Microtubule Nucleation Research Articles

Structure of the native γ-tubulin ring complex capping spindle microtubules

Microtubule (MT) filaments, composed of α/β-tubulin dimers, are fundamental to cellular architecture, function and organismal development. They are nucleated from MT organizing centers by the evolutionarily conserved γ-tubulin ring complex (γTuRC). However, the molecular mechanism of nucleation remains elusive. Here we used cryo-electron tomography to determine the structure of the native γTuRC capping the minus end of a MT in the context of enriched budding yeast spindles. In our structure, γTuRC presents a ring of γ-tubulin subunits to seed nucleation of exclusively 13-protofilament MTs, adopting an active closed conformation to function as a perfect geometric template for MT nucleation. Our cryo-electron tomography reconstruction revealed that a coiled-coil protein staples the first row of α/β-tubulin of the MT to alternating positions along the γ-tubulin ring of γTuRC. This positioning of α/β-tubulin onto γTuRC suggests a role for the coiled-coil protein in augmenting γTuRC-mediated MT nucleation. Based on our results, we describe a molecular model for budding yeast γTuRC activation and MT nucleation.

γ-TuRC asymmetry induces local protofilament mismatch at the RanGTP-stimulated microtubule minus end

The γ-tubulin ring complex (γ-TuRC) is a structural template for de novo microtubule assembly from α/β-tubulin units. The isolated vertebrate γ-TuRC assumes an asymmetric, open structure deviating from microtubule geometry, suggesting that γ-TuRC closure may underlie regulation of microtubule nucleation. Here, we isolate native γ-TuRC-capped microtubules from Xenopus laevis egg extract nucleated through the RanGTP-induced pathway for spindle assembly and determine their cryo-EM structure. Intriguingly, the microtubule minus end-bound γ-TuRC is only partially closed and consequently, the emanating microtubule is locally misaligned with the γ-TuRC and asymmetric. In the partially closed conformation of the γ-TuRC, the actin-containing lumenal bridge is locally destabilised, suggesting lumenal bridge modulation in microtubule nucleation. The microtubule-binding protein CAMSAP2 specifically binds the minus end of γ-TuRC-capped microtubules, indicating that the asymmetric minus end structure may underlie recruitment of microtubule-modulating factors for γ-TuRC release. Collectively, we reveal a surprisingly asymmetric microtubule minus end protofilament organisation diverging from the regular microtubule structure, with direct implications for the kinetics and regulation of nucleation and subsequent modulation of microtubules during spindle assembly.

CFDP1 regulates the stability of pericentric heterochromatin thereby affecting RAN GTPase activity and mitotic spindle formation.

The densely packed centromeric heterochromatin at minor and major satellites is comprised of H3K9me2/3 histones, the heterochromatin protein HP1α, and histone variants. In the present study, we sought to determine the mechanisms by which condensed heterochromatin at major and minor satellites stabilized by the chromatin factor CFDP1 affects the activity of the small GTPase Ran as a requirement for spindle formation. CFDP1 colocalized with heterochromatin at major and minor satellites and was essential for the structural stability of centromeric heterochromatin. Loss of CENPA, HP1α, and H2A.Z heterochromatin components resulted in decreased binding of the spindle nucleation facilitator RCC1 to minor and major satellite repeats. Decreased RanGTP levels as a result of diminished RCC1 binding interfered with chromatin-mediated microtubule nucleation at the onset of mitotic spindle formation. Rescuing chromatin H2A.Z levels in cells and mice lacking CFDP1 through knock-down of the histone chaperone ANP32E not only partially restored RCC1-dependent RanGTP levels but also alleviated CFDP1-knockout-related craniofacial defects and increased microtubule nucleation in CFDP1/ANP32E co-silenced cells. Together, these studies provide evidence for a direct link between condensed heterochromatin at major and minor satellites and microtubule nucleation through the chromatin protein CFDP1.

Abstract 527: Cancer cells in the resistant, endocycling cell state exhibit centrosome amplification

Abstract Therapy resistance is one of the most common underlying causes of poor prognosis in cancer patients, ultimately driving fatal outcome. We have previous demonstrated that following chemotherapeutic stress, a subset of cancer cells undergo a mitotic skip and enter an endocycle, causing whole-genome duplication without division. This endocycling cell state is resistant to cytotoxic therapies and thus is an actuator of therapy resistance. Cells in the endocycling state eventually undergo depolyploidization and repopulate the tumor, observed clinically as a recurrence. We also demonstrated that the minus-end directed motor protein KIFC1, a mediator of centrosome clustering, has higher expression in cells following treatment. Centrosome amplification (CA) involves aberrations in centrosome number, where cells have 3 centrosomes. Cells with extra centrosomes create a multi-polar spindle during mitosis, which presents high risk for cell death and arrest, but cancer cells with CA avoid this by clustering extra centrosomes to create a pseudo-bipolar spindle. Based upon this data, we hypothesize that studying centrosome dynamics and abnormalities presents a window into better understanding endocycle biology and this novel mechanism of therapy resistance. Cancer cells were treated with LD50 dose of cisplatin and released from treatment for varying lengths of time. CA in endocycling cells increased from 59.2% to 100% during the treatment recovery period while maintaining identical levels of clustering. Endocycling cancer cells have a nearly 5-fold increase in declustered centrosomes compared to proliferative cells. Centrosomes in endocycling cells are displaced from the perinuclear region and toward the cell periphery. We hypothesize that centrosome number may indicate the number of times a cell has endocycled and provide insight into the timing and mechanism of mitotic bypass. Centrosomes play a major role in microtubule nucleation, affecting cell motility and polarity, and act as signaling platforms, enhancing signaling activation and specificity. Because of this, we hypothesize that CA may be a critical mechanism for endocycling cancer cells to survive and maintain this resistant cell state. Citation Format: Madison Purkerson, Sarah Amend, Kenneth J. Pienta. Cancer cells in the resistant, endocycling cell state exhibit centrosome amplification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 527.

Abstract 628: RNA binding protein Matrin3 acts as a tumor suppressor by inhibiting microtubule nucleation

Abstract Introduction: Microtubule-associated proteins (MAPs) regulate microtubule dynamics, which is critical for controlling cell division, proliferation, migration, and intracellular transport. Due to their critical role in maintaining cellular integrity, microtubules have served as potent therapeutic targets for cancer treatment. Here, we report “Matrin 3 (MATR3)” as a novel MAP that interacts with β- tubulin and γ-tubulin, which constitutes microtubule organizing center (MTOC) essential for proper spindle formation and chromosomal segregation. Methods: Breast cancer cell lines (MDA-MB-231, MDA-MB-468, SKBR3, MCF7, BT549, HMEC, and MCF10A) were purchased from the American Type Culture Collection and cultured in standard medium. Breast cancer cells were transfected either with MATR3 overexpression plasmid or siRNA specific to MATR3. These overexpression/knockdown breast cancer cells were analyzed for cell viability, migration, invasion, colony formation, cell cycle, apoptosis assays, RNA sequencing, RT-qPCR, western blotting, RNA immunoprecipitation, and in vivo tumor xenograft study. Results: Our findings show that MATR3 binds to RNA encoding MAPs including β-tubulin and γ-tubulin, and regulate their stabilization. MATR3 also interacted with β-tubulin protein. Our data demonstrate that MATR3 functions as a tumor suppressor, as its overexpression inhibits cancer growth, while its depletion increased tumor growth in vivo. Mechanistic studies confirmed that MATR3 mediates its tumor suppressor role by regulating the expression of the MTOC-associated protein-encoding gene MZT2B (Mitotic Spindle Organizing Protein 2B), which is overexpressed in cancers and promotes cancer growth. Further, loss of MATR3 expression or function results in dysregulated microtubule dynamics and uncontrolled expression of the oncogenic proteins including MZT2B. Conclusions: In conclusion, our data strongly suggests that MATR3 serves as a novel RNA-binding protein with tumor suppressor properties. This discovery holds significant importance as MATR3 becomes the second protein, following Adenomatous Polyposis Coli (APC), to be identified for its binding to both MAP RNA and protein. Citation Format: Panneerdoss Subbarayalu, Subapriya Rajamanickam, Santosh Timilsina, Saif Nirzhor, Daisy Medina, Shahad Abdulsahib, Pitta V Prabhakar, Deepika Singh, Fu-Yang Li, Pooja Yadav, Esha Reddy, Krishna Evani, Vijay Eedunuri, Trong Phat Do, Benjamin C. Onyeagucha, Tabrez A. Mohammad, Jae-Hoon Ji, Yidong Chen, Nourhan Abdelfattah, Nicholas Dybdal-Hargreaves, Li-Ju Wang, Yu-Chiao Chiu, Suryavathi Viswanadhapalli, Ratna K. Vadlamudi, Manjeet K. Rao. RNA binding protein Matrin3 acts as a tumor suppressor by inhibiting microtubule nucleation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 628.

The fission yeast NDR kinase Orb6 and its signalling pathway MOR regulate cytoplasmic microtubule organization during the cell cycle.

Microtubule organization and reorganization during the cell cycle are achieved by regulation of the number, distribution and activity of microtubule-organizing centres (MTOCs). In fission yeast, the Mto1/2 complex determines the activity and distribution of cytoplasmic MTOCs. Upon mitosis, cytoplasmic microtubule nucleation ceases; inactivation of the Mto1/2 complex is triggered by Mto2 hyperphosphorylation. However, the protein kinase(s) that phosphorylates Mto2 remains elusive. Here we show that a conserved signalling network, called MOR (morphogenesis Orb6 network) in fission yeast, negatively regulates cytoplasmic MTOCs through Mto2 phosphorylation to ensure proper microtubule organization. Inactivation of Orb6 kinase, the most downstream MOR component, by attenuation of MOR signalling leads to reduced Mto2 phosphorylation, coincident with increased number of both Mto2 puncta and cytoplasmic microtubules. These defects cause the emergence of uncoordinated mitotic cells with cytoplasmic microtubules, resulting in reduced spindle assembly. Thus, the regulation of Mto2 by the MOR is crucial for cytoplasmic microtubule organization and contributes to reorganization of the microtubule cytoskeletons during the cell cycle.

CAMSAPs and nucleation-promoting factors control microtubule release from γ-TuRC

γ-Tubulin ring complex (γ-TuRC) is the major microtubule-nucleating factor. After nucleation, microtubules can be released from γ-TuRC and stabilized by other proteins, such as CAMSAPs, but the biochemical cross-talk between minus-end regulation pathways is poorly understood. Here we reconstituted this process in vitro using purified components. We found that all CAMSAPs could bind to the minus ends of γ-TuRC-attached microtubules. CAMSAP2 and CAMSAP3, which decorate and stabilize growing minus ends but not the minus-end tracking protein CAMSAP1, induced microtubule release from γ-TuRC. CDK5RAP2, a γ-TuRC-interactor, and CLASP2, a regulator of microtubule growth, strongly stimulated γ-TuRC-dependent microtubule nucleation, but only CDK5RAP2 suppressed CAMSAP binding to γ-TuRC-anchored minus ends and their release. CDK5RAP2 also improved selectivity of γ-tubulin-containing complexes for 13- rather than 14-protofilament microtubules in microtubule-capping assays. Knockout and overexpression experiments in cells showed that CDK5RAP2 inhibits the formation of CAMSAP2-bound microtubules detached from the microtubule-organizing centre. We conclude that CAMSAPs can release newly nucleated microtubules from γ-TuRC, whereas nucleation-promoting factors can differentially regulate this process.

The spindle protein CKAP2 regulates microtubule dynamics and ensures faithful chromosome segregation

Regulation of microtubule dynamics by microtubule-associated proteins (MAPs) is essential for mitotic spindle assembly and chromosome segregation. Altered microtubule dynamics, particularly increased microtubule growth rates, were found to be a contributing factor for the development of chromosomal instability, which potentiates tumorigenesis. The MAP XMAP215/CKAP5 is the only known microtubule growth factor, and whether other MAPs regulate microtubule growth in cells is unclear. Our recent in vitro reconstitution experiments have demonstrated that Cytoskeleton-Associated Protein 2 (CKAP2) increases microtubule nucleation and growth rates, and here, we find that CKAP2 is also an essential microtubule growth factor in cells. By applying CRISPR-Cas9 knock-in and knock-out (KO) as well as microtubule plus-end tracking live cell imaging, we show that CKAP2 is a mitotic spindle protein that ensures faithful chromosome segregation by regulating microtubule growth. Live cell imaging of endogenously labeled CKAP2 showed that it localizes to the spindle during mitosis and rapidly shifts its localization to the chromatin upon mitotic exit before being degraded. Cells lacking CKAP2 display reduced microtubule growth rates and an increased proportion of chromosome segregation errors and aneuploidy that may be a result of an accumulation of kinetochore-microtubule misattachments. Microtubule growth rates and chromosome segregation fidelity can be rescued upon ectopic CKAP2 expression in KO cells, revealing a direct link between CKAP2 expression and microtubule dynamics. Our results unveil a role of CKAP2 in regulating microtubule growth in cells and provide a mechanistic explanation for the oncogenic potential of CKAP2 misregulation.

Regulation of microtubule nucleation in mouse bone marrow-derived mast cells by ARF GTPase-activating protein GIT2.

Aggregation of high-affinity IgE receptors (FcϵRIs) on granulated mast cells triggers signaling pathways leading to a calcium response and release of inflammatory mediators from secretory granules. While microtubules play a role in the degranulation process, the complex molecular mechanisms regulating microtubule remodeling in activated mast cells are only partially understood. Here, we demonstrate that the activation of bone marrow mast cells induced by FcϵRI aggregation increases centrosomal microtubule nucleation, with G protein-coupled receptor kinase-interacting protein 2 (GIT2) playing a vital role in this process. Both endogenous and exogenous GIT2 were associated with centrosomes and γ-tubulin complex proteins. Depletion of GIT2 enhanced centrosomal microtubule nucleation, and phenotypic rescue experiments revealed that GIT2, unlike GIT1, acts as a negative regulator of microtubule nucleation in mast cells. GIT2 also participated in the regulation of antigen-induced degranulation and chemotaxis. Further experiments showed that phosphorylation affected the centrosomal localization of GIT2 and that during antigen-induced activation, GIT2 was phosphorylated by conventional protein kinase C, which promoted microtubule nucleation. We propose that GIT2 is a novel regulator of microtubule organization in activated mast cells by modulating centrosomal microtubule nucleation.

Transition of human γ-tubulin ring complex into a closed conformation during microtubule nucleation.

Microtubules are essential for intracellular organization and chromosome segregation. They are nucleated by the γ-tubulin ring complex (γTuRC). However, isolated vertebrate γTuRC adopts an open conformation that deviates from the microtubule structure, raising the question of the nucleation mechanism. In this study, we determined cryo-electron microscopy structures of human γTuRC bound to a nascent microtubule. Structural changes of the complex into a closed conformation ensure that γTuRC templates the 13-protofilament microtubules that exist in human cells. Closure is mediated by a latch that interacts with incorporating tubulin, making it part of the closing mechanism. Further rearrangements involve all γTuRC subunits and the removal of the actin-containing luminal bridge. Our proposed mechanism of microtubule nucleation by human γTuRC relies on large-scale structural changes that are likely the target of regulation in cells.

Understanding microtubule nucleation through the conformational dynamics of α, β, and γ-tubulins

Understanding microtubule nucleation through the conformational dynamics of α, β, and γ-tubulins

Identification of the key genes and mechanisms associated with transcatheter arterial chemoembolisation refractoriness in hepatocellular carcinoma.

Transcatheter arterial embolisation (TACE) is the primary treatment for intermediate-stage hepatocellular carcinoma (HCC) patients while some HCC cases have shown resistance to TACE. To investigate the key genes and potential mechanisms correlated with TACE refractoriness in HCC. The microarray datasets of TACE-treated HCC tissues, HCC and non-HCC tissues were collected by searching multiple public databases. The respective differentially expressed genes (DEGs) were attained via limma R package. Weighted gene co-expression network analysis was employed for identifying the significant modules related to TACE non-response. TACE refractoriness-related genes were obtained by intersecting up-regulated TACE-associated and HCC-associated DEGs together with the genes in significant modules related to TACE non-response. The key genes expression in the above two pairs of samples was compared respectively via Wilcoxon tests and standard mean differences model. The prognostic value of the key genes was evaluated by Kaplan-Meier curve. Multivariate analysis was utilised to investigate the independent prognostic factor in key genes. Single-cell RNA (scRNA) sequencing analysis was conducted to explore the cell types in HCC. TACE refractoriness-related genes activity was calculated via AUCell packages. The CellChat R package was used for the investigation of the cell-cell communication between the identified cell types. HCC tissues of TACE non-responders (n = 66) and TACE responders (n = 81), HCC (n = 3941) and non-HCC (n = 3443) tissues were obtained. The five key genes, DLG associated protein 5 (DLGAP5), Kinesin family member 20A (KIF20A), Assembly factor for spindle microtubules (ASPM), Kinesin family member 11 (KIF11) and TPX2 microtubule nucleation factor (TPX2) in TACE refractoriness-related genes, were identified. The five key genes were all up-regulated in the TACE non-responders group and the HCC group. High expression of the five key genes predicted poor prognosis in HCC. Among the key genes, TPX2 was an independent prognostic factor. Four cell types, hepatocytes, embryonic stem cells, T cells and B cells, were identified in the HCC tissues. The TACE refractoriness-related genes expressed primarily in hepatocytes and embryonic stem cells. Hepatocytes, as the providers of ligands, had the strongest interaction with embryonic stem cells that provided receptors. Five key genes (DLGAP5, KIF20A, ASPM, KIF11 and TPX2) were identified as promoting refractory TACE. Hepatocytes and embryonic stem cells were likely to boost TACE refractoriness.

SiRNA-Mediated BmAurora B Depletion Impedes the Formation of Holocentric Square Spindles in Silkworm Metaphase BmN4 Cells.

Silkworm ovary-derived BmN4 cells rely on chromatin-induced spindle assembly to form microtubule-based square mitotic spindles that ensure accurate segregation of holocentric chromosomes during cell division. The chromosome passenger protein Aurora B regulates chromosomal condensation and segregation, spindle assembly checkpoint activation, and cytokinesis; however, its role in holocentric organisms needs further clarification. This study examined the architecture and dynamics of spindle microtubules during prophase and metaphase in BmN4 cells and those with siRNA-mediated BmAurora B knockdown using immunofluorescence labeling. Anti-α-tubulin and anti-γ-tubulin antibodies revealed faint γ-tubulin signals colocalized with α-tubulin in early prophase during nuclear membrane rupture, which intensified as prophase progressed. At this stage, bright regions of α-tubulin around and on the nuclear membrane surrounding the chromatin suggested the start of microtubules assembling in the microtubule-organizing centers (MTOCs). In metaphase, fewer but larger γ-tubulin foci were detected on both sides of the chromosomes. This resulted in a distinctive multipolar square spindle with holocentric chromosomes aligned at the metaphase plate. siRNA-mediated BmAurora B knockdown significantly reduced the γ-tubulin foci during prophase, impacting microtubule nucleation and spindle structure in metaphase. Spatiotemporal BmAurora B expression analysis provided new insights into the regulation of this mitotic kinase in silkworm larval gonads during gametogenesis. Our results suggest that BmAurora B is crucial for the formation of multipolar square spindles in holocentric insects, possibly through the activation of γ-tubulin ring complexes in multiple centrosome-like MTOCs.

Building on-chip cytoskeletal circuits via branched microtubule networks

Controllable platforms to engineer robust cytoskeletal scaffolds have the potential to create novel on-chip nanotechnologies. Inspired by axons, we combined the branching microtubule (MT) nucleation pathway with microfabrication to develop "cytoskeletal circuits." This active matter platform allows control over the adaptive self-organization of uniformly polarized MT arrays via geometric features of microstructures designed within a microfluidic confinement. We build and characterize basic elements, including turns and divisions, as well as complex regulatory elements, such as biased division and MT diodes, to construct various MT architectures on a chip. Our platform could be used in diverse applications, ranging from efficient on-chip molecular transport to mechanical nano-actuators. Further, cytoskeletal circuits can serve as a tool to study how the physical environment contributes to MT architecture in living cells.

Fe3O4 and Fe3O4core Aushell-based Hyperthermia Reduces Expression of Proliferation Markers Ki-67, TOP2A and TPX2 in a Human Breast Cancer Cell Line.

Hyperthermia represents an adjuvant local anticancer strategy which relies on the increase of temperature beyond the physiological level. In this study, we investigated the anticancer potential of Fe3O4 and Fe3O4core Aushell nanoparticles as hyperthermic agents in terms of cytotoxicity and studied the expression of cellular markers of proliferation (changes in mRNA levels via real-time polymerase chain reaction). The human breast cancer cell line SK-BR-1 was incubated with either Fe3O4 or Fe3O4core Aushell nanoparticles stabilized with tryptophan, prior to hyperthermia treatment. The normal HEK293 cell line was used as a control. Toxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay to estimate possible toxic effects of the tested nanoparticles. After RNA extraction and cDNA synthesis, mRNA expression of three indicators of proliferation, namely marker of proliferation Ki-67, DNA topoisomerase II alpha (TOP2A) and TPX2 microtubule nucleation factor (TPX2), was investigated. At each concentration tested, Fe3O4core Aushell nanoparticles showed greater toxicity compared to Fe3O4, while SK-BR-3 cells were more susceptible to their cytotoxic effects compared to the HEK293 cell line. The expression of Ki-67, TOP2A and TPX2 was reduced in SK-BR-3 cells by both Fe3O4 or Fe3O4core Aushell nanoparticles compared to untreated cells, while the only observed change in HEK293 cells was the up-regulation of TOP2A. Both Fe3O4core Aushell and Fe3O4 NPs exhibit increased cytotoxicity to the cancer cell line tested (SK-BR-3) compared to HEK293 cells. The down-regulation in SK-BR-3 cells of the three proliferative markers studied, Ki-67, TOP2A and TPX2, after incubation with NPs suggests that cells that survived thermal destruction were not actively proliferating.

The microtubule-nucleating factor MACERATOR tethers AUGMIN7 to microtubules and governs phragmoplast architecture.

The plant cytokinetic microtubule array, called the phragmoplast, exhibits higher microtubule dynamics in its center (midzone) than at the periphery (distal zone). This behavior is known as the axial asymmetry. Despite being a major characteristic of the phragmoplast, little is known about regulators of this phenomenon. Here we address the role of microtubule nucleation in axial asymmetry by characterizing MACERATOR (MACET) proteins in Arabidopsis thaliana and Nicotiana benthamiana with a combination of genetic, biochemical, and live-cell imaging assays, using photo-convertible microtubule probes, and modeling. MACET paralogs accumulate at the shrinking microtubule ends and decrease the tubulin OFF rate. Loss of MACET4 and MACET5 function abrogates axial asymmetry by suppressing microtubule dynamicity in the midzone. MACET4 also narrows the microtubule nucleation angle at the phragmoplast leading edge and functions as a microtubule tethering factor for AUGMIN COMPLEX SUBUNIT 7 (AUG7). The macet4 macet5 double mutant shows diminished clustering of AUG7 in the phragmoplast distal zone. Knockout of AUG7 does not affect MACET4 localization, axial asymmetry, or microtubule nucleation angle, but increases phragmoplast length and slows down phragmoplast expansion. The mce4-1 mce5 aug7-1 triple knockout is not viable. Experimental data and modeling demonstrate that microtubule nucleation factors regulate phragmoplast architecture and axial asymmetry directly by generating new microtubules and indirectly by modulating the abundance of free tubulin.

Branched microtubule nucleation and dynein transport organize RanGTP asters in Xenopus laevis egg extract.

Chromosome segregation relies on the correct assembly of a bipolar spindle. Spindle pole self-organization requires dynein-dependent microtubule (MT) transport along other MTs. However, during M-phase RanGTP triggers MT nucleation and branching generating polarized arrays with nonastral organization in which MT minus ends are linked to the sides of other MTs. This raises the question of how branched-MT nucleation and dynein-mediated transport cooperate to organize the spindle poles. Here, we used RanGTP-dependent MT aster formation in Xenopus laevis (X. laevis) egg extract to study the interplay between these two seemingly conflicting organizing principles. Using temporally controlled perturbations of MT nucleation and dynein activity, we found that branched MTs are not static but instead dynamically redistribute over time as poles self-organize. Our experimental data together with computer simulations suggest a model where dynein together with dynactin and NuMA directly pulls and move branched MT minus ends toward other MT minus ends.

Evidence for microtubule nucleation at the Golgi in breast cancer cells.

Golgi-derived microtubule (MT) arrays are essential to directionally persistent cell migration and vesicle transport. In this study, we have examined MT nucleation sites in two breast cancer cell lines, MDA-MB-231 and MCF-7, with the hypothesis that only the migratory invasive MDA-MB-231 cells exhibit MTs originating from the Golgi. MTs were disassembled and allowed to slightly regrow so individual nucleation sites could then be observed via fluorescently tagged antibodies (α-tubulin, cis-Golgi marker GM130, and EB1-a MT plus-end binding protein) and confocal microscopy. To determine if MT nucleation at the Golgi is more apparent during active migration compared to when cells are stationary, cells were treated with the chemoattractant epidermal growth factor (EGF) and examined for colocalizations between the Golgi, α-tubulin, and γ-tubulin. Images were analyzed qualitatively for color overlap, and quantitatively using Manders Colocalization Coefficients. Differences between groups were tested for significance using one-way analysis of variances and Tukey's post hoc test. Significantly higher colocalization values (coloc) in the highly invasive MDA-MB-231 cells (α-tubulin coloc GM130 = 0.39, GM130 coloc α-tubulin = 0.82, GM130 coloc EB1 = 0.24, and EB1 coloc GM130 = 0.38) compared to the weakly invasive MCF-7 cells (0.15, 0.08, 0.02, and 0.16, respectively) were observed. EGF-treated cells exhibited higher colocalization values than control cells for three of the four protein combinations tested, but EGF-treated MDA-MB-231 cells exhibited significantly higher values (α-tubulin coloc GM130 = 0.20, GM130 coloc α-tubulin = 0.89, and γ-tubulin coloc GM130 = 0.47) than both control groups as well as the EGF-treated MCF-7 cells. Results support the hypothesis that MT nucleation at the Golgi occurs more frequently in the invasive MDA-MB-231 cell line compared to the weakly invasive MCF-7 cells. The presence or absence of Golgi-derived MTs may help to explain the difference in migratory potential commonly exhibited by these two cell lines.

Abl2 repairs microtubules and phase separates with tubulin to promote microtubule nucleation

Abl family kinases are evolutionarily conserved regulators of cell migration and morphogenesis. Genetic experiments in Drosophila suggest that Abl family kinases interact functionally with microtubules to regulate axon guidance and neuronal morphogenesis. Vertebrate Abl2 binds to microtubules and promotes their plus-end elongation, both invitro and in cells, but the molecular mechanisms by which Abl2 regulates microtubule (MT) dynamics are unclear. We report here that Abl2 regulates MT assembly via condensation and direct interactions with both the MT lattice and tubulin dimers. We find that Abl2 promotes MT nucleation, which is further facilitated by the ability of the Abl2 C-terminal half to undergo liquid-liquid phase separation (LLPS) and form co-condensates with tubulin. Abl2 binds to regions adjacent to MT damage, facilitates MT repair via fresh tubulin recruitment, and increases MT rescue frequency and lifetime. Cryo-EM analyses strongly support a model in which Abl2 engages tubulin C-terminal tails along an extended MT lattice conformation at damage sites to facilitate repair via fresh tubulin recruitment. Abl2Δ688-790, which closely mimics a naturally occurring splice isoform, retains binding to the MT lattice but does not bind tubulin, promote MT nucleation, or increase rescue frequency. In COS-7 cells, MT reassembly after nocodazole treatment is greatly slowed in Abl2 knockout COS-7 cells compared with wild-type cells, and these defects are rescued by re-expression of Abl2, but not Abl2Δ688-790. We propose that Abl2 locally concentrates tubulin to promote MT nucleation and recruits it to defects in the MT lattice to enable repair and rescue.

Multifaceted modes of γ-tubulin complex recruitment and microtubule nucleation at mitotic centrosomes.

Microtubule nucleation is mediated by γ-tubulin ring complexes (γ-TuRCs). In most eukaryotes, a GCP4/5/4/6 "core" complex promotes γ-tubulin small complex (γ-TuSC) association to generate cytosolic γ-TuRCs. Unlike γ-TuSCs, however, this core complex is non-essential in various species and absent from budding yeasts. In Drosophila, Spindle defective-2 (Spd-2) and Centrosomin (Cnn) redundantly recruit γ-tubulin complexes to mitotic centrosomes. Here, we show that Spd-2 recruits γ-TuRCs formed via the GCP4/5/4/6 core, but Cnn can recruit γ-TuSCs directly via its well-conserved CM1 domain, similar to its homologs in budding yeast. When centrosomes fail to recruit γ-tubulin complexes, they still nucleate microtubules via the TOG domain protein Mini-spindles (Msps), but these microtubules have different dynamic properties. Our data, therefore, help explain the dispensability of the GCP4/5/4/6 core and highlight the robustness of centrosomes as microtubule organizing centers. They also suggest that the dynamic properties of microtubules are influenced by how they are nucleated.