Biology subjects

Lane, E. B.

Publications and source records attributed to Lane, E. B..

4 recordsLinked to original sources

bioRxiv2023

qMaLioffG: A single green fluorescent protein FLIM indicator enabling quantitative imaging of endogenous ATP

The widespread use of fluorescence lifetime imaging microscopy (FLIM) for quantitative imaging is hindered by the limited availability of a FLIM-based genetically encoded indicator using a conventional 488 nm laser. Here, we present qMaLioffG, a single green fluorescent protein FLIM indicator showing a fluorescence lifetime change in ATP concentration within the physiological range. This allows quantitative imaging of endogenous ATP to investigate cellular energy status of different cell types.

biophysics↗

bioRxiv2023

Proximity mapping of desmosomes reveals a striking shift in their molecular neighbourhood associated with maturation

Desmosomes are multiprotein adhesion complexes that link intermediate filaments to the plasma membrane, ensuring the mechanical integrity of cells across tissues, but how they participate in the wider signalling network to exert their full function is unclear. To investigate this we carried out multiplexed protein proximity mapping using biotinylation (BioID). The combined interactomes of the essential desmosomal proteins desmocollin 2a, plakoglobin and plakophilin 2a (Pkp2a) in Madin-Darby canine kidney epithelial cells were mapped and their differences and commonalities characterised as desmosome matured from Ca2+-dependence to the mature, Ca2+-independent, hyperadhesive state, which predominates in tissues. Results suggest that individual desmosomal proteins have distinct roles in connecting to cellular signalling pathways and that these roles alter substantially when cells change their adhesion state. The data provide further support for a dualistic concept of desmosomes in which the properties of Pkp2a differ from those of the other, more stable proteins. This body of data provides an invaluable resource for analysis of desmosome function.

cell biology↗

bioRxiv2021

Desmosomal dualism: the core is stable while plakophilin is dynamic.

Desmosomes, strong cell-cell junctions of epithelia and cardiac muscle, link intermediate filaments to cell membranes and mechanically integrate cells across tissues, dissipating mechanical stress. They comprise 5 major protein classes - desmocollins and desmogleins (the desmosomal cadherins), plakoglobin, plakophilins and desmoplakin - whose individual contribution to the structure and turnover of desmosomes is poorly understood. Using live-cell imaging together with FRAP and FLAP we show that desmosomes consist of two contrasting protein fractions or modules: a very stable desmosomal core of desmosomal cadherins and plakoglobin, and a highly mobile plakophilin. As desmosomes mature from calcium-dependence to calciumindependent hyper-adhesion, core stability increases, but Pkp2a remains highly mobile. Desmoplakin is initially mobile but stabilises with hyper-adhesion. We show that desmosome down-regulation during growth factor-induced cell scattering proceeds by internalisation of whole desmosomes, which still retain a stable core and highly mobile Pkp2a. This molecular mobility of Pkp2a suggests a transient and probably regulatory role for Pkp2a in the desmosome.

cell biology↗

bioRxiv2017

Array-based sequencing of filaggrin gene for comprehensive detection of disease-associated variants

The filaggrin gene (FLG) is essential for skin differentiation and epidermal barrier formation with links to skin diseases, however it has a highly repetitive nucleotide sequence containing very limited stretches of unique nucleotides for precise mapping to reference genomes. Sequencing strategies using polymerase chain reaction (PCR) and conventional Sanger sequencing have been successful for complete FLG coding DNA sequence amplification to identify pathogenic mutations but this time-consuming, labour intensive method has restricted utility. Next-generation sequencing (NGS) offers obvious benefits to accelerate FLG analysis but standard re-sequencing techniques such as oligoprobe-based exome or customized targeted-capture can be expensive, especially for a single target gene of interest. We therefore designed a protocol to improve FLG sequencing throughput using a set of FLG-specific PCR primer assays compatible with microfluidic amplification, multiplexing and current NGS protocols. Using DNA reference samples with known FLG genotypes for benchmarking, this protocol is shown to be concordant for variant detection across different sequencing methodologies. We applied this methodology to analyze cohorts from ethnicities previously not studied for FLG variants and demonstrate usefulness for discovery projects. This comprehensive coverage sequencing protocol is labour-efficient and offers an affordable solution to scale up FLG sequencing for larger cohorts. Robust and rapid FLG sequencing can improve patient stratification for research projects and provide a framework for gene specific diagnosis in the future.

genetics↗