Stokasimov, E. | bioRxiv Science

bioRxiv2022

Transgenerational transcriptional heterogeneity from cytoplasmic chromatin

Transcriptional heterogeneity from plasticity of the epigenetic state of chromatin is thought to contribute to tumor evolution, metastasis, and drug resistance 1-3. However, the mechanisms leading to nongenetic cell-to-cell variation in gene expression remain poorly understood. Here we demonstrate that heritable transcriptional changes can result from the formation of micronuclei, aberrations of the nucleus that are common in cancer4,5. Micronuclei have fragile nuclear envelopes (NE) that are prone to spontaneous rupture, which exposes chromosomes to the cytoplasm and disrupts many nuclear activities 6,7. Using a combination of long-term live-cell imaging and same-cell, single-cell RNA sequencing (Look-Seq2), we identified significant reduction of gene expression in micronuclei, both before and after NE rupture. Furthermore, chromosomes in micronuclei fail to normally recover histone 3 lysine 27 acetylation, a critical step for the reestablishment of normal transcription after mitosis 8-10. These transcription and chromatin defects can persist into the next generation in a subset of cells, even after these chromosomes are incorporated into normal daughter nuclei. Moreover, persistent transcriptional repression is strongly associated with, and may be explained by, surprisingly long-lived DNA damage to these reincorporated chromosomes. Therefore, heritable alterations in transcription can originate from aberrations of nuclear architecture.

cell biology↗

bioRxiv2021

Multimodal tubulin binding by the yeast kinesin-8, Kip3, underlies its motility and depolymerization

The microtubule (MT) cytoskeleton is central to cellular processes including axonal growth, intracellular transport, and cell division, all of which rely on precise spatiotemporal control of MT organization. Kinesin-8s play a key role in regulating MT length by combining highly processive directional motility with MT-end disassembly. However, how kinesin-8 switches between these two apparently opposing activities remains unclear. Here, we define the structural features underlying this molecular switch through cryo-EM analysis of the yeast kinesin-8, Kip3 bound to MTs, and molecular dynamics simulations to approximate the complex of Kip3 with the curved tubulin state found at the MT plus-end. By integrating biochemical and single-molecule biophysical assays, we identified specific intra- and intermolecular interactions that modulate processive motility and MT disassembly. Our findings suggest that Kip3 undergoes conformational changes in response to tubulin curvature that underlie its unique ability to interact differently with the MT lattice than with the MT-end.

biophysics↗