Biology subjects

Thawani, A.

Publications and source records attributed to Thawani, A..

6 recordsLinked to original sources

bioRxiv2026

Single-cell, clonal and spatial atlases of cranial placodes illuminate their specification and evolution

The vertebrate head is defined by complex sensory structures derived from cranial placodes. Placodes arise alongside the neural crest at the neural plate border, yet the mechanisms governing their identity, diversification, and evolutionary origins are unclear. We present an integrated single-cell, spatial, and clonal atlas of placode development to resolve the dynamics of their lineage segregation. Combining single-cell RNA-sequencing, spatial transcriptomics, and high-resolution clonal tracing, we show that placodal and neighboring progenitors form a continuous transcriptional landscape with gradual transitions between domains. Domain boundary cells co-express markers of adjacent territories, suggesting transient bipotent states. Consistent with this, clonal analysis reveals sharing of progenitors between neighboring placodes, supporting a model of competitive segregation. Comparisons with amphioxus suggests that vertebrate olfactory placodes emerged from an ancestral neuroectoderm that later partitioned into distinct neural and olfactory domains. Our findings provide a unified framework for understanding the developmental and evolutionary origins of vertebrate sensory organs.

developmental biology↗

bioRxiv2024

Structures of vertebrate R2 retrotransposon complexes during target-primed reverse transcription and after second strand nicking

R2 retrotransposons are model site-specific eukaryotic non-LTR retrotransposons that copy-and-paste into gene loci encoding ribosomal RNAs. Recently we demonstrated that avian A-clade R2 proteins achieve efficient and precise insertion of transgenes into their native safe-harbor loci in human cells. The features of A-clade R2 proteins that support gene insertion are not characterized. Here, we report high resolution cryo-electron microscopy structures of two vertebrate A-clade R2 proteins, avian and testudine, at the initiation of target-primed reverse transcription and one structure after cDNA synthesis and second strand nicking. Using biochemical and cellular assays we discover the basis for high selectivity of template use and unique roles for each of the expanded A-clade zinc-finger domains in nucleic acid recognition. Reverse transcriptase active site architecture is reinforced by an unanticipated insertion motif in vertebrate A-clade R2 proteins. Our work brings first insights to A-clade R2 protein structure during gene insertion and enables further improvement and adaptation of R2-based systems for precise transgene insertion.

biochemistry↗

bioRxiv2024

Molecular interplay between HURP and Kif18A in mitotic spindle regulation

During mitosis, microtubule dynamics are regulated to ensure proper alignment and segregation of chromosomes. The dynamics of kinetochore-attached microtubules are regulated by hepatoma-upregulated protein (HURP) and the mitotic kinesin-8 Kif18A, but the underlying mechanism remains elusive. Using single-molecule imaging in vitro, we demonstrate that Kif18A motility is regulated by HURP. While sparse decoration of HURP activates the motor, higher concentrations hinder processive motility. To shed light on this behavior, we determined the binding mode of HURP to microtubules using Cryo-EM. The structure reveals that one HURP motif spans laterally across {beta}-tubulin, while a second motif binds between adjacent protofilaments. HURP partially overlaps with the microtubule-binding site of the Kif18A motor domain, indicating that excess HURP inhibits Kif18A motility by steric exclusion. We also observed that HURP and Kif18A function together to suppress dynamics of the microtubule plus-end, providing a mechanistic basis for how they collectively serve in spindle length control.

biophysics↗

bioRxiv2019

Molecular mechanism of microtubule nucleation from gamma-tubulin ring complex

Determining how microtubules (MTs) are nucleated is essential for understanding how the cytoskeleton assembles. While the MT nucleator, {gamma}-tubulin ring complex ({gamma}-TuRC) has been identified, precisely how {gamma}-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 {gamma}-TuRC. We reveal a high {gamma}-/{beta}-tubulin affinity, which facilitates assembly of a MT from {gamma}-TuRC. Whereas spontaneous nucleation requires assembly of 8 {beta}-tubulins, nucleation from {gamma}-TuRC occurs efficiently with a cooperativity of 4 {beta}-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 {beta}-tubulins drive {gamma}-TuRC into a closed conformation. Putative activation domain of CDK5RAP2, NME7 and TPX2 do not enhance {gamma}-TuRC-mediated nucleation, while XMAP215 drastically increases the nucleation efficiency by strengthening the longitudinal {gamma}-/{beta}-tubulin interaction.

cell biology↗

bioRxiv2019

Biochemical reconstitution of branching microtubule nucleation

Microtubules are nucleated from specific locations at precise times in the cell cycle. However, the factors that constitute these microtubule nucleation pathways still need to be identified along with their mode of action. Here, using purified Xenopus laevis proteins we biochemically reconstitute branching microtubule nucleation, a nucleation pathway where microtubules originate from pre-existing microtubules, which is essential for spindle assembly and chromosome segregation. We found that besides the microtubule nucleator gamma-tubulin ring complex ({gamma}-TuRC), the two branching effectors augmin and TPX2 are required to efficiently nucleate branched microtubules. Specifically, TPX2 generates regularly-spaced patches that recruit augmin and {gamma}-TuRC to microtubules, which then nucleate new microtubules at preferred branching angles of less than 90 degrees. Our work demonstrates how {gamma}-TuRC is brought to its nucleation site for branching microtubule nucleation. It provides a blueprint for other microtubule nucleation pathways and for generating a particular microtubule architecture by regulating microtubule nucleation.

cell biology↗

bioRxiv2019

Spatiotemporal organization of branched microtubule networks

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 {gamma}-TuRC, combined with single-molecule observations, we show that first TPX2 is deposited on pre-existing MTs, followed by binding of augmin/{gamma}-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.\n\nImpact StatementA sequential reaction pathway involving TPX2, augmin and {gamma}-TuRC governs the assembly and architecture of branched microtubule networks.

biophysics↗