Biology subjects

Lawrence, E.

Publications and source records attributed to Lawrence, E..

6 recordsLinked to original sources

bioRxiv2026

CLASP2 promotes repair of kinesin-1 damage to the microtubule lattice

Microtubules are cytoskeletal polymers that play essential roles in eukaryotic cells, including structural support, cell division, and intracellular transport. During intracellular transport, kinesin motor proteins move cargo along microtubule tracks via their processive stepping. Recent studies have shown that the kinesin-1 KIF5C can damage the microtubule lattice while stepping. Microtubule damage can be repaired through incorporation of new tubulin subunits, however, excessive lattice damage results in microtubule breakage and disassembly. To identify cellular factors involved in microtubule repair, we performed an siRNA screen targeting microtubule-associated proteins (MAPs) known to regulate microtubule dynamics and stability. Based on the results, we investigated whether the end binding protein EB1 and cytoplasmic linker-associated protein 2 (CLASP2) contribute to repair of microtubule damage. To test this, we used a microtubule destruction assay in which damage was induced in microtubules gliding over surfaces coated with wild-type or mutant KIF5C proteins. Our findings suggest that CLASP2 directly facilitates microtubule repair, whereas EB1 does not. We further examined CLASP function using a microtubule repair assay and found that CLASP2 promotes repair by enhancing tubulin incorporation and reducing microtubule breakage. Together, these findings demonstrate that CLASP proteins play an important role in repairing and protecting against lattice damage caused by kinesin-1 motor activity. Our results further suggest that MAPs can directly regulate microtubule lattice integrity under mechanical stress generated by motor protein-driven intracellular transport.

Cell Biology↗

bioRxiv2024

Transcriptomic alterations including p53 pathway dysregulation prime DNMT3A mutant cells for transformation

DNMT3A mutations are prevalent in haematologic malignancies. Our mouse model introduced the murine homologue (R878H) of the human hotspot R882H mutation into the mouse Dnmt3a locus, resulting in globally reduced DNA methylation in all tissues. Mice with heterozygous R878H mutations developed {gamma}-radiation induced thymic lymphoma more rapidly than control mice, suggesting a vulnerability to stress stimuli in Dnmt3aR878H/+ cells. In competitive transplantations, Dnmt3aR878H/+ Lin-Sca-1+Kit+ (LSK) cells had a competitive advantage over wt cells, indicating a self-renewal phenotype at the expense of differentiation. RNA-sequencing of Dnmt3aR878H/+ LSKs exposed to low dose {gamma}-radiation showed downregulation of the p53 pathway. Accordingly, reduced PUMA expression was observed by flow cytometry in the bone marrow of {gamma}-irradiated Dnmt3aR878H/+ mice due to altered p53 signalling. These findings provide new insights as to how DNMT3A mutations cause subtle changes in the transcriptome of LSK cells which contribute to their increased self-renewal and propensity for malignant transformation. SIGNIFICANCEHotspot DNMT3A R882H mutations are overrepresented in leukaemia suggesting that they confer a susceptibility to disease if further mutations are acquired. To advance therapies for DNMT3A mutant disease, it is essential to understand the mechanisms by which this mutation primes cells for malignant transformation.

cancer biology↗

bioRxiv2024

A flexible, high-throughput system for studying mRNA translation kinetics in vitro and in cellulo with HiBit technology

HiBit is an engineered luciferases 11 amino acid component that can be introduced as a tag at either terminus of a protein of interest. When the LgBit component and a substrate are present, HiBit and LgBit dimerise forming a functional luciferase. The HiBit technology has been extensively used for high-throughput protein turnover studies in cells. Here, we have adapted the use of the HiBit technology to quantify mRNA translation temporally in vitro in the rabbit reticulocyte system and in cellulo in HEK293 cells constitutively expressing LgBit. The assay system can detect differences in Cap, 5UTR, modified nucleotide composition, coding sequence optimisation and poly(A) length. Importantly, using these assays we established the optimal mRNA composition varied depending on the encoded protein of interest, highlighting the importance of screening methods tailored to the protein of interest, and not reliant on reporter proteins. Our findings demonstrated that HiBit can be easily and readily adapted to monitor mRNA translation and offers a novel and highly favourable method for the development of mRNA-based therapeutics. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/600987v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@92cec9org.highwire.dtl.DTLVardef@460f3dorg.highwire.dtl.DTLVardef@192a240org.highwire.dtl.DTLVardef@1da188e_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

A single-cell transposable element atlas of human cell identity

Single cell RNA sequencing (scRNA-seq) is revolutionizing the study of complex biological systems. However, most sequencing studies overlook the contribution of transposable element (TE) expression to the transcriptome. In both scRNA-seq and bulk tissue RNA sequencing (RNA-seq), quantification of TE expression is challenging due to repetitive sequence content and poorly characterized TE gene models. Here, we developed a tool and analysis pipeline for Single cell Transposable Element Locus Level Analysis of scRNA Sequencing (Stellarscope) that reassigns multi-mapped reads to specific genomic loci using an expectation-maximization algorithm. Using Stellarscope, we built an atlas of TE expression in human PBMCs. We found that locus-specific TEs delineate cell types and define new cell subsets not identified by standard mRNA expression profiles. Altogether, this study provides comprehensive insights into the influence of transposable elements in human biology.

genomics↗

bioRxiv2023

A transient modified mRNA encoding Myc and Cyclin T1 induces cardiac regeneration and improves cardiac function after myocardial injury

Cardiac injury, such as myocardial infarction (MI), results in permanent loss of cardiomyocytes and in many cases heart failure. Transgenic expression of the pro-proliferative transcription factor Myc and Cyclin T1 can drive substantial adult cardiomyocyte proliferation to replace lost cardiomyocytes. Herein, we show that Myc and Cyclin T1 induced cardiomyocyte proliferation leads to myocardial repair and functional (long-term) recovery post-MI in mice. To provide a more translational approach, we developed modified mRNA (modRNA) encoding Myc-Ccnt1 as a transient and non-integrating strategy for regeneration. One dose of Myc-Ccnt1 modRNA is sufficient to transiently drives cardiomyocyte proliferation in human pluripotent stem cell-derived cardiomyocytes and a mouse MI model, where it leads to better heart function. Using single nuclei sequencing and proteomics, we show this was functionally mediated by transcriptional activation of cell-cycle regulating genes, which ultimately results in mitosis and cytokinesis of cardiomyocytes. Collectively, these findings indicate that Myc-Ccnt1 modRNA has the potential to be an effective regenerative therapeutic.

molecular biology↗

bioRxiv2018

The mechanical impact of col11a2 loss on joints; col11a2 mutant zebrafish show changes to joint development and function which lead to early onset osteoarthritis

Abstract (max 200 words)Collagen is the major structural component of cartilage and mutations in the genes encoding Type XI collagen are associated with severe skeletal dysplasias (Fibrochondrogenesis and Stickler syndrome) and early onset osteoarthritis. The impact of the lack of Type XI collagen on cell behaviour and mechanical performance during skeleton development is unknown. We studied a zebrafish mutant for col11a2 and evaluated cartilage, bone development and mechanical properties to address this. We show that in col11a2 mutants Type II collagen is made but is prematurely degraded in maturing cartilage and ectopically expressed in the joint. These changes are correlated with increased stiffness of both bone and cartilage; quantified using Atomic Force Microscopy. In the mutants, the skeletal rudiment terminal region in the jaw joint are broader and the interzone smaller. These differences in shape and material properties impact on joint function and mechanical performance, which we modelled using Finite Element Analyses. Finally, we show that col11a2 heterozygous carriers reach adulthood but show signs of severe early onset osteoarthritis. Taken together our data demonstrate a key role for Type XI collagen in maintaining the properties of cartilage matrix; which when lost leads to alterations to cell behaviour that give rise to joint pathologies.

developmental biology↗