Biology subjects

Scarlata, S.

Publications and source records attributed to Scarlata, S..

7 recordsLinked to original sources

bioRxiv2025

Functional classification of GNAI1 disorder variants in C. elegans uncovers conserved and cell-specific mechanisms of dysfunction

Heterotrimeric G proteins transduce signals from G protein coupled receptors, which mediate key aspects of neuronal development and function. Mutations in the GNAI1 gene, which encodes Gi1, cause a disorder characterized by developmental delay, intellectual disability, hypotonia, and epilepsy. However, the mechanistic basis for this disorder remains unknown. Here, we show that GNAI1 is required for ciliogenesis in human cells and use C. elegans as a whole-organism model to determine the functional impact of seven GNAI1-disorder patient variants. Using CRISPR-Cas9 editing in combination with robust cellular (cilia morphology) and behavioral (chemotaxis) assays, we find that T48I, K272R, A328P, and V334E orthologous variants impact both cilia assembly and function in AWC neurons, M88V and I321T have no impact on either phenotype, and D175V exerts neuron-specific effects on cilia-dependent sensory behaviors. Finally, we validate in human ciliated cell lines that D173V, K270R, and A326P GNAI1 variants disrupt ciliary localization of the encoded human Gi1 proteins similarly to their corresponding orthologous substitutions in the C. elegans ODR-3 (D175V, K272R, and A328P). Overall, our findings determine the in vivo effects of orthologous GNAI1 variants and contribute to mechanistic understanding of GNAI1 disorder pathogenesis as well as neuron-specific roles of ODR-3 in sensory biology. ARTICLE SUMMARYG subunits of heterotrimeric G proteins transduce signaling from G protein coupled receptors and play important roles in cell communication and complex behaviors. Mutations in the GNAI1 gene, which encodes Gi1 protein, have been recently linked to a neurodevelopmental disorder; however, it remains unknown how GNAI1 patient mutations disrupt neuronal development or function to manifest in disease. We demonstrate that GNAI1 is required for ciliogenesis and use C. elegans as a whole-animal model in combination with human cells to identify cell-specific and conserved mechanisms of G dysfunction.

developmental biology↗

bioRxiv2024

Human ESCRT-I and ALIX function as scaffolding helical filaments in vivo

The Endosomal Sorting Complex Required for Transport (ESCRT) is an evolutionarily conserved machinery that performs reverse-topology membrane scission in cells universally required from cytokinesis to budding of enveloped viruses. Upstream acting ESCRT-I and ALIX control these events and link recruitment of viral and cellular partners to late-acting ESCRT-III CHMP4 through incompletely understood mechanisms. Using structure-function analyses combined with super-resolution imaging, we show that ESCRT-I and ALIX function as distinct helical filaments in vivo. Together, they are essential for optimal structural scaffolding of HIV-1 nascent virions, the retention of viral and human genomes through defined functional interfaces, and recruitment of CHMP4 that itself assembles into corkscrew-like filaments intertwined with ESCRT-I or ALIX helices. Disruption of filament assembly or their conformationally clustered RNA binding interfaces in human cells impaired membrane abscission, resulted in major structural instability and leaked nucleic acid from nascent virions and nuclear envelopes. Thus, ESCRT-I and ALIX function as helical filaments in vivo and serve as both nucleic acid-dependent structural scaffolds as well as ESCRT-III assembly templates. Significance statementWhen cellular membranes are dissolved or breached, ESCRT is rapidly deployed to repair membranes to restore the integrity of intracellular compartments. Membrane sealing is ensured by ESCRT-III filaments assembled on the inner face of membrane; a mechanism termed inverse topology membrane scission. This mechanism, initiated by ESCRT-I and ALIX, is universally necessary for cytokinesis, wound repair, budding of enveloped viruses, and more. We show ESCRT-I and ALIX individually oligomerize into helical filaments that cluster newly discovered nucleic acid-binding interfaces and scaffold-in genomes within nascent virions and nuclear envelopes. These oligomers additionally appear to serve as ideal templates for ESCRT-III polymerization, as helical filaments of CHMP4B were found intertwined ESCRT-I or ALIX filaments in vivo. Similarly, corkscrew-like filaments of ALIX are also interwoven with ESCRT-I, supporting a model of inverse topology membrane scission that is synergistically reinforced by inward double filament scaffolding.

cell biology↗

bioRxiv2023

Repeated Activation of Gαq by neurotransmitters causes age-dependent accumulation of stress granules in C.elegans

Gq proteins mediate signals from neurotransmitters to transduce calcium signals. In PC12 cells, we have shown that Gq stimulation results in retraction of neurites and induces the formation of stress granules that sequester two specific mRNAs, Chbgb and ATP5f1b. Here, we show that repeated activation of Gq in C. elegans adversely reduces lifespan potentially through accumulation of stress granules and inefficient recovery from neurite retraction. In the absence of stimulation, we could not detect significant changes in number of stress granules from Day 1-15 worms. Single Gq activation increases stress granule size through the enlargement of pre-formed particles with younger worms (Day 1-4) being more responsive than older (Day 8). Repeated Gq stimulation impacts the number of particles through the assembly of nascent particles. Additionally, we a systematic rise in the number of AGL-1 stress granules with repeated Gq stimulation suggesting that stress granules accumulate in neurons and sequester mRNAs. This idea is supported by immunofluorescence studies of ATP5f1b as well as changes in peristaltic speed. In addition to stress granule accumulation, we find that repeated Gq activation results in age-dependent morphological defects in mechanosensory neurons. Taken together, studies show that repeated Gq activation negatively influences the health of C.elegans.

neuroscience↗

bioRxiv2019

IQGAP1 connects phosphoinositide signaling to cytoskeletal reorganization

IQGAP1 is a multi-domain protein that acts as a scaffold for multiple signaling pathways. IQGAP1 generates the lipid messenger PI(3,4,5)P3 by scaffolding the phosphoinositide kinases PIPKIs and PI3K. The dynamics of this scaffolding protein complex in intact, living cells are unknown. Here, we delineate the role of IQGAP1 in PI(3,4,5)P3-mediated signaling in live cells under basal and stimulated conditions using fluorescence lifetime imaging microscopy. We demonstrate that IQGAP1 interacts strongly with PIPKI{gamma} at intracellular entities and on the plasma membrane, and scaffolds PI3K and PIPKI{gamma} in response to physiological changes. Additionally, we show that IQGAP1 scaffolds phosphoinositides with PI3K, PIPKI{gamma} and EGFR, and forms clusters upon cell stimulation with epidermal growth factor. Importantly, we show that IQGAP1 connects PI(3,4,5)P3-mediated signaling and cytoskeletal signaling pathways by binding PIPKI{gamma} in proximity of the cytoskeletal proteins talin and Cdc42. Our results support a model in which IQGAP1 mediates crosstalk between phosphoinositide signaling and the cytoskeleton to promote directed cell movement.

biophysics↗

bioRxiv2019

DECODING THE MECHANOCHEMISTRY OF NEURITE PLASTICITY

The formation and disruption of synaptic connections during development is a fundamental step in neural circuit formation. Subneuronal structures such as neurites are known to be sensitive to the level of spontaneous neuronal activity but the specifics of how neurotransmitter-induced calcium activity regulates neurite homeostasis are not yet fully understood. In response to stimulation by neurotransmitters such as acetylcholine, calcium responses in cells are mediated the Gq/phospholipase C{beta} (PLC{beta})/ phosphatidylinositol 4,5 bisphosphate (PI(4, 5)P2) signaling pathway. Here, we show that prolonged Gq stimulation results in the retraction of neurites in PC12 cells and rupture of neuronal synapses by modulating membrane tension. To understand the underlying cause, we dissected the behavior of individual components of the Gq/PLC{beta}/PI(4, 5)P2 pathway during retraction, and correlated these to the retraction of the membrane and cytoskeletal elements impacted by calcium signaling. We developed a mathematical model that combines biochemical signaling with membrane tension and cytoskeletal mechanics, to show how signaling events are coupled to retraction velocity, membrane tension and actin dynamics. The coupling between calcium and neurite retraction is shown to be operative in the C. elegans nervous system. This study uncovers a novel mechanochemical connection between the Gq/PLC{beta}/(PI(4, 5)P2 pathway that couples calcium responses with neural plasticity.

biophysics↗

bioRxiv2019

Stimulation of Gαq Promotes Stress Granule Formation

During adverse conditions, mammalian cells regulate protein production by sequestering the translational machinery in membrane-less organelles known as stress granules. Here, we found that activation of the G protein subunit Gq promoted the formation of particles that contained stress granule proteins through a mechanism linked to the presence of phospholipase C{beta}1 (PLC{beta}1) in the cytosol. In experiments with PC12 and A10 cells, we showed that under basal conditions, cytosolic PLC{beta}1 bound to stress granule associated proteins, including PABPC1, eIF5A, and Ago2. Knockdown of cytosolic PLC{beta}1 with siRNA or promoting its relocalization to the plasma membrane by activating Gq resulted in the formation of particles containing the stress granule markers, PABPC1, G3BP1, and Ago2. Our studies showed that the composition of these particles resemble those formed under osmotic stress and are distinct from those formed by other stresses. Our results fit a simple thermodynamic model in which cytosolic PLC{beta}1 solubilizes stress granule proteins such that its movement to activated Gq releases these particles to enable the formation of stress granules. Together, our data are suggestive of a link between Gq-coupled signals and protein translation through stress granule formation.

biochemistry↗

bioRxiv2017

Curvature regulates subcellular organelle location to control intracellular signal propagation

The shape of the cell is connected to its function; however, we do not fully understand underlying mechanisms by which global shape regulates a cells functional capabilities. Using theory, experiments and simulation, we investigated how physiologically relevant cell shape changes affect subcellular organization, and consequently intracellular signaling, to control information flow needed for phenotypic function. Vascular smooth muscle cells going from a proliferative and motile circular shape to a contractile fusiform shape show changes in the location of the sarcoplasmic reticulum, inter-organelle distances and differential distribution of receptors in the plasma membrane. These factors together lead to the modulation of signals transduced by the M3 muscarinic receptor/Gq/PLC{beta} pathway at the plasma membrane, amplifying Ca2+ dynamics in the cytoplasm and the nucleus resulting in phenotypic changes, as determined by increased activity of myosin light chain kinase in the cytoplasm and enhanced nuclear localization of the transcription factor NFAT. Taken together, our observations show a systems level phenomenon whereby global cell shape affects subcellular organization to modulate signaling that enables phenotypic changes.

cell biology↗