Cep57, a NEDD1-binding pericentriolar material component, is essential for spindle pole integrity

An Essential Function of the C. elegans Ortholog of TPX2 Is to Localize Activated Aurora A Kinase to Mitotic Spindles

A Developmentally Regulated Two-Step Process Generates a Noncentrosomal Microtubule Network in Drosophila Tracheal Cells

The structure and function of microtubules (MTs) are regulated by post-translational modifications of tubulin subunits, such as acetylation of the Lys40 residue of α-tubulin. Regulation of the organization and dynamics of MTs is essential for the precise formation of the mitotic spindle. Spindle MTs are highly acetylated, but the mechanism regulating this acetylation is largely unknown. Furry (Fry) is an evolutionarily conserved protein that binds to MTs and colocalizes with acetylated MTs in the mitotic spindle. In this study, we examined the role of Fry in the acetylation of MTs in the mitotic spindle. Depletion of Fry significantly reduced the level of MT acetylation in the mitotic spindle. Expression of the N-terminal fragment of Fry induced hyperacetylation of MTs in both mitotic and interphase cells. These results indicate that Fry promotes MT acetylation in the mitotic spindle. We also found that Fry binds to the tubulin deacetylase SIRT2, preferentially in mitotic cells. Cell-free experiments revealed that the N-terminal region of Fry is the domain responsible for binding to and inhibiting the tubulin-deacetylase activity of SIRT2. AGK2, a specific inhibitor of SIRT2, increased the level of MT acetylation in the mitotic spindle, indicating that SIRT2 is involved in the deacetylation of spindle MTs. Furthermore, AGK2 reversed the decrease in MT acetylation induced by Fry depletion. In summary, these results suggest that Fry plays a crucial role in promoting the level of MT acetylation in the mitotic spindle by inhibiting the tubulin-deacetylase activity of SIRT2.

CPAP--a gene mutated in primary microcephaly--is required for procentriole formation. Here we show that CPAP degradation and function is controlled by the poly(ADP-ribose) polymerase tankyrase 1. CPAP is PARsylated by tankyrase 1 in vitro and in vivo. Overexpression of tankyrase 1 leads to CPAP proteasomal degradation, preventing centriole duplication, whereas depletion of tankyrase 1 stabilizes CPAP in G1, generating elongated procentrioles and multipolarity. Tankyrase 1 localizes to centrosomes exclusively in G1, coinciding with CPAP degradation. Hence, tankyrase 1-mediated PARsylation regulates CPAP levels during the cell cycle to limit centriole elongation and ensure normal centrosome function.

The functional role of centrioles or basal bodies in mitotic spindle assembly and function is currently unclear. Although supernumerary centrioles have been associated with multipolar spindles in cancer cells, suggesting centriole number might dictate spindle polarity, bipolar spindles are able to assemble in the complete absence of centrioles, suggesting a level of centriole-independence in the spindle assembly pathway. In this report we perturb centriole number using mutations in Chlamydomonas reinhardtii, and measure the response of the mitotic spindle to these perturbations in centriole number. Although altered centriole number increased the frequency of monopolar and multipolar spindles, the majority of spindles remained bipolar regardless of the centriole number. But even when spindles were bipolar, abnormal centriole numbers led to asymmetries in tubulin distribution, half-spindle length and spindle pole focus. Half spindle length correlated directly with number of centrioles at a pole, such that an imbalance in centriole number between the two poles of a bipolar spindle correlated with increased asymmetry between half spindle lengths. These results are consistent with centrioles playing an active role in regulating mitotic spindle length. Mutants with centriole number alteration also show increased cytokinesis defects, but these do not correlate with centriole number in the dividing cell and may therefore reflect downstream consequences of defects in preceding cell divisions.

Segregation of chromosomes during cell division requires correct formation of mitotic spindles. Here, we show that a scaffold attachment factor A (SAF-A), also known as heterogeneous nuclear ribonucleoprotein-U, contributes to the attachment of spindle microtubules (MTs) to kinetochores and spindle organization. During mitosis, SAF-A was localized at the spindles, spindle midzone and cytoplasmic bridge. Depletion of SAF-A by RNA interference induced mitotic delay and defects in chromosome alignment and spindle assembly. We found that SAF-A specifically co-immunoprecipitated with the chromosome peripheral protein nucleolin and the spindle regulators Aurora-A and TPX2, indicating that SAF-A is associated with nucleolin and the Aurora-A-TPX2 complex. SAF-A was colocalized with TPX2 and Aurora-A in spindle poles and MTs. Elimination of TPX2 or Aurora-A from cells abolished the association of SAF-A with the mitotic spindle. Interestingly, SAF-A can bind to MTs and contributes to the targeting of Aurora-A to mitotic spindle MTs. Our finding indicates that SAF-A is a novel spindle regulator that plays an essential role in kinetochore-MT attachment and mitotic spindle organization.

Kif18A Uses a Microtubule Binding Site in the Tail for Plus-End Localization and Spindle Length Regulation

<div>Abstract<p>Drugs that perturb microtubules are commonly used to treat breast cancers of all subtypes in both early stage and metastatic disease, but they are effective in only approximately 50% of patients. High concentrations of microtubule-targeting agents can elicit mitotic arrest in cell culture models; however, recent evidence from primary and metastatic breast cancers has revealed that these agents only accumulate at intratumoral levels capable of inducing abnormal multipolar mitotic spindles, not mitotic arrest. Although the maintenance of multipolar spindles can generate cytotoxic rates of chromosomal instability (CIN), focusing of aberrant multipolar spindles into normal bipolar spindles can dramatically reduce CIN and confer resistance to microtubule poisons. Here, we showed that inhibition of the mitotic kinesin centromeric-associated protein-E (CENP-E) overcomes resistance caused by focusing multipolar spindles. Clinically relevant microtubule-targeting agents used a mechanistically conserved pathway to induce multipolar spindles without requiring centrosome amplification. Focusing could occur at any point in mitosis, with earlier focusing conferring greater resistance to antimicrotubule agents. CENP-E inhibition increased CIN on focused spindles by generating chromosomes that remained misaligned at spindle poles during anaphase, which substantially increased death in the resulting daughter cells. CENP-E inhibition synergized with diverse, clinically relevant microtubule poisons to potentiate cell death in cell lines and suppress tumor growth in orthotopic tumor models. These results suggest that primary resistance to microtubule-targeting drugs can be overcome by simultaneous inhibition of CENP-E.</p><p><b>Significance:</b> The increased incidence of polar chromosomes induced by inhibition of the mitotic kinesin CENP-E exacerbates chromosomal instability, reduces daughter cell viability, and improves sensitivity to microtubule-targeting therapies.</p></div>

Cells with blocked microtubule polymerization are delayed in mitosis, but eventually manage to proliferate despite substantial chromosome missegregation. While several studies have analyzed the first cell division after microtubule depolymerization, we have asked how cells cope long-term with microtubule impairment. We allowed 24 clonal populations of yeast cells with beta-tubulin mutations preventing proper microtubule polymerization, to evolve for ˜150 generations. At the end of the laboratory evolution experiment, cells had regained the ability to form microtubules and were less sensitive to microtubule-depolymerizing drugs. Whole-genome sequencing identified recurrently mutated genes, in particular for tubulins and kinesins, as well as pervasive duplication of chromosome VIII. Recreating these mutations and chromosome VIII disomy prior to evolution confirmed that they allow cells to compensate for the original mutation in beta-tubulin. Most of the identified mutations did not abolish function, but rather restored microtubule functionality. Analysis of the temporal order of resistance development in independent populations repeatedly revealed the same series of events: disomy of chromosome VIII followed by a single additional adaptive mutation in either tubulins or kinesins. Since tubulins are highly conserved among eukaryotes, our results have implications for understanding resistance to microtubule-targeting drugs widely used in cancer therapy.

Influence of Centriole Behavior on the First Spindle Formation in Zygotes of the Brown AlgaFucus distichus(Fucales, Phaeophyceae)

Myosin 10 (Myo10) has the ability to link actin filaments to integrin-based adhesions and to microtubules by virtue of its integrin-binding FERM domain and microtubule-binding MyTH4 domain, respectively. Here we used Myo10 knockout cells to define Myo10’s contribution to the maintenance of spindle bipolarity, and complementation to quantitate the relative contributions of its MyTH4 and FERM domains. Myo10 knockout HeLa cells and mouse embryo fibroblasts (MEFs) both exhibit a pronounced increase in the frequency of multipolar spindles. Staining of unsynchronized metaphase cells showed that the primary driver of spindle multipolarity in knockout MEFs and knockout HeLa cells lacking supernumerary centrosomes is pericentriolar material (PCM) fragmentation, which creates γ-tubulin-positive acentriolar foci that serve as additional spindle poles. For HeLa cells possessing supernumerary centrosomes, Myo10 depletion further accentuates spindle multipolarity by impairing the clustering of the extra spindle poles. Complementation experiments show that Myo10 must interact with both integrins and microtubules to promote PCM/pole integrity. Conversely, Myo10’s ability to promote the clustering of supernumerary centrosomes only requires that it interact with integrins. Importantly, images of Halo-Myo10 knock-in cells show that the myosin localizes exclusively within adhesive retraction fibers during mitosis. Based on these and other results, we conclude that Myo10 promotes PCM/pole integrity at a distance, and that it facilitates supernumerary centrosome clustering by promoting retraction fiber-based cell adhesion, which likely provides an anchor for the microtubule-based forces driving pole focusing.

Microtubules (MTs) are a major component of the mammalian cytoskeleton. MTs are essential for cell morphogenesis and cellular functions including cell motility, cell division and intra-cellular transport. MT functions are tightly regulated by post-translational modifications (PTMs) and MT-associated proteins (MAPs). However, the underlying mechanisms are still elusive. Septins are a family of GTP-binding proteins that can form hetero-oligomeric and polymeric structures, and function as scaffolds or diffusion barriers, controlling the localization of membrane and cytoplasmic proteins. Mammalian septins interact with MTs and actin filaments, Septins are also involved in Golgi-to-plasma membrane vesicle transport and chromosome alignment. Interestingly, septins have been shown to interact with the centromere-associated protein E (CENP-E), a mitotic kinesin-like motor that links kinetochores to the ends of spindle MTs. However, it is unknown whether septins interact directly with MTs and how they affect MT organization and intracellular transport. In the first part of this thesis, I studied how MT organization is regulated by septins. I showed that the N-terminal domain of SEPT9 contains the novel repeat motifs K/R-x-x-E/D and R/K-R-x-E, which bind and bundle MTs by interacting with the acidic C-terminal tails of [beta]-tubulin. Alanine scanning mutagenesis revealed that the K/R-R/x-x-E/D motifs pair electrostatically with one another and the C-terminal tails of [beta]-tubulin, enabling septin-septin interactions that link MTs together. SEPT9 is the only gene linked to hereditary neuralgic amyotrophy (HNA), a rare autosomal-dominant neuropathy. SEPT9 isoforms lacking repeat motifs or containing the HNA-linked mutation R88W, which maps to the R/K-R-x-E motif, diminished intracellular MT bundling and impaired asymmetric neurite growth in PC-12 cells. These findings provide the first insight into the mechanism of septin interaction with MTs and the molecular and cellular basis of HNA. In the second part of this thesis, I discovered a novel interaction between SEPT9 and KIF17, a kinesin 2 family motor that is important for learning and memory, mediating the transport of the NMDA glutamate receptor in hippocampal neurons. I found that SEPT9 associates directly with the cargo-binding C-terminal tail of KIF17 and competes with mLin-10/Mint1, a cargo adaptor/scaffold protein, which links KIF17 to the NMDA receptor subunit 2B (NR2B). Significantly, SEPT9 down-regulates NR2B transport into the dendrites of hippocampal neurons. Because SEPT9 does not affect the microtubule-dependent motility of KIF17, my results suggest that SEPT9 modulates the interaction of KIF17 with NR2B cargo. These results provide the first evidence of an interaction between septins and a non-mitotic kinesin, and suggest that SEPT9 modulates specifically the interactions of KIF17 with membrane cargo. These findings advanced our knowledge of how septins associate with MTs and revealed the mechanism of MT binding and bundling by septins. My studies also provided the first clue about the etiology of HNA, which might be helpful in developing therapies for this disease in the future. In addition, the results of this work provided the first insight into how septins may regulate kinesin-dependent transport and the transport of a neurotransmitter to dendrite membrane. This knowledge could potentially be helpful in developing treatment for diseases associated with misregulation of MT organization and MT based transport.

Previous studies revealed the presence of several tight junction (TJ) proteins in the centrosome and their interaction with various centriolar proteins, prompting us to analyze whether this also applies to the TJ protein ZO-2. Here, we found that ZO-2 colocalizes with CEP164 in the distal appendage of the mother centriole and is also present in the pericentriolar region, mitotic spindle poles, the basal body of primary cilia, and the tail of spermatozoa. The absence of ZO-2 altered the cellular content of centriolar proteins CEP164, centriolin, and CEP135, but did not change the morphology of centrioles. ZO-2 depletion inhibits the development of astral and mitotic spindle microtubules expressing EB1. At the spindle poles, ZO-2 depletion increases the accumulation of NuMA while reducing the levels of kinesin KIF14 and the TPX2 scaffold, and the accumulation of the kinase p-Aurora, leading to a decrease in mitotic spindle length, microtubule instability, and abnormal chromosome congression. KIF14, NuMA, and p-Aurora co-immunoprecipitate with ZO-2, and NuMA and Aurora-A bind to different segments of ZO-2. At the ciliary basal body, ZO-2 depletion reduces the content of CEP164, KIF14, and IFT-B protein IFT57, while increasing the expression of p-Aurora and pAKT. These changes block primary cilium development and the response to Sonic Hedgehog signaling pathway stimulation. These results suggest that, rather than being a centrosomal architectural component, ZO-2 enhances microtubule stability and serves as a scaffold that facilitates the adequate accumulation of spindle pole and centriole proteins, allowing proper poleward spindle microtubule flux and cilia development.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00441-025-03992-0.

Three-dimensional electron microscopy analysis of ndc10-1 mutant reveals an aberrant organization of the mitotic spindle and spindle pole body defects in Saccharomyces cerevisiae

Current approaches for the analysis of spindle organization