Spatial regulation of Aurora A activity during mitotic spindle assembly requires RHAMM to correctly localize TPX2

Construction of a mitotic spindle requires biochemical pathways to assemble spindle microtubules and structural proteins to organize these microtubules into a bipolar array. Through a complex with dynein, the receptor for hyaluronan-mediated motility (RHAMM) cross-links mitotic microtubules to provide structural support, maintain spindle integrity, and correctly orient the mitotic spindle. Here, we locate RHAMM to sites of microtubule assembly at centrosomes and non-centrosome sites near kinetochores and demonstrate that RHAMM is required for the activation of Aurora kinase A. Silencing of RHAMM delays the kinetics of spindle assembly, mislocalizes targeting protein for XKlp2 (TPX2), and attenuates the localized activation of Aurora kinase A with a consequent reduction in mitotic spindle length. The RHAMM–TPX2 complex requires a C-terminal basic leucine zipper in RHAMM and a domain that includes the nuclear localization signal in TPX2. Together, our findings identify RHAMM as a critical regulator for Aurora kinase A signaling and suggest that RHAMM ensures bipolar spindle assembly and mitotic progression through the integration of biochemical and structural pathways.     

Aurora A kinase and its substrate TACC3 are required for central spindle assembly

Cell division entails a marked reorganization of the microtubule network to form the spindle, a molecular machine that ensures accurate chromosome segregation to the daughter cells. Spindle organization is highly dynamic throughout mitosis and requires the activity of several kinases and complex regulatory mechanisms. Aurora A (AurA) kinase is essential for the assembly of the metaphase bipolar spindle and, thus, it has been difficult to address its function during the last phases of mitosis. Here, we examine the consequences of inhibiting AurA in cells undergoing anaphase, and show that AurA kinase activity is necessary for the assembly of a robust central spindle during anaphase. We also identify TACC3 as an AurA substrate essential in central spindle formation.     

Age‐dependent integrity of the meiotic spindle assembly checkpoint in females requires Aurora kinase B

A hallmark of advanced maternal age is a significant increase in meiotic chromosome segregation errors, resulting in early miscarriages and congenital disorders. These errors most frequently occur during meiosis I (MI). The spindle assembly checkpoint (SAC) prevents chromosome segregation errors by arresting the cell cycle until proper chromosome alignment is achieved. Unlike in mitosis, the SAC in oocytes is desensitized, allowing chromosome segregation in the presence of improperly aligned chromosomes. Whether SAC integrity further deteriorates with advancing maternal age, and if this decline contributes to increased segregation errors remains a fundamental question. In somatic cells, activation of the SAC depends upon Aurora kinase B (AURKB), which functions to monitor kinetochore–microtubule attachments and recruit SAC regulator proteins. In mice, oocyte‐specific deletion of AURKB (Aurkb cKO) results in an increased production of aneuploid metaphase II‐arrested eggs and premature age‐related infertility. Here, we aimed to understand the cause of the short reproductive lifespan and hypothesized that SAC integrity was compromised. In comparing oocytes from young and sexually mature Aurkb cKO females, we found that SAC integrity becomes compromised rapidly with maternal age. We show that the increased desensitization of the SAC is driven by reduced expression of MAD2, ZW10 and Securin proteins, key contributors to the SAC response pathway. The reduced expression of these proteins is the result of altered protein homeostasis, likely caused by the accumulation of reactive oxygen species. Taken together, our results demonstrate a novel function for AURKB in preserving the female reproductive lifespan possibly by protecting oocytes from oxidative stress.     

Regulation of Op18 during spindle assembly in Xenopus egg extracts

Oncoprotein 18 (Op18) is a microtubule-destabilizing protein that is negatively regulated by phosphorylation. To evaluate the role of the three Op18 phosphorylation sites in Xenopus (Ser 16, 25, and 39), we added wild-type Op18, a nonphosphorylatable triple Ser to Ala mutant (Op18-AAA), and to mimic phosphorylation, a triple Ser to Glu mutant (Op18-EEE) to egg extracts and monitored spindle assembly. Op18-AAA dramatically decreased microtubule length and density, while Op18-EEE did not significantly affect spindle microtubules. Affinity chromatography with these proteins revealed that the microtubule-destabilizing activity correlated with the ability of Op18 to bind tubulin. Since hyperphosphorylation of Op18 is observed upon addition of mitotic chromatin to extracts, we reasoned that chromatin-associated proteins might play a role in Op18 regulation. We have performed a preliminary characterization of the chromatin proteins recruited to DNA beads, and identified the Xenopus polo-like kinase Plx1 as a chromatin-associated kinase that regulates Op18 phosphorylation. Depletion of Plx1 inhibits chromatin-induced Op18 hyperphosphorylation and spindle assembly in extracts. Therefore, Plx1 may promote microtubule stabilization and spindle assembly by inhibiting Op18.     

The budding yeast Ipl1/Aurora protein kinase regulates mitotic spindle disassembly

Ipl1p is the budding yeast member of the Aurora family of protein kinases, critical regulators of genomic stability that are required for chromosome segregation, the spindle checkpoint, and cytokinesis. Using time-lapse microscopy, we found that Ipl1p also has a function in mitotic spindle disassembly that is separable from its previously identified roles. Ipl1-GFP localizes to kinetochores from G1 to metaphase, transfers to the spindle after metaphase, and accumulates at the spindle midzone late in anaphase. Ipl1p kinase activity increases at anaphase, and ipl1 mutants can stabilize fragile spindles. As the spindle disassembles, Ipl1p follows the plus ends of the depolymerizing spindle microtubules. Many Ipl1p substrates colocalize with Ipl1p to the spindle midzone, identifying additional proteins that may regulate spindle disassembly. We propose that Ipl1p regulates both the kinetochore and interpolar microtubule plus ends to regulate its various mitotic functions.     

The KinI kinesin Kif2a is required for bipolar spindle assembly through a functional relationship with MCAK

Although the microtubule-depolymerizing KinI motor Kif2a is abundantly expressed in neuronal cells, we now show it localizes to centrosomes and spindle poles during mitosis in cultured cells. RNAi-induced knockdown of Kif2a expression inhibited cell cycle progression because cells assembled monopolar spindles. Bipolar spindle assembly was restored in cells lacking Kif2a by treatments that altered microtubule assembly (nocodazole), eliminated kinetochore-microtubule attachment (loss of Nuf2), or stabilized microtubule plus ends at kinetochores (loss of MCAK). Thus, two KinI motors, MCAK and Kif2a, play distinct roles in mitosis, and MCAK activity at kinetochores must be balanced by Kif2a activity at poles for spindle bipolarity. These treatments failed to restore bipolarity to cells lacking the activity of the kinesin Eg5. Thus, two independent pathways contribute to spindle bipolarity, with the Eg5-dependent pathway using motor force to drive spindle bipolarity and the Kif2a-dependent pathway relying on microtubule polymer dynamics to generate force for spindle bipolarity.     

Direct patterning of centrosome arrays as templates for the assembly of microtubules

We have achieved, for the first time, the selective patterning of centrosomes onto solid substrates. The use of such patterned centrosome arrays as templates for the directed polymerization of microtubules was also demonstrated. Centrosomes are small organelles in animal cells that serve as nucleation and organization centers of microtubules. Directed assembly of microtubules on the patterned centrosome arrays provides a new route to control the positions and directions of microtubules on surfaces. Combining the patterning of the isolated centrosomes and the directed growth of microtubules may lead to the generation of desired microtubule networks for bio-based nanodevices.     

Cep72 regulates the localization of key centrosomal proteins and proper bipolar spindle formation

Microtubule‐nucleation activity and structural integrity of the centrosome are critical for various cellular functions. The γ‐tubulin ring complexes (γTuRCs) localizing to the pericentriolar matrix (PCM) of the centrosome are major sites of microtubule nucleation. The PCM is thought to be created by two cognate large coiled‐coil proteins, pericentrin/kendrin and CG‐NAP/AKAP450, and its stabilization by Kizuna is essential for bipolar spindle formation. However, the mechanisms by which these proteins are recruited and organized into a proper structure with microtubule‐organizing activity are poorly understood. Here we identify a centrosomal protein Cep72 as a Kizuna‐interacting protein. Interestingly, Cep72 is essential for the localization of CG‐NAP and Kizuna. Cep72 is also involved in γTuRC recruitment to the centrosome and CG‐NAP confers the microtubule‐nucleation activity on the γTuRCs. During mitosis, Cep72‐mediated microtubule organization is important for converging spindle microtubules to the centrosomes, which is needed for chromosome alignment and tension generation between kinetochores. Our findings show that Cep72 is the key protein essential for maintaining microtubule‐organizing activity and structural integrity of the centrosome.     

癌症高表达蛋白--Hec1在纺锤体组装检查点中的作用

细胞增殖依赖于细胞分裂前染色体的复制及随后的姐妹染色单体分离到达两极。纺锤体组装检查点具有确保染色体信息传递保真性的作用,检查点的缺失可能导致染色体的分离异常和肿瘤形成。癌症高表达蛋白(Hec1)通过与调控G2/M期的蛋白间的相互作用而在染色体的分离中发挥重要作用。Hec1与Nuf2的复合物,在G2/M期与动粒相结合,Hec1的缺失将导致严重的染色体分离错误。Hec1具有召集Mps1和Mad1/Mad2复合物结合到动粒上的作用,这种结合可以激活纺锤体组装检查点途径中非常重要的APCCdc20途径。但是Hec1、Mps1、Mad1三者之间的相互作用仍未明了。Hec1还可以通过与26S蛋白酶复合物的不同亚基结合调控其功能。Hec1是一种丝氨酸磷酸化蛋白,其磷酸化是由Nek2在G2/M期完成的。     

Drosophila Aurora A kinase is required to localize D-TACC to centrosomes and to regulate astral microtubules

Disruption of the function of the A-type Aurora kinase of Drosophila by mutation or RNAi leads to a reduction in the length of astral microtubules in syncytial embryos, larval neuroblasts, and cultured S2 cells. In neuroblasts, it can also lead to loss of an organized centrosome and its associated aster from one of the spindle poles, whereas the centrosome at the other pole has multiple centrioles. When centrosomes are present at the poles of aurA mutants or aurA RNAi spindles, they retain many antigens but are missing the Drosophila counterpart of mammalian transforming acidic coiled coil (TACC) proteins, D-TACC. We show that a subpopulation of the total Aurora A is present in a complex with D-TACC, which is a substrate for the kinase. We propose that one of the functions of Aurora A kinase is to direct centrosomal organization such that D-TACC complexed to the MSPS/XMAP215 microtubule-associated protein may be recruited, and thus modulate the behavior of astral microtubules.     

Interaction of spindle assembly factor TPX2 with importins-α/β inhibits protein phase separation

The microtubule-based mitotic spindle is responsible for equally partitioning the genome during each cell division, and its assembly is executed via several microtubule nucleation pathways. Targeting Protein for XKlp2 (TPX2) stimulates the branching microtubule nucleation pathway, where new microtubules are nucleated from preexisting ones within mitotic or meiotic spindles. TPX2, like other spindle assembly factors, is sequestered by binding to nuclear importins-α/β until the onset of mitosis, yet the molecular nature of this regulation remains unclear. Here we demonstrate that TPX2 interacts with importins-α/β with nanomolar affinity in a 1:1:1 monodispersed trimer. We also identify a new nuclear localization sequence in TPX2 that contributes to its high-affinity interaction with importin-α. In addition, we establish that TPX2 interacts with importin-β via dispersed, weak interactions. We show that interactions of both importin-α and -β with TPX2 inhibit its ability to undergo phase separation, which was recently shown to enhance the kinetics of branching microtubule nucleation. In summary, our study informs how importins regulate TPX2 to facilitate spindle assembly, and provides novel insight into the functional regulation of protein phase separation.     

The spindle assembly checkpoint promotes chromosome bi-orientation: A novel Mad1 role in chromosome alignment

Faithful chromosome segregation relies on dynamic interactions between spindle microtubules and chromosomes. Especially, all chromosomes must be aligned at the equator of the spindle to establish bi-orientation before they start to segregate. The spindle assembly checkpoint (SAC) monitors this process, inhibiting chromosome segregation until all chromosomes achieve bi-orientation. The original concept of ‘checkpoints’ was proposed as an external surveillance system that does not play an active role in the process it monitors. However, accumulating evidence from recent studies suggests that SAC components do play an active role in chromosome bi-orientation. In this review, we highlight a novel Mad1 role in chromosome alignment, which is the first conserved mechanism that links the SAC and kinesin-mediated chromosome gliding.     

Spatial Compartmentalization Specializes the Function of Aurora A and Aurora B

Aurora kinase A and B share great similarity in sequences, structures, and phosphorylation motif, yet they show different localizations and play distinct crucial roles. The factors that determine such differences are largely unknown. Here we targeted Aurora A to the localization of Aurora B and found that Aurora A phosphorylates the substrate of Aurora B and substitutes its function in spindle checkpoint. In return, the centrosome targeting of Aurora B substitutes the function of Aurora A in the mitotic entry. Expressing the chimera proteins of the Auroras with exchanged N termini in cells indicates that the divergent N termini are also important for their spatiotemporal localizations and functions. Collectively, we demonstrate that functional divergence of Aurora kinases is determined by spatial compartmentalization, and their divergent N termini also contribute to their spatial and functional differentiation.     

Positioning centrosomes and spindle poles: looking at the periphery to find the centre

Centrosome positioning is tightly controlled throughout the cell cycle and probably shares common regulatory mechanisms with spindle-pole positioning. In this article, we detail the possible mechanisms controlling centrosome and spindle positioning in various organisms both in interphase and mitotic cells, and discuss recent findings showing how microtubule plus-end-associated proteins interact with the cell cortex. We suggest that microtubule plus-end complexes simultaneously regulate microtubule dynamics and microtubule anchoring at the cell periphery to allow proper centrosome and spindle-pole positioning.     

Aurora Kinase A proximity map reveals centriolar satellites as regulators of its ciliary function

Aurora kinase A (AURKA) is a conserved kinase that plays crucial roles in numerous cellular processes. Although AURKA overexpression is frequent in human cancers, its pleiotropic functions and multifaceted regulation present challenges in its therapeutic targeting. Key to overcoming these challenges is to identify and characterize the full range of AURKA interactors, which are often weak and transient. Previous proteomic studies were limited in monitoring dynamic and non‐mitotic AURKA interactions. Here, we generate the proximity interactome of AURKA in asynchronous cells, which consists of 440 proteins involving multiple biological processes and cellular compartments. Importantly, AURKA has extensive proximate and physical interactions to centriolar satellites, key regulators of the primary cilium. Loss‐of‐function experiments identify satellites as negative regulators of AURKA activity, abundance, and localization in quiescent cells. Notably, loss of satellites activates AURKA at the basal body, decreases centrosomal IFT88 levels, and causes ciliogenesis defects. Collectively, our results provide a resource for dissecting spatiotemporal regulation of AURKA and uncover its proteostatic regulation by satellites as a new mechanism for its ciliary functions.     

AIBp regulates mitotic entry and mitotic spindle assembly by controlling activation of both Aurora-A and Plk1

We previously reported that Aurora-A and the hNinein binding protein AIBp facilitate centrosomal structure maintenance and contribute to spindle formation. Here, we report that AIBp also interacts with Plk1, raising the possibility of functional similarity to Bora, which subsequently promotes Aurora-A–mediated Plk1 activation at Thr210 as well as Aurora-A activation at Thr288. In kinase assays, AIBp acts not only as a substrate but also as a positive regulator of both Aurora-A and Plk1. However, AIBp functions as a negative regulator to block phosphorylation of hNinein mediated by Aurora-A and Plk1. These findings suggest a novel AIBp-dependent regulatory machinery that controls mitotic entry. Additionally, knockdown of hNinein caused failure of AIBp to target the centrosome, whereas depletion of AIBp did not affect the localization of hNinein and microtubule nucleation. Notably, knockdown of AIBp in HeLa cells impaired both Aurora-A and Plk1 kinase, resulting in phenotypes with multiple spindle pole formation and chromosome misalignment. Our data show that depletion of AIBp results in the mis-localization of TACC3 and ch-TOG, but not CEP192 and CEP215, suggesting that loss of AIBp dominantly affects the Aurora-A substrate to cause mitotic aberrations. Collectively, our data demonstrate that AIBp contributes to mitotic entry and bipolar spindle assembly and may partially control localization, phosphorylation, and activation of both Aurora-A and Plk1 via hNinein during mitotic progression.