Breaking NGF-TrkA immunosuppression in melanoma sensitizes immunotherapy for durable memory T cell protection.

Melanoma cells, deriving from neuroectodermal melanocytes, may exploit the nervous system's immune privilege for growth. Here we show that nerve growth factor (NGF) has both melanoma cell intrinsic and extrinsic immunosuppressive functions. Autocrine NGF engages tropomyosin receptor kinase A (TrkA) on melanoma cells to desensitize interferon γ signaling, leading to T and natural killer cell exclusion. In effector T cells that upregulate surface TrkA expression upon T cell receptor activation, paracrine NGF dampens T cell receptor signaling and effector function. Inhibiting NGF, either through genetic modification or with the tropomyosin receptor kinase inhibitor larotrectinib, renders melanomas susceptible to immune checkpoint blockade therapy and fosters long-term immunity by activating memory T cells with low affinity. These results identify the NGF-TrkA axis as an important suppressor of anti-tumor immunity and suggest larotrectinib might be repurposed for immune sensitization. Moreover, by enlisting low-affinity T cells, anti-NGF reduces acquired resistance to immune checkpoint blockade and prevents melanoma recurrence.

Targeting YAP-mediated HSC death susceptibility and senescence for treatment of liver fibrosis.

BACKGROUND AND AIMS: Liver fibrosis results from the accumulation of myofibroblasts (MFs) derived from quiescent HSCs, and yes-associated protein (YAP) controls this state transition. Although fibrosis is also influenced by HSC death and senescence, whether YAP regulates these processes and whether this could be leveraged to treat liver fibrosis are unknown. APPROACH AND RESULTS: YAP activity was manipulated in MF-HSCs to determine how YAP impacts susceptibility to pro-apoptotic senolytic agents or ferroptosis. Effects of senescence on YAP activity and susceptibility to apoptosis versus ferroptosis were also examined. CCl 4 -treated mice were treated with a ferroptosis inducer or pro-apoptotic senolytic to determine the effects on liver fibrosis. YAP was conditionally disrupted in MFs to determine how YAP activity in MF-HSC affects liver fibrosis in mouse models. Silencing YAP in cultured MF-HSCs induced HSC senescence and vulnerability to senolytics, and promoted ferroptosis resistance. Conversely, inducing HSC senescence suppressed YAP activity, increased sensitivity to senolytics, and decreased sensitivity to ferroptosis. Single-cell analysis of HSCs from fibrotic livers revealed heterogeneous sensitivity to ferroptosis, apoptosis, and senescence. In mice with chronic liver injury, neither the ferroptosis inducer nor senolytic improved fibrosis. However, selectively depleting YAP in MF-HSCs induced senescence and decreased liver injury and fibrosis. CONCLUSION: YAP determines whether MF-HSCs remain activated or become senescent. By regulating this state transition, Yap controls both HSC fibrogenic activity and susceptibility to distinct mechanisms for cell death. MF-HSC-specific YAP depletion induces senescence and protects injured livers from fibrosis. Clarifying determinants of HSC YAP activity may facilitate the development of novel anti-fibrotic therapies.

Targeting senescent hepatocytes using the thrombomodulin-PAR1 inhibitor vorapaxar ameliorates NAFLD progression.

BACKGROUND AND AIMS: Senescent hepatocytes accumulate in parallel with fibrosis progression during NASH. The mechanisms that enable progressive expansion of nonreplicating cell populations and the significance of that process in determining NASH outcomes are unclear. Senescing cells upregulate thrombomodulin-protease-activated receptor-1 (THBD-PAR1) signaling to remain viable. Vorapaxar blocks the activity of that pathway. We used vorapaxar to determine if and how THBD-PAR1 signaling promotes fibrosis progression in NASH. APPROACH AND RESULTS: We evaluated the THBD-PAR1 pathway in liver biopsies from patients with NAFLD. Chow-fed mice were treated with viral vectors to overexpress p16 in hepatocytes and induce replicative senescence. Effects on the THBD-PAR1 axis and regenerative capacity were assessed; the transcriptome of p16-overexpressing hepatocytes was characterized, and we examined how conditioned medium from senescent but viable (dubbed "undead") hepatocytes reprograms HSCs. Mouse models of NASH caused by genetic obesity or Western diet/CCl 4 were treated with vorapaxar to determine effects on hepatocyte senescence and liver damage. Inducing senescence upregulates the THBD-PAR1 signaling axis in hepatocytes and induces their expression of fibrogenic factors, including hedgehog ligands. Hepatocyte THBD-PAR1 signaling increases in NAFLD and supports sustained hepatocyte senescence that limits effective liver regeneration and promotes maladaptive repair. Inhibiting PAR1 signaling with vorapaxar interrupts this process, reduces the burden of 'undead' senescent cells, and safely improves NASH and fibrosis despite ongoing lipotoxic stress. CONCLUSION: The THBD-PAR1 signaling axis is a novel therapeutic target for NASH because blocking this pathway prevents accumulation of senescing but viable hepatocytes that generate factors that promote maladaptive liver repair.

Activation of Mitofusin2 by Smad2-RIN1 Complex during Mitochondrial Fusion.

Smads are nuclear-shuttling transcriptional mediators of transforming growth factor-ß (TGF-ß) signaling. Although their essential nuclear roles in gene regulation during development and carcinogenesis are well established, whether they have important cytoplasmic functions remains unclear. Here we report that Smad2 is a critical determinant of mitochondrial dynamics. We identified mitofusin2 (MFN2) and Rab and Ras Interactor 1 (RIN1) as new Smad2 binding partners required for mitochondrial fusion. Unlike TGF-ß-induced Smad2/3 transcriptional responses underlying mitochondrial fragmentation and apoptosis, inactive cytoplasmic Smad2 rapidly promotes mitochondrial fusion by recruiting RIN1 into a complex with MFN2. We demonstrate that Smad2 is a key scaffold, allowing RIN1 to act as a GTP exchange factor for MFN2-GTPase activation to promote mitochondrial ATP synthesis and suppress superoxide production. These results reveal functional implications between Smads and mitochondrial dysfunction in cancer and metabolic and neurodegenerative disorders.

EPDR1 is a noncanonical effector of insulin-mediated angiogenesis regulated by an endothelial-specific TGF-ß receptor complex.

Insulin signaling in blood vessels primarily functions to stimulate angiogenesis and maintain vascular homeostasis through the canonical PI3K and MAPK signaling pathways. However, angiogenesis is a complex process coordinated by multiple other signaling events. Here, we report a distinct crosstalk between the insulin receptor and endoglin/activin receptor-like kinase 1 (ALK1), an endothelial cell-specific TGF-ß receptor complex essential for angiogenesis. While the endoglin-ALK1 complex normally binds to TGF-ß or bone morphogenetic protein 9 (BMP9) to promote gene regulation via transcription factors Smad1/5, we show that insulin drives insulin receptor oligomerization with endoglin-ALK1 at the cell surface to trigger rapid Smad1/5 activation. Through quantitative proteomic analysis, we identify ependymin-related protein 1 (EPDR1) as a major Smad1/5 gene target induced by insulin but not by TGF-ß or BMP9. We found endothelial EPDR1 expression is minimal at the basal state but is markedly enhanced upon prolonged insulin treatment to promote cell migration and formation of capillary tubules. Conversely, we demonstrate EPDR1 depletion strongly abrogates these angiogenic effects, indicating that EPDR1 is a crucial mediator of insulin-induced angiogenesis. Taken together, these results suggest important therapeutic implications for EPDR1 and the TGF-ß pathways in pathologic angiogenesis during hyperinsulinemia and insulin resistance.

CD36 initiates the secretory phenotype during the establishment of cellular senescence.

Cellular senescence is a unique cell fate characterized by stable proliferative arrest and the extensive production and secretion of various inflammatory proteins, a phenomenon known as the senescence-associated secretory phenotype (SASP). The molecular mechanisms responsible for generating a SASP in response to senescent stimuli remain largely obscure. Here, using unbiased gene expression profiling, we discover that the scavenger receptor CD36 is rapidly upregulated in multiple cell types in response to replicative, oncogenic, and chemical senescent stimuli. Moreover, ectopic CD36 expression in dividing mammalian cells is sufficient to initiate the production of a large subset of the known SASP components via activation of canonical Src-p38-NF-κB signaling, resulting in the onset of a full senescent state. The secretome is further shown to be ligand-dependent, as amyloid-beta (Aß) is sufficient to drive CD36-dependent NF-κB and SASP activation. Finally, loss-of-function experiments revealed a strict requirement for CD36 in secretory molecule production during conventional senescence reprogramming. Taken together, these results uncover the Aß-CD36-NF-κB signaling axis as an important regulator of the senescent cell fate via induction of the SASP.

An Approach to Characterize the Impact Absorption Performance of Construction Helmets in Top Impact.

The helmets used by construction site workers are mainly designed for head protection when objects are dropped from heights. Construction helmets are also casually called "hard hats" in industries. Common construction helmets are mostly categorized as type 1 according to different standards. All type 1 helmets have to pass type 1 standard impact tests, which are top impact tests-the helmet is fixed and is impacted by a free falling impactor on the top crown of the helmet shell. The purpose of this study was to develop an approach that can determine the performance characterization of a helmet. A total of 31 drop impact tests using a representative type 1 helmet model were performed at drop heights from 0.30 to 2.23 m, which were estimated to result in impact speeds from 2.4 to 6.6 m/s. Based on our results, we identified a critical drop height that was used to evaluate the performance of helmets. The peak impact forces and peak accelerations varied nonproportionally with the drop height. When the drop height is less than the critical height, the peak force and peak acceleration increase gradually and slowly with increasing drop height. When the drop height is greater than the critical height, the peak force and peak acceleration increase steeply with even a slight increase in drop height. Based on the critical drop height, we proposed an approach to determine the safety margin of a helmet. The proposed approach would make it possible to determine the performance characteristics of a helmet and to estimate the safety margin afforded by the helmet, if the helmet first passes the existing standardized tests. The proposed test approach would provide supplementary information for consumers to make knowledgeable decisions when selecting construction helmets.

Evaluation of the shock absorption performance of construction helmets under repeated top impacts.

It is accepted in industries that an industrial helmet should be disposed of when it is subjected to a significant impact. There is no scientific evidence that supports this well-accepted belief. The current study was intended to evaluate the shock absorption performance of industrial helmets under repeated impacts. Common industrial or construction helmets are categorized as Type I according to ANSI Z89.1 and they are designed to mainly protect top impacts. A representative basic Type I construction helmet model was selected in the study. Helmets were repeatedly impacted ten times using a commercial drop tower tester with an impactor (mass 3.6 kg) at different drop heights from 0.30 to 2.03 m. A total of 80 impact trials were performed in the study. The relationships of the transmitted force with the drop height and with impact number were analyzed. A new parameter - the endurance limit - was proposed to evaluate the shock absorption performance of a helmet. The helmets were observed to experience cumulative structural damage with increasing impact number, resulting in a degrading shock absorption performance, when being impacted repeatedly with magnitudes greater than the endurance limit. Repeated impacts with magnitudes smaller than the endurance limit did not cause measurable cumulative structural damage to the helmets in our study.

Accelerated Evaluation of Automated Vehicles Safety in Lane-Change Scenarios Based on Importance Sampling Techniques.

Automated vehicles (AVs) must be thoroughly evaluated before their release and deployment. A widely used evaluation approach is the Naturalistic-Field Operational Test (N-FOT), which tests prototype vehicles directly on the public roads. Due to the low exposure to safety-critical scenarios, N-FOTs are time consuming and expensive to conduct. In this paper, we propose an accelerated evaluation approach for AVs. The results can be used to generate motions of the other primary vehicles to accelerate the verification of AVs in simulations and controlled experiments. Frontal collision due to unsafe cut-ins is the target crash type of this paper. Human-controlled vehicles making unsafe lane changes are modeled as the primary disturbance to AVs based on data collected by the University of Michigan Safety Pilot Model Deployment Program. The cut-in scenarios are generated based on skewed statistics of collected human driver behaviors, which generate risky testing scenarios while preserving the statistical information so that the safety benefits of AVs in nonaccelerated cases can be accurately estimated. The cross-entropy method is used to recursively search for the optimal skewing parameters. The frequencies of the occurrences of conflicts, crashes, and injuries are estimated for a modeled AV, and the achieved accelerated rate is around 2000 to 20 000. In other words, in the accelerated simulations, driving for 1000 miles will expose the AV with challenging scenarios that will take about 2 to 20 million miles of real-world driving to encounter. This technique thus has the potential to greatly reduce the development and validation time for AVs.

Endoglin Regulation of Smad2 Function Mediates Beclin1 Expression and Endothelial Autophagy.

Autophagy is the targeted degradation of proteins and organelles critical for homeostasis and cell survival. Transforming growth factor ß (TGF-ß) differentially regulates autophagy in a context-specific manner, although the precise intracellular mechanisms remain less clear. Importantly, how TGF-ß controls autophagic responses in endothelial cells (EC) during angiogenesis is unknown. Here we identified endoglin, an EC-specific TGF-ß co-receptor essential for angiogenesis, as a key determinant of autophagy. Among the two opposing TGF-ß Smad pathways in the EC system (Smad1/5/8 and Smad2/3), we found Smad2 as the major transcriptional regulator of autophagy that targets beclin1 (BECN1) gene expression. Smad2, but not Smad3, acts as a repressor upstream of the BECN1 promoter region. Overall, endoglin promotes autophagy by impeding Smad2 transcriptional repressor activity. Notably, increased beclin1 levels upon Smad2 knockdown directly correlated with enhanced autophagy during angiogenesis. Taken together, these results establish endoglin as a critical mediator of autophagy and demonstrate a new transcriptional mechanism by which Smad2 inhibits angiogenesis.

Physiological correspondence dictates cortical long-term potentiation and depression by thalamic induction.

The auditory cortex exhibits frequency-specific plasticity over a life cycle. Although thalamocortical long-term potentiation (LTP) and depression (LTD) are components of a widely held model underlying the receptive field (RF) plasticity of cortical neurons, the model lacks direct supporting evidence. We show here that conventional high-frequency tetanic stimulation (TS) of the auditory thalamus induced long-term changes in cortical field excitatory postsynaptic potentials, including both LTP and LTD, in mice. Thalamic TS induced LTP when the stimulated thalamic and recorded cortical neurons were tuned to the same frequency and induced LTD when they were tuned to different frequencies. The thalamocortical LTP was N-methyl-d-aspartate-dependent, but the LTD also involved cortical γ-amino-butyric acidergic inhibition. Notably, the frequency-specificity of cortical LTP/LTD was in accordance with the frequency-specific plasticity of spike-based RFs of cortical neurons. Our results suggest that cortical LTP and LTD induced by thalamic induction can be a consequence of identical stimuli, occur in an input-specific manner, and account for frequency-specific remodeling of RFs of auditory cortical neurons.

Gap Acceptance During Lane Changes by Large-Truck Drivers-An Image-Based Analysis.

This paper presents an analysis of rearward gap acceptance characteristics of drivers of large trucks in highway lane change scenarios. The range between the vehicles was inferred from camera images using the estimated lane width obtained from the lane tracking camera as the reference. Six-hundred lane change events were acquired from a large-scale naturalistic driving data set. The kinematic variables from the image-based gap analysis were filtered by the weighted linear least squares in order to extrapolate them at the lane change time. In addition, the time-to-collision and required deceleration were computed, and potential safety threshold values are provided. The resulting range and range rate distributions showed directional discrepancies, i.e., in left lane changes, large trucks are often slower than other vehicles in the target lane, whereas they are usually faster in right lane changes. Video observations have confirmed that major motivations for changing lanes are different depending on the direction of move, i.e., moving to the left (faster) lane occurs due to a slower vehicle ahead or a merging vehicle on the right-hand side, whereas right lane changes are frequently made to return to the original lane after passing.

Src-mediated post-translational regulation of endoglin stability and function is critical for angiogenesis.

Endoglin is a transforming growth factor ß (TGF-ß) co-receptor essential for angiogenesis and tumor vascularization. Endoglin modulates the crucial balance between pro- and anti-angiogenic signaling by activin receptor-like kinase (ALK) 1, 5, and TGF-ß type II (TßRII) receptors. Despite its established role in physiology and disease, the mechanism of endoglin down-regulation remains unknown. Here we report that the conserved juxtamembrane cytoplasmic tyrosine motif ((612)YIY(614)) is a critical determinant of angiogenesis. Src directly phosphorylates this motif to induce endoglin internalization and degradation via the lysosome. We identified epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) as Src-activators that induce endoglin turnover following (612)YIY(614) phosphorylation. Interestingly, Src phosphorylation of endoglin-(612)YIY(614) was also an important process for receptor down-regulation by TRACON105 (TRC105), an endoglin-targeting antibody currently in clinical trials. The regulation of (612)YIY(614) phosphorylation was critical for angiogenesis, as both the phosphomimetic and unphosphorylatable mutants impaired endothelial functions including proliferation, migration, and capillary tube formation. Collectively, these findings establish Src and pro-angiogenic mitogens as critical mediators of endoglin stability and function.

Testing the shock protection performance of Type I construction helmets using impactors of different masses.

BACKGROUND: Wearing protective helmets is an important prevention strategy to reduce work-related traumatic brain injuries. The existing standardized testing systems are used for quality control and do not provide a quantitative measure of the helmet performance. OBJECTIVE: To analyze the failure characterizations of Type I industrial helmets and develop a generalized approach to quantify the shock absorption performance of Type I industrial helmets based on the existing standardized setups. METHODS: A representative basic Type I construction helmet model was selected for the study. Top impact tests were performed on the helmets at different drop heights using two different impactor masses (3.6 and 5.0 kg). RESULTS: When the helmets were impacted with potential impact energies smaller than the critical potential impact energy values, there was a consistent relationship between the peak impact force and the potential impact energy. When the helmets were impacted under potential impact energies greater than the critical potential impact energy values, the peak impact forces increased steeply with increasing potential impact energy. CONCLUSION: A concept of safety margin for construction helmets based on potential impact energy was introduced to quantify the helmets' shock absorption performance. The proposed method will help helmet manufacturers improve their product quality.

Branched-Chain Amino Acid Accumulation Fuels the Senescence-Associated Secretory Phenotype.

The essential branched-chain amino acids (BCAAs) leucine, isoleucine, and valine play critical roles in protein synthesis and energy metabolism. Despite their widespread use as nutritional supplements, BCAAs' full effects on mammalian physiology remain uncertain due to the complexities of BCAA metabolic regulation. Here a novel mechanism linking intrinsic alterations in BCAA metabolism is identified to cellular senescence and the senescence-associated secretory phenotype (SASP), both of which contribute to organismal aging and inflammation-related diseases. Altered BCAA metabolism driving the SASP is mediated by robust activation of the BCAA transporters Solute Carrier Family 6 Members 14 and 15 as well as downregulation of the catabolic enzyme BCAA transaminase 1 during onset of cellular senescence, leading to highly elevated intracellular BCAA levels in senescent cells. This, in turn, activates the mammalian target of rapamycin complex 1 (mTORC1) to establish the full SASP program. Transgenic Drosophila models further indicate that orthologous BCAA regulators are involved in the induction of cellular senescence and age-related phenotypes in flies, suggesting evolutionary conservation of this metabolic pathway during aging. Finally, experimentally blocking BCAA accumulation attenuates the inflammatory response in a mouse senescence model, highlighting the therapeutic potential of modulating BCAA metabolism for the treatment of age-related and inflammatory diseases.

Antagonizing the irreversible thrombomodulin-initiated proteolytic signaling alleviates age-related liver fibrosis via senescent cell killing.

Cellular senescence is a stress-induced, stable cell cycle arrest phenotype which generates a pro-inflammatory microenvironment, leading to chronic inflammation and age-associated diseases. Determining the fundamental molecular pathways driving senescence instead of apoptosis could enable the identification of senolytic agents to restore tissue homeostasis. Here, we identify thrombomodulin (THBD) signaling as a key molecular determinant of the senescent cell fate. Although normally restricted to endothelial cells, THBD is rapidly upregulated and maintained throughout all phases of the senescence program in aged mammalian tissues and in senescent cell models. Mechanistically, THBD activates a proteolytic feed-forward signaling pathway by stabilizing a multi-protein complex in early endosomes, thus forming a molecular basis for the irreversibility of the senescence program and ensuring senescent cell viability. Therapeutically, THBD signaling depletion or inhibition using vorapaxar, an FDA-approved drug, effectively ablates senescent cells and restores tissue homeostasis in liver fibrosis models. Collectively, these results uncover proteolytic THBD signaling as a conserved pro-survival pathway essential for senescent cell viability, thus providing a pharmacologically exploitable senolytic target for senescence-associated diseases.

Endoglin inhibits ERK-induced c-Myc and cyclin D1 expression to impede endothelial cell proliferation.

Endoglin is an endothelial-specific transforming growth factor beta (TGF-ß) co-receptor essential for angiogenesis and vascular remodeling. Endoglin regulates a wide range of cellular processes, including cell adhesion, migration, and proliferation, through TGF-ß signaling to canonical Smad and Smad-independent pathways. Despite its overall pro-angiogenic role in the vasculature, the underlying mechanism of endoglin action is poorly characterized. We previously identified ß-arrestin2 as a binding partner that causes endoglin internalization from the plasma membrane and inhibits ERK signaling towards endothelial migration. In the present study, we examined the mechanistic role of endoglin and ß-arrestin2 in endothelial cell proliferation. We show that endoglin impedes cell growth through sustained inhibition of ERK-induced c-Myc and cyclin D1 expression in a TGF-ß-independent manner. The down-regulation of c-Myc and cyclin D1, along with growth-inhibition, are reversed when the endoglin/ß-arrestin2 interaction is disrupted. Given that TGF-ß-induced Smad signaling potently represses c-Myc in most cell types, our findings here show a novel mechanism by which endoglin augments growth-inhibition by targeting ERK and key downstream mitogenic substrates.

Evaluation of the Fall Protection of Type I Industrial Helmets.

The performance of Type I industrial helmets for fall protection is not required to be tested in standardized tests. The current study analyzed the fall protection performance of Type I industrial helmets and evaluated if the use of a chin strap and the suspension system tightness have any effect on protection performance. Head impact tests were performed using an instrumented manikin. There were 12 combinations of test conditions: with or without chin strap usage, three levels of suspension system tightness, and two impact surfaces. Four representative helmet models (two basic and two advanced models) were selected for the study. Impact tests without a helmet under all other applicable test conditions were used as a control group. There were four replicates for each test condition-a total of 192 impact tests with helmets and eight impact tests for the control group. The peak acceleration and the calculated head impact criteria (HIC) were used to evaluate shock absorption performance of the helmets. The results showed that all four helmet models demonstrated excellent performance for fall protection compared to the barehead control group. The fall protection performance of the advanced helmet models was substantially better than the basic helmet models. However, the effects of the use of chin straps and suspension system tightness on the helmets' fall protection performance were statistically not significant.

Cancer-cell-derived GABA promotes ß-catenin-mediated tumour growth and immunosuppression.

Many cancers have an unusual dependence on glutamine. However, most previous studies have focused on the contribution of glutamine to metabolic building blocks and the energy supply. Here, we report that cancer cells with aberrant expression of glutamate decarboxylase 1 (GAD1) rewire glutamine metabolism for the synthesis of γ-aminobutyric acid (GABA)-a prominent neurotransmitter-in non-nervous tissues. An analysis of clinical samples reveals that increased GABA levels predict poor prognosis. Mechanistically, we identify a cancer-intrinsic pathway through which GABA activates the GABAB receptor to inhibit GSK-3ß activity, leading to enhanced ß-catenin signalling. This GABA-mediated ß-catenin activation both stimulates tumour cell proliferation and suppresses CD8+ T cell intratumoural infiltration, such that targeting GAD1 or GABABR in mouse models overcomes resistance to anti-PD-1 immune checkpoint blockade therapy. Our findings uncover a signalling role for tumour-derived GABA beyond its classic function as a neurotransmitter that can be targeted pharmacologically to reverse immunosuppression.

ßIV-spectrin as a stalk cell-intrinsic regulator of VEGF signaling.

Defective angiogenesis underlies over 50 malignant, ischemic and inflammatory disorders yet long-term therapeutic applications inevitably fail, thus highlighting the need for greater understanding of the vast crosstalk and compensatory mechanisms. Based on proteomic profiling of angiogenic endothelial components, here we report ßIV-spectrin, a non-erythrocytic cytoskeletal protein, as a critical regulator of sprouting angiogenesis. Early loss of endothelial-specific ßIV-spectrin promotes embryonic lethality in mice due to hypervascularization and hemorrhagic defects whereas neonatal depletion yields higher vascular density and tip cell populations in developing retina. During sprouting, ßIV-spectrin expresses in stalk cells to inhibit their tip cell potential by enhancing VEGFR2 turnover in a manner independent of most cell-fate determining mechanisms. Rather, ßIV-spectrin recruits CaMKII to the plasma membrane to directly phosphorylate VEGFR2 at Ser984, a previously undefined phosphoregulatory site that strongly induces VEGFR2 internalization and degradation. These findings support a distinct spectrin-based mechanism of tip-stalk cell specification during vascular development.