Biology subjects

Lohse, M. J.

Publications and source records attributed to Lohse, M. J..

9 recordsLinked to original sources

bioRxiv2026

Dynamic visualization of physiological CaMKII activity using sensitive FRET biosensors

Calcium-calmodulin (CaM)-dependent protein kinase II (CaMKII) is a key mediator of complex physiological processes throughout the body, from the brain to the reproductive system, where CaMKII translates spatiotemporally dynamic calcium elevations into specific biological functions. Directly visualizing CaMKII activity dynamics in living cells using genetically encoded fluorescent biosensors can thus provide crucial insights into the molecular regulation of health and disease. Yet the ability to sensitively and specifically monitor endogenous CaMKII activity in physiologically relevant contexts is limited by the lack of sensors that can achieve robust, quantitative visualization of CaMKII responses. Here, we leveraged a recent serine/threonine kinome-wide substrate atlas to rationally engineer a powerful suite of Forster resonance energy transfer (FRET)-based CaMKII kinase activity reporters with high specificity, sensitivity, and signal-to-noise ratio. Using these biosensors, we were able to sensitively and robustly visualize endogenous CaMKII activity dynamics in both cultured cell lines and primary cells, including cardiomyocytes, oocytes, and neurons. We further utilized 2pFLIM imaging of organotypic hippocampal slices to quantitatively track LTP-induced CaMKII activity within single dendritic spines, highlighting a major advance in the study of physiological CaMKII signaling.

bioengineering↗

bioRxiv2026

Nuclear βactin dependent chromatin accessibility governs stem cell pluripotency and extracellular matrix gene programs to maintain cellular biomechanics for cell lineage decisions

Pluripotency requires coordinated regulation of chromatin state, transcription and extracellular matrix (ECM) mechanics, but how these layers are integrated remains unclear. Here, using {beta}-actin knockout mouse embryonic stem cells (mESCs) and a nuclear-targeted {beta}-actin rescue, we identify nuclear {beta}-actin as a key regulator linking chromatin accessibility to mechanosensitive control of cell fate. {beta}-actin loss reduced OCT4, SOX2 and NANOG, broadly rewired the transcriptome and proteome and decreased accessibility at pluripotency regulatory regions. Integrated RNA-seq and ATAC-seq revealed coordinated dysregulation of stemness, ECM, mechanotransduction and early-lineage programs. These changes were accompanied by fibronectin and collagen upregulation, altered nuclear morphology, reduced Lamin A/C and mechanosensing proteins, increased nuclear YAP1 and greater ECM stiffness heterogeneity. Functionally, knockout cells displayed biased lineage specification, failed neuronal differentiation, ectopic cardiomyocyte-like differentiation, and markedly reduced teratoma growth with diminished ectodermal representation. Nuclear {beta}-actin re-expression restored many molecular, mechanical and differentiation defects, although chromatin rescue remained incomplete. Together, these findings establish nuclear {beta}-actin as an integrator of chromatin regulation and ECM-dependent mechanotransduction that preserves pluripotency and developmental competence.

cell biology↗

bioRxiv2024

Disruption of basal CXCR4 oligomers impairs oncogenic properties in lymphoid neoplasms

The chemokine receptor CXCR4 is overexpressed in many cancers and contributes to pathogenesis, disease progression, and resistance to therapies. CXCR4 is known to form oligomers, but the potential functional relevance in malignancies remain elusive. Using a newly established nanobody-based BRET method, we demonstrate that oligomerization of endogenous CXCR4 on lymphoid cancer cell lines correlates with enhanced expression levels. Specific disruption of CXCR4 oligomers reduced basal cell migration and pro-survival signaling via changes in the phosphoproteome, indicating the existence of basal CXCR4-oligomer-mediated signaling. Oligomer disruption also inhibited growth of primary CLL 3D spheroids and sensitized primary malignant cells to clinically used Bcl-2 inhibitor venetoclax. Given its limited efficacy in some patients and the ability to develop resistance, sensitizing malignant B-cells to venetoclax is of clinical relevance. Taken together, we established a new, non-canonical and critical role for CXCR4 oligomers in lymphoid neoplasms and demonstrated that selective targeting thereof has clinical potential. Significance statementClass A GPCRs, including the chemokine receptor CXCR4, can form oligomers, but their functional relevance remains poorly understood. This study provides evidence for the role of basal CXCR4 oligomers in lymphoid neoplasms, where they drive pro-survival signaling, migration, and tumor growth. We use a novel nanobody-based BRET method to demonstrate that endogenous CXCR4 constitutively oligomerizes in lymphoid cancer cells, correlating with receptor expression levels. Pharmacological disruption of these oligomers reduces tumor- associated signaling, impairs spheroid growth, and sensitizes patient-derived malignant cells to the apoptosis-inducing drug Venetoclax. Since CXCR4 is frequently overexpressed and potentially clustered in various malignancies, this work offers broader implications for enhancing treatment efficacy, overcoming drug resistance, and potentially reducing side effects across multiple cancer types.

biochemistry↗

bioRxiv2024

MRAP2 modifies signaling and oligomerization state of the melanocortin-4 receptor

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor (GPCR) and a key regulator of appetite and metabolism. It can interact with the melanocortin-receptor accessory protein 2 (MRAP2), a single transmembrane helix protein known to interact with several different GPCRs. However, the consequences of this interaction are not completely understood. Here we report that co-expression of MRAP2 has multiple effects on the MC4R: it enhances G protein-mediated signaling and simultaneously impairs {beta}-arrestin2 recruitment and, consequently, internalization. In addition, co-expression of MRAP2 leads to an increased number of monomers of MC4R by disrupting receptor oligomers. A structural homology model of the active state MC4R-MRAP2-Gs complex suggests interaction sites between MRAP2 and MC4R that are relevant for receptor activation. Taken together, our data indicate that MRAP2 is an accessory protein that interacts with and influences MC4R structure, biasing its signaling towards G protein-mediated effects.

pharmacology and toxicology↗

bioRxiv2022

Fluorescence spectroscopy of low-level endogenous β-Adrenergic Receptor expression at the plasma membrane of differentiating human iPSC-derived cardiomyocytes

The potential of human induced pluripotent stem cells (hiPSCs) to be differentiated into cardiomyocytes (CMs) mimicking the adult CMs functional morphology, marker genes and signaling characteristics has been investigated since over a decade. The evolution of the membrane localization of CM-specific G protein-coupled receptors throughout differentiation has received, however, only limited attention to date. We employ here advanced fluorescent spectroscopy, namely linescan Fluorescence Correlation Spectroscopy (FCS), to observe how the plasma membrane abundance of the {beta}1- and {beta}2-adrenergic receptors ({beta}1/2-ARs), labelled using a bright and photostable fluorescent antagonist, evolves during long-term monolayer culture of hiPSC-derived CMs. We compare it to the kinetic of observed mRNA levels in wildtype (WT) hiPSCs and in two CRISPR/Cas9 knock-in clones. We conduct these observations against the backdrop of our recent report that {beta}2-ARs, as opposed to {beta}1-ARs, specifically segregate to the T-Tubular system of adult CMs.

developmental biology↗

bioRxiv2021

Proteolytic cleavage of the extracellular domain affects signaling of parathyroid hormone receptor 1

Parathyroid hormone 1 receptor (PTH1R) is a member of the class B family of G protein-coupled receptors, which are characterized by a large extracellular domain required for ligand binding. We have previously shown that the extracellular domain of PTH1R is subject to metalloproteinase cleavage in vivo that is regulated by ligand-induced receptor trafficking and leads to impaired stability of PTH1R. In this work, we localize the cleavage site in the first loop of the extracellular domain using amino-terminal protein sequencing of purified receptor and by mutagenesis studies. We further show, that a receptor mutant not susceptible to proteolytic cleavage exhibits reduced signaling to Gs and increased activation of Gq compared to wild-type PTH1R. These findings indicate that the extracellular domain modulates PTH1R signaling specificity, and that its cleavage affects receptor signaling.

pharmacology and toxicology↗

bioRxiv2021

Functional modulation of PTH1R activation and signalling by RAMP2

Receptor-activity-modifying proteins (RAMPs) are ubiquitously expressed membrane proteins that associate with different G protein-coupled receptors (GPCRs) including the parathyroid hormone 1 receptor (PTH1R), a class B GPCR and an important modulator of mineral ion homeostasis and bone metabolism. However, it is unknown whether and how RAMP proteins may affect PTH1R function. Using different optical biosensors to measure the activation of PTH1R and its downstream signalling, we describe here that RAMP2 acts as a specific allosteric modulator of PTH1R, shifting PTH1R to a unique pre-activated state that permits faster activation in a ligand-specific manner. Moreover, RAMP2 modulates PTH1R downstream signalling in an agonist-dependent manner, most notably increasing the PTH-mediated Gi3 signalling sensitivity. Additionally, RAMP2 increases both PTH- and PTHrP-triggered {beta}-arrestin2 recruitment to PTH1R. Employing homology modelling we describe the putative structural molecular basis underlying our functional findings. These data uncover a critical role of RAMPs in the activation and signalling of a GPCR that may provide a new venue for highly specific modulation of GPCR function and advanced drug design.

pharmacology and toxicology↗

bioRxiv2021

One gene - two proteins: The C-terminus of the prototypical M2 muscarinic receptor localizes to the mitochondria

Muscarinic acetylcholine receptors are prototypical G protein-coupled receptors (GPCRs), members of a large family of seven transmembrane receptors mediating a wide variety of extracellular signals. We show here, in cultured cells and in a murine model, that the carboxyl terminal fragment of the muscarinic M2 receptor, comprising the transmembrane regions 6 and 7 (M2tail), is expressed by virtue of an internal ribosome entry site localized in the third intracellular loop. Single cell imaging and import in isolated yeast mitochondria reveals that M2tail, whose expression is upregulated in cells undergoing integrated stress response, does not follow the normal route to the plasma membrane, but is almost exclusively sorted to the mitochondria inner membrane: here it controls oxygen consumption, cell proliferation and the formation of reactive oxygen species by reducing oxidative phosphorylation. Crispr/Cas9 editing of the key methionine where cap-independent translation begins in human induced pluripotent stem cells, reveals the physiological role of this process in influencing cell proliferation and oxygen consumption at the endogenous level. The expression of the C-terminal domain of a GPCR, capable of regulating mitochondrial function, constitutes a hitherto unknown mechanism notably unrelated to its canonical signalling function as a GPCR at the plasma membrane. This work thus highlights a potential novel mechanism thar cells may use for controlling their metabolism under variable environmental conditions, notably as a negative regulator of cell respiration.

cell biology↗

bioRxiv2017

Visualization of class A GPCR oligomerization by image-based fluorescence fluctuation spectroscopy

G protein-coupled receptors (GPCRs) represent the largest class of cell surface receptors conveying extracellular information into intracellular signals. Many GPCRs have been shown to be able to oligomerize and it is firmly established that Class C GPCRs (e.g. metabotropic glutamate receptors) function as obligate dimers. However, the oligomerization capability of the larger Class A GPCRs (e.g. comprising the {beta}-adrenergic receptors ({beta}-ARs)) is still, despite decades of research, highly debated.\n\nHere we assess the oligomerization behavior of three prototypical Class A GPCRs, the {beta}1-ARs, {beta}2-ARs, and muscarinic M2Rs in single, intact cells. We combine two image correlation spectroscopy methods based on molecular brightness, i.e. the analysis of fluorescence fluctuations over space and over time, and thereby provide an assay able to robustly and precisely quantify the degree of oligomerization of GPCRs. In addition, we provide a comparison between two labelling strategies, namely C-terminally-attached fluorescent proteins and N-terminally-attached SNAP-tags, in order to rule out effects arising from potential fluorescent protein-driven oligomerization. The degree of GPCR oligomerization is expressed with respect to a set of previously reported as well as newly established monomeric or dimeric control constructs. Our data reveal that all three prototypical GPRCs studied display, under unstimulated conditions, a prevalently monomeric fingerprint. Only the {beta}2-AR shows a slight degree of oligomerization.\n\nFrom a methodological point of view, our study suggests three key aspects. First, the combination of two image correlation spectroscopy methods allows addressing cells transiently expressing high concentrations of membrane receptors, far from the single molecule regime, at a density where the kinetic equilibrium should favor dimers and higher-order oligomers. Second, our methodological approach, allows to selectively target cell membrane regions devoid of artificial oligomerization hot-spots (such as vesicles). Third, our data suggest that the {beta}1-AR appears to be a superior monomeric control than the widely used membrane protein CD86.\n\nTaken together, we suggest that our combined image correlation spectroscopy method is a powerful approach to assess the oligomerization behavior of GPCRs in intact cells at high expression levels.

biophysics↗