX2 and tubulin (Fig. 5, A to D). We have also identified Eg5 as a component of the purified matrix by peptide mass fingerprinting (42). These LB3 matrices nucleated MTs in vitro when incubated with pure tubulin. The matrix-nucleated MT arrays were tethered to the matrices, and no MTs were assembled in the absence of the matrices (Fig. 5, E and F). When LB3 matrices were assembled in egg extracts from which the SAF XMAP215 was immunodepleted, these matrices were unable to promote MT assembly (Fig. 5, E and F). This suggests that the LB3 matrices could promote spindle assembly by tethering SAFs.

Fig. 5.

Isolated LB3 matrices nucleate MT assembly. (A) Isolation of LB3 matrix. AurA beads and RanGTP were used to induce spindle assembly in egg extracts (a). Spindles were separated from the egg extracts either by centrifugation through a glycerol cushion onto coverslips (b) or by retrieval with a magnet (c). The magnet-retrieved spindles were washed with buffer containing nocodazole. LB3 matrices were retained on the beads (d). To separate the LB3 matrices from the beads, the sample was pipetted repeatedly. Beads (e) were then retrieved with a magnet, leaving LB3 matrices (f) in the supernatant. Scale bar, 10 μm. (B) Commassie blue staining of the samples described in (A). 1 μl of egg extracts, or the equivalent of 5, 30, 120, 600, or 600 μl of extract was loaded in lanes a, b, c, d, e, or f, respectively. (C) Western blotting of the samples in (A) with antibody to PAR. Similar amounts of materials were loaded as in (B), except in lanes e and f, where only the equivalent of 120 μl of the extracts was loaded in each lane. (D) Western blotting of the samples in (B) with antibody to tubulin, TPX2, Eg5, XMAP215, or LB3. (E) Isolation of LB3 matrices from mock-depleted or XMAP215-depleted egg extracts. Top, isolated LB3 matrices (green) with little tubulin (red). Bottom, isolated LB3 matrices (green) from the XMAP215-depleted egg extracts without XMAP215 (red). Scale bars, 5 μm. (F) MT assembly induced by isolated LB3 matrices. The LB3 matrices isolated as described in (E) were used in MT assembly assays with pure tubulin. LB3 matrices, green; MTs, red. The graph at the bottom shows the quantification of matrices that nucleated MTs. Scale bar, 10 μm.

The requirement for proper assembly of the LB3 matrix in spindle formation. Immunodepletion of LB3 from M-phase egg extracts severely disrupted spindle assembly and prevented the assembly of LB3 matrices containing SAFs (Fig. 2, E to G, and Fig. 4D). Lamins assemble into polymers of various organizations, which is important for nuclear assembly and function. To determine whether lamin assembly is required for the formation of mitotic LB3 matrix and spindle assembly, we used three LB3 mutants [ΔNLB3, LB3T, or LB3T(–)nls] that have dominant negative effects on the assembly and organization of nuclear lamina and proper nuclear structure and function (1719). Both the N and C termini of LB3 (583 amino acids in length) are required for the polymerization of lamins in vitro (18, 36). These mutant lamins disassemble lamin structures in fully assembled nuclei in vivo and prevent nuclear assembly on sperm chromatin in Xenopus interphase egg extracts (1719, 36). The ΔNLB3 mutant lacks the N-terminal 32 amino acids of LB3. LB3T contains the C-terminal 200 amino acids of LB3, and LB3T(–)nls is made from LB3T by mutating the NLS (32). Wild-type and mutant LB3 proteins were expressed and purified from bacteria as 6His fusions (Fig. 6A) (32).

Fig. 6.

Effects of mutant LB3 proteins to disrupt mitotic spindles and LB3 matrices. (A) Purified LB3 and mutant LB3 proteins were analyzed by SDS–polyacrylamide gel electrophoresis and Coomassie blue staining. (B) Effects of mutant LB3 to disrupt spindle assembly induced by Xenopus sperm chromatin. Examples of normal and defective spindles or MT asters are shown. The graph shows the quantification of different MT structures under the indicated conditions. (C) Effects of mutant LB3 proteins to disrupt spindle assembly induced by AurA beads and RanGTP. Examples of normal and defective spindles as well as MT asters are shown. The graph at the bottom shows the quantification of different MT structures under the indicated conditions. (D) Effects of mutant LB3 proteins to disrupt the assembly of LB3 matrices around AurA beads. Spindle assembly was induced with AurA beads plus RanGTP in the presence of buffer, wild-type, or mutant LB3. MTs were depolymerized, and LB3 matrices were detected using LB3 antibody. Examples of beads with associated LB3 matrices are shown. The graph on the right shows the quantification of AurA beads associated with LB3 matrices under the indicated conditions. Scales: white bar, 10 μm; magnetic beads, 2.8 μm.

All three mutant LB3 proteins disrupted spindle assembly in extracts incubated with sperm chromatin or with AurA beads with RanGTP, whereas neither buffer alone nor wild-type LB3 affected spindle assembly (Fig. 6, B and C) (32). Localization of SAFs on MT structures was abnormal in the presence of all mutant LB3 proteins. Eg5 and XMAP215 localized to the spindles in the presence of wild-type LB3, but the two SAFs formed aggregates outside the MTs in the presence of LB3 mutants (fig. S6). LB3 mutants may perturb the organization of LB3 matrices, which may in turn disrupt the interaction between the SAFs and MTs. Indeed, we found that all three LB3 mutants disrupted the assembly of the LB3 matrix that is normally associated with either sperm chromatin or AurA beads (Fig. 6D). Thus, proper assembly of the LB3 matrix appears to be required to organize and localize SAFs on spindles in mitosis.

Discussion. Our studies demonstrate that RanGTP regulates the assembly of an LB3 matrix in M-phase Xenopus egg extracts. Moreover, similar to MT aster and spindle assembly, the assembly of LB3 matrix is sensitive to increased amounts of importin α and β. Therefore, RanGTP might activate the assembly of the LB3 matrix by displacing importin α and β from proteins that are required for the formation of the matrix.

We propose that Xenopus LB3 is one such protein regulated by RanGTP during matrix assembly. LB3 contains an NLS at its C terminus, which has an essential role in the assembly of LB into higher order structures in vitro and the nuclear lamina in vivo (1719, 36). Importin α bound to the NLS in the C-terminal domain of LB3 (fig. S5A), and in the egg extracts, the interaction of LB3 with importin α and β was sensitive to RanGTP (fig. S5B). The release of importins from LB3 by RanGTP may make the C-terminal domain accessible for its assembly into the LB3 matrix.

The assembly of the LB3 matrix did not require MT polymerization. However, LB3 matrices assembled around AurA beads or sperm chromatin in the absence of MTs were smaller (Figs. 3 and 4). This suggests a reciprocal regulatory mechanism; with the LB matrix regulating MT assembly and organization and the MTs in turn regulating the assembly of the LB matrix. Such a reciprocal regulatory mechanism could be mediated by the SAFs that interact with both MTs and the LB matrix (Fig. 7). We have not detected specific interactions between LB3 and any of the SAFs by coimmunoprecipitation from M-phase egg extracts, but SAFs could interact with other proteins on the LB3 matrices.

Fig. 7.

Model for the mechanism of RanGTP-mediated spindle assembly. RanGTP independently stimulates assembly of MTs and lamin B–containing matrix, which reciprocally regulate each other and SAFs, leading to spindle assembly.

Immunodepletion of LB3 or addition of truncated LB3 proteins with dominant negative activity that prevents lamin polymerization prevented the assembly of LB3 matrices containing any of the SAFs (Figs. 4 and 5). Thus, we propose that LB3 is a necessary structural component of the mitotic spindle.

The role of nuclear structural proteins such as intermediate filament proteins in mitosis may not be limited to human cells and Xenopus egg extracts. Like all other intermediate filament proteins, nuclear lamins assemble into higher order structures through the interactions of their highly alpha-helical central rod domains as well as their non–alpha-helical N- and C-terminal domains (36). Although lamins exist only in metazoans, coiled-coil proteins that can self-assemble are also found in the nucleus of plants and fungi. Some of these latter proteins may form nuclear structures similar to lamins (4347). These nuclear proteins might become part of the spindle matrix in mitosis. Indeed, the yeast nuclear protein FIN1p contains coiled-coil domains and associates with spindles during mitosis (46). Furthermore, purified FIN1p self-assembles into 10-nm filaments resembling the cytoskeletal intermediate filaments formed in vitro (46).

In interphase nuclei of vertebrate cells, LB is concentrated at the nuclear lamina and is also distributed throughout the nucleoplasm. During interphase, the lamins interact with a wide range of nuclear proteins to regulate many nuclear functions as well as nuclear structural integrity. At the onset of mitosis, lamins are phosphorylated by Cdk1, which leads to the disassembly of nuclear lamina (48, 49). The prevailing idea is that the disassembled LB is dispersed throughout the cytoplasm during mitosis. However, a fraction of LB is associated with the mitotic spindle and/or mitotic chromosomes (19, 2628). Our studies suggest that LB might perform functions analogous to those of the nuclear lamina to regulate spindle integrity and chromosome organization in mitosis.

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