Kinetochore-microtubule Attachments Research Articles

The Ndc80-Cdt1-Ska1 complex is a central processive kinetochore-microtubule coupling unit.

It is known that microtubule-binding proteins including the Ska1 complex and the DNA replication licensing factor, Cdt1, enable the kinetochore-localized Ndc80 complex to form robust kinetochore-microtubule attachments. However, it is not clear how the Ndc80 complex is stably coupled to dynamic spindle microtubule plus-ends. Here, we have developed a conditional auxin-inducible degron approach to reveal a function for Cdt1 in chromosome segregation and kinetochore-microtubule interactions that is separable from its role in DNA replication licensing. Further, we demonstrate that a direct interaction between Cdt1 and Ska1 is required for recruiting Cdt1 to kinetochores and spindle microtubules. Cdt1 phosphorylation by Cdk1 kinase is critical for Ska1 binding, kinetochore-microtubule attachments, and mitotic progression. Furthermore, we show that Cdt1 synergizes with Ndc80 and Ska1 for microtubule binding, including forming a diffusive, tripartite Ndc80-Cdt1-Ska1 complex that can processively track dynamic microtubule plus-ends in vitro. Taken together, our data identify the Ndc80-Cdt1-Ska1 complex as a central molecular unit that can promote processive bidirectional tip-tracking of microtubules by kinetochores.

Stable kinetochore-microtubule attachment requires loop-dependent Ndc80-Ndc80 binding.

During cell division, kinetochores link chromosomes to spindle microtubules. The Ndc80 complex, a crucial microtubule binder, populates each kinetochore with dozens of copies. Whether adjacent Ndc80 complexes cooperate to promote microtubule binding remains unclear. Here we demonstrate that the Ndc80 loop, a short sequence that interrupts the Ndc80 coiled-coil at a conserved position, folds into a more rigid structure than previously assumed and promotes direct interactions between full-length Ndc80 complexes on microtubules. Mutations in the loop impair these Ndc80-Ndc80 interactions, prevent the formation of force-resistant kinetochore-microtubule attachments, and cause cells to arrest in mitosis for hours. This arrest is not due to an inability to recruit the kinetochore-microtubule stabilizing SKA complex and cannot be overridden by mutations in the Ndc80 tail that strengthen microtubule attachment. Thus, loop-mediated organization of adjacent Ndc80 complexes is crucial for stable end-on kinetochore-microtubule attachment and spindle assembly checkpoint satisfaction.

The role of kinetochore dynein in checkpoint silencing is restricted to disassembly of the corona.

During mitosis, kinetochore-microtubule attachments are monitored by a molecular surveillance system known as the spindle assembly checkpoint. The prevailing model posits that dynein evicts checkpoint proteins (e.g., Mad1, Mad2) from stably attached kinetochores by transporting them away from kinetochores, thus contributing to checkpoint silencing. However, the mechanism by which dynein performs this function, and its precise role in checkpoint silencing remain unresolved. Here, we find that dynein's role in checkpoint silencing is restricted to evicting checkpoint effectors from the fibrous corona, and not the outer kinetochore. Dynein evicts these molecules from the corona in a manner that does not require stable, end-on microtubule attachments. Thus, by disassembling the corona through indiscriminate microtubule encounters, dynein primes the checkpoint signaling apparatus so it can respond to stable end-on microtubule attachments and permit cells to progress through mitosis. Accordingly, we find that dynein function in checkpoint silencing becomes largely dispensable in cells in which checkpoint effectors are excluded from the corona.

Structural basis of centromeric cohesion protection

In the early stages of mitosis, cohesin is released from chromosome arms but not from centromeres. The protection of centromeric cohesin by SGO1 maintains the sister chromatid cohesion that resists the pulling forces of microtubules until all chromosomes are attached in a bipolar manner to the mitotic spindle. Here we present the X-ray crystal structure of a segment of human SGO1 bound to a conserved surface of the cohesin complex. SGO1 binds to a composite interface formed by the SA2 and SCC1RAD21 subunits of cohesin. SGO1 shares this binding interface with CTCF, indicating that these distinct chromosomal regulators control cohesin through a universal principle. This interaction is essential for the localization of SGO1 to centromeres and protects centromeric cohesin against WAPL-mediated cohesin release. SGO1–cohesin binding is maintained until the formation of microtubule–kinetochore attachments and is required for faithful chromosome segregation and the maintenance of a stable karyotype.

Coordination between the Ndc80 complex and dynein is essential for microtubule plus-end capture by kinetochores during early mitosis

Mitotic kinetochores are initially captured by dynamic microtubules via a "search-and-capture" mechanism. The microtubule motor, dynein, is critical for kinetochore capture as it has been shown to transport microtubule-attached chromosomes toward the spindle pole during prometaphase. The microtubule-binding nuclear division cycle 80 (Ndc80) complex that is recruited to kinetochores in prophase is known to play a central role in forming kinetochore-microtubule (kMT) attachments in metaphase. It is not yet clear, however, how Ndc80 contributes to initial kMT capture during prometaphase. Here, by combining CRISPR/Cas9-mediated knockout and RNAi technology with assays specific to study kMT capture, we show that mitotic cells lacking Ndc80 exhibit substantial defects in this function during prometaphase. Rescue experiments show that Ndc80 mutants deficient in microtubule-binding are unable to execute proper kMT capture. While cells inhibited of dynein alone are predominantly able to make initial kMT attachments, cells co-depleted of Ndc80 and dynein show severe defects in kMT capture. Further, we use an in vitro total internal reflection fluorescence microscopy assay to reconstitute microtubule capture events, which suggest that Ndc80 and dynein coordinate with each other for microtubule plus-end capture and that the phosphorylation status of Ndc80 is critical for productive kMT capture. A novel interaction between Ndc80 and dynein that we identify in prometaphase extracts might be critical for efficient plus-end capture. Thus, our studies, for the first time, identify a distinct event in the formation of initial kMT attachments, which is directly mediated by Ndc80 and in coordination with dynein is required for efficient kMT capture and chromosome alignment.

Ru360 protects against vitrification-induced oocyte meiotic defects by restoring mitochondrial function

Oocyte vitrification has been widely application in female fertility preservation. Recent studies found that vitrification of immature (germinal vesicle stage, GV) oocytes increased the risk of aneuploidy during meiotic maturation; however, the underlying mechanisms and the strategies to prevent this defect remain unexplored. In this study, we found that vitrification of GV oocytes decreased the first polarbody extrusion rate (90.51 ± 1.04% vs. 63.89 ± 1.39%, p < 0.05) and increased the aneuploid rate (2.50% vs. 20.00%, p < 0.05), accompanied with a series of defects during meiotic maturation, including aberrant spindle morphology, chromosome misalignment, incorrect Kinetochore-Microtubule attachments (KT-MTs) and weakened spindle assembly checkpoint protein complex (SAC) function. We also found that vitrification disrupted mitochondrial function by increasing mitochondrial Ca2+ levels. Importantly, inhibition of mitochondrial Ca2+ entry by 1 μM Ru360 significantly restored mitochondrial function and rescued the meiotic defects, indicating that the increase of mitochondrial Ca2+, at least, was a cause of meiotic defects in vitrified oocytes. These results shed light on the molecular mechanisms of oocyte vitrification-induced adverse effects of meiotic maturation and provided a potential strategy to improve oocyte cryopreservation protocols further.

Double-checking chromosome segregation.

Enduring chromosome segregation errors represent potential threats to genomic stability due to eventual chromosome copy number alterations (aneuploidy) and formation of micronuclei-key intermediates of a rapid mutational process known as chromothripsis that is found in cancer and congenital disorders. The spindle assembly checkpoint (SAC) has been viewed as the sole surveillance mechanism that prevents chromosome segregation errors during mitosis and meiosis. However, different types of chromosome segregation errors stemming from incorrect kinetochore-microtubule attachments satisfy the SAC and are more frequent than previously anticipated. Remarkably, recent works have unveiled that most of these errors are corrected during anaphase and only rarely result in aneuploidy or formation of micronuclei. Here, we discuss recent progress in our understanding of the origin and fate of chromosome segregation errors that satisfy the SAC and shed light on the surveillance, correction, and clearance mechanisms that prevent their transmission, to preserve genomic stability.

Abstract 1663: Development of a novel oral microtubule targeting agent with pan-cancer efficacy

Abstract Microtubule-targeting agents (MTAs) are among the most effective chemotherapeutics used in the treatment of cancer. However, the clinical utility of current MTAs, such as paclitaxel and vinblastine, are often limited due to adverse side effects or multidrug resistance (MDR) driving the continuous pursuit for the development of novel microtubule interactors. Here, we report the development of a novel non P-gp substrate MTA that is metabolically stable, and that displays broad-spectrum anti-cancer activity in vitro and ex vivo. Anti-cancer activity was assessed by screening a panel of 102 cancer cell lines and in a multi-tumor type panel of patient-derived organoids (PDOs). In vitro, double to triple digit nanomolar (ranging from 17nM to 318 nM) potency was observed in 101 cell lines, while viability was reduced by more than 70% in 76 cell lines out of 102. Similarly, 23 out of 34 (68%) of ex vivo treated PDOs were highly or partially responsive as indicated by a 37-73% decrease in viability. Studies using spindle assembly checkpoint (SAC) inhibitors demonstrated a mechanism at or before microtubule-kinetochore attachment in M phase of the cell cycle. Subsequent microscopic evaluation of kinetochore assembly revealed a lack of proper mitotic spindle formation highlighted with an increase in Mad1 signal intensity emphasizing metaphase arrest. Furthermore, in vitro tubulin polymerization assays demonstrated that the anti-cancer effect is a result of cytoskeleton targeting to inhibit microtubule assembly. Additionally, the antimicrotubule agent inhibited the formation of EBI (N,N’-ethylene-bis(iodoacetamide)):β-tubulin adducts indicating an occupied colchicine-binding site of tubulin. Six multi-tumor preclinical oncology models were evaluated for in vivo anti-tumor efficacy by oral administration of the compound. Further, tumor growth inhibition (%) was demonstrated in preclinical murine models of Acute Myeloid Leukemia (AML) (MV-4-11, 51%), colorectal (COLO205, 35%), prostate (DU 145, 38%), and gastric cancer (HS746T, 74%; SNU-5, 48%). Moreover, tumor tissue analysis for prominent mitotic cell cycle markers, Cyclin B1 and pHH3, revealed a dose-dependent elevation of both markers revealing mitotic arrest phenotype. Together, our data supports further preclinical and clinical development of a novel oral MTA notably in Taxane resistant and/or Taxane sensitive tumors. Citation Format: Maria-Dorothea Nastke, Jacob Matson, Andressa Mota, Archna Ravi, Alex Batrouni, Marcus O'Hara, Shiva Kazerounian, Arcan Guven, Nicole Pellegrino, Kashni Grover, Kayleigh Gray, Anne Diers, Michael Kiebish, Vivek Vishnudas, Stephane Gesta. Development of a novel oral microtubule targeting agent with pan-cancer efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1663.

Abstract 317: Particulate matter accumulates chromosomal instability during mitosis

Abstract Particulate matter (PM), a major component of outdoor air pollution triggers genotoxicity as well as cytotoxicity. Although the harmful effects of PM on DNA damage and its carcinogenic risk have been thoroughly studied, the detailed mechanism by which PM affects chromosomal stability remains unclear. Maintenance of chromosome is finely monitored during mitotic phase through multiple proteins and structures. The present study shows that treatment of urban particulate matter (UPM) inhibited proliferation of lung cells through the accumulation of chromosomal instability which is determined by the formation of multinucleation and macronucleation. In particular, PM treatment prolonged mitotic duration. This delay in mitotic progression resulted from inactivation of Aurora B at prometaphase and metaphase, causing a failure in dynamic stabilization of kinetochore-microtubule attachment. In addition, ingression rate of cleavage furrow and actin clearing from polar cortex were significantly reduced at telophase. It indicates that dysregulation of spatiotemporal dynamics of mitotic proteins and structures finally disturbs cytokinetic division. Overall, PM leads to accumulation of chromosomal instability, finally contributing to its carcinogenicity. Citation Format: Jaewook Ryu, Ja-Eun Kim. Particulate matter accumulates chromosomal instability during mitosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 317.

Distinct Aurora B pools at the inner centromere and kinetochore have different contributions to meiotic and mitotic chromosome segregation.

Proper chromosome segregation depends on the establishment of bioriented kinetochore-microtubule attachments, which often requires multiple rounds of release and reattachment. Aurora B and C kinases phosphorylate kinetochore proteins to release tensionless attachments. Multiple pathways recruit Aurora B/C to the centromere and kinetochore. We studied how these pathways contribute to anaphase onset timing and correction of kinetochore-microtubule attachments in budding yeast meiosis and mitosis. We find that the pool localized by the Bub1/Bub3 pathway sets the normal duration of meiosis and mitosis, in differing ways. Our meiosis data suggests a model that disruption of this pathway leads to PP1 kinetochore localization, which dephosphorylates Cdc20 for premature anaphase onset. For error correction, the Bub1/Bub3 and COMA pathways are individually important in meiosis but compensatory in mitosis. Finally, we find that the haspin and Bub1/3 pathways function together to ensure error correction in mouse oogenesis. Our results suggest that each recruitment pathway localizes spatially distinct kinetochore-localized Aurora B/C pools that function differently between meiosis and mitosis.

Initial spindle positioning at the oocyte center protects against incorrect kinetochore-microtubule attachment and aneuploidy in mice.

Spindle positioning within the oocyte must be tightly regulated. In mice, the spindle is predominantly assembled at the oocyte center before its migration toward the cortex to achieve the highly asymmetric division, a characteristic of female meiosis. The significance of the initial central positioning of the spindle is largely unknown. We show that initial spindle positioning at the oocyte center is an insurance mechanism to avoid the premature exposure of the spindle to cortical CDC42 signaling, which perturbs proper kinetochore-microtubule attachments, leading to the formation of aneuploid gametes. These findings contribute to understanding why female gametes are notoriously associated with high rates of aneuploidy, the leading genetic cause of miscarriage and congenital abnormalities.

SH2D4A promotes centrosome maturation to support spindle microtubule formation and mitotic progression

Mitotic progression requires the precise formation of spindle microtubules based on mature centrosomes. During the G2/M transition, centrosome maturation progresses, and associated microtubules bundle to form mitotic spindle fibers and capture the chromosomes for alignment at the cell equator. Mitotic kinases-induced phosphorylation signaling is necessary for these processes. Here, we identified SH2 domain-containing protein 4A (SH2D4A/PPP1R38) as a new mitotic regulator. SH2D4A knockdown delays mitotic progression. The time-lapse imaging analysis showed that SH2D4A specifically contributes to the alignment of chromosomes. The cold treatment assay and microtubule regrowth assay indicated that SH2D4A promotes microtubule nucleation to support kinetochore–microtubule attachment. This may be due to the centrosome maturation by SH2D4A via centrosomal recruitment of pericentriolar material (PCM) such as cep192, γ-tubulin, and PLK1. SH2D4A was found to be a negative regulator of PP1 phosphatase. Consistently, treatment with a PP1 inhibitor rescues SH2D4A-knockdown-induced phenotypes, including the microtubule nucleation and centrosomal recruitment of active PLK1. These results suggest that SH2D4A is involved in PCM recruitment to centrosomes and centrosome maturation through attenuation of PP1 phosphatases, accelerating the spindle formation and supporting mitotic progression.

The importance of microtubule-dependent tension in accurate chromosome segregation.

Accurate chromosome segregation is vital for cell and organismal viability. The mitotic spindle, a bipolar macromolecular machine composed largely of dynamic microtubules, is responsible for chromosome segregation during each cell replication cycle. Prior to anaphase, a bipolar metaphase spindle must be formed in which each pair of chromatids is attached to microtubules from opposite spindle poles. In this bipolar configuration pulling forces from the dynamic microtubules can generate tension across the sister kinetochores. The tension status acts as a signal that can destabilize aberrant kinetochore-microtubule attachments and reinforces correct, bipolar connections. Historically it has been challenging to isolate the specific role of tension in mitotic processes due to the interdependency of attachment and tension status at kinetochores. Recent technical and experimental advances have revealed new insights into how tension functions during mitosis. Here we summarize the evidence that tension serves as a biophysical signal that unifies multiple aspects of kinetochore and centromere function to ensure accurate chromosome segregation.

The fission yeast kinetochore complex Mhf1-Mhf2 regulates the spindle assembly checkpoint and faithful chromosome segregation.

The outer kinetochore serves as a platform for the initiation of the spindle assembly checkpoint (SAC) and for mediating kinetochore-microtubule attachments. How the inner kinetochore subcomplex CENP-S-CENP-X is involved in regulating the SAC and kinetochore-microtubule attachments has not been well characterized. Using live-cell microscopy and yeast genetics, we found that Mhf1-Mhf2, the CENP-S-CENP-X counterpart in the fission yeast Schizosaccharomyces pombe, plays crucial roles in promoting the SAC and regulating chromosome segregation. The absence of Mhf2 attenuates the SAC, impairs the kinetochore localization of most of the components in the constitutive centromere-associated network (CCAN), and alters the localization of the kinase Ark1 (yeast homolog of Aurora B) to the kinetochore. Hence, our findings constitute a model in which Mhf1-Mhf2 ensures faithful chromosome segregation by regulating the accurate organization of the CCAN complex, which is required for promoting SAC signaling and for regulating kinetochore-microtubule attachments. This article has an associated First Person interview with the first author of the paper.

CCDC86 is a novel Ki-67-interacting protein important for cell division.

The chromosome periphery is a network of proteins and RNAs that coats the outer surface of mitotic chromosomes. Despite the identification of new components, the functions of this complex compartment are poorly characterised. In this study, we identified a novel chromosome periphery-associated protein, CCDC86 (also known as cyclon). Using a combination of RNA interference, microscopy and biochemistry, we studied the functions of CCDC86 in mitosis. CCDC86 depletion resulted in partial disorganisation of the chromosome periphery with alterations in the localisation of Ki-67 (also known as MKI67) and nucleolin (NCL), and the formation of abnormal cytoplasmic aggregates. Furthermore, CCDC86-depleted cells displayed errors in chromosome alignment, altered spindle length and increased apoptosis. These results suggest that, within the chromosome periphery, different subcomplexes that include CCDC86, nucleolin and B23 (nucleophosmin or NPM1) are required for mitotic spindle regulation and correct kinetochore-microtubule attachments, thus contributing to chromosome segregation in mitosis. Moreover, we identified CCDC86 as a MYCN-regulated gene, the expression levels of which represent a powerful marker for prognostic outcomes in neuroblastoma.

Kinetochore-microtubule attachment in human cells is regulated by the interaction of a conserved motif of Ska1 with EB1

The kinetochore establishes the linkage between chromosomes and the spindle microtubule plus ends during mitosis. In vertebrates, the spindle-kinetochore-associated (Ska1,2,3) complex stabilizes kinetochore attachment with the microtubule plus ends, but how Ska is recruited to and stabilized at the kinetochore-microtubule interface is not understood. Here, our results show that interaction of Ska1 with the general microtubule plus end-associated protein EB1 through a conserved motif regulates Ska recruitment to kinetochores in human cells. Ska1 forms a stable complex with EB1 via interaction with the motif in its N-terminal disordered loop region. Disruption of this interaction either by deleting or mutating the motif disrupts Ska complex recruitment to kinetochores and induces chromosome alignment defects, but it does not affect Ska complex assembly. Atomic-force microscopy imaging revealed that Ska1 is anchored to the C-terminal region of the EB1 dimer through its loop and thereby promotes formation of extended structures. Furthermore, our NMR data showed that the Ska1 motif binds to the residues in EB1 that are the binding sites of other plus end targeting proteins that are recruited to microtubules by EB1 through a similar conserved motif. Collectively, our results demonstrate that EB1-mediated Ska1 recruitment onto the microtubule serves as a general mechanism for the formation of vertebrate kinetochore-microtubule attachments and metaphase chromosome alignment.

The deubiquitinating enzyme complex BRISC regulates Aurora B activation via lysine-63-linked ubiquitination in mitosis

Faithful chromosome segregation requires bi-oriented kinetochore-microtubule attachment on the metaphase spindle. Aurora B kinase, the catalytic core of the chromosome passage complex (CPC), plays a crucial role in this process. Aurora B activation has widely been investigated in the context of protein phosphorylation. Here, we report that Aurora B is ubiquitinated in mitosis through lysine-63 ubiquitin chains (K63-Ub), which is required for its activation. Mutation of Aurora B at its primary K63 ubiquitin site inhibits its activation, reduces its kinase activity, and disrupts the association of Aurora B with other components of CPC, leading to severe mitotic defects and cell apoptosis. Moreover, we identify that BRCC36 isopeptidase complex (BRISC) is the K63-specific deubiquitinating enzyme for Aurora B. BRISC deficiency augments the accumulation of Aurora B K63-Ubs, leading to Aurora B hyperactivation and erroneous chromosome–microtubule attachments. These findings define the role of K63-linked ubiquitination in regulating Aurora B activation and provide a potential site for Aurora B-targeting drug design.

Kinetochore- and chromosome-driven transition of microtubules into bundles promotes spindle assembly

Mitotic spindle assembly is crucial for chromosome segregation and relies on bundles of microtubules that extend from the poles and overlap in the middle. However, how these structures form remains poorly understood. Here we show that overlap bundles arise through a network-to-bundles transition driven by kinetochores and chromosomes. STED super-resolution microscopy reveals that PRC1-crosslinked microtubules initially form loose arrays, which become rearranged into bundles. Kinetochores promote microtubule bundling by lateral binding via CENP-E/kinesin-7 in an Aurora B-regulated manner. Steric interactions between the bundle-associated chromosomes at the spindle midplane drive bundle separation and spindle widening. In agreement with experiments, theoretical modeling suggests that bundles arise through competing attractive and repulsive mechanisms. Finally, perturbation of overlap bundles leads to inefficient correction of erroneous kinetochore-microtubule attachments. Thus, kinetochores and chromosomes drive coarsening of a uniform microtubule array into overlap bundles, which promote not only spindle formation but also chromosome segregation fidelity.

MPS1 localizes to end-on microtubule-attached kinetochores to promote microtubule release

In eukaryotes, the spindle assembly checkpoint protects genome stability in mitosis by preventing chromosome segregation until incorrect microtubule-kinetochore attachment geometries have been eliminated and chromosome biorientation has been completed. These error correction and checkpoint processes are linked by the conserved Aurora B and MPS1 Ser/Thr kinases.1,2 MPS1-dependent checkpoint signaling is believed to be initiated by kinetochores without end-on microtubule attachments,3,4 including those generated by Aurora B-mediated error correction. The current model posits that MPS1 competes with microtubules for binding sites at the kinetochore.3,4 MPS1 is thought to first recognize kinetochores not blocked by microtubules and then initiate checkpoint signaling. However, MPS1 is also required for chromosome biorientation and correction of microtubule-kinetochore attachment errors.5,6,7,8,9 This latter function, which must require direct interaction with microtubule-attached kinetochores, is not readily explained within the constraints of the current model. Here, we show that MPS1 transiently localizes to end-on attached kinetochores and that this recruitment depends on the relative activities of Aurora B and its counteracting phosphatase PP2A-B56 rather than microtubule-attachment state per se. MPS1 autophosphorylation also regulates MPS1 kinetochore levels but does not determine the response to microtubule attachment. At end-on attached kinetochores, MPS1 actively promotes microtubule release together with Aurora B. Furthermore, in live cells, MPS1 is detected at attached kinetochores before the removal of microtubules. During chromosome alignment, MPS1, therefore, coordinates both the resolution of incorrect microtubule-kinetochore attachments and the initiation of spindle checkpoint signaling.

Multi-Photon Laser Ablation of Cytoplasmic Microtubule Organizing Centers in Mouse Oocytes.

The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at prophase I of the first meiotic division. After puberty and upon meiotic resumption, the nuclear membrane disassembles (nuclear envelope breakdown), and the spindle is assembled mainly at the oocyte center. Initial central spindle positioning is essential to protect against abnormal kinetochore-microtubule (MT) attachments and aneuploidy. The centrally positioned spindle migrates in a time-sensitive manner toward the cortex, and this is a necessary process to extrude a tiny polar body. In mitotic cells, spindle positioning relies on the interaction between centrosome-mediated astral MTs and the cell cortex. On the contrary, mouse oocytes lack classic centrosomes and, instead, contain numerous acentriolar MT organizing centers (MTOCs). At the metaphase I stage, mouse oocytes have two different sets of MTOCs: (1) MTOCs that are clustered and sorted to assemble spindle poles (polar MTOCs), and (2) metaphase cytoplasmic MTOCs (mcMTOCs) that remain in the cytoplasm and do not contribute directly to spindle formation but play a crucial role in regulating spindle positioning and timely spindle migration. Here, a multi-photon laser ablation method is described to selectively deplete endogenously labeled mcMTOCs in oocytes collected from Cep192-eGfp reporter mice. This method contributes to the understanding of the molecular mechanisms underlying spindle positioning and migration in mammalian oocytes.