Signaling Compartments Research Articles

TcCARP3 modulates compartmentalized cAMP signals involved in osmoregulation, infection of mammalian cells, and colonization of the triatomine vector in the human pathogen Trypanosoma cruzi.

Cyclic AMP signaling pathways are poorly understood in the stercorarian parasite Trypanosoma cruzi . Specifically, the mechanisms driving the activation of TcACs in response to microenvironmental stress are completely unknown. This study unveils the role of TcCARP3 in modulating the content of cAMP through the interaction with several TcACs and putative cAMP effectors in T. cruzi . Particularly, TcCARP3 interacts with TcAC1 in the main developmental stages of this parasite's life cycle, where both proteins display a dual localization pattern. These results provide new evidence supporting the compartmentalization of cAMP signals in trypanosomes. Moreover, our data unequivocally demonstrates that TcCARP3 is required for key cellular processes for parasite survival, such as response to osmotic stress, host cell invasion, intracellular replication, and the ability to colonize the hindgut of the triatomine vector. In summary, we found that TcCARP3 is an adenylate cyclase regulator, necessary for the life cycle progression of T. cruzi .

Functional Interdependence of Anoctamins May Influence Conclusions from Overexpression Studies.

Anoctamin 6 (ANO6, TMEM16F) is a phospholipid (PL) scramblase that moves PLs between both plasma membrane (PM) leaflets and operates as an ion channel. It plays a role in development and is essential for hemostasis, bone mineralization and immune defense. However, ANO6 has also been shown to regulate cellular Ca2+ signaling and PM compartments, thereby controlling the expression of ion channels such as CFTR. Given these pleiotropic effects, we investigated the functional interdependence of the ubiquitous ANO6 with other commonly co-expressed anoctamins. As most expression studies on anoctamins use HEK293 human embryonic kidney cells, we compared ion currents, PL scrambling and Ca2+ signals induced by the overexpression of anoctamins in HEK293 wild-type parental and ANO6-knockout cells. The data suggest that the endogenous expression of ANO6 significantly affects the results obtained from overexpressed anoctamins, particularly after increasing intracellular Ca2+. Thus, a significant interdependence of anoctamins may influence the interpretation of data obtained from the functional analysis of overexpressed anoctamins.

A dendrite is a dendrite is a dendrite? Dendritic signal integration beyond the "antenna" model.

Neurons in central nervous systems receive multiple synaptic inputs and transform them into a largely standardized output to their target cells-the action potential. A simplified model posits that synaptic signals are integrated by linear summation and passive propagation towards the axon initial segment, where the threshold for spike generation is either crossed or not. However, multiple lines of research during past decades have shown that signal integration in individual neurons is much more complex, with important functional consequences at the cellular, network, and behavioral-cognitive level. The interplay between concomitant excitatory and inhibitory postsynaptic potentials depends strongly on the relative timing and localization of the respective synapses. In addition, dendrites contain multiple voltage-dependent conductances, which allow scaling of postsynaptic potentials, non-linear input processing, and compartmentalization of signals. Together, these features enable a rich variety of single-neuron computations, including non-linear operations and synaptic plasticity. Hence, we have to revise over-simplified messages from textbooks and use simplified computational models like integrate-and-fire neurons with some caution. This concept article summarizes the most important mechanisms of dendritic integration and highlights some recent developments in the field.

Inhibition of Cdc42-Interacting Protein 4-calcineurin complexes attenuate pathological cardiac remodeling after myocardial infarction

Myocardial infarction (MI) is a major cause of mortality and morbidity worldwide. Heart failure due to MI results from both acute ischemia/reperfusion (I/R) injury and subsequent post-MI pathological cardiac remodeling. The Ca2+/calmodulin-dependent protein phosphatase calcineurin (CaN) is a critical mediator of pathological cardiac remodeling. Along with its role in the immune system, the pro-survival function of CaN complicates the targeting of the phosphatase in heart disease. CaNAβ is a CaN isoform uniquely associated with pathological cardiac hypertrophy. We have discovered that pools of cardiomyocyte CaNAβ are anchored via the unique CaNAβ N-terminal polyproline domain (PP) to scaffold proteins such as CIP4 (Cdc42 interacting protein 4, TRIP10), creating discrete Ca2+-CaN signaling compartments. Pathological cardiac remodeling induced by pressure overload was inhibited by CIP4 gene knock-out and by inhibition of CaNAβ binding to CIP4 using an adeno-associated virus (AAV) vector that expresses the CaNAβ PP peptide. It remains unknown whether PP-anchored CaNAβ also participates in CaN-dependent cardioprotective signaling in I/R injury. We now consider whether CIP4-CaNAβ signalosomes are required for myocyte survival in ischemic cardiomyopathy. To test the relevance of CIP4 to ischemic reperfusion (IR) injury, tamoxifen-inducible, cardiac-specific CIP4 knock-out (CIP4 CKO), control Tg(Myh6-cre/Esr1*), and control CIP4f/f mice were subjected to 30 minutes of ischemia by transient left anterior descending coronary artery (LAD) ligation. Cardiac function and structure were evaluated 24 hours after I/R by echocardiography and histology. Left ventricular ejection fraction was significantly preserved, while infarct size was significantly decreased by CIP4 CKO when compared to the two control cohorts. Separate mice subjected to permanent LAD ligation showed that CIP4 CKO resulted in long-term preservation of cardiac function. To disrupt CaNAβ-CIP4 complexes in vivo, the CaNAβ PP domain peptide was expressed again using AAV. In contrast to the expression of the VIVIT pan-CaN inhibitory peptide, CaNAβ PP peptide expression did not exacerbate I/R injury and in fact preserved cardiac function immediately after infarction. Moreover, VIVIT peptide expression worsened, while CaNAβ PP peptide expression improved cardiac function long term after induction of MI by permanent LAD ligation. Together, these results suggest that CIP4-CaNAβ signalosomes do not contribute to cardioprotection, while inducing pathological cardiac remodeling. These results suggest that targeting of CIP4-CaNAβ signalosomes constitute a novel strategy for inhibiting I/R injury and preventing the development of heart failure in response to diverse forms of cardiovascular disease. This work was supported by National Institutes of Health R01 grants HL126950 and HL158052. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Guanylate cyclase-C Signaling Axis as a theragnostic target in colorectal cancer: a systematic review of literature

IntroductionColorectal cancer (CRC) is a devastating disease that affects millions of people worldwide. Recent research has highlighted the crucial role of the guanylate cyclase-C (GC-C) signaling axis in CRC, from the early stages of tumorigenesis to disease progression. GC-C is activated by endogenous peptides guanylin (GU) and uroguanylin (UG), which are critical in maintaining intestinal fluid homeostasis. However, it has been found that these peptides may also contribute to the development of CRC. This systematic review focuses on the latest research on the GC-C signaling axis in CRC.MethodsAccording to the aim of the study, a systematic literature search was conducted on Medline and PubMed databases. Ultimately, a total of 40 articles were gathered for the systematic review.ResultsOur systematic literature search revealed that alterations in GC-C signaling compartments in CRC tissue have demonstrated potential as diagnostic, prognostic, and therapeutic markers. This research highlights a potential treatment for CRC by targeting the GC-C signaling axis. Promising results from recent studies have explored the use of this signaling axis to develop new vaccines and chimeric antigen receptors that may be used in future clinical trials.ConclusionThe findings presented in this review provide compelling evidence that targeting the GC-C signaling axis may be an advantageous approach for treating CRC.

Primary cilia control oligodendrocyte precursor cell proliferation in white matter injury via Hedgehog-independent CREB signaling

Remyelination after white matter injury (WMI) often fails in diseases such as multiple sclerosis because of improper recruitment and repopulation of oligodendrocyte precursor cells (OPCs) in lesions. How OPCs elicit specific intracellular programs in response to a chemically and mechanically diverse environment to properly regenerate myelin remains unclear. OPCs construct primary cilia, specialized signaling compartments that transduce Hedgehog (Hh) and G-protein-coupled receptor (GPCR) signals. We investigated the role of primary cilia in the OPC response to WMI. Removing cilia from OPCs genetically via deletion of Ift88 results in OPCs failing to repopulate WMI lesions because of reduced proliferation. Interestingly, loss of cilia does not affect Hh signaling in OPCs or their responsiveness to Hh signals but instead leads to dysfunctional cyclic AMP (cAMP)-dependent cAMP response element-binding protein (CREB)-mediated transcription. Because inhibition of CREB activity in OPCs reduces proliferation, we propose that a GPCR/cAMP/CREB signaling axis initiated at OPC cilia orchestrates OPC proliferation during development and in response to WMI.

RAS GTPases and Interleaflet Coupling in the Plasma Membrane.

RAS genes are frequently mutated in cancer. The primary signaling compartment of wild-type and constitutively active oncogenic mutant RAS proteins is the inner leaflet of the plasma membrane (PM). Thus, a better understanding of the unique environment of the PM inner leaflet is important to shed further light on RAS function. Over the past few decades, an integrated approach of superresolution imaging, molecular dynamic simulations, and biophysical assays has yielded new insights into the capacity of RAS proteins to sort lipids with specific headgroups and acyl chains, to assemble signaling nanoclusters on the inner PM. RAS proteins also sense and respond to changes in components of the outer PM leaflet, including glycophosphatidylinositol-anchored proteins, sphingophospholipids, glycosphingolipids, and galectins, as well as cholesterol that translocates between the two leaflets. Such communication between the inner and outer leaflets of the PM, called interleaflet coupling, allows RAS to potentially integrate extracellular mechanical and electrostatic information with intracellular biochemical signaling events, and reciprocally allows mutant RAS-transformed tumor cells to modify tumor microenvironments. Here, we review RAS-lipid interactions and speculate on potential mechanisms that allow communication between the opposing leaflets of the PM.

Cell lineage analysis reveals signal tracing and compartment characterisation in Drosophila haltere.

Insect halteres, as specialised hind wings, play an important role during aerial manoeuvres. In Drosophila, halteres and wings are homologous appendages with different morphology. Previous studies have focused on the metamorphosis of halteres, while current knowledge about its cell lineage and regional compartmentalization is still limited. In this study, we performed cell-lineage tracing of canonical landmark signals in halteres and present a simple model for haltere development. Cell lineage tracing in wings was used as a reference. The nub showed wing-like expressions in halteres, whereas hth and pnr exhibited different expressions in adult wings and halteres. The lineage tracing revealed that the pouch region gives rise to end-bulb, and hinge cells contribute to proximal haltere formation. Moreover, we demonstrated that twi-expressing cells participate in the cell population of the distal end-bulb. Haematoxylin and eosin staining indicated that muscle cells were present at the distal end-bulb. These results indicated that adult halteres displayed unique cell lineage patterns and the muscle cells are important components of end-bulbs.

Mutations in Phosphodiesterase 3A (PDE3A) Cause Hypertension Without Cardiac Damage.

Hypertension with brachydactyly (HTNB) represents an autosomal dominant form of hypertension. It is a rare syndrome, in which the blood pressure can rise by more than 50 mmHg. If untreated, the patients die of stroke by the age of 50 years. In HTNB, vascular smooth muscle cell proliferation is increased, vasodilation compromised, and the kidney not affected. Surprisingly, after decades of hypertension, HTNB is not associated with hypertension-induced cardiac damage. HTNB is caused by gain-of-function mutations in the PDE3A (phosphodiesterase 3A) gene. The mutant enzymes are hyperactive. PDE3A (phosphodiesterase 3A) hydrolyzes and thereby terminates cyclic adenosine monophosphate signaling in defined cellular compartments. The cardioprotective effect involves local changes of cyclic adenosine monophosphate signaling and inhibition of Ca2+ reuptake into the sarcoplasmic reticulum of cardiac myocytes. This review introduces HTNB and discusses how insight into the molecular mechanisms underlying HTNB could contribute to a better understanding of blood pressure control and lead to PDE3A-directed strategies for the treatment of essential hypertension and the prevention of hypertension-induced cardiac damage. A focus will be on cAMP (cyclic adenosine monophosphate) signaling compartments.

Chemigenetic kinase biosensors for multiplexed activity sensing and super-resolution imaging.

Genetically encoded fluorescent biosensors have revolutionized the study of dynamic biochemical processes in living cells. They enable to investigate intertwined signaling pathways both under physiological and pathological conditions. For instance, kinase activity is often regulated through tight spatiotemporal control via compartmentation. Specifically, protein kinase A (PKA) is organized into plasma membrane nanodomains or sequestered in cytoplasmic phase separated bodies. Due to the small size of these compartments, little is known about the PKA activity organization and regulation within them, even though this knowledge would be crucial to untangle intricately regulated PKA signaling. To investigate these signaling compartments with the necessary spatial resolution and under more physiological conditions new biosensors with enhanced capabilities need to be developed.

Atheroprone fluid shear stress-regulated ALK1-Endoglin-SMAD signaling originates from early endosomes

BackgroundFluid shear stress enhances endothelial SMAD1/5 signaling via the BMP9-bound ALK1 receptor complex supported by the co-receptor Endoglin. While moderate SMAD1/5 activation is required to maintain endothelial quiescence, excessive SMAD1/5 signaling promotes endothelial dysfunction. Increased BMP signaling participates in endothelial-to-mesenchymal transition and inflammation culminating in vascular diseases such as atherosclerosis. While the function of Endoglin has so far been described under picomolar concentrations of BMP9 and short-term shear application, we investigated Endoglin under physiological BMP9 and long-term pathophysiological shear conditions.ResultsWe report here that knock-down of Endoglin leads to exacerbated SMAD1/5 phosphorylation and atheroprone gene expression profile in HUVECs sheared for 24 h. Making use of the ligand-trap ALK1-Fc, we furthermore show that this increase is dependent on BMP9/10. Mechanistically, we reveal that long-term exposure of ECs to low laminar shear stress leads to enhanced Endoglin expression and endocytosis of Endoglin in Caveolin-1-positive early endosomes. In these endosomes, we could localize the ALK1-Endoglin complex, labeled BMP9 as well as SMAD1, highlighting Caveolin-1 vesicles as a SMAD signaling compartment in cells exposed to low atheroprone laminar shear stress.ConclusionsWe identified Endoglin to be essential in preventing excessive activation of SMAD1/5 under physiological flow conditions and Caveolin-1-positive early endosomes as a new flow-regulated signaling compartment for BMP9-ALK1-Endoglin signaling axis in atheroprone flow conditions.

The function of IRAP+ endosomes in T cells

T lymphocytes form an immunological synapse with cells infected by pathogens or cancer cells, after recognition of MHC-epitope complexes by the TCR. The expression of the TCR on the T cell surface, its internalization and its recycling are essential for the TCR signaling pathway. While the proteins implicated in this pathway are well known, there is controversy on the role of TCR endocytosis and recycling in TCR signaling. We identified IRAP+ endosomes as a novel compartment in T cells, that controls TCR signaling. Using mice that are deficient for IRAP only in T cells (IRAP-lox-lox mice crossed with Lck-cre mice) we observed a decreased effector CD8+ T cell number 12 days after immunization with a tumor cell line, as well as increased tumor weight. Since IRAP+ endosomes had never been studied before in T cells, we proceeded to the characterization of this compartment by immunoprecipitations and immunofluorescence not only in mouse transgenic OTI T cells but also in the Jurkat human T cell line. We demonstrated that IRAP colocalizes and coimmunoprecipitates with Rab4, Lck and CD3ζ, the limiting TCR component for signal transduction. In addition, the absence of IRAP inhibits the polarized recruitment of signaling proteins to the synapse, alters the intracellular distribution of CD3ζ, leads to a strong reduction in TCR signaling and results in diminished IL-2 production. According to our results, we suggest that CD3ζ recycles and can signal through IRAP endosomes and that this signaling compartment is essential for the engagement of the TCR-dependent signaling pathways. Our results will help to the understanding of molecular mechanisms implicated in T cell activation, a crucial step of the adaptive immune response on which is based the organism’s defense towards pathogens and tumors.

Subtype-Selective Positive Modulation of KCa2.3 Channels Increases Cilia Length.

Small-conductance Ca2+-activated potassium (KCa2.x) channels are gated exclusively by intracellular Ca2+. The activation of KCa2.3 channels induces hyperpolarization, which augments Ca2+ signaling in endothelial cells. Cilia are specialized Ca2+ signaling compartments. Here, we identified compound 4 that potentiates human KCa2.3 channels selectively. The subtype selectivity of compound 4 for human KCa2.3 over rat KCa2.2a channels relies on an isoleucine residue in the HA/HB helices. Positive modulation of KCa2.3 channels by compound 4 increased flow-induced Ca2+ signaling and cilia length, while negative modulation by AP14145 reduced flow-induced Ca2+ signaling and cilia length. These findings were corroborated by the increased cilia length due to the expression of Ca2+-hypersensitive KCa2.3_G351D mutant channels and the reduced cilia length resulting from the expression of Ca2+-hyposensitive KCa2.3_I438N channels. Collectively, we were able to associate functions of KCa2.3 channels and cilia, two crucial components in the flow-induced Ca2+ signaling of endothelial cells, with potential implications in vasodilation and ciliopathic hypertension.

A perinuclear calcium compartment regulates cardiac myocyte hypertrophy

The pleiotropic Ca2+/calmodulin-dependent phosphatase calcineurin is a key regulator of pathological cardiac myocyte hypertrophy. The selective activation of hypertrophic calcineurin signaling under stress conditions has been attributed to compartmentation of Ca2+ signaling in cardiac myocytes. Here, perinuclear signalosomes organized by the scaffold protein muscle A-Kinase Anchoring Protein β (mAKAPβ/AKAP6β) are shown to orchestrate local Ca2+ transients, inducing calcineurin-dependent NFATc nuclear localization and myocyte hypertrophy in response to β-adrenergic receptor activation. Fluorescent biosensors for Ca2+ and calcineurin and protein kinase A (PKA) activity, both diffusely expressed and localized by nesprin-1α to the nuclear envelope, are used to define an autonomous mAKAPβ signaling compartment in adult and neonatal rat ventricular myocytes. Notably, β-adrenergic-stimulated perinuclear Ca2+ and PKA and CaN activity transients depended upon mAKAPβ expression, while Ca2+ elevation and PKA and CaN activity in the cytosol were mAKAPβ independent. Buffering perinuclear cAMP and Ca2+ prevented calcineurin-dependent NFATc nuclear translocation and myocyte hypertrophy, without affecting cardiac myocyte contractility. Additional findings suggest that the perinuclear Ca2+ transients were mediated by signalosome-associated ryanodine receptors regulated by local PKA phosphorylation. These results demonstrate the existence of a functionally independent Ca2+ signaling compartment in the cardiac myocyte regulating hypertrophy and provide a premise for targeting mAKAPβ signalosomes to prevent selectively cardiac hypertrophy in disease.

Post-Synapses in the Brain: Role of Dendritic and Spine Structures.

Brain synapses are neuronal structures of the greatest interest. For a long time, however, the knowledge about them was variable, and interest was mostly focused on their pre-synaptic portions, especially neurotransmitter release from axon terminals. In the present review interest is focused on post-synapses, the structures receiving and converting pre-synaptic messages. Upon further modulation, such messages are transferred to dendritic fibers. Dendrites are profoundly different from axons; they are shorter and of variable thickness. Their post-synapses are of two types. Those called flat/intended/aspines, integrated into dendritic fibers, are very frequent in inhibitory neurons. The spines, small and stemming protrusions, connected to dendritic fibers by their necks, are present in almost all excitatory neurons. Several structures and functions including the post-synaptic densities and associated proteins, the nanoscale mechanisms of compartmentalization, the cytoskeletons of actin and microtubules, are analogous in the two post-synaptic forms. However other properties, such as plasticity and its functions of learning and memory, are largely distinct. Several properties of spines, including emersion from dendritic fibers, growth, change in shape and decreases in size up to disappearance, are specific. Spinal heads correspond to largely independent signaling compartments. They are motile, their local signaling is fast, however transport through their thin necks is slow. When single spines are activated separately, their dendritic effects are often lacking; when multiple spines are activated concomitantly, their effects take place. Defects of post-synaptic responses, especially those of spines, take place in various brain diseases. Here alterations affecting symptoms and future therapy are shown to occur in neurodegenerative diseases and autism spectrum disorders.

Fixation of Embryonic Mouse Tissue for Cytoneme Analysis

Developmental tissue patterning and postdevelopmental tissue homeostasis depend upon controlled delivery of cellular signals called morphogens. Morphogens act in a concentration- and time-dependent manner to specify distinct transcriptional programs that instruct and reinforce cell fate. One mechanism by which appropriate morphogen signaling thresholds are ensured is through delivery of the signaling proteins by specialized filopodia called cytonemes. Cytonemes are very thin (≤200 nm in diameter) and can grow to lengths of several hundred microns, which makes their preservation for fixed-image analysis challenging. This paper describes a refined method for delicate handling of mouse embryos for fixation, immunostaining, and thick sectioning to allow for visualization of cytonemes using standard confocal microscopy. This protocol has been successfully used to visualize cytonemes that connect distinct cellular signaling compartments during mouse neural tube development. The technique can also be adapted to detect cytonemes across tissue types to facilitate the interrogation of developmental signaling at unprecedented resolution.

Fixation of Embryonic Mouse Tissue for Cytoneme Analysis.

Developmental tissue patterning and postdevelopmental tissue homeostasis depend upon controlled delivery of cellular signals called morphogens. Morphogens act in a concentration- and time-dependent manner to specify distinct transcriptional programs that instruct and reinforce cell fate. One mechanism by which appropriate morphogen signaling thresholds are ensured is through delivery of the signaling proteins by specialized filopodia called cytonemes. Cytonemes are very thin (≤200 nm in diameter) and can grow to lengths of several hundred microns, which makes their preservation for fixed-image analysis challenging. This paper describes a refined method for delicate handling of mouse embryos for fixation, immunostaining, and thick sectioning to allow for visualization of cytonemes using standard confocal microscopy. This protocol has been successfully used to visualize cytonemes that connect distinct cellular signaling compartments during mouse neural tube development. The technique can also be adapted to detect cytonemes across tissue types to facilitate the interrogation of developmental signaling at unprecedented resolution.

Phosphodiesterase 7B regulates prostanoid‐ but not ß‐adrenergic‐stimulated cAMP levels in primary human airway smooth muscle cells

Human airway smooth muscle (HASM) cells contain G‐protein coupled receptor (GPCR) signal transduction pathways that are organized in the plasma membrane and form cAMP signaling compartments. One such compartment contains ß2‐adrenergic receptors (ß2AR) coupled to adenylyl cyclase (AC)6 and is principally localized in lipid rafts. Another compartment expresses E prostanoid receptors 2 and 4 (EP2/4) coupled to AC2 and is restricted to non‐raft regions of the plasma membrane. Various phosphodiesterase (PDE) isozymes modulate cyclic AMP (cAMP) levels in response to these pathways. Non‐specific or PDE‐family specific PDE inhibitors have been used to treat asthma and chronic obstructive pulmonary disease with limited success, in part due to expression of these PDE isoforms throughout the body. Drugs that inhibit specific PDE isozymes might produce therapeutic effects in airways with fewer off‐target effects. RNA‐sequencing data in HASM cells indicates that PDE7B is the third most highly expressed PDE isoform and includes expression of both the PDE7B‐201 and PDE7B‐203 splice variants. We sought to characterize the role of PDE7B in the lipid raft and non‐raft cAMP signaling compartments. We used siRNA to knock down both PDE7B splice variants, confirmed protein reduction via Western Blot analysis, and then observed live cells’ real‐time cAMP levels in response to either isoproterenol (ßAR agonist) or PGE2 (EP agonist). The cADDis‐FMP biosensor, which localizes in lipid raft regions, did not report altered cAMP dynamics in response to ß2AR or EPR agonists following PDE7B knockdown. However, the cADDis‐FS15 biosensor, which reports cAMP levels in non‐raft plasma membrane regions, reported significant increases in cAMP levels stimulated by EPR agonists but no change in cAMP levels stimulated by ß2AR agonist. PDE7B may play a unique role in regulating non‐raft associated EP2/4 signaling without altering cAMP levels in other areas of the cell, thereby presenting a novel therapeutic target for pulmonary diseases.

Spatially encoded GPCR signaling depends on the precise subcellular positioning of receptor‐containing endosomes

G protein‐coupled receptors (GPCRs), which comprise the largest class of drug targets, are critical signaling proteins that translate extracellular stimuli to mediate human physiology. As such, understanding GPCR signaling cascades will provide insight into the molecular processes underlying complex physiologies, how these are perturbed in disease, and inform our ability to design more efficient therapeutics. In recent years, our lab and others have made the paradigm‐shifting discovery that GPCRs can be activated after drug‐induced internalization into endosomal compartments to generate spatially biased responses. We have found that transcriptional signaling downstream of GPCR activation is initiated strictly from intracellular receptors. However, it remains unknown how the endosome can promote such selective signaling outcomes. One possibility is that the biophysical positioning of endosomal GPCRs within the cell, specifically their proximity to the nucleus, may allow for cAMP signaling cascades that result in gene transcription. To address this, I devised a strategy to rapidly redistribute endosomes in intact cells and coupled the approach with two novel microscopy‐based optical transcriptional readouts. Specifically, I developed and optimized a chemically induced heterodimerization system that fuses endosomal embedding proteins to plus‐ or minus‐end directed molecular motors and allows their relocalization ‘on command’. To determine the impact of endosomal redistribution on transcription, I utilized either accumulation of a fluorescent cAMP transcriptional reporter or single‐molecule RNA in situ hybridization of endogenous target genes. With these complementary readouts, I show that redistributing receptor‐containing endosomes toward the cell periphery significantly inhibits GPCR‐mediated gene transcription without perturbing the biochemical composition of the endosome. In an orthogonal approach, I utilized localized optogenetic adenylyl cyclases to demonstrate that cAMP produced at or proximal to the cell periphery similarly failed to induce transcription. Lastly, I uncovered that this critical role of endosomal positioning stems in part from enhanced PDE activity around the plasma membrane. This study supports a key role for endosomal positioning in the transduction of the GPCR‐initiated signal. Ultimately, this work helps dissect what makes the endosome a unique signaling compartment and illuminate the molecular mechanisms underlying spatial selectivity of GPCR signaling.

Dendritic branch structure compartmentalizes voltage-dependent calcium influx in cortical layer 2/3 pyramidal cells.

Back-propagating action potentials (bAPs) regulate synaptic plasticity by evoking voltage-dependent calcium influx throughout dendrites. Attenuation of bAP amplitude in distal dendritic compartments alters plasticity in a location-specific manner by reducing bAP-dependent calcium influx. However, it is not known if neurons exhibit branch-specific variability in bAP-dependent calcium signals, independent of distance-dependent attenuation. Here, we reveal that bAPs fail to evoke calcium influx through voltage-gated calcium channels (VGCCs) in a specific population of dendritic branches in mouse cortical layer 2/3 pyramidal cells, despite evoking substantial VGCC-mediated calcium influx in sister branches. These branches contain VGCCs and successfully propagate bAPs in the absence of synaptic input; nevertheless, they fail to exhibit bAP-evoked calcium influx due to a branch-specific reduction in bAP amplitude. We demonstrate that these branches have more elaborate branch structure compared to sister branches, which causes a local reduction in electrotonic impedance and bAP amplitude. Finally, we show that bAPs still amplify synaptically-mediated calcium influx in these branches because of differences in the voltage-dependence and kinetics of VGCCs and NMDA-type glutamate receptors. Branch-specific compartmentalization of bAP-dependent calcium signals may provide a mechanism for neurons to diversify synaptic tuning across the dendritic tree.