Biology subjects

Morstein, J.

Publications and source records attributed to Morstein, J..

10 recordsLinked to original sources

bioRxiv2026

Chemical Genetic Targeting of the LRRK2 GTPase Domain

Genetic variants throughout the multi-domain protein leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of autosomal dominant Parkinson's disease, and the most prevalent Parkinson's-associated LRRK2 variants enhance kinase activity. Hence, the kinase domain has been extensively targeted for therapeutic development. However, clinical progression of LRRK2 kinase inhibitors has been limited by on-target peripheral toxicities linked to strong kinase suppression, raising the question of whether targeting LRRK2 kinase activity through other means could offer a more tunable therapeutic window. LRRK2 is one of two proteins in the human proteome that possess a Roc-COR GTPase domain in tandem with a kinase domain, and the GTPase domain has been shown to intramolecularly regulate kinase output. Here, we ask whether the Roc-COR GTPase can be targeted as an alternative approach to downregulate kinase activity. We utilize a chemical genetic approach to sensitize LRRK2 to existing GTPase inhibitors and show that pharmacologically engaging the Roc-COR GTPase domain in cells decreases but does not fully inhibit LRRK2-mediated Rab10 T73 phosphorylation. This study lays the groundwork for future efforts directed at the development of pharmacological inhibitors for the LRRK2 GTPase, providing an alternative approach for therapies targeting LRRK2-driven Parkinson's disease.

molecular biology↗

bioRxiv2026

Selective Editing and Functionalization of the Mammalian Lipidome

Lipids exhibit extraordinary molecular diversity, yet tools to selectively manipulate defined lipid classes in living cells are lacking. Here we show that lipid tail structure biases metabolic fate, enabling the design of synthetic lipid analogs with programmable metabolic selectivity. This approach enables selective cellular production of distinct lipid species or subclasses, including types of neutral lipids, phospholipids, sphingolipids, and ether lipids, without genetic or enzymatic perturbation. We further couple metabolic selectivity to chemical functionalization using bifunctional lipids, in which one modification directs metabolic flux and a second enables bioorthogonal tagging. Using this strategy, we achieve selective in situ labeling of different lipid pools in living cells. Together, our work establishes a chemical biology strategy that enables unprecedented precision in modulating, functionalizing, and rewiring the mammalian lipidome.

cell biology↗

bioRxiv2024

Optical control of sphingolipid biosynthesis using photoswitchable sphingosines

Sphingolipid metabolism comprises a complex interconnected web of enzymes, metabolites and modes of regulation that influence a wide range of cellular and physiological processes. Deciphering the biological relevance of this network is challenging as numerous intermediates of sphingolipid metabolism are short-lived molecules with often opposing biological activities. Here, we introduce clickable, azobenzene-containing sphingosines, termed caSphs, as light-sensitive substrates for sphingolipid biosynthesis. Photo-isomerization of the azobenzene moiety enables reversible switching between a straight trans- and curved cis-form of the lipids hydrocarbon tail. Combining in vitro enzyme assays with metabolic labeling studies, we demonstrate that trans-to-cis isomerization of caSphs profoundly stimulates their metabolic conversion by ceramide synthases and downstream sphingomyelin synthases. These light-induced changes in sphingolipid production rates are acute, reversible, and can be implemented with great efficiency in living cells. Our findings establish caSphs as versatile tools with unprecedented opportunities to manipulate sphingolipid biosynthesis and function with the spatiotemporal precision of light.

biochemistry↗

bioRxiv2023

Selective activation of intracellular β1AR using a spatially restricted antagonist

G-protein-coupled receptors (GPCRs) regulate several physiological and pathological processes and represent the target of approximately 30% of FDA-approved drugs. GPCR-mediated signaling was thought to occur exclusively at the plasma membrane. However, recent studies have unveiled their presence and function at subcellular membrane compartments. There is a growing interest in studying compartmentalized signaling of GPCRs. This requires development of novel tools to separate GPCRs signaling at the plasma membrane from the ones initiated at intracellular compartments. We took advantage of the structural and pharmacological information available for {beta}1-adrenergic receptor ({beta}1AR), an exemplary GPCR that functions at subcellular compartments, and rationally designed spatially restricted antagonists. We generated a cell impermeable {beta}1AR antagonist by conjugating a suitable pharmacophore to a sulfonate-containing fluorophore. This cell-impermeable antagonist only inhibited {beta}1AR on the plasma membrane. In contrast, a cell permeable {beta}1AR agonist containing a non-sulfonated fluorophore, efficiently inhibited both the plasma membrane and Golgi pools of {beta}1ARs. Furthermore, the cell impermeable antagonist selectively inhibited the phosphorylation of downstream effectors of PKA proximal to the plasma membrane in adult cardiomyocytes while {beta}1AR intracellular pool remained active. Our tools offer promising avenues for investigating compartmentalized {beta}1AR signaling in various context, potentially advancing our understanding of {beta}1AR-mediated cellular responses in health and disease. They also offer a general strategy to study compartmentalized signaling for other GPCRs in various biological systems.

cell biology↗

bioRxiv2022

Optical Control of Translation with a Puromycin-Photoswitch

Translation is an elementary cellular process that involves a large number of factors interacting in a concerted fashion with the ribosome. Numerous natural products have emerged that interfere with ribosomal function, such as puromycin, which mimics an aminoacyl tRNA and causes premature chain termination. Here, we introduce a photoswitchable version of puromycin that, in effect, puts translation under optical control. Our compound, termed puroswitch, features a diazocine that allows for reversible and nearly quantitative isomerization and pharmacological modulation. Its synthesis involves a new photoswitchable amino acid building block. Puroswitch shows little activity in the dark and becomes substantially more active and cytotoxic, in a graded fashion, upon irradiation with various wavelengths of visible light. In vitro translation assays confirm that puroswitch inhibits translation with a mechanism similar to that of puromycin itself. Once incorporated into nascent proteins, puroswitch, reacts with standard puromycin antibodies, which allows for tracking de novo protein synthesis using western blots and immunohistochemistry. As a cell-permeable small molecule, puroswitch can be used for nascent proteome profiling in a variety of cell types, including primary mouse neurons. We envision puroswitch as a useful biochemical tool for the optical control of translation and for monitoring newly synthesized proteins in defined locations and at precise time points.

biochemistry↗

bioRxiv2022

Optical Control Of Neuronal Activities With Photoswitchable Nanovesicles

Precise modulation of neuronal activity by neuroactive molecules is essential for understanding brain circuits and behavior. However, tools for highly controllable molecular release are lacking. Here, we developed a photoswitchable nanovesicle with azobenzene-containing phosphatidylcholine (azo-PC), coined azosome, for neuromodulation. Irradiation with 365 nm light triggers the trans-to-cis isomerization of azo-PC, resulting in a disordered lipid bilayer with decreased thickness and cargo release. Irradiation with 455 nm light induces reverse isomerization and switches the release off. Real-time fluorescence imaging shows controllable and repeatable cargo release within seconds (< 3 s). Importantly, we demonstrate that SKF-81297, a dopamine D1-receptor agonist, can be released from the azosome to activate cultures of primary striatal neurons. Azosome shows promise in precise optical control over the molecular release and can be a valuable tool for molecular neuroscience studies.

bioengineering↗

bioRxiv2022

Optical Control of Membrane Fluidity Modulates Protein Secretion

The lipid composition of cellular membranes is dynamic and undergoes remodelling affecting biophysical properties, such as membrane fluidity, which are critical to biological function. Here, we introduce an optical approach to manipulate membrane fluidity based on exogenous synthetic fatty acid with an azobenzene photoswitch, termed FAAzo4. Cells rapidly incorporate FAAzo4 into phosphatidylcholine (PC), the major phospholipid in mammalian cells, in a concentration- and cell type-dependent manner. This generates photoswitchable PC analogs (AzoPC), which are predominantly located in the endoplasmic reticulum (ER). Irradiation causes a rapid photoisomerization that increases membrane fluidity with high spatiotemporal precision. We use these PhotoCells to study the impact of membrane mechanics on protein export from the ER and demonstrate that this two-step process has distinct membrane fluidity requirements. Our approach represents an unprecedented way of manipulating membrane fluidity in cellulo and opens novel avenues to probe roles of fluidity in a wide variety of biological processes.

cell biology↗

bioRxiv2021

Cholesterol and sphingomyelin are critical for Fcγ receptor-mediated phagocytosis of Cryptococcus neoformans by macrophages

Cryptococcus neoformans is a fungal pathogen that causes life-threatening meningoencephalitis in lymphopenic patients. Pulmonary macrophages comprise the first line of host defense upon inhalation of fungal spores, whereby macrophages either aid in clearance or serve as a niche for its dissemination. Given that macrophages play a key role in the outcome of a cryptococcal infection, it is crucial to understand factors that mediate phagocytosis of C. neoformans. Since lipid rafts (high order plasma membrane domains enriched in cholesterol and sphingomyelin) have been implicated in facilitating phagocytosis, we evaluated whether these ordered domains govern macrophages ability to phagocytose C. neoformans. We found that cholesterol or sphingomyelin depletion resulted in significantly deficient IgG-mediated phagocytosis of the fungus. Moreover, repletion of macrophage cells with a raft-promoting sterol (7-dehydrocholesterol) rescued this phagocytic deficiency while a raft-inhibiting sterol (coprostanol) significantly decreased IgG-mediated phagocytosis of C. neoformans. Using a photoswitchable sphingomyelin (AzoSM), we observed that the raft-promoting conformation (trans-AzoSM) resulted in efficient phagocytosis whereas raft-inhibiting conformation (cis-AzoSM) significantly blunted phagocytosis in a reversible manner. We observed that the effect on phagocytosis may be mediated by facilitating Fc{gamma} receptor (Fc{gamma}R) function, whereby IgG immune complexes cross-link to Fc{gamma}RIII, resulting in tyrosine phosphorylation of FcR {gamma}-subunit (FcR{gamma}), an important accessory protein in the Fc{gamma}R signaling cascade. Correspondingly, cholesterol or sphingomyelin depletion resulted in decreased FcR{gamma} phosphorylation. Repletion with 7-dehydrocholesterol restored phosphorylation, whereas repletion with coprostanol showed FcR{gamma} phosphorylation comparable to unstimulated cells. Together, these data suggest that lipid rafts are critical for facilitating Fc{gamma}RIII-mediated phagocytosis of C. neoformans.

microbiology↗

bioRxiv2020

Evolutionarily related small viral fusogens hijack distinct but modular actin nucleation pathways to drive cell-cell fusion

Fusion-associated small transmembrane (FAST) proteins are a diverse family of non-structural viral proteins that, once expressed on the plasma membrane of infected cells, drive fusion with neighboring cells, increasing viral spread and pathogenicity. Unlike viral fusogens with tall ectodomains that pull two membranes together through conformational changes, FAST proteins have short fusogenic ectodomains that cannot bridge the inter-membrane gap between neighboring cells. One orthoreovirus FAST protein, p14, has been shown to hijack the actin cytoskeleton to drive cell-cell fusion, but the actin adaptor-binding motif identified in p14 is not found in any other FAST protein. Here, we report that an evolutionarily divergent FAST protein, p22 from aquareovirus, also hijacks the actin cytoskeleton but does so through different adaptor proteins, Intersectin-1 and Cdc42, that trigger N-WASP-mediated branched actin assembly. We show that despite using different pathways, the cytoplasmic tails of p22 and p14 can be exchanging to create a potent chimeric fusogen, suggesting they are modular and play similar functional roles. When we replace p22s branched actin nucleator, N-WASP, with the parallel filament nucleator, formin, its ability to drive fusion is maintained, indicating that localized mechanical pressure on the plasma membrane coupled to a membrane-disruptive ectodomain is sufficient to drive cell-cell fusion. This work points to a common biophysical strategy used by FAST proteins to push rather than pull membranes together to drive fusion, one that may be harnessed by other short fusogens responsible for physiological cell-cell fusion.

cell biology↗

bioRxiv2018

Carbon monoxide, a retrograde messenger generated in post-synaptic mushroom body neurons evokes local dopamine release

Dopaminergic neurons innervate extensive areas of the brain and release dopamine (DA) onto a wide range of target neurons. However, DA release is also precisely regulated, and in Drosophila, DA is released specifically onto mushroom body (MB) neurons, which have been coincidentally activated by cholinergic and glutamatergic inputs. The mechanism for this precise release has been unclear. Here we found that coincidentally activated MB neurons generate carbon monoxide (CO) which functions as a retrograde signal evoking local DA release from presynaptic terminals. CO production depends on activity of heme oxygenase in post-synaptic MB neurons, and CO-evoked DA release requires Ca2+ efflux through ryanodine receptors in DA terminals. CO is only produced in MB areas receiving coincident activation, and removal of CO using scavengers blocks DA release. We propose that DA neurons utilize two distinct modes of transmission to produce global and local DA signaling.\n\nSIGNIFICANCE STATEMENTDopamine (DA) is needed for various higher brain functions including memory formation. However, DA neurons form extensive synaptic connections, while memory formation requires highly specific and localized DA release. Here we identify a mechanism through which DA release from presynaptic terminals is controlled by postsynaptic activity. Postsynaptic neurons activated by cholinergic and glutamatergic inputs generate carbon monoxide, which acts as a retrograde messenger inducing presynaptic DA release. Released DA is required for memory-associated plasticity. Our work identifies a novel mechanism that restricts DA release to the specific postsynaptic sites that require DA during memory formation.

neuroscience↗