Petrie, S. K. | Search bioRxiv

bioRxiv2023

CG-SLENP: A chemical genetics strategy to selectively label existing proteins and newly synthesized proteins

Protein synthesis and subsequent delivery to the target locations in cells are essential for their proper functions. Methods to label and distinguish newly synthesized proteins from existing ones are critical to assess their differential properties, but such methods are lacking. We describe the first chemical genetics-based approach for selective labeling of existing and newly synthesized proteins that we termed as CG-SLENP. Using HaloTag in-frame fusion with lamin A (LA), we demonstrate that the two pools of proteins can be selectively labeled using CG-SLENP in living cells. We further employ our recently developed selective small molecule ligand LBL1 for LA to probe the potential differences between newly synthesized and existing LA. Our results show that LBL1 can differentially modulate these two pools of LA. These results indicate that the assembly states of newly synthesized LA are distinct from existing LA in living cells. The CG-SLENP method is potentially generalizable to study any cellular proteins. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/565346v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@d18268org.highwire.dtl.DTLVardef@c8b9ddorg.highwire.dtl.DTLVardef@226bf5org.highwire.dtl.DTLVardef@1dda80c_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion

Precise and efficient insertion of large DNA fragments into somatic cells using gene editing technologies to label or modify endogenous proteins remains challenging. Non-specific insertions/deletions (INDELs) resulting from the non-homologous end joining pathway make the process error-prone. Further, the insert is not readily removable. Here, we describe a method called CRISPR-mediated insertion of exon (CRISPIE) that can precisely and reversibly label endogenous proteins using CRISPR/Cas9-based editing. CRISPIE inserts a designer donor module, which consists of an exon encoding the protein sequence flanked by intron sequences, into an intronic location in the target gene. INDELs at the insertion junction will be spliced out, leaving mRNAs nearly error-free. We used CRISPIE to fluorescently label endogenous proteins in neurons in vivo with previously unachieved efficiency. We demonstrate that this method is broadly applicable, and that the insert can be readily removed later. CRISPIE permits protein sequence insertion with high fidelity, efficiency, and flexibility.

cell biology↗

bioRxiv2020

Insulin potentiates the synchronous firing of arcuate nucleus Kiss1 neurons that protects against diet-induced obesity

Kisspeptin (Kiss1) neurons are essential for reproduction, but their role in the control of energy balance and other homeostatic functions remains unclear. High frequency firing of hypothalamic arcuate Kiss1 (Kiss1ARH) neurons releases kisspeptin into the median eminence, and neurokinin B (NKB) and dynorphin onto neighboring Kiss1ARH neurons to generate a slow excitatory postsynaptic potential (EPSP) mediated by TRPC5 channels that entrains intermittent, synchronous firing of Kiss1ARH neurons. High frequency optogenetic stimulation of Kiss1ARH neurons releases glutamate to excite the anorexigenic proopiomelanocortin (POMC) neurons and inhibit the orexigenic neuropeptide Y/agouti-related peptide (AgRP) neurons via metabotropic glutamate receptors. At the molecular level, the endoplasmic reticulum calcium-sensing protein stromal interaction molecule 1 (STIM1) is critically involved in the regulation of neuronal Ca2+ signaling and neuronal excitability through its interaction with plasma membrane calcium (e.g., TRPC) channels. 17{beta}-estradiol (E2) downregulates Stim1 mRNA expression in female arcuate neurons. Therefore, we hypothesized that deletion of Stim1 in Kiss1ARH neurons would increase neuronal excitability and their synchronous firing, which ultimately would affect energy homeostasis. Using optogenetics in combination with whole-cell recording and GCaMP6 imaging in slices, we discovered that the deletion of Stim1 in Kiss1 neurons significantly increased the amplitude of the slow EPSP and augmented synchronous [Ca2+]i oscillations in Kiss1ARH neurons. Deletion of Stim1 in Kiss1ARH neurons amplified the actions of NKB and protected ovariectomized female mice from developing obesity and glucose intolerance with high-fat dieting. Therefore, STIM1 appears to play a critical role in regulating synchronous firing of Kiss1ARH neurons, which ultimately affects energy homeostasis. Significance StatementHypothalamic arcuate kisspeptin (Kiss1ARH) neurons are essential for stimulating the pulsatile release of gonadotropin releasing hormone (GnRH) and maintaining fertility. However, Kiss1ARH neurons appear to be a key player in coordinating energy balance with reproduction. The regulation of calcium channels and hence calcium signaling is critically dependent on the endoplasmic reticulum calcium-sensing protein stromal interaction molecule 1 (STIM1), which interacts with the plasma membrane calcium channels. We have conditionally deleted Stim1 in Kiss1ARH neurons and found that it significantly increased the excitability of Kiss1ARH neurons and protected ovariectomized female mice from developing obesity and glucose intolerance with high-fat dieting.

neuroscience↗