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Abstract Microtubules are generated at centrosomes, chromosomes, and within spindles during cell division. Whereas microtubule nucleation at the centrosome is well characterized, much remains unknown about where, when, and how microtubules are nucleated at chromosomes. To address these questions, we reconstitute microtubule nucleation from purified chromosomes in meiotic Xenopus egg extract and find that chromosomes alone can form spindles. We visualize microtubule nucleation near chromosomes using total internal reflection fluorescence microscopy to find that this occurs through branching microtubule nucleation. By inhibiting molecular motors, we find that the organization of the resultant polar branched networks is consistent with a theoretical model where the effectors for branching nucleation are released by chromosomes, forming a concentration gradient that spatially biases branching microtbule nucleation. In the presence of motors, these branched networks are ultimately organized into functional spindles, where the number of emergent spindle poles scales with the number of chromosomes and total chromatin area. 

Multifocal microscopes (MFMs) are becoming increasingly popular in fluorescence microscopy due to their high speed three-dimensional (3D) imaging capabilities. Conventional MFMs use a fixed fabricated grating as the multifocal grating but these are limited to a restricted wavelength range and a fixed object-plane separation. Spatial light modulators (SLMs) represent an alternative to fabricated gratings due to their real-time programmability, providing complete control over emission wavelength range and object plane separations. However, algorithms commonly used to obtain multifocal grating patterns which provide uniform intensity across the subimages are not directly applicable to SLM-based MFMs due to inherent pixel-to-pixel crosstalk effects present in the SLM chip. We recently developed an in-situ iterative algorithm which generates grating patterns that provide near-uniform illumination of the subimages in SLM-based MFMs. This algorithm is universal across wavelengths, object-plane separations, and SLM manufacturers. As part of our efforts to develop an SLM-based MFM that can respond rapidly to changing experimental parameters, we implement a gradient descent-based optimization method. We evaluate its performance in comparison with a grid search based routine. Experimental results obtained on a custom-made SLM-based MFM indicate that the grid-search optimized grating patterns provide superior subimage intensity uniformity versus the gradient-descent method. These experiments also provide an insight into the energy landscape involved in these optimizations. This study increases the utility of SLM-based MFMs in high-speed imaging. 

Determining how microtubules (MTs) are nucleated is essential for understanding how the cytoskeleton assembles. While the MT nucleator, γ-tubulin ring complex (γ-TuRC) has been identified, precisely how γ-TuRC nucleates a MT remains poorly understood. Here, we developed a single molecule assay to directly visualize nucleation of a MT from purified Xenopus laevis γ-TuRC. We reveal a high γ-/αβ-tubulin affinity, which facilitates assembly of a MT from γ-TuRC. Whereas spontaneous nucleation requires assembly of 8 αβ-tubulins, nucleation from γ-TuRC occurs efficiently with a cooperativity of 4 αβ-tubulin dimers. This is distinct from pre-assembled MT seeds, where a single dimer is sufficient to initiate growth. A computational model predicts our kinetic measurements and reveals the rate-limiting transition where laterally associated αβ-tubulins drive γ-TuRC into a closed conformation. NME7, TPX2, and the putative activation domain of CDK5RAP2 h γ-TuRC-mediated nucleation, while XMAP215 drastically increases the nucleation efficiency by strengthening the longitudinal γ-/αβ-tubulin interaction. 

To understand how chromosomes are segregated, it is necessary to explain the precise spatiotemporal organization of microtubules (MTs) in the mitotic spindle. We use Xenopus egg extracts to study the nucleation and dynamics of MTs in branched networks, a process that is critical for spindle assembly. Surprisingly, new branched MTs preferentially originate near the minus-ends of pre-existing MTs. A sequential reaction model, consisting of deposition of nucleation sites on an existing MT, followed by rate-limiting nucleation of branches, reproduces the measured spatial profile of nucleation, the distribution of MT plus-ends and tubulin intensity. By regulating the availability of the branching effectors TPX2, augmin and γ-TuRC, combined with single-molecule observations, we show that first TPX2 is deposited on pre-existing MTs, followed by binding of augmin/γ-TuRC to result in the nucleation of branched MTs. In sum, regulating the localization and kinetics of nucleation effectors governs the architecture of branched MT networks.