Repression of TPXL3 expression leads to retarded growth
To repress the expression of TPXL3 in A. thaliana, an artificial microRNA gene was designed to specifically target at this gene but not other related genes (Fig. 1A). These amiR-TPXL3 transformants produced dwarf plant that exhibited different degrees of growth inhibition when compared to the control plant (Fig. 1B). To test whether the growth phenotype was correlated to the expression level of the target gene TPXL3, real-time RT-PCR experiments were carried out and showed that greater reduction of the mRNA level was associated with more severe growth defects (Fig. 1C). To further prove the linkage of the amiR-TPXL3 expression with the phenotype, we constructed an amiR-TPXL3-resistant version of the TPXL3 gene (TXPL3R) by introduction of silent mutations and delivered it into one of the amiR-TPXL3 mutant. The expression of the TPXL3R gene under the control of the TPXL3 native promoter and in fusion with GFP (green fluorescent protein) fully suppressed the growth phenotype in the mutant to render plants resembling the wild-type control (Supplemental Fig. 1). Therefore, we concluded that the growth phenotypes exhibited by the amiR-TPXL3 mutants were caused by the repression of TXPL3 expression and these mutants could serve as the genetic material for subcellular phenotypic analysis of the consequence of compromised TPXL3 expression.
TPXL3 exhibits a distinct dynamic localization pattern during mitosis
A previous interaction assay in yeast revealed that TPXL3 barely binds to α Aurora but that the homologous TPXL2 interacts strongly 15. However, it was hypothesized that the TPXL3 protein serves as the primary activator of α Aurora (AUR1 and AUR2) in A. thaliana in part because of the lethality caused by the loss of this gene. To learn their in vivo activities, we compared the localization patterns of TPXL3 and TPXL2 when fused with GFP and expressed in the corresponding homozygous mutant background. When surveyed by fluorescent microscopy, the two proteins exhibited different localization dynamics in dividing root cells (Fig. 2A, B). While TPXL3 showed pronounced association with mitotic microtubule arrays, TPXL2 did not show distinguishable localization patterns other than residing in some interphase nuclei. Therefore, this result was in line with the lack of growth phenotype upon the loss of the TPXL2 gene highlighted the essential contribution of TPXL3 15.
To gain insights into the dynamic localization of TPXL3 during mitosis, we observed the functional TPXL3-GFP fusion protein expressed in the homozygous tpxl3-1 mutant background 15, by time-lapse live-cell imaging. The TPXL3-GFP signal became concentrated on the nuclear envelope at prophase and gradually polarized towards two poles (3:00 & 9:00, Fig. 2C). Around the time of nuclear envelope breakdown (10:30), the GFP signal was largely concentrated at the poles. Concomitant with the development of the mitotic spindle, the TPXL3 signal spread along spindle microtubules (12:00–15:30). Following the anaphase onset (17:30), the signal retrieved from the middle zone together with the shortening of kinetochore fiber microtubules and eventually it became highly concentrated at the poles again (19:30). When the phragmoplast formed, TPXL3 became enriched in a region flanking the phragmoplast microtubules (22:00–27:00 and Supplemental Fig. 4). Such a pattern was later replaced by the returning of the protein to the reforming nuclear envelope (35:00–41:00). Therefore, TPXL3 exhibited microtubule-associated (along spindle microtubules) and microtubule-independent (phragmoplast flanking) patterns of distribution during mitosis and cytokinesis.
To discern whether TPXL3 dynamics mirrored that of α Aurora, we then performed co-localization experiments. To do so, the Aurora 1 (AUR1) gene was expressed in fusion with the FLAG peptide in the TPXL3-GFP transgenic lines. Like the GFP-AUR1 fusion, this FLAG-AUR1 fusion protein was fully functional as the transgene restored growth of the aur1 aur2 double mutant to the wild-type level (Supplemental Fig. 2). Throughout the mitotic cell cycle, the FLAG-AUR1 and TPXL3-GFP exhibited identical localization patterns on the prophase spindle and kinetochore fiber microtubules, and on the reforming nuclear envelope, but not associated with microtubules in developing phragmoplasts (Fig. 3 and Supplemental Fig. 4). Therefore, the data supported the in vivo association of the two proteins.
To test whether the distinct localization of TPXL3 was dependent on dynamic microtubules, we challenged cells with low doses of the microtubule-depolymerizing agent oryzalin. The TPXL3-GFP signal became largely overlapped with that of microtubules and associated with microtubules in the spindle midzone during late anaphase and phragmoplast (Supplemental Fig. 3). Therefore, we concluded that the polarized localization pattern of TPXL3 was dependent on the dynamic microtubule arrays during mitosis.
Compromised TPXL3 expression leads to delocalization of α Aurora and γ-tubulin
Because of the colocalization of TPXL3 and AUR1, we asked whether compromised expression of TPXL3 would affect the localization of AUR1. To do so, we had the GFP-AUR1 fusion protein expressed in the amiR-TPXL3 mutant and compared its localization to that in the control cells. In the control cells, GFP-AUR1 prominently decorated spindle microtubules but was absent from the microtubule segments in the vicinity of chromosomes (Fig. 4A). In the amiR-TPXL3 mutant, however, the GFP-AUR1 signal became largely diffuse in the cytoplasm and its signal on spindle microtubules did not stand out significantly (Fig. 4B). The contrasted difference was obvious when the spindle-associated signal was referenced to the cytoplasmic one (Fig. 4C). Therefore, we concluded that TPXL3 is required for AUR1 localization on spindle microtubules in A. thaliana.
Conversely, we asked whether compromised α Aurora function would impact TPXL3 localization. We compared the TPXL3-GFP signal in the control cells of the complemented line and in the aur1 aur2 double mutant background. The localization in the prophase spindle on the nuclear envelope, mitotic spindle, and the phragmoplast-devoid region was comparable in both genetic backgrounds (Supplemental Fig. 4). Therefore, we concluded that TPXL3 likely achieves its localization independently to α Aurora, and in turn dictates the localization of α Aurora.
The spindle pole-biased localization of TPXL3 and α Aurora resembled that of microtubule-nucleating factors represented by γ-tubulin in A. thaliana 16. To discern the relationship between α Aurora/TPXL3 and γ-tubulin, we first carried out dual localization experiments by detecting TPXL3-GFP and γ-tubulin with respective antibodies (Fig. 4D). Prior to nuclear envelope breakdown in prophase, TPXL3 prominently accumulated on the nuclear envelope when γ-tubulin was barely detectable (Fig. 4D). Starting from late prophase, the TPXL3 signal was largely overlapping with γ-tubulin on kinetochore fibers of the mitotic spindle (Fig. 4E). Striking differences were discovered in the spindle midzone and developing phragmoplasts where γ-tubulin prominently decorated microtubule minus ends (Fig. 4F, G). TPXL3, however, was barely detected and later accumulated at the distal edges of the two groups of γ-tubulin signals (Fig. 4F, G). This finding was surprising because γ-tubulin is generally thought to be at the minus end of phragmoplast microtubules. Therefore, α Aurora/TPXL3 are associated with subcellular structures that flank the phragmoplast microtubules.
Because TPXL3 appeared earlier than γ-tubulin on mitotic arrays, we then examined γ-tubulin localization in amiR-TPXL3 mutant cells. Compared to the conspicuous localization of γ-tubulin on prophase spindle poles and developing spindles in the control cells, such polarized pattern was largely lost and replaced by weak signals clouding on microtubules (Fig. 4H, I). The γ-tubulin signal associated with spindle microtubules were seriously reduced while the diffuse signal in the cytoplasm became more noticeable (Fig. 4J). Therefore, the results support the notion that TPXL3 plays a critical role in regulating the localization of α Aurora and perhaps consequently γ-tubulin complex on the spindle microtubule arrays.
TPXL3 directly binds to microtubules in vivo and is phosphorylated by α Aurora
Because the TPXL3 polypeptide exhibits significant sequence divergence from the canonical TPX2 protein 15, we dissected the structure-function relationship by transiently expressing truncated versions in tobacco cells (Fig. 5). To do so, we divided TPXL3 into the following five segments (I-V): the N-terminal Aurora-binding motif (I), first novel domain (II), domain III with the first predicted nuclear localization signal, and domain IV with the second nuclear localization signal and importin-binding site, and the C-terminal novel domain (V) (Fig. 5A). When the full length TPXL3 was expressed in fusion with GFP under the control of the constitutive viral 35S promoter, the fusion protein was nuclear with stronger signals in the nucleolus (Fig. 5B). The deletion of either domain V or IV did not alter such a localization pattern (Fig. 5B). The removal of domains III to V, however, resulted in the fusion protein decorating cortical microtubule-like network, and domain II was sufficient for this localization pattern (Fig. 5B). To ascertain whether the cytoskeletal or nuclear localization would dominate when both features were included, domains II-V were expressed and exhibited nuclear localization like others seen above (Fig. 5B). In fact, domains IV plus V, III plus IV, or I plus III-V also rendered similar localization patterns (Fig. 5B). Finally, we tested whether domain V had a localization determinant and found its GFP fusion was uniformly diffuse in the cytoplasm and nucleus (Fig. 5B). To ascertain whether domain II interacted with cortical microtubules, we expressed the truncated protein together with a microtubule marker of CKL6 and detected completely overlapping patterns either with domains I and II or domain II alone (Fig. 5C). Therefore, we concluded that domain II constitutes a microtubule-binding site in TPXL3 while domains III and IV have nuclear localization activities. The essential function of TPXL3 is perhaps brought about by these domains that make respective contributions.
Both α Aurora and TPXL3 exhibited nuclear localization in interphase cells 15. When co-expressed, the full length TPXL3 fused with GFP had exclusive nuclear localization with an emphasis in the nucleolus, as the TagRFP-AUR1 fusion protein (Fig. 6A). To test whether α Aurora’s nuclear localization was dependent on TPXL3, we had TagRFP-AUR1 co-expressed with truncated TPXL3 containing only domains I and II corresponding to the Aurora-binding motif and microtubule-binding domain, respectively. Consequently, TagRFP-AUR1 decorated cortical microtubules and completely overlapped with TPXL3I + II-GFP (Fig. 6B). Such microtubule localization was completely dependent on the domain I as the loss of this AUR-binding site abolished the microtubule-association (Fig. 6C).
The AUR1-TPXL3 interaction was further tested in vitro using fusion proteins expressed in and purified from bacterial hosts. When compared to the interaction between the full-length GST-TPXL3 and 6xHis-AUR1, it was lost when the GST-TPXL3II − V fusion protein was used (Supplemental Fig. 5). The deletion of domains III to V did not affect the interaction (Supplemental Fig. 5).
We then tested how specific domain(s) of TPXL3 contributed to the kinase activity of α Aurora. AUR1 exhibited some autophosphorylation activities, while full-length TPXL3, TPXL3II − V, or TPXL3I + II alone did not (Fig. 6D). The addition of full-length TPXL3 enhanced AUR1 phosphorylation and had TPXL3 also phosphorylated, and such an activity was completely dependent on the Aurora-binding domain at the N-terminus (Fig. 6D). Furthermore, the results also showed that the phosphorylation sites were largely included in the Aurora- and microtubule-binding sites within the first two domains (Fig. 6D).
TPXL3 regulates spindle morphogenesis and axial growth
To link TPXL3 function to microtubule remodeling during mitosis, we delivered a GFP-TUB6 marker of microtubules into the amiR-TPXL3 mutant to monitor mitotic arrays and compared them to those in control cells by live-cell imaging (Supplemental Movies 2 and 3). Microtubules are assembled into a bipolar prophase spindle around the timing of nuclear envelope breakdown in the control cell expressing TPXL3 (times 00:00 to 01:00, Fig. 7A). This array was followed by the spindle arrays of prominent kinetochore fibers converged towards obvious poles (02:00 to 06:00, Fig. 7A). Following the anaphase onset, converged kinetochore fibers shortened and microtubules in the spindle midzone emerged and coalesced before the appearance of two mirrored microtubule sets joined by the fluorescently dark midline (06:00 to 08:30, Fig. 7A). Such a dynamic pattern was largely altered in the amiR-TPXL3 mutant cell although microtubules continued to undergo rapid reorganization (Fig. 7B). Unlike the fusiform appearance of spindle microtubule in the control cells, mutant cells assembled discrete microtubule bundles running in parallel to each other upon nuclear envelope breakdown (00:00, Fig. 7B). These microtubule bundles were not integrated into a spindle with converged spindle poles when bundles freely splayed outwards (01:00 to 03:00, Fig. 7B). Although a bipolar spindle array was formed later with obvious kinetochore fibers terminating at the metaphase plate, it lacked converging poles (04:00 to 07:00, Fig. 7B). Anaphase onset was delayed and sometimes anaphase spindle elongation was minimized, and microtubules in the spindle midzone were developed into robust bundles that later coalesced into the phragmoplast array (09:00 to 13:00, Fig. 7B).
To quantitatively evaluate convergence of spindle microtubules, we measured spindle solidity, which is defined as the ratio of the spindle area to the convex hull area (Fig. 7C, Supplemental Fig. 6A, B). Converged and splayed microtubules result in high and low ratios, respectively. Repression of TPXL3 expression significantly decreased solidity, leading to splayed organization of spindle microtubules (Fig. 7C). To quantitatively evaluate spindle elongation, we also measured spindle aspect ratio, which is a morphometric parameter that is defined as the ratio of the major axis length to the minor axis length of the fitted ellipse (Fig. 7D, Supplemental Fig. 6A, C). Knockdown of TPXL3 significantly decreased aspect ratio, leading to decreased spindle elongation (Fig. 7D).
Although the amiRNA-TPXL3 mutant cell formed distorted spindle microtubule arrays, it produced a bipolar phragmoplast array that underwent robust expansion towards the cell cortex during cytokinesis, similar to what was observed in the control cell (09:30 to 16:30 in the control and 13:00 to 22:00 in amiR-TPXL3, Fig. 7A, B). Therefore, we concluded that down regulation of TPXL3 expression had greater impacts on spindle microtubule remodeling than on the phragmoplast array.
To explore the functionality of different TPXL3 domains in spindle morphogenesis, we expressed different derivatives of TPXL3 under the control of the native TPXL3 promoter in the amiR-TPXL3 mutant plants. The amiR-TPXL3 (5) mutant line as the host for the expression of different TPXL3 derivatives. First, we used anti-tubulin immunofluorescence to examine metaphase spindle microtubule arrays and detected disorganized poles in mutant cells, similar to what was observed by live-cell imaging (Fig. 8A). When the full-length microRNA-resistant form of TPXL3 (mTPXL3) was expressed, metaphase cells restored typical spindles with converged poles (Fig. 8A). Concomitantly, the transgene suppressed the stunted growth phenotype of the host plant and rendered adult plants that resembled the wild-type control (Fig. 8B). Then, we had the AUR-binding motif (domain I) or the microtubule-binding site (domain II) removed and found that the truncated TPXL3II − V and TPXL3ΔII proteins did not restore the spindle morphology, neither did they improve seedling growth (Fig. 8A, B). These results supported the notion that TPXL3’s function was inseparable from its interactions with α Aurora or microtubules. Surprisingly, the mTPXL3I − IV derivative with the C-terminal domain V removed was able to restore the converged spindle microtubule arrays in metaphase cells and growth and reproduction almost as robust as the wild-type control (Fig. 8A, B). The derivative with domain IV removed, however, enhanced spindle defects with kinetochore microtubule fibers arranged in a palisade-like fashion and resulted in great retardation of plant growth (Fig. 8A, B). Similarly, expression of a truncated version of TPXL3 containing only domains I and II resulted in a similar if not more exaggerated negative impact in both spindle morphogenesis and seedling growth as mTPXL3I − IV (Fig. 8A, B). Collectively, these results affirmed that defects in spindle microtubule organization are translated into deficiencies in overall growth besides demonstrating the indispensability of domains I (AUR-binding), II (microtubule-binding), and IV (importin binding) for TPXL3 function.
Because the mTPXL3ΔI+II protein had a stronger negative impact in spindle morphogenesis and plant growth when compared to the deletion of either domain, we asked whether TPXL3’s function was completely aligned with α Aurora. To do so, we introduced amiR-TPXL3 into the aur1 aur2 double mutant. Down regulation of TPXL3 in the aur mutant further enhanced the growth defects (Fig. 8C). Therefore, TPXL3 may possess novel function(s) in addition to targeting and activating the α Aurora kinase.