Biology subjects

Popchock, A. R.

Publications and source records attributed to Popchock, A. R..

5 recordsLinked to original sources

bioRxiv2025

Direct observation of interdependent and hierarchical kinetochore assembly on individual centromeres

Kinetochores are megadalton protein machines that harness microtubules to segregate chromosomes during cell division. The kinetochores must assemble after DNA replication during every cell cycle onto specialized regions of chromosomes called centromeres, but the order and regulation of their assembly remains unclear due to the complexity of kinetochore composition and the difficulty resolving individual kinetochores in vivo. Here, by adapting a prior single-molecule method for monitoring kinetochore assembly in budding yeast lysates, we identify a sequential order of assembly and uncover previously unknown interdependencies between subcomplexes. We show that inner kinetochore assembly depends partly on outer kinetochore components, and that outer kinetochore branches do not assemble independently of one another. Notably, Mif2 assembly is a rate-limiting step that can be accelerated by binding to the Mtw1 subcomplex, thereby promoting rapid assembly of many inner and outer kinetochore components. The importance of controlling kinetochore assembly kinetics is supported by a Mif2 mutant lacking both autoinhibition and Mtw1 subcomplex binding activity, which leads to defective kinetochore-microtubule attachments when the centromeric histone variant Cse4 is overexpressed. Altogether, our work provides a direct view of kinetochore assembly and reveals highly interdependent regulatory events that control its order and timing. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/661565v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@bcc252org.highwire.dtl.DTLVardef@79b6b2org.highwire.dtl.DTLVardef@c974e0org.highwire.dtl.DTLVardef@184689_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

TinA enables kinesin-14/KlpA to exhibit processive minus-end-directed motility

Kinesin-14 motors contribute to spindle assembly by localizing to spindle poles and anchoring the minus ends of spindle microtubules. Unlike other kinesin-14 motors, KlpA uniquely exhibits plus-end-directed motility on single microtubules as individual homodimers. However, the mechanism by which KlpA achieves minus-end-directed motility on single microtubules remains elusive. Here, we report that TinA, a highly conserved microtubule-anchoring protein, serves as an activator of KlpA for minus-end-directed motility. TinA directly interacts with KlpA to form minus-end-directed complexes that exhibit continuous movement on microtubules with two distinct velocity modes. The assembly of KlpA-TinA complexes depends on TinA binding to the central stalk of KlpA. Furthermore, TinA is a microtubule-binding protein, with its C-terminal region playing a critical role in microtubule interaction. Deletion of the C-terminus of TinA markedly reduces its microtubule-binding ability and severely impairs the formation of KlpA-TinA complexes. Nonetheless, KlpA-TinA complexes formed without the C-terminus of TinA still exhibit minus-end-directed motility, albeit with a single velocity mode. Collectively, these findings provide critical mechanistic insights into how TinA modulates KlpA, enabling the kinesin-14 motor to achieve minus-end-directed motility.

biophysics↗

bioRxiv2024

Probing mechanical selection in diverse eukaryotic genomes through accurate prediction of 3D DNA mechanics

Connections between the mechanical properties of DNA and biological functions have been speculative due to the lack of methods to measure or predict DNA mechanics at scale. Recently, a proxy for DNA mechanics, cyclizability, was measured by loop-seq and enabled genome-scale investigation of DNA mechanics. Here, we use this dataset to build a computational model predicting bias-corrected intrinsic cyclizability, with near-perfect accuracy, solely based on DNA sequence. Further, the model predicts intrinsic bending direction in 3D space. Using this tool, we aimed to probe mechanical selection - that is, the evolutionary selection of DNA sequence based on its mechanical properties - in diverse circumstances. First, we found that the intrinsic bend direction of DNA sequences correlated with the observed bending in known protein-DNA complex structures, suggesting that many proteins co-evolved with their DNA partners to capture DNA in its intrinsically preferred bent conformation. We then applied our model to large-scale yeast population genetics data and showed that centromere DNA element II, whose consensus sequence is unknown, leaving its sequence-specific role unclear, is under mechanical selection to increase the stability of inner-kinetochore structure and to facilitate centromeric histone recruitment. Finally, in silico evolution under strong mechanical selection discovered hallucinated sequences with cyclizability values so extreme that they required experimental validation, yet, found in nature in the densely packed mitochondrial(mt) DNA of Namystynia karyoxenos, an ocean-dwelling protist with extreme mitochondrial gene fragmentation. The need to transmit an extraordinarily large amount of mtDNA, estimated to be > 600 Mb, in combination with the absence of mtDNA compaction proteins may have pushed mechanical selection to the extreme. Similarly extreme DNA mechanics are observed in bird microchromosomes, although the functional consequence is not yet clear. The discovery of eccentric DNA mechanics in unrelated unicellular and multicellular eukaryotes suggests that we can predict extreme natural biology which can arise through strong selection. Our methods offer a way to study the biological functions of DNA mechanics in any genome and to engineer DNA sequences with desired mechanical properties.

biophysics↗

bioRxiv2024

Stable centromere association of the yeast histone variant Cse4 requires its essential N-terminal domain

Chromosome segregation relies on kinetochores that assemble on specialized centromeric chromatin containing a histone H3 variant. In budding yeast, a single centromeric nucleosome containing Cse4 assembles at a sequence-defined 125 bp centromere. Yeast centromeric sequences are poor templates for nucleosome formation in vitro, suggesting the existence of mechanisms that specifically stabilize Cse4 nucleosomes in vivo. The extended Cse4 N-terminal tail binds to the chaperone Scm3, and a short essential region called END within the N-terminal tail binds the inner kinetochore complex OA. To address the roles of these interactions, we utilized single molecule fluorescence assays to monitor Cse4 during kinetochore assembly. We found that OA and Scm3 independently stabilize Cse4 at centromeres via their END interaction. Scm3 binding to the Cse4 END is enhanced by Ipl1/Aurora B phosphorylation, identifying a previously unknown role for Ipl1 in ensuring Cse4 stability. Strikingly, an Ipl1 phosphomimetic mutation in the Cse4 END enhances Scm3 binding and can restore Cse4 recruitment in mutants defective in OA binding. Together, these data suggest that a key function of the essential Cse4 N-terminus is to ensure Cse4 localization at centromeres.

cell biology↗

bioRxiv2024

Kinetochores grip microtubules with directionally asymmetric strength

For accurate mitosis, all chromosomes must achieve bi-orientation, with replicated sister chromatids coupled via kinetochores to the plus ends of opposing microtubules. However, kinetochores first bind the sides of microtubules and subsequently find plus ends by directed transport or when side-attached microtubules shorten and bring their ends to the kinetochores. Mitotic accuracy depends on the selective release of erroneous attachments and proposed mechanisms have focused mainly on plus-end attachments. Whether erroneous side-attachments are distinguished from correct side-attachments is unknown. Here we show that side-attached kinetochores are very sensitive to microtubule polarity, gripping six-fold more strongly when pulled toward plus versus minus ends. This directionally asymmetric grip correlates with changes in the axial arrangement of subcomplexes within the kinetochores, suggesting that internal architecture dictates attachment strength. We propose that the kinetochores directional grip promotes accuracy specifically during early mitosis, by stabilizing correct attachments even before both sisters have found plus ends.

biophysics↗