Biology subjects

Clements, K.

Publications and source records attributed to Clements, K..

3 recordsLinked to original sources

bioRxiv2024

Genetically-encoded markers for confocal visualization of single dense core vesicles

Neuronal dense core vesicles (DCVs) store and release a diverse array of neuromodulators, trophic factors and bioamines. The analysis of single DCVs has largely been possible only using electron microscopy, which makes understanding cargo segregation and DCV heterogeneity difficult. To address these limitations, we developed genetically-encoded markers for DCVs that can be used in combination with standard immunohistochemistry and expansion microscopy, to enable single-vesicle resolution with confocal microscopy.

neuroscience↗

bioRxiv2024

Repurposing CRISPR-Cas13 systems for robust mRNA trans-splicing

Type VI CRISPR enzymes have been developed as programmable RNA-guided Cas proteins for eukaryotic RNA editing. Notably, Cas13 has been utilized for site-targeted single base edits, demethylation, RNA cleavage or knockdown and alternative splicing. However, the ability to edit large stretches of mRNA transcripts remains a significant challenge. Here, we demonstrate that CRISPR-Cas13 systems can be repurposed to assist trans-splicing of exogenous RNA fragments into an endogenous pre-mRNA transcript, a method termed CRISPR Assisted mRNA Fragment Trans-splicing (CRAFT). Using split reporter-based assays, we evaluate orthogonal Cas13 systems, optimize guide RNA length and screen for optimal trans-splicing site(s) across a range of intronic targets. We achieve markedly improved editing of large 5 and 3 segments in different endogenous mRNAs across various mammalian cell types compared to other spliceosome-mediated trans-splicing methods. CRAFT can serve as a versatile platform for attachment of protein tags, studying the impact of multiple mutations/single nucleotide polymorphisms, modification of untranslated regions (UTRs) or replacing large segments of mRNA transcripts.

bioengineering↗

bioRxiv2019

A luciferase-based assay identifies niclosamide derivatives antagonizing Mcl-1 through post-translational down-regulation

The up-regulation of Mcl-1 expression is a major mechanism of cancer cell survival and therapy resistance. However, the underlying molecular mechanism remains incompletely understood, limiting the number of druggable approaches to selectively inhibit Mcl-1 function. In this study, we designed and employed a novel mechanistic high-throughput screening system to selectively uncover post-translational modulators of Mcl-1. We generated a cell-based high-throughput screening assay in which myeloid leukemia K562 cells constitutively express Mcl-1 or Bcl-xL fused with luciferase (Luc-Mcl-1 or Luc-Bcl-xL, respectively) under a viral promoter. 1,650 bioactive compounds were screened for their ability to selectively induce Mcl-1 down-regulation in a 2-hour assay. A family of niclosamide derivatives were eventually identified for their remarkable ability to decrease Mcl-1 protein stability, exemplified by N007. These salicylate derivatives did not alter Mcl-1 mRNA levels, but selectively induced proteasome-dependent Mcl-1 down-regulation independent of Noxa, Mule, or GSK3{beta}. We also demonstrate that N007 potently induced cell death in leukemia cell lines, including those resistant to Bcl-2 inhibitors. Our work highlights the versatility of the mechanistic high-throughput screening approach as a valuable tool in identifying novel agents with the ability to down-regulate proteins crucial to human diseases.

biochemistry↗