Biology subjects

Lim, C. J.

Publications and source records attributed to Lim, C. J..

14 recordsLinked to original sources

bioRxiv2026

Diversifying particle-orientation distributions in cryo-EM with LEA protein additives

Preferential orientation at the air-water interface (AWI) remains a persistent challenge in cryo-electron microscopy, often requiring extensive optimization or specialized grid preparation strategies. Here, we show that late embryogenesis abundant (LEA) proteins, previously shown to protect fragile samples from AWI-induced damage, can be leveraged to resolve sample orientation bias. The addition of LEA proteins can yield either new orientation distributions or more isotropic ones in samples suffering from orientation biases. These findings establish LEA proteins as a practical class of cryo-EM additives that can reduce AWI-induced damage and improve particle-orientation distributions in single-particle analysis.

biophysics↗

bioRxiv2026

Fission yeast RPA-TERT-Tpz1TPP1 complex promotes telomere extension and suppresses telomere recombination

Telomerase maintains chromosome ends by extending telomeric DNA, yet how recruited telomerase becomes productively engaged remains poorly understood. Recent studies found that Replication Protein A (RPA) contributes to telomerase stimulation through interaction with TERT in humans and with the TPP1 ortholog Est3 in budding yeast, suggesting a direct role in telomerase activation. Here, we provide genetic and structural modeling evidence for a RPA-Trt1TERT-Tpz1TPP1 ternary complex that promotes telomere extension while suppressing recombination in fission yeast. Guided by results from genetic screen, followed by AlphaFold3 modeling and systematic mutagenesis of RPA, Trt1, and Tpz1, we identify four key interfaces supporting telomerase function: Ssb1RPA1-Trt1, Ssb2RPA2-Trt1, Ssb2RPA2-Tpz1, and the TEL-patch-mediated Trt1-Tpz1 interaction. Notably, Tpz1-R81, previously assigned as the TEL patch, instead contacts Ssb2 in the complex. Epistasis and suppressor analyses indicate that the newly identified RPA-Trt1 and RPA-Tpz1 interfaces collaborate with the Trt1-Tpz1 interface to allow telomerase activation after recruitment. Furthermore, comparative analyses using AlphaFold3 suggest that these interactions are likely conserved in budding yeast and humans. Collectively, these findings support a model in which RPA serves as an essential component of the active telomerase complex, coordinating TERT and TPP1-like factors to enable productive telomerase engagement. Author SummaryTelomeres are specialized dynamic protective structures at the ends of eukaryotic chromosomes that must be properly maintained to preserve genome stability. Telomerase extends telomeric DNA, but how recruited telomerase becomes fully activated to promote telomere extension remains poorly understood. In this study, we use fission yeast to investigate the role of the conserved single-stranded DNA-binding protein complex Replication Protein A (RPA) in this process. We find that RPA forms a functional complex with the telomerase catalytic subunit TERT and Tpz1, a component of the telomere protection complex shelterin and the fission yeast ortholog of human TPP1. Genetic and structural analyses identify multiple interactions within the RPA-TERT-Tpz1 complex that are required for efficient telomere extension. Disrupting these interactions allows telomerase recruitment but prevents productive telomerase action at chromosome ends. Our results further suggest that similar mechanisms may operate in other organisms, including budding yeast and humans. These findings provide insight into how telomerase activity is regulated at chromosome ends.

molecular biology↗

bioRxiv2026

Aurora kinase A enables collective invasion and metastasis by endowing a leader cell phenotype and stabilizing Eplin-mediated cohesion with follower cells

The metastatic process initiates with collective cell invasion into surrounding tissues and axillary nodes, and subsequent colonization at a distant site. Previously, we found collective invasion is augmented during the G2 cell cycle phase, facilitated through Aurora kinase A (AURKA)-mediated centrosome polarization in the leader cell. Here, we identify cell cycle-associated gene signatures as overrepresented in axilla and liver metastatic sites, with AURKA expression strongly correlated with breast cancer metastasis signatures, and pan-cancer patient survival. Then, we show GFP-AURKA expression endows breast epithelia cells with the ability to form metastatic outgrowths within immune-incompetent chicken embryos. Multi-parametric imaging of wound closure assays reveals phenotypes enabled by, and dependent upon AURKA expression. We discover leader cells express AURKA and acquire front-polarized centrosomes, which differentiates them from other cells in the migrating group. Ectopic expression of GFP-AURKA induces a leader cell phenotype. Conversely, inhibition of AURKA activity alters actin dynamics, promotes turnover of cell contacts, and reduces coordination within migrating groups. Specifically, AURKA interacts with the actin regulator EPLIN, and AURKA inhibition localizes EPLIN to lamellipodia and away from E-cadherin-positive contacts. Inhibiting these necessary roles for AURKA may provide a critical barrier against the metastatic spread of human breast carcinoma cells.

cancer biology↗

bioRxiv2025

Structural Basis of Herpesvirus Helicase-Primase Inhibition by Pritelivir and Amenamevir

Widespread herpesvirus infections are associated with various diseases. DNA replication of human herpes simplex virus type 1 (HSV-1) requires a helicase-primase (HP) complex of three core proteins: UL5, UL52, and UL8. This complex unwinds viral DNA and synthesizes primers for DNA replication, making it an attractive antiviral target. Although HP inhibitors pritelivir and amenamevir were identified through screening, their binding mechanisms remain unclear. Here, we report cryo-EM structures of HSV-1 HP bound to a forked DNA template alone, and in complex with pritelivir or amenamevir. The structures reveal a bilobed architecture highlighting helicase-primase coordinated action at the replication fork and providing a structural basis for HP inhibition by illustrating precisely how pritelivir and amenamevir block helicase activity. Data lays a solid foundation for the development of improved antiviral therapies.

biochemistry↗

bioRxiv2024

Human Replication Protein A complex is a Telomerase Processivity Factor Essential for Telomere Maintenance

Telomerase is crucial for maintaining telomere length and safeguarding genome stability. In this study, we identified Replication Protein A (RPA) as a novel telomerase processivity factor, functioning alongside the telomerase recruitment factor TPP1-POT1. AlphaFold2 predictions revealed that RPA and TPP1 interact with telomerase at distinct binding sites. Using separation- of-function mutants, we discovered that RPA-mediated telomerase stimulation is indispensable for telomere elongation, while TPP1-POT1 primarily functions in recruiting telomerase to telomeres. Furthermore, we demonstrated that short telomere disease-associated telomerase mutations compromise RPAs ability to stimulate telomerase, establishing a link between impaired RPA-dependent processivity and telomeropathies. Our findings redefine human telomerase regulation by establishing RPA as a critical regulator and provide new insights into the molecular basis of telomere-related diseases.

molecular biology↗

bioRxiv2024

Small LEA proteins as an effective air-water interface protectant for fragile samples during cryo-EM grid plunge freezing

Sample loss due to air-water interface (AWI) interactions is a significant challenge during cryo-electron microscopy (cryo-EM) sample grid plunge freezing. We report that small Late Embryogenesis Abundant (LEA) proteins, which naturally bind to AWI, can protect samples from AWI damage during plunge freezing. This protection is demonstrated with two LEA proteins from nematodes and tardigrades, which rescued the cryo-EM structural determination outcome of two fragile multisubunit protein complexes.

biophysics↗

bioRxiv2021

Structure of human CST-pol-α/primase bound to a telomeric overhang poised for initiation of telomere C-strand synthesis

Telomere replication and regulation protect mammalian chromosome ends and promote genome stability. An essential step in telomere maintenance is the C-strand fill-in process, which is the de novo synthesis of the complementary strand of the telomere overhang. This step is catalyzed by polymerase-alpha/primase complex (pol-/primase) and coordinated by an accessory factor, CTC1-STN1-TEN1 (CST). Using cryogenic-electron microscopy single-particle analysis, we report the structure of the human telomere C-strand fill-in preinitiation complex (PIC) at 3.9 [A] resolution. The structure reveals a CST and a pol-/primase co-bound to a single telomere overhang, poised for de novo RNA primer synthesis. Upon PIC assembly, the pol-/primase undergoes large conformation change from its apo-state; CST partitions the DNA and RNA catalytic centers of pol-/primase into two separate domains and positions the 3' end of an extended telomere single-stranded DNA template towards the RNA catalytic center (PRIM1 or p49). The telomeric single-stranded DNA template is further positioned by the POLA1 (or p180) catalytically dead exonuclease domain. Together with CST, the exonuclease domain forms a tight-fit molecular tunnel for template direction. Given the structural homology of CST to Replication Protein A (RPA), our structure provides the structural basis for a new model of how pol-/primase lagging-strand DNA synthesis is coordinated by single-stranded DNA-binding accessory factors.

biochemistry↗

bioRxiv2021

CST does not evict elongating telomerase but prevents initiation by ssDNA binding

The CST complex (CTC1-STN1-TEN1) has been shown to inhibit telomerase extension of the G-strand of telomeres and facilitate the switch to C-strand synthesis by DNA polymerase alpha-primase (pol -primase). Recently the structure of human CST was solved by cryo-EM, allowing the design of mutant proteins defective in telomeric ssDNA binding and prompting the reexamination of CST inhibition of telomerase. The previous proposal that human CST inhibits telomerase by sequestration of the DNA primer was tested with a series of DNA-binding mutants of CST and modeled by a competitive binding simulation. The DNA-binding mutants had substantially reduced ability to inhibit telomerase, as predicted from their reduced affinity for telomeric DNA. These results provide strong support for the previous primer sequestration model. We then tested whether addition of CST to an ongoing processive telomerase reaction would terminate DNA extension. Pulse-chase telomerase reactions with addition of either wild-type CST or DNA-binding mutants showed that CST has no detectable ability to terminate ongoing telomerase extension in vitro. The same lack of inhibition was observed with or without pol -primase bound to CST. These results suggest how the switch from telomerase extension to C-strand synthesis may occur.

biochemistry↗

bioRxiv2021

A Calcium/Palmitoylation Switch Interfaces the Signaling Networks of Stress Response and Transition to Flowering

The precise timing of flowering in adverse environments is critical for plants to secure reproductive success. We report a novel mechanism controlling the time of flowering by which the palmitoylation-dependent nuclear import of protein SOS3/CBL4, a Ca2+-signaling intermediary in the plant response to salinity, results in the selective stabilization of the flowering time regulator GIGANTEA inside the nucleus under salt stress, while degradation of GIGANTEA in the cytosol releases the protein kinase SOS2 to achieve salt tolerance. S-acylation of SOS3 was critical for its nuclear localization and the promotion of flowering, but dispensable for salt tolerance. SOS3 interacted with the photoperiodic flowering components GIGANTEA and FKF1 on the CONSTANS gene promoter to sustain the transcription of CO and FT under salinity. Thus, SOS3 acts as a Ca2+- and palmitoylation-dependent molecular switch that fine-tunes flowering in a saline environment through the shared spatial separation and selective stabilization of GIGANTEA. The SOS3 protein connects two signaling networks to co-regulate stress adaptation and time of flowering. Short summaryS-acylation promoted the nuclear import of SOS3/CBL4 for the selective stabilization of the photoperiodic floral regulator GIGANTEA to fine-tune flowering time in a saline environment. Spatial separation of SOS3 acts as a molecular switch co-regulating stress adaptation and time of flowering.

plant biology↗

bioRxiv2021

HIV-1 Vpu promotes phagocytosis of infected CD4+ T cells by macrophages through downregulation of CD47

Human immunodeficiency virus (HIV) remodels the cell surface of infected cells to facilitate viral dissemination and promote immune evasion. The membrane-associated Vpu accessory protein encoded by HIV-1 plays a key role in this process by altering cell surface levels of multiple host proteins. Using an unbiased quantitative plasma membrane profiling approach, we previously identified CD47 as a putative host target downregulated by Vpu. CD47 is a ubiquitously-expressed cell surface protein that interacts with the myeloid cell inhibitory receptor SIRP to deliver a "dont-eat-me" signal, thus protecting cells from phagocytosis. In this study, we investigate whether CD47 modulation by HIV-1 Vpu might promote the susceptibility of macrophages to viral infection via phagocytosis of infected CD4+ T cells. Indeed, we find that Vpu downregulates CD47 expression on infected CD4+ T cells leading to an enhanced capture and phagocytosis by macrophages. Interestingly, it is through this process that a CCR5-tropic transmitted/founder (T/F) virus, which otherwise poorly infects macrophages in its cell-free form, becomes infectious in macrophages. Importantly, we show that HIV-1-infected cells expressing a Vpu-resistant CD47 mutant are less prone to infect macrophages through phagocytosis. Mechanistically, Vpu forms a physical complex with CD47 through its transmembrane domain and targets the latter for lysosomal degradation. These results reveal a novel role of Vpu in modulating macrophage infection, which has important implications for HIV-1 transmission in early stages of infection and the establishment of viral reservoir. IMPORTANCEMacrophages play critical roles in HIV transmission, viral spread early in infection, and as a reservoir of virus. Selective capture and engulfment of HIV-1 infected T cells was shown to drive efficient macrophage infection suggesting that this mechanism represents an important mode of infection notably for weakly macrophage-tropic T/F viruses. In this study, we provide insight into the signals that regulate this process. We show that the HIV-1 accessory protein Vpu downregulates cell surface levels of CD47, a host protein that interacts with the inhibitory receptor SIRP to deliver a "dont-eat-me" signal to macrophages. This allows for enhanced capture and phagocytosis of infected T cells by macrophages, ultimately leading to their productive infection even with T/F virus. These findings provide new insights into the mechanisms governing the intercellular transmission of HIV-1 to macrophages with implications for the establishment of the macrophage reservoir and early HIV-1 dissemination in vivo.

microbiology↗

bioRxiv2020

Single cell RNA-seq reveals immunosuppressive gastric stem-like cancer cells as a poor prognostic factor

ObjectiveGastric cancer (GC) tumors are highly heterogenous with different subpopulations of epithelial cells. We employed single cell RNA sequencing (scRNA-seq) to dissect the heterogeneity and identified subpopulations of cancer cells with stem-like properties. We further investigated their resistance to oxaliplatin chemotherapy and their contribution to gastric cancer outcome. DesignWe performed scRNA-seq on FACS sorted epithelial and immune cells from paired samples of GC tumors and normal adjacent tissues. We identified two epithelial subpopulations (STMN1+IQGAP3+ and STMN1+IQGAP3-) with stem-like properties. We characterized and compared them to known healthy gastric stem cell populations. We also cultivated GC derived organoids to study the chemoresistance of similarly marked populations. Lastly, we employed immunohistochemistry (IHC) staining to ascertain the predicted immunosuppressive interactions. ResultsThe STMN1+IQGAP3+ subpopulation showed a higher tumor mutation burden, upregulated proliferative pathways and transcriptomically resembled proliferative healthy gastric isthmus stem cells. The STMN1+IQGAP3- subpopulation were comparatively quiescent and transcriptomically resembled enteroendocrine cells. Both transcriptomic signatures were associated with worse mortality than other epithelial subpopulations with the quiescent being associated with the poorest patient survival. GC tissue derived organoids were dominated by STMN1+IQGAP3+ cells but the STMN1+IQGAP3- compartment was more resistant to chemotherapy. We also verified the likely suppression of CD8 T cell cytotoxicity by STMN1+IQGAP3+ cells through the NECTIN2/TIGIT interaction. ConclusionsCancer cells with stem-like characteristics are associated with poor survival through chemoresistance and immunosuppression. Reactivating the immune system through checkpoint blockade is an opportunity to eliminate these cells. What is already known on this topicMultiple gastric stem cell populations have been identified and linked to tumor initiation in rodent-based studies. However, none of them have been conclusively proven in human tumors. Isolating and characterizing tumor cells with stem-like properties will help shed light on their possible origin and possible mitigation strategies. What this study addsHere we identified two sets of stem-like gastric cancer cells that are associated with poorer patient prognosis. One set is highly proliferative and exhibits oxaliplatin susceptibility. It also engages in immunosuppressive interactions such as NECTIN2/TIGIT. The other set is quiescent and highly resistant to oxaliplatin. How this study might affect research, practice or policyThe transcriptome signatures of the identified stem-like cells can aid in patient prognosis and identify patients who can benefit from checkpoint blockade therapy to reactivate their immune response towards gastric cancer cells.

immunology↗

bioRxiv2019

PDX models reflect the proteome landscape of pediatric acute lymphoblastic leukemia but divert in select pathways

Murine xenografts of pediatric leukemia are known to accurately recapitulate genomic aberrations. How this translates to the functional capacity of the proteome is unknown. Here, we studied global protein abundance, phosphorylation, and proteolytic processing in 11 pediatric B- and T-cell acute lymphoblastic leukemia patients and 19 corresponding xenografts. Protein level differences that stratified pediatric disease subtypes at diagnostic and relapse stages were largely recapitulated in xenograft models. Patient xenografts lacked multiple human leukocyte antigens, and complement proteins, and presented incomplete response mechanisms to the host immune system which is absent in the murine model. The dominant expression of MKI67 and cell cycle proteins indicated a high proliferative capacity of xenografted cells residing in the spleen. Structural genomic changes and mutations found in patients were reflected at the protein level. The post-translational modification landscape is shaped by leukemia type and host and only to a limited degree by the patient of origin. This study portrays how genomic and host factors shape protein and post-translational modification landscapes differently, and confirms murine patient-derived xenograft as competent model system while highlighting important areas of diverging biology.

cancer biology↗

bioRxiv2019

The cryo-EM structure of human CST reveals a two-megadalton decameric assembly bound to telomeric DNA

The single-stranded DNA-binding CTC1-STN1-TEN1 (CST) complex is essential for telomere maintenance and genome-wide replication recovery, processes that are critical for genome stability. Here, we report the 2.95 [A] cryo-EM structure of human CST bound to telomeric single-stranded DNA, which unexpectedly assembles as a decameric supercomplex. The atomic model of the 134 kDa CTC1, built almost entirely de novo, reveals the overall architecture of CST and the DNA-binding anchor site. In situ arrangements of STN1 and TEN1 are revealed, with STN1 interacting with CTC1 at two separated sites, allowing allosteric mediation of CST decameric assembly. Surprisingly, CTC1 lacks the anticipated structural homology to yeast Cdc13 but instead shares similarity with a form of Replication Protein A. The atomic-resolution model of human CST provides crucial mechanistic understanding of CST mutations associated with human diseases. Moreover, the decameric form of CST suggests the intriguing possibility of ssDNA architectural organization similar to what the nucleosome provides for dsDNA.\n\nOne Sentence SummaryHuman telomeric single-stranded DNA triggers the assembly of a decameric protein supercomplex solved by cryo-EM.

biophysics↗

bioRxiv2019

Set2-mediated alternative splicing of Srsf11 regulates cocaine reward behavior

Alternative splicing is a key mechanism for neuronal gene regulation, and is grossly altered in mouse brain reward regions following investigator-administered cocaine. It is well established that cocaine epigenetically regulates transcription, yet mechanism(s) by which cocaine-induced epigenetic modifications regulate alternative splicing is largely unexplored. Our group and others have previously identified the histone modification, H3K36me3, as a putative splicing regulator. However, it has not yet been possible to establish the direct causal relevance of this modification to alternative splicing in brain or any other context. We found that mouse cocaine self-administration caused widespread alternative splicing, concomitant with enrichment of H3K36me3 at splice junctions. Differentially spliced genes were enriched in the motif for splice factor, Srsf11, which was both differentially spliced and enriched in H3K36me3. Epigenetic editing led us to conclude that H3K36me3 functions directly in alternative splicing of Srsf11, and that Set2 mediated H3K36me3 bidirectionally regulates cocaine intake.

neuroscience↗