Biology subjects

Kim, C. K.

Publications and source records attributed to Kim, C. K..

9 recordsLinked to original sources

bioRxiv2025

Acute in vivo proximity labeling for membrane targeted proteomics in neuronal circuits

MOTIVATIONDespite the growing use of proximity labeling tools, there are limited techniques available to identify subcellularly localized proteins within targeted neuronal circuits on acute timescales in vivo. We engineered membrane targeted versions of the proximity labeling enzyme TurboID and validated their use for proteomic discovery in the mouse brain. We optimized an in vivo protocol to identify proteins in cell bodies and long range neuronal projections, and proteins differentially detected during acute drug labeling windows. SUMMARYA major goal within molecular systems neuroscience is to bridge the study of neuronal circuit function with changes in protein expression and localization in awake behaving animals. However, there are limited tools for capturing changes in subcellularly-defined proteomes within neuronal circuits during activity-gated timescales in vivo. Here, we engineered targeted versions of the proximity labeling enzyme TurboID, to tag proteins at the neuronal membrane during a user-delivered biotin injection. We optimized a labeling strategy that enables a one-to-two-hour labeling window and tagged proteins in medial prefrontal cortex (mPFC) cell bodies and corresponding axons in a downstream projection. We performed proteomics to identify proteins enriched in mPFC cell bodies and terminals, and upregulated in mPFC cell bodies following an acute cocaine injection. These advancements enable the detection of proteins at the subcellular level within short labeling windows, allowing identification of stimulus-specific proteomes in behaving mice.

neuroscience↗

bioRxiv2025

High-Speed Neural Imaging with Multiplexed Miniature Two-Photon Microscopy (M-MINI2P)

Head-mounted miniaturized two-photon microscopes are powerful tools to record neural activity with cellular resolution deep in the mouse brain during unrestrained, free-moving behavior. Two-photon microscopy, however, is traditionally limited in imaging frame rate due to the necessity of raster scanning the laser excitation spot over a large field-of-view (FOV). Here, we present two multiplexed miniature two-photon microscopes (M-MINI2Ps) to increase the imaging frame rate while preserving the spatial resolution. Two different FOVs are imaged simultaneously and then demixed temporally or computationally. We demonstrate large-scale (500x500 m2 FOV) multiplane calcium imaging in visual cortex and prefrontal cortex in freely moving mice for spontaneous activity and auditory stimulus evoked responses. Furthermore, the increased speed of M-MINI2Ps also enables two-photon voltage imaging at 400 Hz over a 380x150 m2 FOV in freely moving mice. M-MINI2Ps have compact footprints and are compatible with the open-source MINI2P. M-MINI2Ps, together with their design principles, allow the capture of faster physiological dynamics and population recordings over a greater volume than currently possible in freely moving mice, and will be a powerful tool in systems neuroscience.

neuroscience↗

bioRxiv2024

Deconstruction of a memory engram reveals distinct ensembles recruited at learning

How are associative memories formed? Which cells represent a memory, and when are they engaged? By visualizing and tagging cells based on their calcium influx with unparalleled temporal precision, we identified non-overlapping dorsal CA1 neuronal ensembles that are differentially active during associative fear memory acquisition. We dissected the acquisition experience into periods during which salient stimuli were presented or certain mouse behaviors occurred and found that cells associated with specific acquisition periods are sufficient alone to drive memory expression and contribute to fear engram formation. This study delineated the different identities of the cell ensembles active during learning, and revealed, for the first time, which ones form the core engram and are essential for memory formation and recall.

neuroscience↗

bioRxiv2023

Rapid, biochemical tagging of cellular activity history in vivo

Intracellular calcium (Ca2+) is ubiquitous to cell signaling across all biology. While existing fluorescent sensors and reporters can detect activated cells with elevated Ca2+ levels, these approaches require implants to deliver light to deep tissue, precluding their noninvasive use in freely-behaving animals. Here we engineered an enzyme-catalyzed approach that rapidly and biochemically tags cells with elevated Ca2+ in vivo. Ca2+-activated Split-TurboID (CaST) labels activated cells within 10 minutes with an exogenously-delivered biotin molecule. The enzymatic signal increases with Ca2+ concentration and biotin labeling time, demonstrating that CaST is a time-gated integrator of total Ca2+ activity. Furthermore, the CaST read-out can be performed immediately after activity labeling, in contrast to transcriptional reporters that require hours to produce signal. These capabilities allowed us to apply CaST to tag prefrontal cortex neurons activated by psilocybin, and to correlate the CaST signal with psilocybin-induced head-twitch responses in untethered mice.

neuroscience↗

bioRxiv2023

Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells

The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions, and function with light. We integrated optogenetic control into proximity labeling (PL), a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the PL enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. "LOV-Turbo" works in multiple contexts and dramatically reduces background in biotin-rich environments such as neurons. We used LOV-Turbo for pulse-chase labeling to discover proteins that traffick between endoplasmic reticulum, nuclear, and mitochondrial compartments under cellular stress. We also showed that instead of external light, LOV-Turbo can be activated by BRET from luciferase, enabling interaction-dependent PL. Overall, LOV-Turbo increases the spatial and temporal precision of PL, expanding the scope of experimental questions that can be addressed with PL.

molecular biology↗

bioRxiv2020

A molecular calcium integrator reveals a striatal cell-type driving aversion

The ability to record transient cellular events in the DNA or RNA of cells would enable precise, large-scale analysis, selection, and reprogramming of heterogeneous cell populations. Here we report a molecular technology for stable genetic tagging of cells that exhibit activity-related increases in intracellular calcium concentration (FLiCRE). We used FLiCRE to transcriptionally label activated neural ensembles in the nucleus accumbens of the mouse brain during brief stimulation of aversive inputs. Using single-cell RNA sequencing, we detected FLiCRE transcripts among the endogenous transcriptome, providing simultaneous readout of both cell-type and calcium activation history. We identified a cell-type in the nucleus accumbens activated downstream of long-range excitatory projections. Taking advantage of FLiCREs modular design, we expressed an optogenetic channel selectively in this cell-type, and showed that direct recruitment of this otherwise genetically-inaccessible population elicits behavioral aversion. The specificity and minute-resolution of FLiCRE enables molecularly-informed characterization, manipulation, and reprogramming of activated cellular ensembles.

neuroscience↗

bioRxiv2019

Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions

Technologies that convert transient protein-protein interactions (PPIs) into stable expression of a reporter gene are useful for genetic selections, high-throughput screening, and multiplexing with omics technologies. We previously reported SPARK (Kim et al., 2017), a transcription factor that is activated by the coincidence of blue light and a PPI. Here, we report an improved, second-generation SPARK2 that incorporates a luciferase moiety to control the light-sensitive LOV domain. SPARK2 can be temporally gated by either external light or addition of a small-molecule luciferin, which causes luciferase to open LOV via proximity-dependent BRET. Furthermore, the nested \"AND\" gate design of SPARK2--in which both protease recruitment to the membrane-anchored transcription factor and LOV domain opening are regulated by the PPI of interest--yields a lower-background system and improved PPI specificity. We apply SPARK2 to high-throughput screening for GPCR agonists and for the detection of trans-cellular contacts, all with versatile transcriptional readout.

bioengineering↗

bioRxiv2018

Overexpression of RsMYB1 enhances heavy metal stress tolerance in transgenic petunia by elevating the transcript levels of stress tolerant and antioxidant genes

The RsMYB1 transcription factor (TF) controls the regulation of anthocyanin in radish (Raphanus sativus), and its overexpression in tobacco and petunia strongly enhances anthocyanin production. However, no data exists on whether RsMYB1 is involved in the mechanism that leads to abiotic stress tolerance. Under normal conditions, transgenic petunia plants expressing RsMYB1 and WT were able to thrive by producing well-developed broad leaves and regular roots. In contrast, a reduction in plant growth was observed when they were exposed to heavy metals (CuSO4, ZnSO4, MnSO4, and K2Cr2O7). However, RsMYB1-overexpressing plants were found to be more tolerant to the stresses than the WT plants because the expressions of stress tolerant genes (GSH and PCs) and antioxidant genes (SOD, CAT, and POX) were enhanced. In addition, according to the phylogenetic analysis, RsMYB1 has a strong sequence similarity with other MYB TFs that confer different abiotic stresses. These results suggest that overexpression of RsMYB1 enhances the expression levels of metal-induced stress tolerance genes and antioxidant genes, and the resultant increase in gene expression improves heavy metal stress tolerance in petunia.

plant biology↗

bioRxiv2017

Identification Of Cellular-Activity Dynamics Across Large Tissue Volumes In The Mammalian Brain

Tracking the coordinated activity of cellular events through volumes of intact tissue is a major challenge in biology that has inspired significant technological innovation. Yet scanless measurement of the high-speed activity of individual neurons across three dimensions in scattering mammalian tissue remains an open problem. Here we develop and validate a computational imaging approach (SWIFT) that integrates high-dimensional, structured statistics with light field microscopy to allow the synchronous acquisition of single-neuron resolution activity throughout intact tissue volumes as fast as a camera can capture images (currently up to 100 Hz at full camera resolution), attaining rates needed to keep pace with emerging fast calcium and voltage sensors. We demonstrate that this large field-of-view, single-snapshot volume acquisition method--which requires only a simple and inexpensive modification to a standard fluorescence microscope--enables scanless capture of coordinated activity patterns throughout mammalian neural volumes. Further, the volumetric nature of SWIFT also allows fast in vivo imaging, motion correction, and cell identification throughout curved subcortical structures like the dorsal hippocampus, where cellular-resolution dynamics spanning hippocampal subfields can be simultaneously observed during a virtual context learning task in a behaving animal. SWIFTs ability to rapidly and easily record from volumes of many cells across layers opens the door to widespread identification of dynamical motifs and timing dependencies among coordinated cell assemblies during adaptive, modulated, or maladaptive physiological processes in neural systems.

neuroscience↗