Biology subjects

Torvi, J. R.

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

4 recordsLinked to original sources

bioRxiv2024

Enhanced endogenous gene tagging in human induced pluripotent stem cells via AAV6-mediated donor delivery

Systematically tagging endogenous proteins with fluorescent markers in human induced pluripotent stem cells (hiPSCs) allows observation of live cell dynamics in different cell states. However, the precise insertion of fluorescent proteins into live cells via CRISPR/Cas9-induced editing relies on homology-directed repair (HDR). The nonhomologous end-joining (NHEJ) DNA repair pathway often outcompetes HDR, resulting in irreversible insertions and deletions (INDELs) and low knock-in efficiency. Recognizing successful HDR-mediated tagging events is an additional challenge when the target gene is not expressed in stem cells and successful tagging cannot be immediately observed. To address these challenges, we used: 1) adeno-associated virus serotype 6 (AAV6) mediated DNA donors at optimized multiplicity of infection (MOI) to deliver tag payloads at maximal efficiency; 2) titrated, multiplexed Cas9:gRNA ribonucleo-protein (RNP) amounts to assure balanced HDR/INDEL frequency among conditions; 3) long-amplicon droplet digital PCR (ddPCR) to measure the frequency of HDR-generated alleles in edited pools; and 4) simultaneous Inference of CRISPR Edits (ICE) to detect and thereby avoid conditions significantly saturated (>50%) with INDELs. These approaches enabled us to identify efficient and accurate editing conditions and recover tagged cells, including cells tagged at loci not expressed in stem cells. Together these steps allowed us to develop an efficient methodology and workflow to clonally isolate directly from an ideal cell pool with optimal HDR and minimized INDEL frequencies. Using this approach, we achieved both monoallelic and biallelic insertion of fluorescent markers into four genes that are turned on during differentiation but not initially expressed in hiPSCs, where direct selection of tagged cells based on fluorescence was impossible: TBR2, TBXT, CDH2 (pro-differentiation and pro-migratory genes), and CDH5 (endothelial specific gene). Through a systematic evaluation of various gRNA sequences and RNP concentrations, we identified conditions for each gene that achieved high HDR frequencies, peaking at 38.6%, while also avoiding conditions saturated with INDELs, where isolation of clones with a tagged allele in trans with an unedited allele is difficult. Over-all, this methodology enhances the efficiency of fluorescent tag knock-in at genes not expressed in hiPSCs, facilitating reliable image-based observation of cellular processes, and enables recovery of accurately edited mono- and biallelically tagged clones. We standardized these approaches to yield an efficient and general workflow for introducing large HDR mediated knock-ins into hiPSCs.

cell biology↗

bioRxiv2023

Interdependence of a microtubule polymerase and a motor protein in establishment of kinetochore end-on attachments

Faithful segregation of chromosomes into daughter cells during mitosis requires formation of attachments between kinetochores and mitotic spindle microtubules. Chromosome alignment on the mitotic spindle, also referred to as congression, is facilitated by translocation of side-bound chromosomes along the microtubule surface, which allows the establishment of end-on attachment of kinetochores to microtubule plus ends. Spatial and temporal constraints hinder observation of these events in live cells. Therefore, we used our previously developed reconstitution assay to observe dynamics of kinetochores, the yeast kinesin-8, Kip3, and the microtubule polymerase, Stu2, in lysates prepared from metaphase-arrested budding yeast, Saccharomyces cerevisiae. Using total internal reflection fluorescence (TIRF) microscopy to observe kinetochore translocation on the lateral microtubule surface toward the microtubule plus end, motility was shown to be dependent on both Kip3, as we reported previously, and Stu2. These proteins were shown to have distinct dynamics on the microtubule. Kip3 is highly processive and moves faster than the kinetochore. Stu2 tracks both growing and shrinking microtubule ends but also colocalizes with moving lattice-bound kinetochores. In cells, we observed that both Kip3 and Stu2 are important for establishing chromosome biorientation, Moreover, when both proteins are absent, biorientation is completely defective. All cells lacking both Kip3 and Stu2 had declustered kinetochores and about half also had at least one unattached kinetochore. Our evidence argues that despite differences in their dynamics, Kip3 and Stu2 share roles in chromosome congression to facilitate proper kinetochore-microtubule attachment.

cell biology↗

bioRxiv2022

Stimulating microtubule growth is not the essential function of the microtubule polymerase Stu2

ch-TOG family proteins, including the budding yeast Stu2, are essential for spindle formation and chromosome segregation. Such functions depend on an array of activities ranging from microtubule nucleation, polymerization and depolymerization, to conferring tension sensitivity to kinetochores. This functional diversity makes it challenging to dissect these various functions and understand their relative importance. Here, we developed separation-of-function mutants and used artificial tethering tools to elucidate several important mechanistic insights into Stu2s essential role. We show that Stu2s microtubule polymerization activity depends on its basic linker region but is surprisingly dispensable for viability; that in fact, Stu2 carries out an essential kinetochore-associated function; and finally, that Stu2s precise location within the kinetochore is critical for its function, suggesting a spatial separation mode of action may underlie its ability to confer tension sensitivity. Our findings highlight the significance of Stu2s kinetochore role and provide insights into the molecular mechanisms by which it performs its various functions.

cell biology↗

bioRxiv2022

Reconstitution of Kinetochore and Microtubule Dynamics Reveals a Role for a Kinesin-8 in Establishing End-on Attachments

During mitosis, individual microtubules make attachments to chromosomes via a specialized protein complex called the kinetochore to faithfully segregate the chromosomes to daughter cells. Translocation of kinetochores on the lateral surface of the microtubule has been proposed to contribute to high fidelity chromosome capture and alignment at the mitotic midzone, but has been difficult to observe in vivo because of spatial and temporal constraints. To overcome these barriers, we used total internal reflection fluorescence (TIRF) microscopy to track the interactions between endogenously tagged tubulin, kinetochore proteins, and other microtubule-associated proteins in lysates from metaphase-arrested Saccharomyces cerevisiae. Using both TIRF microscopy and cryo-correlative light microscopy and electron tomography, we successfully reconstituted microtubule-bound, intact kinetochores. These kinetochores translocate on the lateral microtubule surface toward the microtubule plus end and transition to end-on attachment, whereupon microtubule depolymerization commences. The directional kinetochore movement is dependent on the highly processive kinesin-8, Kip3. We propose that Kip3 facilitates stable kinetochore attachment to microtubule plus ends through its abilities to move the kinetochore laterally on the surface of the microtubule and to regulate microtubule plus end dynamics.

cell biology↗