Biology subjects

Lara-Gonzalez, P.

Publications and source records attributed to Lara-Gonzalez, P..

11 recordsLinked to original sources

bioRxiv2026

TMEM209/NPP-28 partitions PLK-1 between the nuclear envelope and nuclear interior during mitosis

The onset of mitosis in animal cells is marked by nuclear envelope breakdown (NEBD), which requires coordinated disassembly of nuclear pore complexes (NPCs) and the nuclear lamina. Polo-like kinase 1 (Plk1/PLK-1) initiates NEBD through phosphorylation of NPC components, but mechanisms that spatially regulate PLK-1 between NPCs and the nuclear interior are unclear. Here, we identify the Caenorhabditis elegans TMEM209 homolog NPP-28 as a transmembrane nucleoporin that concentrates PLK-1 at the nuclear envelope while limiting its nuclear accumulation to promote NEBD. We identify docking sites within NPP-28 that mediate PLK-1 nuclear envelope-association. NPP-28 is phosphorylated by PLK-1 in vitro, and mutational analysis suggests that NPP-28-mediated docking facilitates NEBD, whereas phosphorylation prevents premature NEBD. Simultaneous loss of npp-28 and the transmembrane nucleoporin npp-12/gp210 exacerbates mitotic NEBD defects in early embryos and causes severe defects in germline development. Together, our findings identify NPP-28 as a spatial regulator of PLK-1 during mitosis with functional relationships with other conserved transmembrane nucleoporins depending on context.

cell biology↗

bioRxiv2026

A meiotic B-type cyclin selectively drives mitotic progression and fidelity in cancer cells

Cell cycle transitions are driven by cyclins in complex with cyclin-dependent kinases (CDK). Here, we demonstrate that cyclin B3, an evolutionarily divergent B-type cyclin previously thought to function exclusively in female meiosis, is expressed in mitotic cells and interacts with both CDK1 and CDK2. We show that cyclin B3-CDK1/2 is essential for timely mitotic progression by ensuring proper chromosome alignment and stable kinetochore-microtubule interactions, while its overexpression accelerates mitosis and improves chromosome alignment kinetics. Through phospho-mass spectrometry, we identify several kinetochore and mitotic spindle components as substrates of cyclin B3-CDK1/2 in mitosis. Notably, cyclin B3 depletion disrupts mitotic progression in most cancer cell lines tested - including colorectal, osteosarcoma, and breast cancer models - while non-transformed cells are largely unaffected. These findings identify cyclin B3-CDK1/2 as a novel driver of mitotic progression and reveal a preferential dependency in a subset of cancer cells that may represent a potential therapeutic vulnerability.

cancer biology↗

bioRxiv2026

Cyclin B3-CDK1 couples translational de-repression to embryonic mitoses via OMA protein degradation in C. elegans.

During early embryogenesis, gene expression relies on maternally loaded mRNAs whose translation is controlled by RNA binding proteins. Here, we identify a critical role for the cyclin B3-CDK1 complex, known for its function in mitosis, in driving early embryonic gene expression in C. elegans. The cyclin B3-CDK1 complex works by marking the RNA binding OMA proteins (OMA-1 and OMA-2) for degradation, which ensures the de-repression and translation of their target mRNAs. OMA protein degradation relies on cyclin B3s conserved phosphate-binding pocket, which promotes multi-site OMA phosphorylation and the generation of phospho-degrons. Notably, the phosphate-binding pocket of cyclin B3 does not substantially contribute to its mitotic roles, indicating that the mitotic and translational functions of the cyclin B3-CDK1 complex are separable. These findings establish that embryonic activation of the cyclin B3-CDK1 complex drives both mitotic divisions and mRNA de-repression, which ensures that cell division is coupled to the early gene expression program in development.

developmental biology↗

bioRxiv2025

Mammalian Cells Integrate Endoplasmic Reticulum and Nuclear Envelope signals to time mitotic entry

Accurate cell division requires coordination between organelle organization and cell-cycle progression, but how architectural and functional cues from the endoplasmic reticulum (ER) and nuclear envelope (NE)--a continuous membrane network--interface with mitotic control remains unclear. Here, we demonstrate that mammalian cells integrate ER/NE structure and functions to regulate the onset and progression of mitosis. Perturbing ER function with diverse stressors causes a selective delay at the metaphase-anaphase transition, accompanied by defective spindle assembly, chromosome misalignment, and loss of coordinated ER-chromosome organization. Under these conditions, the checkpoint protein MAD1 fails to efficiently dissociate from the NE. ER stress also disrupts microtubule-organizing centers and the centriculum, an ER-derived compartment surrounding centrosomes. Restoring ER structure by expressing the shaping proteins CLIMP63(1-192) or REEP4 rescues spindle organization and mitotic progression. Conversely, transient metaphase arrest induced by partial APC/C inhibition remodels ER morphology independently of stress, and this remodeling is reversed by CLIMP63(1-192). These findings uncover a bidirectional link between ER structure function and the spindle assembly checkpoint, identifying the organelle architecture as an instructive signal that modulates mitotic timing in mammalian cell.

cell biology↗

bioRxiv2024

The CYK-4 GAP domain regulates cortical targeting of centralspindlin to promote contractile ring assembly and facilitate ring dissolution

During cytokinesis, an equatorial contractile ring partitions the cell contents. Contractile ring assembly requires an equatorial zone of active GTP-bound RhoA generated by the guanine nucleotide exchange factor ECT21,2. ECT2 is activated by centralspindlin, a complex composed of two molecules each of kinesin-6 and CYK4. During anaphase, Centralspindlin is activated at the central spindle between the separating chromosomes and diffuses to the plasma membrane, where it engages with ECT2 via its N-terminal half3,4. The C-terminal half of CYK4 contains a lipid-binding C1 domain that contributes to plasma membrane targeting5 and a GTPase-activating protein (GAP) domain that has an interaction surface for a Rho family GTPase, whose functions have remained unclear 1,3,4,6,7. Here, using the one-cell stage C. elegans embryo as a model, we show that RhoA and the Rho-binding interface of the CYK4 GAP domain drive the recruitment of centralspindlin to the equatorial cortex. By contrast, a point mutant that selectively disrupts GAP activity does not prevent cortical centralspindlin recruitment but instead substantially delays dissipation of centralspindlin from the cortex. These findings suggest that positive feedback, in which centralspindlin recruitment promotes the generation of active RhoA and active RhoA drives centralspindlin recruitment, is central to the rapid assembly of the contractile ring within a narrow time window. They also indicate that the CYK4 GAP catalytic activity contributes to release of centralspindlin from the cortex, potentially to ensure timely dissolution of the contractile ring.

cell biology↗

bioRxiv2024

Phospho-KNL-1 recognition by a TPR domain targets the BUB-1/BUB-3 complex to C. elegans kinetochores

During mitosis, the Bub1-Bub3 complex concentrates at kinetochores, the microtubule-coupling interfaces on chromosomes, where it contributes to spindle checkpoint activation, kinetochore-spindle microtubule interactions, and protection of centromeric cohesion. Bub1 has a conserved N-terminal tetratricopeptide (TPR) domain followed by a binding motif for its conserved interactor Bub3. The current model for Bub1-Bub3 localization to kinetochores is that Bub3, along with its bound motif from Bub1, recognizes phosphorylated "MELT" motifs in the kinetochore scaffold protein Knl1. Motivated by the greater phenotypic severity of BUB-1 versus BUB-3 loss in C. elegans, we show that the BUB-1 TPR domain directly recognizes a distinct class of phosphorylated motifs in KNL-1 and that this interaction is essential for BUB-1-BUB-3 localization and function. BUB-3 recognition of phospho-MELT motifs additively contributes to drive super-stoichiometric accumulation of BUB-1-BUB-3 on its KNL-1 scaffold during mitotic entry. Bub1s TPR domain interacts with Knl1 in other species, suggesting that collaboration of TPR-dependent and Bub3-dependent interfaces in Bub1-Bub3 localization and functions may be conserved.

cell biology↗

bioRxiv2023

Cyclin B3 is a dominant fast-acting cyclin that drives rapid early embryonic mitoses

In many species, early embryonic mitoses proceed at a very rapid pace, but how this pace is achieved is not understood. Here we show that in the early C. elegans embryo, cyclin B3 is the dominant driver of rapid embryonic mitoses. Metazoans typically have three cyclin B isoforms that associate with and activate Cdk1 kinase to orchestrate mitotic events: the related cyclins B1 and B2 and the more divergent cyclin B3. We show that whereas embryos expressing cyclins B1 and B2 support slow mitosis (NEBD to Anaphase [~] 600s), the presence of cyclin B3 dominantly drives the [~]3-fold faster mitosis observed in wildtype embryos. CYB-1/2-driven mitosis is longer than CYB-3-driven mitosis primarily because the progression of mitotic events itself is slower, rather than delayed anaphase onset due to activation of the spindle checkpoint or inhibitory phosphorylation of the anaphase activator CDC-20. Addition of cyclin B1 to cyclin B3-only mitosis introduces an [~]60s delay between the completion of chromosome alignment and anaphase onset, which likely ensures segregation fidelity; this delay is mediated by inhibitory phosphorylation on CDC-20. Thus, the dominance of cyclin B3 in driving mitotic events, coupled to introduction of a short cyclin B1-dependent delay in anaphase onset, sets the rapid pace and ensures fidelity of mitoses in the early C. elegans embryo.

cell biology↗

bioRxiv2022

PLK-1 tethered on BUB-1 directs CDC-20 kinetochore recruitment to ensure timelyembryonic mitoses

During mitosis chromosomes assemble kinetochores in order to dynamically couple with spindle microtubules (Cheeseman, 2014; Musacchio & Desai, 2017). Kinetochores also function as signaling hubs directing mitotic progression by recruiting and controlling the fate of the Anaphase Promoting Complex/Cyclosome (APC/C) activator CDC-20 (Lara-Gonzalez et al., 2017; Lara-Gonzalez, Pines, et al., 2021; Musacchio, 2015). Kinetochores either incorporate CDC-20 into checkpoint complexes that inhibit the APC/C or dephosphorylate CDC-20, which allows it to interact with and activate the APC/C (Kim et al., 2017; Lara-Gonzalez et al., 2017). The importance of these two CDC-20 fates likely depends on biological context. In somatic cells the major mechanism controlling mitotic progression is the spindle checkpoint. By contrast, progression through mitosis during the cell cycles of early embryos is largely checkpoint-independent (Clute & Masui, 1995; Duro & Nilsson, 2021; Gerhart et al., 1984; Zhang et al., 2015). Here, by manipulating CDC-20 phosphorylation status, we show that CDC-20 phosphoregulation controls mitotic duration in the C. elegans embryo and defines a checkpoint-independent temporal mitotic optimum for robust embryogenesis. Flux of CDC-20 through kinetochores for local dephosphorylation requires an ABBA motif on BUB-1 that directly interfaces with the structured WD40 domain of CDC-20 (Di Fiore et al., 2015; Diaz-Martinez et al., 2015; He et al., 2013; Kim et al., 2017). We show that a conserved "STP" motif in BUB-1 that docks the mitotic kinase PLK-1 (Qi et al., 2006) is also necessary to recruit CDC-20 to kinetochores and for timely mitotic progression. The kinase activity of PLK-1 is required for CDC-20 to localize to kinetochores and targets a site within the CDC-20-binding ABBA motif of BUB-1; phosphorylation of this site promotes BUB-1-CDC-20 interaction and mitotic progression. Thus, the BUB-1-bound pool of PLK-1 ensures timely mitosis during embryonic cell cycles by promoting CDC-20 recruitment to the vicinity of kinetochore-localized phosphatase activity.

cell biology↗

bioRxiv2022

Nuclear-enriched protein phosphatase 4 ensures outer kinetochore assemblyprior to nuclear dissolution

A landmark event in the transition from interphase to mitosis in metazoans is nuclear envelope breakdown (NEBD). Many events important for mitosis occur prior to NEBD, including condensation of replicated chromosomes and assembly of kinetochores to rapidly engage spindle microtubules. Here we show that nuclear-enriched protein phosphatase 4 (PP4) ensures robust assembly of the microtubule-coupling outer kinetochore prior to NEBD. In the absence of PP4, chromosomes exhibit extended monopolar orientation after NEBD and subsequently mis-segregate. A secondary consequence of diminished outer kinetochore assembly is defective sister chromatid resolution. After NEBD, a cytoplasmic activity compensates for PP4 loss, leading to outer kinetochore assembly and recovery of chromosomes from monopolar orientation to significant biorientation. The Ndc80-Ska microtubule-binding module of the outer kinetochore is required for this recovery. PP4 associates with the inner kinetochore protein CENP-C; however, disrupting the PP4-CENP-C interaction does not perturb chromosome segregation. These results establish that PP4-dependent outer kinetochore assembly prior to NEBD is critical for timely and proper engagement of chromosomes with spindle microtubules.

cell biology↗

bioRxiv2020

PP2A:B56 Regulates Meiotic Chromosome Segregation in C. elegans Oocytes

Protein Phosphatase 2A (PP2A) is an heterotrimer composed of scaffolding (A), catalytic (C), and regulatory (B) subunits with various key roles during cell division. While A and C subunits form the core enzyme, the diversity generated by interchangeable B subunits dictates substrate specificity. Within the B subunits, B56-type subunits play important roles during meiosis in yeast and mice by protecting centromeric cohesion and stabilising the kinetochore-microtubule attachments. These functions are achieved through targeting of B56 subunits to centromere and kinetochore by Shugoshin and BUBR1. In the nematode Caenorhabditis elegans (C. elegans) the closest BUBR1 ortholog lacks the B56 interaction domain and the Shugoshin orthologue is not required for normal segregation during oocyte meiosis. Therefore, the role of PP2A in C. elegans female meiosis is not known. Here, we report that PP2A is essential for meiotic spindle assembly and chromosome dynamics during C. elegans female meiosis. Specifically, B56 subunits PPTR-1 and PPTR-2 associate with chromosomes during prometaphase I and regulate chromosome congression. The chromosome localization of B56 subunits does not require shugoshin orthologue SGO-1. Instead we have identified the kinase BUB-1 as the key B56 targeting factor to the chromosomes during meiosis. PP2A BUB-1 recruits PP2A:B56 to the chromosomes via dual mechanism: 1) PPTR-1/2 interacts with the newly identified LxxIxE short linear motif (SLiM) within a disordered region in BUB-1 in a phosphorylation-dependent manner; and 2) PPTR-2 can also be recruited to chromosomes in a BUB-1 kinase domain-dependent manner. Our results highlight a novel, BUB-1-dependent mechanism for B56 recruitment, essential for recruiting a pool of PP2A required for proper chromosome congression during meiosis I.

cell biology↗

bioRxiv2017

A toolkit for tissue-specific protein degradation in C. elegans

Proteins essential for embryo production, cell division, and early embryonic events are frequently re-utilized later in embryogenesis, during organismal development, or in the adult. Examining protein function across these different biological contexts requires tissue-specific perturbation. Here, we describe a method that utilizes expression of a fusion between a GFP-targeting nanobody and SOCS-box containing ubiquitin ligase adaptor to target GFP tagged proteins for degradation. When combined with endogenous locus GFP tagging by CRISPR-Cas9 or rescue of a null mutant with a GFP fusion, this approach enables routine and efficient tissue-specific protein ablation. We show that this approach works in multiple tissues--the epidermis, intestine, body wall muscle, sensory neurons, and touch neurons--where it recapitulates expected loss-of-function mutant phenotypes. The transgene toolkit and the strain set described here will complement existing approaches to enable routine analysis of the tissue-specific roles of C. elegans proteins.

developmental biology↗