CCDC74A/B are K-fiber crosslinkers required for chromosomal alignment.

BACKGROUND: Spindle microtubule organization, regulated by microtubule-associated proteins, is critical for cell division. Proper organization of kinetochore fiber (K-fiber), connecting spindle poles and kinetochores, is a prerequisite for precise chromosomal alignment and faithful genetic material transmission. However, the mechanisms of K-fiber organization and dynamic maintenance are still not fully understood. RESULTS: We reveal that two previously uncharacterized coiled-coil domain proteins CCDC74A and CCDC74B (CCDC74A/B) are spindle-localized proteins in mammalian cells. They bind directly to microtubules through two separate domains and bundle microtubules both in vivo and in vitro. These functions are required for K-fiber organization, bipolar spindle formation, and chromosomal alignment. Moreover, CCDC74A/B form homodimers in vivo, and their self-association activity is necessary for microtubule bundling and K-fiber formation. CONCLUSIONS: We characterize CCDC74A and CCDC74B as microtubule-associated proteins that localize to spindles and are important K-fiber crosslinkers required for bipolar spindle formation and chromosome alignment.

The microtubule-associated protein HURP recruits the centrosomal protein TACC3 to regulate K-fiber formation and support chromosome congression.

Kinetochore fibers (K-fibers) are microtubule bundles attached to chromosomes. Efficient K-fiber formation is required for chromosome congression, crucial for faithful chromosome segregation in cells. However, the mechanisms underlying K-fiber formation before chromosome biorientation remain unclear. Depletion of hepatoma up-regulated protein (HURP), a RanGTP-dependent microtubule-associated protein localized on K-fibers, has been shown to result in low-efficiency K-fiber formation. Therefore, here we sought to identify critical interaction partners of HURP that may modulate this function. Using co-immunoprecipitation and bimolecular fluorescence complementation assays, we determined that HURP interacts directly with the centrosomal protein transforming acidic coiled coil-containing protein 3 (TACC3), a centrosomal protein, both in vivo and in vitro through the HURP1-625 region. We found that HURP is important for TACC3 function during kinetochore microtubule assembly at the chromosome region in prometaphase. Moreover, HURP regulates stable lateral kinetochore attachment and chromosome congression in early mitosis by modulation of TACC3. These findings provide new insight into the coordinated regulation of K-fiber formation and chromosome congression in prometaphase by microtubule-associated proteins.

CLIP-170 recruits PLK1 to kinetochores during early mitosis for chromosome alignment.

The cytoplasmic linker protein (CLIP)-170, an outer kinetochore protein, has a role in kinetochore-microtubule attachment and chromosome alignment during mitosis. However, the mechanism by which CLIP-170 is involved in chromosome alignment is not known. Here, we show that CLIP-170 colocalizes with Polo-like kinase 1 (PLK1) at kinetochores during early mitosis. Depletion of CLIP-170 results in a significant reduction in PLK1 recruitment to kinetochores and causes kinetochore-fiber (K-fiber) instability and defects in chromosome alignment at the metaphase plate. These phenotypes are dependent on the phosphorylation of CLIP-170 at a CDK1-dependent site, T287, as ectopic expression of wild-type CLIP-170, but not the expression of a non-phosphorylatable mutant, CLIP-170-T287A, restores PLK1 localization at kinetochores and rescues K-fiber stability and chromosome alignment in CLIP-170-depleted cells. These data suggest that CLIP-170 acts as a novel recruiter and spatial regulator of PLK1 at kinetochores during early mitosis, promoting K-fiber stability and chromosome alignment for error-free chromosome segregation.

Expansion microscopy of the mitotic spindle.

The mitotic spindle is a dynamic and complex cellular structure made of microtubules and associated proteins. Although the general localization of most proteins has been identified, the arrangement of the microtubules in the mitotic spindle and precise localization of various proteins are still under intensive research. However, techniques used previously to decipher such puzzles are resolution limited or require complex microscopy systems. On the other hand, expansion microscopy is a novel super-resolution microscopy technique that uses physical expansion of fixed specimens to allow features closer than the diffraction limit of light (~250nm) to become resolvable in the expanded specimen on a conventional confocal microscope. This chapter focuses on expansion microscopy of the mitotic spindle, specifically using tubulin labeling to visualize all microtubule subpopulations within the spindle. Furthermore, we discuss a protocol for expansion of GFP-tagged proteins, such as protein regulator of cytokinesis 1 (PRC1). We also discuss various approaches for image analysis pointing out main advantages of expansion microscopy when compared to previously used techniques. This approach is currently used in our laboratory to study the architecture of the microtubules in the mitotic spindle after perturbations of various proteins important for the structural and dynamical properties of the mitotic spindle.

Microneedle manipulation of the mammalian spindle reveals specialized, short-lived reinforcement near chromosomes.

The spindle generates force to segregate chromosomes at cell division. In mammalian cells, kinetochore-fibers connect chromosomes to the spindle. The dynamic spindle anchors kinetochore-fibers in space and time to move chromosomes. Yet, how it does so remains poorly understood as we lack tools to directly challenge this anchorage. Here, we adapt microneedle manipulation to exert local forces on the spindle with spatiotemporal control. Pulling on kinetochore-fibers reveals the preservation of local architecture in the spindle-center over seconds. Sister, but not neighbor, kinetochore-fibers remain tightly coupled, restricting chromosome stretching. Further, pulled kinetochore-fibers pivot around poles but not chromosomes, retaining their orientation within 3 µm of chromosomes. This local reinforcement has a 20 s lifetime, and requires the microtubule crosslinker PRC1. Together, these observations indicate short-lived, specialized reinforcement in the spindle center. This could help protect chromosome attachments from transient forces while allowing spindle remodeling, and chromosome movements, over longer timescales.

Mapping Load-Bearing in the Mammalian Spindle Reveals Local Kinetochore Fiber Anchorage that Provides Mechanical Isolation and Redundancy.

Active forces generated at kinetochores move chromosomes, and the dynamic spindle must robustly anchor kinetochore fibers (k-fibers) to bear this load. The mammalian spindle bears the load of chromosome movement far from poles, but we do not know where and how-physically and molecularly-this load distributes across the spindle. In part, this is because probing spindle mechanics in live cells is difficult. Yet answering this question is key to understanding how the spindle generates and responds to force and performs its diverse mechanical functions. Here, we map load-bearing across the mammalian spindle in space-time and dissect local anchorage mechanics and mechanism. To do so, we laser-ablate single k-fibers at different spindle locations and in different molecular backgrounds and quantify the immediate relaxation of chromosomes, k-fibers, and microtubule speckles. We find that load redistribution is locally confined in all directions: along the first 3-4 µm from kinetochores, scaling with k-fiber length, and laterally within ∼2 µm of k-fiber sides, without detectable load sharing between neighboring k-fibers. A phenomenological model suggests that dense, transient crosslinks to the spindle along k-fibers bear the load of chromosome movement but that these connections do not limit the timescale of spindle reorganization. The microtubule crosslinker NuMA is needed for the local load-bearing observed, whereas Eg5 and PRC1 are not detectably required, suggesting specialization in mechanical function. Together, the data and model suggest that NuMA-mediated crosslinks locally bear load, providing mechanical isolation and redundancy while allowing spindle fluidity. These features are well suited to support robust chromosome segregation.

Microscopy Methods for Analysis of Spindle Dynamics in Meiotic Drosophila Spermatocytes.

The spindle is a microtubule-based structure whose remodeling is required for partitioning the chromosomes and cytoplasm during meiosis. Characterizing microtubule behavior is fundamental to understanding how these tubulin polymers contribute to successful cell division. Here, a procedure is described for the imaging and analysis of spindle microtubule dynamics in cultures of living Drosophila melanogaster primary spermatocytes expressing tubulin tagged with enhanced green fluorescent protein. It employs time-lapse scanning confocal microscopy and the photobleaching of fiduciary marks onto fluorescently tagged microtubules. These labels are subsequently used to determine the sites and rates of kinetochore fiber growth and shrinkage during metaphase. This method can be readily applied to different microtubule populations, meiotic stages, and genetic backgrounds.

Studying kinetochore-fiber ultrastructure using correlative light-electron microscopy.

Electron microscopy (EM) has dominated high-resolution cellular imaging for over 50 years, thanks to its ability to resolve on nanometer-scale intracellular structures such as the microtubules of the mitotic spindle. It is advantageous to view the cell of interest prior to processing the sample for EM. Correlative light-electron microscopy (CLEM) is a technique that allows one to visualize cells of interest by light microscopy (LM) before being transferred to EM for ultrastructural examination. Here, we describe how CLEM can be applied as an effective tool to study the spindle apparatus of mitotic cells. This approach allows transfected cells of interest, in desirable stages of mitosis, to be followed from LM to EM. CLEM has often been considered as a technically challenging and laborious technique. In this chapter, we provide step-by-step pictorial guides that allow successful CLEM to be achieved. In addition, we explain how it is possible to vary the sectioning plane, allowing spindles and microtubules to be analyzed from different angles, and the outputs that can be obtained from these methods when applied to the study of kinetochore fiber ultrastructure.