Biology subjects

Climer, L.

Publications and source records attributed to Climer, L..

4 recordsLinked to original sources

bioRxiv2026

KDM6A is enzymatically active at the Golgi apparatus where it is necessary for maintenance of Golgi apparatus morphology

KDM6A and KDM6B, lysine demethylases, play a central role in regulation of transcription through their demethylation activity towards H3K27me3 and have also been shown to be dynamically regulated upon epithelial-mesenchymal transition (EMT). In particular, KDM6A has been implicated in a variety of malignancies and is frequently mutated in cancers including those in kidney, bladder, colorectal, and breast. KDM6A and KDM6B function as part of the COMPASS-like protein complex (CLC) which includes methyltransferases KMT2C/KMT2D and the regulatory WRAD-subcomplex. KDM6A total protein is known to be suppressed by TGF-{beta} in MCF10A breast cells; however, our examination of protein localization by immunofluorescence and LC-MS/MS reveals that KDM6A expression is lost in the nucleus but retained in the Golgi apparatus, independent of other CLC complex members. Importantly, KDM6A isolated from a biochemically-separated subcellular membranous fraction possesses the catalytic JmjC domain and is enzymatically active, indicating that KDM6A may have non-nuclear substrates. Further, knockdown of KDM6A and treatment with TGF-{beta} similarly alter Golgi apparatus morphology, in a cell type specific manner, suggesting context-dependent regulatory functions. These findings support a model in which KDM6A is necessary to maintain Golgi apparatus morphology and is enzymatically active outside of the nucleus.

cell biology↗

bioRxiv2024

Lack of Oncomodulin Increases ATP-Dependent Calcium Signaling and Susceptibility to Noise in Adult Mice

Tight regulation of Ca2+ is crucial for the function of cochlear outer hair cells (OHCs). Dysregulation of Ca2+ homeostasis in OHCs is associated with impaired hearing and contributes to increased vulnerability to insults such as noise exposure. Ca2+ signaling in developing OHCs is modulated by oncomodulin (OCM), an EF-hand calcium-binding protein. Here, we investigated whether the lack of OCM disrupts the control of intracellular Ca2+ in mature OHCs, and influences vulnerability to acoustic injury. Using young adult CBA/CaJ mice, we found that OHCs from Ocm-knockout (Ocm-/-) mice showed normal biophysical profiles, electromotile responses, and synaptic innervation compared to littermate controls. Moderate noise exposure (95 dB SPL, 2 hrs) caused temporary threshold shifts in Ocm+/+ and Ocm-/- mice. However, while Ocm+/+ fully recovered thresholds 2 weeks after noise exposure, Ocm-/- mice showed permanent threshold shifts. Additionally, Ocm-/- mice had auditory brainstem responses with highly variable latencies and amplitudes both before and after noise exposure compared to Ocm+/+ mice. Using a genetically encoded Ca2+ sensor (GCaMP6s) expressed in Ocm+/+ and Ocm-/- OHCs, we found that prolonged noise exposure (95 dB SPL, 9 hrs) significantly increased GCaMP6s fluorescence, ATP-induced Ca2+ signaling and also caused greater threshold shifts in Ocm-/- compared to Ocm+/+ OHCs. However, prolonged noise exposure had no significant change in the number of presynaptic OHC ribbons in either Ocm+/+ or Ocm-/- mice. We assessed whether the ATP-induced responses were due to changes in P2X2 receptor expression. Prior to noise exposure, P2X2 expression was higher in the cochlea of Ocm-/- mice compared to Ocm+/+ mice. Following prolonged noise, P2X2 receptors were upregulated in the cochlea of Ocm+/+ but not in the Ocm-/- mice, which retained their pre-noise expression level. We propose that the lack of OCM increases susceptibility to cochlear pathology and that purinergic signaling and dysregulation of cytosolic Ca2+ homeostasis likely contribute to early onset hearing loss in the Ocm-/- mice.

neuroscience↗

bioRxiv2023

A fluorogenic complementation tool kit for interrogating lipid droplet-organelle interaction

Contact sites between lipid droplets and other organelles are essential for cellular lipid and energy homeostasis. Detection of these contact sites at nanometer scale over time in living cells is challenging. Here, we developed a tool kit for detecting contact sites based on Fluorogen- Activated Bimolecular complementation at CONtact sites, FABCON, using a reversible, low affinity split fluorescent protein, splitFAST. FABCON labels contact sites with minimal perturbation to organelle interaction. Via FABCON, we quantitatively demonstrated that endoplasmic reticulum (ER)- and mitochondria (mito)-lipid droplet contact sites are dynamic foci in distinct metabolic conditions, such as during lipid droplet biogenesis and consumption. An automated analysis pipeline further classified individual contact sites into distinct subgroups based on size, likely reflecting differential regulation and function. Moreover, FABCON is generalizable to visualize a repertoire of organelle contact sites including ER-mito. Altogether, FABCON reveals insights into the dynamic regulation of lipid droplet-organelle contact sites and generates new hypotheses for further mechanistical interrogation during metabolic switch.

cell biology↗

bioRxiv2022

Oncomodulin (OCM) uniquely regulates calcium signaling in neonatal cochlear outer hair cells.

In cochlear outer hair cells (OHCs), a network of Ca2+ channels, pumps and Ca2+-binding proteins (CaBPs) regulates the localization, spread, and magnitude of free Ca2+ ions. During early postnatal development, OHCs express three prominent mobile EF-hand CaBPs: oncomodulin (OCM), -parvalbumin (APV) and sorcin. We have previously shown that deletion of Ocm (Ocm-/-) gives rise to progressive cochlear dysfunction in young adult mice. Here, we show that changes in Ca2+ signaling begin early in postnatal development of Ocm-/- mice. While mutant OHCs exhibit normal electrophysiological profiles compared to controls, their intracellular Ca2+ signaling is altered. The onset of OCM expression at postnatal day 3 (P3) causes a developmental change in KCl-induced Ca2+ transients in OHCs and leads to slower KCl-induced Ca2+ transients than those elicited in cells from Ocm-/- littermates. We compared OCM buffering kinetics with other CaBPs in animal models and cultured cells. In a double knockout of Ocm and Apv (Ocm-/-;Apv-/-), mutant OHCs show even faster Ca2+ kinetics, suggesting that APV may also contribute to early postnatal Ca2+ signaling. In transfected HEK293T cells, OCM slows Ca2+ kinetics more so than either APV or sorcin. We conclude that OCM controls the intracellular Ca2+ environment by lowering the amount of freely available [Ca2+]i in OHCs and in transfected HEK293T cells. We propose that OCM plays an important role in shaping the development of early OHC Ca2+ signals through its inimitable Ca2+ buffering capacity.

developmental biology↗