Biology subjects

Carballo, J. A.

Publications and source records attributed to Carballo, J. A..

5 recordsLinked to original sources

bioRxiv2025

Nucleolar Cdc14 Splitting Reflects Recombination Context and Meiotic Chromosome Dynamics

Chromosome dynamics, recombination, and nucleolar organization intersect during meiotic prophase I, yet how recombination context influences nucleolar architecture remains unclear. We analyzed the nucleolar pool of Cdc14 in Saccharomyces cerevisiae under matched prophase-I gating. In a recombination-competent reference (ndt80{Delta}), Cdc14-mCherry formed a predominant single focus with occasional, reversible two-focus episodes that Nop56-GFP placed within the nucleolar compartment ("nucleolar splitting"). Splitting rose sharply in dmc1{Delta} ndt80{Delta} and remained high in spo11-y135f dmc1{Delta} ndt80{Delta}, demonstrating that elevated DSB formation is not required and pointing instead to homolog-engagement state as the key variable. Population checkpoint readouts did not map onto the phenotype: Hop1 phosphorylation was strong in dmc1{Delta}, weak/transient in ndt80{Delta}, and undetectable in spo11-y135f, yet splitting was high in dmc1{Delta} and spo11-y135f and low in ndt80{Delta}. Persistence metrics showed that events concentrated early and waned in ndt80{Delta}, whereas dmc1{Delta} and spo11 backgrounds retained activity into later windows, consistently across thresholds. We propose that nucleolar splitting reflects rheological responses of a nucleolar condensate to chromosome-scale forces that vary with homolog engagement. This view is consistent with DSB-independent contributions from telomere clustering into the bouquet configuration, telomere-led rapid prophase movements, and centromere coupling/pairing as routes that shape force transmission to the rDNA territory. In this sense, the nucleolus emerges as a mesoscale, mechanically sensitive readout of meiotic chromosome dynamics.

genetics↗

bioRxiv2023

Exportin-mediated nucleocytoplasmic transport maintains Pch2 homeostasis during meiosis

The meiotic recombination checkpoint reinforces the order of events during meiotic prophase I, ensuring the accurate distribution of chromosomes to the gametes. The AAA+ ATPase Pch2 remodels the Hop1 axial protein enabling adequate levels of Hop1-T318 phosphorylation to support the ensuing checkpoint response. While these events are focalized at chromosome axes, the checkpoint activating function of Pch2 relies on its cytoplasmic population. In contrast, forced nuclear accumulation of Pch2 leads to checkpoint inactivation. Here, we reveal the mechanism by which Pch2 travels from the cell nucleus to the cytoplasm to maintain Pch2 cellular homeostasis. Leptomycin B treatment provokes the nuclear accumulation of Pch2, indicating that its nucleocytoplasmic transport is mediated by the Crm1 exportin recognizing proteins containing Nuclear Export Signals (NESs). Consistently, leptomycin B leads to checkpoint inactivation and impaired Hop1 axial localization. Pch2 nucleocytoplasmic traffic is independent of its association with Zip1 and Orc1. We also identify a conserved functional NES in the non-catalytic N-terminal domain of Pch2 that is required for its nucleocytoplasmic traffic and proper checkpoint activity. In sum, we unveil another layer of control of Pch2 function during meiosis involving the nuclear export via the exportin pathway that is crucial to maintain the critical balance of Pch2 distribution among different cellular compartments.

cell biology↗

bioRxiv2021

Pch2 orchestrates the meiotic recombination checkpoint from the cytoplasm

During meiosis, defects in critical events trigger checkpoint activation and restrict cell cycle progression. The budding yeast Pch2 AAA+ ATPase orchestrates the checkpoint response launched by synapsis deficiency; deletion of PCH2 or mutation of the ATPase catalytic sites suppress the meiotic block of the zip1{Delta} mutant lacking the central region of the synaptonemal complex. Pch2 action enables adequate levels of phosphorylation of the Hop1 axial component at threonine 318, which in turn promotes activation of the Mek1 effector kinase and the ensuing checkpoint response. In zip1{Delta} chromosomes, Pch2 is exclusively associated to the rDNA region, but this nucleolar fraction is not required for checkpoint activation, implying that another yet uncharacterized Pch2 population must be responsible for this function. Here, we have artificially redirected Pch2 to different subcellular compartments by adding ectopic NES or NLS sequences or by trapping Pch2 in an immobile extranuclear domain, and we have evaluated the effect on Hop1 chromosomal distribution and checkpoint activity. We have also deciphered the spatial and functional impact of Pch2 regulators including Orc1, Dot1 and Nup2. We conclude that the cytoplasmic pool of Pch2 is sufficient to support the meiotic recombination checkpoint involving the subsequent Hop1-Mek1 activation on chromosomes, whereas the nuclear accumulation of Pch2 has pathological consequences. We propose that cytoplasmic Pch2 provokes a conformational change in Hop1 that poises it for its chromosomal incorporation and phosphorylation. Our discoveries shed light into the intricate regulatory network controlling the accurate balance of Pch2 distribution among different cellular compartments, which is essential for proper meiotic outcomes.

cell biology↗

bioRxiv2020

SWR1-independent association of H2A.Z to the LINC complex promotes meiotic chromosome motion

ABSTRACTThe H2A.Z histone variant is deposited into chromatin by the SWR1 complex affecting multiple aspects of meiosis. Here we describe a SWR1-independent localization of H2A.Z at meiotic telomeres and the centrosome. We demonstrate that H2A.Z colocalizes and interacts with Mps3, the SUN component of the LINC complex that spans the nuclear envelope and links meiotic telomeres to the cytoskeleton promoting meiotic chromosome movement. H2A.Z also interacts with the meiosis-specific Ndj1 protein that anchors telomeres to the nuclear periphery via Mps3. Telomeric localization of H2A.Z depends on Ndj1 and the N-terminal domain of Mps3. Although telomeric attachment to the nuclear envelope is maintained in the absence of H2A.Z, the distribution of Mps3 is altered. The velocity of chromosome movement during meiotic prophase I is reduced in the htz1Δ mutant lacking H2A.Z, but it is unaffected in swr1Δ cells. We reveal that H2A.Z is an additional LINC-associated factor that contributes to promote telomere-driven chromosome motion critical for error-free gametogenesis.Competing Interest StatementThe authors have declared no competing interest.Non-standard abbreviationsBiFCBimolecular Fluorescence ComplementationDICDifferential Interference ContrastKAcPotassium AcetateNENuclear EnvelopeLINCLinker of the Nucleoskeleton and CytoskeletonrDNARibosomal DNASCSynaptonemal ComplexSIMStructured Illumination MicroscopySPBSpindle Pole BodyVCC-terminal moiety of the Venus fluorescent proteinVNN-terminal moiety of the Venus fluorescent proteinWCEWhole cell extractsView Full Text

cell biology↗

bioRxiv2019

Cdc14 phosphatase ensures resolution of aberrant meiotic recombination outcomes partly through activating Yen1/GEN1

Meiotic defects derived from incorrect DNA repair during gametogenesis can lead to mutations, aneuploidies and infertility. Coordinated resolution of meiotic recombination intermediates is required for crossover formation, ultimately necessary for accurate completion of both rounds of chromosome segregation. Numerous master kinases orchestrate the correct assembly and activity of the repair machinery. Although much less is known, reversal of phosphorylation events in meiosis must also be key to coordinate the timing and functionality of repair enzymes. Cdc14 is an evolutionarily conserved phosphatase required for the dephosphorylation of multiple CDK1 targets. Mutations that inactivate this phosphatase lead to meiotic failure, but until now it was unknown if Cdc14 plays a direct role in meiotic recombination. Here, we show that elimination of Cdc14 leads to severe defects in the processing and resolution of recombination intermediates, causing a drastic depletion of crossovers when other repair pathways are compromised. We also show that Cdc14 is required for correct activity and localization of the Holliday Junction resolvase Yen1/GEN1. We reveal that Cdc14 regulates Yen1 activity from meiosis I onwards, and this function is essential for crossover resolution in the absence of other repair pathways. We also demonstrate that Cdc14 and Yen1 are required to safeguard sister chromatid segregation during the second meiotic division, a late action that is independent of the earlier role in crossover formation. Thus, this work uncovers previously undescribed functions of Cdc14 in the regulation of meiotic recombination.

genetics↗