Biology subjects

Daum, J. R.

Publications and source records attributed to Daum, J. R..

3 recordsLinked to original sources

bioRxiv2026

Functional Interaction of SKA and NDC80 Complexes at Kinetochores Promoting Anaphase Onset in Mitosis

The kinetochore and spindle complex (SKA) and NDC80 complexes are essential kinetochore elements that ensure highly accurate chromosome segregation and successful progression through mitosis. The SKA heterodimer complex consists of SKA1, SKA2, and SKA3 subunits, and the NDC80 complex contains NDC80, NUF2, SPC24, and SPC25 subunits. Through live cell fluorescence timelapse imaging assays and expression of RNAi-resistant SKA3 constructs, we rescue SKA complex function in cells lacking endogenous SKA3. These assays reveal a critical span within SKA3s C-terminus required for successful mitotic progression. Structural protein modeling shows that this span encompasses the majority of a roughly 40 amino acid SKA3 C-terminal structural element that promotes interaction with the coiled-coil NDC80 and NUF2 subunits of the NDC80 complex. Thus, although spindle and kinetochore concentration of the SKA complex is mediated in part by the tubulin and tip-tracking capabilities provided by the SKA1 component of the SKA complex, transition from metaphase to anaphase requires the contribution of SKA3s C-terminal structural interface to mediate interaction between the SKA and NDC80 complexes. Significance StatementAccurate chromosome segregation is essential for genomic stability, and its failure underlies developmental defects, and cancer. The kinetochore-microtubule interface, where the SKA and NDC80 complexes converge, is central, yet the of these two complexes is incompletely defined. We identify a critical segment within the C-terminus of SKA3 required for the metaphase-to-anaphase transition. Using live-cell imaging with RNAi-resistant rescue constructs and structural modeling, we demonstrate that this region mediates SKA engagement with the coiled-coil domains of NDC80 and NUF2. Our findings establish that a small segment of SKA3s C-terminus provides an essential physical bridge between the SKA and NDC80 complexes. This work refines understanding kinetochore-microtubule interaction, functionally identifying a discrete contact whose disruption may be relevant to chromosomal instability in disease.

cell biology↗

bioRxiv2022

Dynamic Features of Chromosomal Instability during Culture of Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) hold great potential for regenerative medicine. By reprogramming a patient"s own cells, immunological rejection can be avoided during transplantation. For expansion and gene editing, iPSCs are grown in artificial culture for extended times. Culture affords potential danger for accumulation of genetic aberrations. To study these, two iPS cell lines were cultured and periodically analyzed using advanced optical mapping to detect and classify chromosome numerical and segmental changes that included deletions, insertions, balanced translocations and inversions. In one of the lines, a population trisomic for chromosome 12 gained dominance over a small number of passages. This appearance and dominance of the culture by chromosome 12 trisomic cells was tracked through intermediate passages by analysis of chromosome spreads. Mathematical modeling suggested that the proliferation rates of diploid versus trisomic cells could not account for the rapid dominance of the trisomic population. In addition, optical mapping revealed hundreds of structural variations distinct from those generally found within the human population. Many of these structural variants were detected in samples taken early in the culturing process and were maintained in late passage samples, while others were acquired over the course of culturing.

cell biology↗

bioRxiv2022

Four novel, immortal cell lines derived from embryos of Xenopus tropicalis

The diploid anuran, Xenopus tropicalis, has emerged as a key research model in cell and developmental biology. To enhance the usefulness of this species, we developed methods for generating immortal cell lines from Nigerian strain (NXR_1018, RRID:SCR_013731) X. tropicalis embryos. We generated 14 cell lines that were propagated for several months. We selected four morphologically distinct lines, XTN-6, XTN-8, XTN-10, and XTN-12 for further characterization. Karyotype analysis revealed that three of the lines, XTN-8, XTN-10, and XTN-12 were primarily diploid. XTN-6 cultures showed a consistent mixed population of diploid cells, cells with chromosome 8 trisomy, and cells containing a tetraploid content of chromosomes. The lines were propagated using conventional culture methods as adherent cultures at 30{degrees}C in a simple, diluted L-15 medium containing fetal bovine serum without use of a high CO2 incubator. Transcriptome analysis indicated that the four lines were distinct lineages. These methods will be useful in the generation of cell lines from normal and mutant strains of X. tropicalis as well as other species of Xenopus.

cell biology↗