Crosstalk between biochemical signaling and trafficking determines AMPAR dynamics in synaptic plasticity

Synaptic plasticity involves the modification of both biochemical and structural components of neurons. Many studies have revealed that the change in the number density of the glutamatergic receptor AMPAR at the synapse is proportional to synaptic weight update; increase in AMPAR corresponds to strengthening of synapses while decrease in AMPAR density weakens synaptic connections. The dynamics of AMPAR are thought to be regulated by upstream signaling, primarily the calcium-CaMKII pathway, trafficking to and from the synapse, and influx from extrasynaptic sources. Here, we have developed a set of models using compartmental ordinary differential equations to systematically investigate contributions of signaling and trafficking variations on AMPAR dynamics at the synaptic site. We find that the model properties including network architecture and parameters significantly affect the integration of fast upstream species by slower downstream species. Furthermore, we predict that the model outcome, as determined by bound AMPAR at the synaptic site, depends on (a) the choice of signaling model (bistable CaMKII or monostable CaMKII dynamics), (b) trafficking versus influx contributions, and (c) frequency of stimulus. Therefore, AMPAR dynamics can have unexpected dependencies when upstream signaling dynamics (such as CaMKII and PP1) are coupled with trafficking modalities.

Glucocorticoid-Mediated Developmental Programming of Vertebrate Stress Responsivity

Glucocorticoids, vertebrate steroid hormones produced by cells of the adrenal cortex or interrenal tissue, function dynamically to maintain homeostasis under constantly changing and occasionally stressful environmental conditions. They do so by binding and thereby activating nuclear receptor transcription factors, the Glucocorticoid and Mineralocorticoid Receptors (MR and GR, respectively). The GR, by virtue of its lower affinity for endogenous glucocorticoids (cortisol or corticosterone), is primarily responsible for transducing the dynamic signals conveyed by circadian and ultradian glucocorticoid oscillations as well as transient pulses produced in response to acute stress. These dynamics are important determinants of stress responsivity, and at the systemic level are produced by feedforward and feedback signaling along the hypothalamus-pituitary–adrenal/interrenal axis. Within receiving cells, GR signaling dynamics are controlled by the GR target gene and negative feedback regulator fkpb5. Chronic stress can alter signaling dynamics via imperfect physiological adaptation that changes systemic and/or cellular set points, resulting in chronically elevated cortisol levels and increased allostatic load, which undermines health and promotes development of disease. When this occurs during early development it can “program” the responsivity of the stress system, with persistent effects on allostatic load and disease susceptibility. An important question concerns the glucocorticoid-responsive gene regulatory network that contributes to such programming. Recent studies show that klf9, a ubiquitously expressed GR target gene that encodes a Krüppel-like transcription factor important for metabolic plasticity and neuronal differentiation, is a feedforward regulator of GR signaling impacting cellular glucocorticoid responsivity, suggesting that it may be a critical node in that regulatory network.

Synthetic Condensed-Phase Signaling Expands Kinase Specificity and Responds to Macromolecular Crowding

Liquid–liquid phase separation (LLPS) can concentrate biomolecules and accelerate reactions within membraneless organelles. For example, the nucleolus and PML-nuclear bodies are thought to create network hubs by bringing signaling molecules such as kinases and substrates together. However, the mechanisms and principles connecting mesoscale organization to signaling dynamics are difficult to dissect due to the pleiotropic effects associated with disrupting endogenous condensates. Here, we recruited multiple distinct kinases and substrates into synthetic LLPS systems to create new phosphorylation reactions within condensates, and generally found increased activity and broadened specificity. Dynamic phosphorylation within condensates could drive cell-cycle-dependent localization changes. Detailed comparison of phosphorylation of clients with varying recruitment valency and affinity into condensates comprised of either flexible or rigid scaffolds revealed unexpected principles. First, high client concentration within condensates is important, but is not the main factor for efficient multi-site phosphorylation. Rather, the availability of a large number of excess client binding sites, together with a flexible scaffold is crucial. Finally, phosphorylation within a suboptimal, flexible condensate was modulated by changes in macromolecular crowding. Thus, condensates readily generate new signaling connections and can create sensors that respond to perturbations to the biophysical properties of the cytoplasm.

Spatial-proteomics reveals phospho-signaling dynamics at subcellular resolution

Dynamic change in subcellular localization of signaling proteins is a general concept that eukaryotic cells evolved for eliciting a coordinated response to stimuli. Mass spectrometry-based proteomics in combination with subcellular fractionation can provide comprehensive maps of spatio-temporal regulation of protein networks in cells, but involves laborious workflows that does not cover the phospho-proteome level. Here we present a high-throughput workflow based on sequential cell fractionation to profile the global proteome and phospho-proteome dynamics across six distinct subcellular fractions. We benchmark the workflow by studying spatio-temporal EGFR phospho-signaling dynamics in vitro in HeLa cells and in vivo in mouse tissues. Finally, we investigate the spatio-temporal stress signaling, revealing cellular relocation of ribosomal proteins in response to hypertonicity and muscle contraction. Proteomics data generated in this study can be explored through https://SpatialProteoDynamics.github.io.

CBIO-27. INTEGRATED FUNCTIONAL AND MOLECULAR CHARACTERIZATION OF THE INTRINSIC APOPTOTIC MACHINERY IDENTIFIES THERAPEUTIC VULNERABILITIES IN GLIOBLASTOMA

Molecular profiling frequently fails to predict therapeutic outcomes in cancer. This is in part due to the myriad of genetic alterations comprising a tumor which enable rapid rewiring of cancer signaling pathways. Functional profiling ascertains signaling dynamics and has emerged as an alternative method to predict drug responses. It is unknown whether incorporating molecular with functional profiling offers superior insight into specific tumor dependencies and consequently therapeutic susceptibilities. Here we performed integrated molecular and functional characterization (via BH3 profiling) of the intrinsic apoptotic machinery in 50 GBM patient specimens. We found that treatment with standard of care (e.g., temozolomide or radiation) rewired the apoptotic machinery in a p53 dependent manner, eliciting an exclusive survival dependency on BCL-xL in a genetically defined subset of GBM tumors. However, functional BH3 profiling of ‘apoptotic priming’ was required to predict response to combined IR/TMZ and BCL-xL inhibition in p53 WT GBM tumors. Accordingly, a composite of genetic and functional profiling predicted the response of patient derived GBM models to IR/TMZ with a novel GBM specific BCLxL antagonist. Collectively, these studies identify the mechanisms of intrinsic apoptosis resistance in GBM and demonstrate how functional and molecular data can be complementary to robustly predict therapy-induced cell death.

From spikes to intercellular waves: Tuning intercellular calcium signaling dynamics modulates organ size control

Information flow within and between cells depends significantly on calcium (Ca2+) signaling dynamics. However, the biophysical mechanisms that govern emergent patterns of Ca2+ signaling dynamics at the organ level remain elusive. Recent experimental studies in developing Drosophila wing imaginal discs demonstrate the emergence of four distinct patterns of Ca2+ activity: Ca2+ spikes, intercellular Ca2+ transients, tissue-level Ca2+ waves, and a global “fluttering” state. Here, we used a combination of computational modeling and experimental approaches to identify two different populations of cells within tissues that are connected by gap junction proteins. We term these two subpopulations “initiator cells,” defined by elevated levels of Phospholipase C (PLC) activity, and “standby cells,” which exhibit baseline activity. We found that the type and strength of hormonal stimulation and extent of gap junctional communication jointly determine the predominate class of Ca2+ signaling activity. Further, single-cell Ca2+ spikes are stimulated by insulin, while intercellular Ca2+ waves depend on Gαq activity. Our computational model successfully reproduces how the dynamics of Ca2+ transients varies during organ growth. Phenotypic analysis of perturbations to Gαq and insulin signaling support an integrated model of cytoplasmic Ca2+ as a dynamic reporter of overall tissue growth. Further, we show that perturbations to Ca2+ signaling tune the final size of organs. This work provides a platform to further study how organ size regulation emerges from the crosstalk between biochemical growth signals and heterogeneous cell signaling states.

Tissue-wide coordination of calcium signaling regulates the epithelial stem cell pool during homeostasis

Regenerative processes in the mammalian skin require coordinated cell-cell communication. Ca2+ signaling can coordinate tissue-level responses in developing and wounded epithelia in tissue explants and invertebrates. However, its role in the homeostatic, regenerative basal layer of the skin epithelium is unknown due to significant challenges in studying signaling dynamics in a spatially complex tissue context in live mice. Here we combine in vivo imaging of dynamic Ca2+ signaling at the single cell level across thousands of cells with a novel computational approach, Geometric Scattering Trajectory Homology (GSTH). GSTH models Ca2+ as signals over a cell adjacency graph and uses a multi-level wavelet-like transform (called a scattering transform) to extract signaling patterns from our high dimensional in vivo datasets. We discover local Ca2+ signaling patterns are orchestrated so that signals flow in a coordinated and directed manner across the tissue, distinct from topographically uncoordinated Ca2+ signaling in excitatory tissues. Directed Ca2+ signaling is regulated by the major gap junction protein in the epidermal stem cell layer, Connexin 43 (Cx43). Cx43 gap junctions are dissociated as cells progress through the cell cycle out of G1 and play an essential role in the progression of stem cells from G2 towards mitosis. Finally, G2 cells display related signaling patterns and are essential for tissue-level signaling coordination. Together, our results provide insight into how such a ubiquitous signaling pathway regulates highly specific behaviors and outcomes at a tissue-wide level to maintain proper homeostasis.

DNA origami patterning of synthetic T cell receptors reveals spatial control of the sensitivity and kinetics of signal activation

Receptor clustering plays a key role in triggering cellular activation, but the relationship between the spatial configuration of clusters and the elicitation of downstream intracellular signals remains poorly understood. We developed a DNA-origami–based system that is easily adaptable to other cellular systems and enables rich interrogation of responses to a variety of spatially defined inputs. Using a chimeric antigen receptor (CAR) T cell model system with relevance to cancer therapy, we studied signaling dynamics at single-cell resolution. We found that the spatial arrangement of receptors determines the ligand density threshold for triggering and encodes the temporal kinetics of signaling activities. We also showed that signaling sensitivity of a small cluster of high-affinity ligands is enhanced when surrounded by nonstimulating low-affinity ligands. Our results suggest that cells measure spatial arrangements of ligands, translate that information into distinct signaling dynamics, and provide insights into engineering immunotherapies.