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Item does not contain fulltext Aging is characterized by declines in physiological function that increase risk of age-associated diseases and limit healthspan, mediated in part by chronic low-grade inflammation. Interleukin (IL)-37 suppresses inflammation in pathophysiological states but has not been studied in the context of aging in otherwise healthy humans. Thus, we investigated associations between IL-37 and markers of healthspan in 271 young (18-39 years; n = 41), middle-aged (40-64 years; n = 162), and older (65 + years; n = 68) adults free of overt clinical disease. After conducting a thorough validation of AdipoGen's IL-37 ELISA, we found that plasma IL-37 is lower in older adults (young: 339 ± 240, middle-aged: 345 ± 234; older: 258 ± 175 pg/mL; P = 0.048), despite elevations in pro-inflammatory markers. As such, the ratios of circulating IL-37 to pro-inflammatory markers were considerably lower in older adults (e.g., IL-37 to C-reactive protein: young, 888 ± 918 vs. older, 337 ± 293; P = 0.02), indicating impaired IL-37 responsiveness to a pro-inflammatory state with aging and consistent with the notion of immunosenescence. These ratios were related to multiple indicators of healthspan, including positively to cardiorespiratory fitness (P

Vascular dysfunction: develops progressively with ageing; increases the risk of cardiovascular diseases (CVD); and is characterized by endothelial dysfunction and arterial stiffening, which are primarily mediated by superoxide-driven oxidative stress and consequently reduced nitric oxide (NO) bioavailability and arterial structural changes. Interventions initiated before vascular dysfunction manifests may have more promise for reducing CVD risk than interventions targeting established dysfunction. Gut microbiome-derived trimethylamine N-oxide (TMAO) induces vascular dysfunction, is associated with higher CV risk, and can be suppressed by 3,3-dimethyl-1-butanol (DMB). We investigated whether DMB supplementation could prevent age-related vascular dysfunction in C57BL/6N mice when initiated prior to development of dysfunction. Mice received drinking water with 1% DMB or normal drinking water (control) from midlife (18 months) until being studied at 21, 24 or 27 months of age, and were compared to young adult (5 month) mice. Endothelial function [carotid artery endothelium-dependent dilatation (EDD) to acetylcholine; pressure myography] progressively declined with age in control mice, which was fully prevented by DMB via higher NO-mediated EDD and lower superoxide-related suppression of EDD (normalization of EDD with the superoxide dismutase mimetic TEMPOL). In vivo aortic stiffness (pulse wave velocity) increased progressively with age in controls, but DMB attenuated stiffening by ∼ 70%, probably due to preservation of endothelial function, as DMB did not affect aortic intrinsic mechanical (structural) stiffness (stress-strain testing) nor adventitial abundance of the arterial structural protein collagen. Our findings indicate that long-term DMB supplementation prevents/attenuates age-related vascular dysfunction, and therefore has potential for translation to humans for reducing CV risk with ageing. KEY POINTS: Vascular dysfunction, characterized by endothelial dysfunction and arterial stiffening, develops progressively with ageing and increases the risk of cardiovascular diseases (CVD). Interventions aimed at preventing the development of CV risk factors have more potential for preventing CVD relative to those aimed at reversing established dysfunction. The gut microbiome-derived metabolite trimethylamine N-oxide (TMAO) induces vascular dysfunction, is associated with higher CV risk and can be suppressed by supplementation with 3,3-dimethyl-1-butanol (DMB). In mice, DMB prevented the development of endothelial dysfunction and delayed and attenuated in vivo arterial stiffening with ageing when supplementation was initiated in midlife, prior to the development of dysfunction. DMB supplementation or other TMAO-suppressing interventions have potential for translation to humans for reducing CV risk with ageing.

We provide novel evidence that increased circulating concentrations of the gut microbiome-derived metabolite trimethylamine N-oxide (TMAO) contribute to vascular dysfunction associated with consumption of a Western-style diet and that this dysfunction can be prevented by suppressing TMAO with DMB, thereby supporting translation of this compound to humans. Furthermore, to our knowledge, we present the first evidence of the role of TMAO in mediating impairments in endurance exercise tolerance and increased frailty in any context.

Aging is associated with stiffening of the large elastic arteries and consequent increases in systolic blood pressure (SBP), which together increase cardiovascular disease risk; however, the upstream mechanisms are incompletely understood. Using complementary translational approaches in mice and humans, we investigated the role of the gut microbiome-derived metabolite trimethylamine N-oxide (TMAO) in age-related aortic stiffening and increased SBP. Aortic stiffness was measured using carotid-femoral or aortic pulse wave velocity (PWV) in humans and mice, respectively. Study 1: Plasma TMAO concentrations were elevated ( P r 2 =0.15, P r 2 =0.09, P

Aortic stiffening is a major independent risk factor for cardiovascular diseases, kidney dysfunction, and cognitive impairment. Doxorubicin chemotherapy-treated cancer survivors have greater aortic stiffness relative to healthy controls, but the mechanisms by which doxorubicin induces arterial stiffening are unknown. We tested the hypothesis that doxorubicin increases aortic stiffness by increasing intrinsic mechanical wall stiffness due to proinflammatory signaling-induced adverse structural changes, including collagen deposition (fibrosis), elastin fragmentation, and formation of AGEs (advanced glycation end products). In vivo aortic stiffness (assessed via aortic pulse wave velocity), aortic intrinsic wall stiffness (ex vivo assessment of elastic modulus), and potential underlying mechanisms were assessed 4 weeks after administration of doxorubicin (10 mg/kg) or vehicle (saline) in young adult male C57BL6/J mice. Aortic pulse wave velocity increased by ~30% following doxorubicin (pre: 341±18 versus post: 431±28 cm/s, mean±SEM, P =0.001) and aortic elastic modulus was ~100% higher following doxorubicin (5438±445 kPa) versus vehicle (2659±433 kPa; P =0.003). These effects of doxorubicin were associated with an ~3-fold greater formation of AGEs ( P =0.01) and an ~50% reduction in elastin ( P =0.01), whereas collagen deposition was unaffected. Doxorubicin increased aortic proinflammatory cytokines ( P =0.03) without a compensatory increase in the anti-inflammatory cytokine interleukin-10. Direct ex vivo exposure of aorta rings to doxorubicin mimicked the increase in aortic elastic modulus observed in vivo with doxorubicin, whereas TNF-α (tumor necrosis factor-α) inhibition prevented this response. Doxorubicin induces aortic stiffening in vivo due, in part, to an increase in intrinsic wall stiffness associated with elastin degradation and AGEs formation and mediated by TNF-α-dependent vascular inflammation.

To determine the efficacy of inorganic nitrite supplementation on endothelial function in humans and mechanisms of action, we performed (1) a randomized, placebo-controlled, parallel-group clinical trial with sodium nitrite (80 mg/day, 12 weeks) in older adults (N=49, 68±1 year) and (2) reverse-translation experiments in young (6 months) and old (27 months) c57BL/6 mice. In the clinical trial, sodium nitrite increased plasma nitrite ( P P P P P P P Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT02393742.

KEY POINTS The results of the present study establish the temporal pattern of age-related vascular dysfunction across the adult lifespan in sedentary mice consuming a non-Western diet, and the underlying mechanisms The results demonstrate that consuming a Western diet accelerates and exacerbates vascular ageing across the lifespan in sedentary mice They also show that lifelong voluntary aerobic exercise has remarkable protective effects on vascular function throughout the lifespan, in the setting of ageing alone, as well as ageing compounded by Western diet consumption Overall, the results indicate that amelioration of mitochondrial oxidative stress and inflammation are key mechanisms underlying the voluntary aerobic exercise-associated preservation of vascular function across the lifespan in both the presence and absence of a Western dietary pattern ABSTRACT: Advancing age is the major risk factor for cardiovascular diseases, driven largely by vascular endothelial dysfunction (impaired endothelium-dependent dilatation, EDD) and aortic stiffening (increased aortic pulse wave velocity, aPWV). In humans, vascular ageing occurs in the presence of differences in diet and physical activity, but the interactive effects of these factors are unknown. We assessed carotid artery EDD and aPWV across the lifespan in mice consuming standard (normal) low-fat chow (NC) or a high-fat/high-sucrose Western diet (WD) in the absence (sedentary, SED) or presence (voluntary wheel running, VWR) of aerobic exercise. Ageing impaired nitric oxide-mediated EDD (peak EDD 88 ± 12% 6 months P = 0.003 vs. 59 ± 9% 27 months NC-SED), which was accelerated by WD (60 ± 18% 6 months WD-SED). In NC mice, aPWV increased 32% with age (423 ± 13 cm/s at 24 months P

BACKGROUND Doxorubicin (DOXO) chemotherapy increases risk for cardiovascular disease in part by inducing endothelial dysfunction in conduit arteries. However, the mechanisms mediating DOXO-associated endothelial dysfunction in (intact) arteries and treatment strategies are not established. OBJECTIVES We tested the hypothesis that DOXO impairs endothelial function in conduit arteries via excessive mitochondrial reactive oxygen species (ROS) and that these effects could be prevented by treatment with a mitochondrial-targeted antioxidant (MitoQ). METHODS Endothelial function (endothelium-dependent dilation [EDD] to acetylcholine) and vascular mitochondrial ROS were assessed 4 weeks following administration (10 mg/kg intraperitoneal injection) of DOXO. A separate cohort of mice received chronic (4 weeks) oral supplementation with MitoQ (drinking water) for 4 weeks following DOXO. RESULTS EDD in isolated pressurized carotid arteries was 55% lower 4 weeks following DOXO (peak EDD, DOXO: 42 ± 7% vs. sham: 94 ± 3%; p = 0.006). Vascular mitochondrial ROS was 52% higher and manganese (mitochondrial) superoxide dismutase was 70% lower after DOXO versus sham (p = 0.0008). Endothelial function was rescued by administration of the mitochondrial-targeted antioxidant, MitoQ, to the perfusate. Exposure to plasma from DOXO-treated mice increased mitochondrial ROS in cultured endothelial cells. Analyses of plasma showed differences in oxidative stress-related metabolites and a marked reduction in vascular endothelial growth factor A in DOXO mice, and restoring vascular endothelial growth factor A to sham levels normalized mitochondrial ROS in endothelial cells incubated with plasma from DOXO mice. Oral MitoQ supplementation following DOXO prevented the reduction in EDD (97 ± 1%; p = 0.002 vs. DOXO alone) by ameliorating mitochondrial ROS suppression of EDD. CONCLUSIONS DOXO-induced endothelial dysfunction in conduit arteries is mediated by excessive mitochondrial ROS and ameliorated by mitochondrial-specific antioxidant treatment. Mitochondrial ROS is a viable therapeutic target for mitigating arterial dysfunction with DOXO. (J Am Coll Cardiol CardioOnc 2020;2:475–88) © 2020 The Authors. Published by Elsevier on behalf of the American College of Cardiology Foundation.

Age-related vascular endothelial dysfunction is a major antecedent to cardiovascular diseases. We investigated whether increased circulating levels of the gut microbiome-generated metabolite trimethylamine-N-oxide induces endothelial dysfunction with aging. In healthy humans, plasma trimethylamine-N-oxide was higher in middle-aged/older (64±7 years) versus young (22±2 years) adults (6.5±0.7 versus 1.6±0.2 µmol/L) and inversely related to brachial artery flow-mediated dilation ( r 2 =0.29, P P G -nitro-L-arginine methyl ester). Acute incubation of carotid arteries with trimethylamine-N-oxide recapitulated these events. Next, treatment with 3,3-dimethyl-1-butanol for 8 to 10 weeks to suppress trimethylamine-N-oxide selectively improved endothelium-dependent dilation in old mice to young levels (peak: 90±2%) by normalizing vascular superoxide production, restoring nitric oxide-mediated dilation, and ameliorating superoxide-related suppression of endothelium-dependent dilation. Lastly, among healthy middle-aged/older adults, higher plasma trimethylamine-N-oxide was associated with greater nitrotyrosine abundance in biopsied endothelial cells, and infusion of the antioxidant ascorbic acid restored flow-mediated dilation to young levels, indicating tonic oxidative stress-related suppression of endothelial function with higher circulating trimethylamine-N-oxide. Using multiple experimental approaches in mice and humans, we demonstrate a clear role of trimethylamine-N-oxide in promoting age-related endothelial dysfunction via oxidative stress, which may have implications for prevention of cardiovascular diseases.

Leukocyte migration is controlled by a leading-edge chemosensory pathway that generates the regulatory lipid phosphatidylinositol-3,4,5-trisphosphate (PIP(3)), a growth signal, thereby driving leading-edge expansion up attractant gradients toward sites of infection, inflammation, or tissue damage. PIP(3) also serves as an important growth signal in growing cells and oncogenesis. The kinases PDK1, AKT1 or PKB, and PKCα are key components of a plasma-membrane-based PIP(3) and Ca(2+) signaling circuit that regulates these processes. PDK1 and AKT1 are recruited to the membrane by PIP(3), whereas PKCα is recruited to the membrane by Ca(2+). All three of these master kinases phosphoregulate an array of protein targets. For example, PDK1 activates AKT1, PKCα, and other AGC kinases by phosphorylation at key sites. PDK1 is believed to form PDK1-AKT1 and PDK1-PKCα heterodimers stabilized by a PDK1-interacting fragment (PIF) interaction between the PDK1 PIF pocket and the PIF motif of the AGC binding partner. Here, we present the first, to our knowledge, single-molecule studies of full-length PDK1 and AKT1 on target membrane surfaces, as well as their interaction with full-length PKCα. These studies directly detect membrane-bound PDK1-AKT1 and PDK1-PKCα heterodimers stabilized by PIF interactions formed at physiological ligand concentrations. PKCα exhibits eightfold higher PDK1 affinity than AKT1 and can competitively displace AKT1 from PDK1-AKT1 heterodimers. Ensemble activity measurements under matched conditions reveal that PDK1 activates AKT1 via a cis mechanism by phosphorylating an AKT1 molecule in the same PDK1-AKT1 heterodimer, whereas PKCα acts as a competitive inhibitor of this phosphoactivation reaction by displacing AKT1 from PDK1. Overall, the findings provide insights into the binding and regulatory interactions of the three master kinases on their target membrane and suggest that a recently described tumor suppressor activity of PKC isoforms may arise from its ability to downregulate PDK1-AKT1 phosphoactivation in the PIP(3)-PDK1-AKT1-mTOR pathway linked to cell growth and oncogenesis.

Background High‐resistance inspiratory muscle strength training (IMST) is a novel, time‐efficient physical training modality. Methods and Results We performed a double‐blind, randomized, sham‐controlled trial to investigate whether 6 weeks of IMST (30 breaths/day, 6 days/week) improves blood pressure, endothelial function, and arterial stiffness in midlife/older adults (aged 50–79 years) with systolic blood pressure ≥120 mm Hg, while also investigating potential mechanisms and long‐lasting effects. Thirty‐six participants completed high‐resistance IMST (75% maximal inspiratory pressure, n=18) or low‐resistance sham training (15% maximal inspiratory pressure, n=18). IMST was safe, well tolerated, and had excellent adherence (≈95% of training sessions completed). Casual systolic blood pressure decreased from 135±2 mm Hg to 126±3 mm Hg ( P P P =0.03); blood pressure was unaffected by sham training (all P >0.05). Twenty‐four hour systolic blood pressure was lower after IMST versus sham training ( P =0.01). Brachial artery flow‐mediated dilation improved ≈45% with IMST ( P P =0.73). Human umbilical vein endothelial cells cultured with subject serum sampled after versus before IMST exhibited increased NO bioavailability, greater endothelial NO synthase activation, and lower reactive oxygen species bioactivity ( P P =0.05) and altered select circulating metabolites (targeted plasma metabolomics) associated with cardiovascular function. Neither IMST nor sham training influenced arterial stiffness ( P >0.05). Conclusions High‐resistance IMST is a safe, highly adherable lifestyle intervention for improving blood pressure and endothelial function in midlife/older adults with above‐normal initial systolic blood pressure. Registration URL: https://www.clinicaltrials.gov ; Unique identifier: NCT03266510.

Leukocyte migration is controlled by a leading edge chemosensory pathway that generates the regulatory lipid PIP3, a growth signal, thereby driving leading edge expansion up attractant gradients toward sites of infection, inflammation, or tissue damage. PIP3 also serves as an important growth signal in growing cells and oncogenesis. The kinases PDK1, AKT1/PKB and PKCα are key components of a plasma membrane-based PIP3 and Ca2+ signaling circuit that regulates these processes. PDK1 and AKT1 are recruited to the membrane by PIP3, while PKCα is recruited to the membrane by Ca2+. All three of these master kinases phosphoregulate an array of protein targets. For example, PDK1 activates AKT1, PKCα and other AGC kinases by phosphorylation at key sites. PDK1 is known to form PDK1:AKT1 and PDK1:PKCα heterodimers stabilized by a PIF interaction between the PDK1 PIF pocket and the PIF motif of the AGC binding partner. Here we present the first, to our knowledge, single molecule studies of full length PDK1 and AKT1 on target membrane surfaces, as well as their interaction with full length PKCα. The findings show that membrane-bound PDK1:AKT1 and PDK1:PKCα heterodimers form under physiological conditions, and are stabilized by PIF interaction. PKCα exhibits 8-fold higher PDK1 affinity than AKT1, thus PKCα competitively displaces AKT1 from PDK1:AKT1 heterodimers. Ensemble activity measurements under matched conditions reveal that PDK1 activates AKT1 via a cis mechanism by phosphorylating an AKT1 molecule in the same PDK1:AKT1 heterodimer, while PKCα acts as a competitive inhibitor of this phosphoactivation reaction by displacing AKT1 from PDK1. Overall, the findings provide new insights into molecular and regulatory interactions of the three master kinases on their target membrane, and suggest that the recently described tumor suppressor activity of PKC may arise from its ability to downregulate PDK1-AKT1 phosphoactivation in the PIP3-PDK1-AKT1-mTOR pathway linked to cell growth and oncogenesis.STATEMENT OF SIGNIFICANCEThis work investigates three master kinases that play central roles in guiding white blood cell migration to sites of infection, inflammation or tissue damage. More broadly, the same kinases help regulate production of a cell growth signal, and may trigger cancer when dysregulated. Using powerful single molecule methods, the work detects and analyzes the interactions between the three purified kinases on their target membrane surface. The findings reveal functionally important differences between pairwise binding affinities of different binding partners. Additional studies reveal that the highest affinity kinase can disrupt and inhibit the activated complex formed by association of the other two kinases. Such inhibition is proposed to help prevent cancer by limiting growth signal production by the activated complex.

We assessed the efficacy of oral supplementation with the flavanoid apigenin on arterial function during aging and identified critical mechanisms of action. Young (6 mo) and old (27 mo) C57BL/6N mice (model of arterial aging) consumed drinking water containing vehicle (0.2% carboxymethylcellulose; 10 young and 7 old) or apigenin (0.5 mg/mL in vehicle; 10 young and 9 old) for 6 wk. In vehicle-treated animals, isolated carotid artery endothelium-dependent dilation (EDD), bioassay of endothelial function, was impaired in old versus young (70% ± 9% vs. 92% ± 1%, P < 0.0001) due to reduced nitric oxide (NO) bioavailability. Old mice had greater arterial reactive oxygen species (ROS) production and oxidative stress (higher nitrotyrosine) associated with greater nicotinamide adenine dinucleotide phosphate oxidase (oxidant enzyme) and lower superoxide dismutase 1 and 2 (antioxidant enzymes); ex vivo administration of Tempol (antioxidant) restored EDD to young levels, indicating ROS-mediated suppression of EDD. Old animals also had greater aortic stiffness as indicated by higher aortic pulse wave velocity (PWV, 434 ± 9 vs. 346 ± 5 cm/s, P < 0.0001) due to greater intrinsic aortic wall stiffness associated with lower elastin levels and higher collagen, advanced glycation end products (AGEs), and proinflammatory cytokine abundance. In old mice, apigenin restored EDD (96% ± 2%) by increasing NO bioavailability, normalized arterial ROS, oxidative stress, and antioxidant expression, and abolished ROS inhibition of EDD. Moreover, apigenin prevented foam cell formation in vitro (initiating step in atherosclerosis) and mitigated age-associated aortic stiffening (PWV 373 ± 5 cm/s) by normalizing aortic intrinsic wall stiffness, collagen, elastin, AGEs, and inflammation. Thus, apigenin is a promising therapeutic for arterial aging. NEW & NOTEWORTHY Our study provides novel evidence that oral apigenin supplementation can reverse two clinically important indicators of arterial dysfunction with age, namely, vascular endothelial dysfunction and large elastic artery stiffening, and prevents foam cell formation in an established cell culture model of early atherosclerosis. Importantly, our results provide extensive insight into the biological mechanisms of apigenin action, including increased nitric oxide bioavailability, normalization of age-related increases in arterial ROS production and oxidative stress, reversal of age-associated aortic intrinsic mechanical wall stiffening and adverse remodeling of the extracellular matrix, and suppression of vascular inflammation. Given that apigenin is commercially available as a dietary supplement in humans, these preclinical findings provide the experimental basis for future translational studies assessing the potential of apigenin to treat arterial dysfunction and reduce cardiovascular disease risk with aging.

Chronic calorie restriction (CR) improves cardiovascular function and several other physiological markers of healthspan. However, CR is impractical in non-obese older humans due to potential loss of lean mass and bone density, poor adherence, and risk of malnutrition. Time-restricted feeding (TRF), which limits the daily feeding period without requiring a reduction in calorie intake, may be a promising alternative healthspan—extending strategy for midlife and older adults; however, there is limited evidence for its feasibility and efficacy in humans. We conducted a randomized, controlled pilot study to assess the safety, tolerability, and overall feasibility of short-term TRF (eating

Leukocyte migration is controlled by a membrane-based chemosensory pathway on the leading edge pseudopod that guides cell movement up attractant gradients during the innate immune and inflammatory responses. This study employed single cell and population imaging to investigate drug-induced perturbations of leading edge pseudopod morphology in cultured, polarized RAW macrophages. The drugs tested included representative therapeutics (acetylsalicylic acid, diclofenac, ibuprofen, acetaminophen) as well as control drugs (PDGF, Gö6976, wortmannin). Notably, slow addition of any of the four therapeutics to cultured macrophages, mimicking the slowly increasing plasma concentration reported for standard oral dosage in patients, yielded no detectable change in pseudopod morphology. This finding is consistent with the well established clinical safety of these drugs. However, rapid drug addition to cultured macrophages revealed four distinct classes of effects on the leading edge pseudopod: (i) non-perturbing drug exposures yielded no detectable change in pseudopod morphology (acetylsalicylic acid, diclofenac); (ii) adaptive exposures yielded temporary collapse of the extended pseudopod and its signature PI(3,4,5)P3 lipid signal followed by slow recovery of extended pseudopod morphology (ibuprofen, acetaminophen); (iii) disruptive exposures yielded long-term pseudopod collapse (Gö6976, wortmannin); and (iv) activating exposures yielded pseudopod expansion (PDGF). The novel observation of adaptive exposures leads us to hypothesize that rapid addition of an adaptive drug overwhelms an intrinsic or extrinsic adaptation system yielding temporary collapse followed by adaptive recovery, while slow addition enables gradual adaptation to counteract the drug perturbation in real time. Overall, the results illustrate an approach that may help identify therapeutic drugs that temporarily inhibit the leading edge pseudopod during extreme inflammation events, and toxic drugs that yield long term inhibition of the pseudopod with negative consequences for innate immunity. Future studies are needed to elucidate the mechanisms of drug-induced pseudopod collapse, as well as the mechanisms of adaptation and recovery following some inhibitory drug exposures.

Cellular pathways controlling chemotaxis, growth, survival, and oncogenesis are activated by receptor tyrosine kinases and small G-proteins of the Ras superfamily that stimulate specific isoforms of phosphatidylinositol-3-kinase (PI3K). These PI3K lipid kinases phosphorylate the constitutive lipid phosphatidylinositol-4,5-bisphosphate (PIP2) to produce the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3). Progress has been made in understanding direct, moderate PI3K activation by receptors. In contrast, the mechanism by which receptors and Ras synergistically activate PI3K to much higher levels remains unclear, and two competing models have been proposed: membrane recruitment versus activation of the membrane-bound enzyme. To resolve this central mechanistic question, this study employs single-molecule imaging to investigate PI3K activation in a six-component pathway reconstituted on a supported lipid bilayer. The findings reveal that simultaneous activation by a receptor activation loop (from platelet-derived growth factor receptor, a receptor tyrosine kinase) and H-Ras generates strong, synergistic activation of PI3Kα, yielding a large increase in net kinase activity via the membrane recruitment mechanism. Synergy requires receptor phospho-Tyr and two anionic lipids (phosphatidylserine and PIP2) to make PI3Kα competent for bilayer docking, as well as for subsequent binding and phosphorylation of substrate PIP2 to generate product PIP3. Synergy also requires recruitment to membrane-bound H-Ras, which greatly speeds the formation of a stable, membrane-bound PI3Kα complex, modestly slows its off rate, and dramatically increases its equilibrium surface density. Surprisingly, H-Ras binding significantly inhibits the specific kinase activity of the membrane-bound PI3Kα molecule, but this minor enzyme inhibition is overwhelmed by the marked enhancement of membrane recruitment. The findings have direct impacts for the fields of chemotaxis, innate immunity, inflammation, carcinogenesis, and drug design.

Aging is associated with vascular endothelial dysfunction, reduced exercise tolerance, and impaired whole‐body glucose metabolism. Interleukin‐37 (IL‐37), an anti‐inflammatory cytokine of the interleukin‐1 family, exerts salutary physiological effects in young mice independent of its inflammation‐suppressing properties. Here, we assess the efficacy of IL‐37 treatment for improving physiological function in older age. Old mice (26–28 months) received daily intraperitoneal injections of recombinant human IL‐37 (recIL‐37; 1 µg/200 ml PBS) or vehicle (200 ml PBS) for 10–14 days. Vascular endothelial function (ex vivo carotid artery dilation to increasing doses of acetylcholine, ACh) was enhanced in recIL‐37 vs. vehicle‐treated mice via increased nitric oxide (NO) bioavailability (all p, Physiological function declines with aging, increasing risk of chronic diseases and disability. We show for the first time that treatment with a novel anti‐inflammatory compound interleukin‐37 (IL‐37) improves multiple physiological functions in old mice, and identify potential roles of reduced superoxide production, improved oxidative metabolism and circulating factors in mediating improvements. Our findings support IL‐37 therapy as a novel strategy for improving diverse physiological functions in old age.

In chemotaxing ameboid cells, a complex leading-edge signaling circuit forms on the cytoplasmic leaflet of the plasma membrane and directs both actin and membrane remodeling to propel the leading edge up an attractant gradient. This leading-edge circuit includes a putative amplification module in which Ca2+-protein kinase C (Ca2+-PKC) is hypothesized to phosphorylate myristoylated alanine-rich C kinase substrate (MARCKS) and release phosphatidylinositol-4,5-bisphosphate (PIP2), thereby stimulating production of the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3) by the lipid kinase phosphoinositide-3-kinase (PI3K). We investigated this hypothesized Ca2+-PKC-MARCKS-PIP2-PI3K-PIP3 amplification module and tested its key predictions using single-molecule fluorescence to measure the surface densities and activities of its protein components. Our findings demonstrate that together Ca2+-PKC and the PIP2-binding peptide of MARCKS modulate the level of free PIP2, which serves as both a docking target and substrate lipid for PI3K. In the off state of the amplification module, the MARCKS peptide sequesters PIP2 and thereby inhibits PI3K binding to the membrane. In the on state, Ca2+-PKC phosphorylation of the MARCKS peptide reverses the PIP2 sequestration, thereby releasing multiple PIP2 molecules that recruit multiple active PI3K molecules to the membrane surface. These findings 1) show that the Ca2+-PKC-MARCKS-PIP2-PI3K-PIP3 system functions as an activation module in vitro, 2) reveal the molecular mechanism of activation, 3) are consistent with available in vivo data, and 4) yield additional predictions that are testable in live cells. More broadly, the Ca2+-PKC-stimulated release of free PIP2 may well regulate the membrane association of other PIP2-binding proteins, and the findings illustrate the power of single-molecule analysis to elucidate key dynamic and mechanistic features of multiprotein signaling pathways on membrane surfaces.

Cellular pathways controlling chemotaxis, growth, survival, and oncogenesis are activated by receptor tyrosine kinases and small G-proteins of the Ras superfamily that stimulate specific isoforms of phosphatidylinositol-3-kinase (PI3K). These PI3K lipid kinases phosphorylate the constitutive lipid phosphatidylinositol-4,5-bisphosphate (PIP2) to produce the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3). Progress has been made in understanding direct, moderate PI3K activation by receptors. In contrast, the mechanism by which receptors and Ras synergistically activate PI3K to much higher levels remains unclear, and two competing models have been proposed: membrane recruitment versus activation of the membrane-bound enzyme. To resolve this central mechanistic question, this study employs single-molecule imaging to investigate PI3K activation in a six-component pathway reconstituted on a supported lipid bilayer. The findings reveal that simultaneous activation by a receptor activation loop (from platelet-derived growth factor receptor, a receptor tyrosine kinase) and H-Ras generates strong, synergistic activation of PI3Kα, yielding a large increase in net kinase activity via the membrane recruitment mechanism. Synergy requires receptor phospho-Tyr and two anionic lipids (phosphatidylserine and PIP2) to make PI3Kα competent for bilayer docking, as well as for subsequent binding and phosphorylation of substrate PIP2 to generate product PIP3. Synergy also requires recruitment to membrane-bound H-Ras, which greatly speeds the formation of a stable, membrane-bound PI3Kα complex, modestly slows its off rate, and dramatically increases its equilibrium surface density. Surprisingly, H-Ras binding significantly inhibits the specific kinase activity of the membrane-bound PI3Kα molecule, but this minor enzyme inhibition is overwhelmed by the marked enhancement of membrane recruitment. The findings have direct impacts for the fields of chemotaxis, innate immunity, inflammation, carcinogenesis, and drug design.

Protein kinase C-α (PKCα) has been studied widely as a paradigm for conventional PKCs, with two C1 domains (C1A and C1B) being important for the regulation and function of the kinase. However, it is challenging to explore these domains in membrane-bound environments with either simulations or experiments alone. In this work, we have combined modeling, simulations, and experiments to understand the molecular basis of the PKCα C1A and C1B domain interactions with membranes. Our atomistic simulations of the PKCα C1 domains reveal the dynamic interactions of the proteins with anionic lipids, as well as the conserved hydrogen bonds and the distinct nonpolar contacts formed with lipid activators. Corroborating evidence is obtained from additional simulations and experiments in terms of lipid binding and protein diffusion. Overall, our study, for the first time, explains with atomistic detail how the PKCα C1A and C1B domains interact differently with various lipids. On the molecular level, the information provided by our study helps to shed light on PKCα regulation and activation mechanism. The combined computational/experimental approach demonstrated in this work is anticipated to enable further studies to explore the roles of C1 domains in many signaling proteins and to better understand their molecular mechanisms in normal cellular function and disease development.

Phosphoinositide-dependent kinase-1 (PDK1) is an essential master kinase recruited to the plasma membrane by the binding of its C-terminal PH domain to the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3). Membrane binding leads to PDK1 phospho-activation, but despite the central role of PDK1 in signaling and cancer biology, this activation mechanism remains poorly understood. PDK1 has been shown to exist as a dimer in cells, and one crystal structure of its isolated PH domain exhibits a putative dimer interface. It has been proposed that phosphorylation of PH domain residue T513 (or the phospho-mimetic T513E mutation) may regulate a novel PH domain dimer-monomer equilibrium, thereby converting an inactive PDK1 dimer to an active monomer. However, the oligomeric states of the PH domain on the membrane have not yet been determined, nor whether a negative charge at position 513 is sufficient to regulate its oligomeric state. This study investigates the binding of purified wild-type (WT) and T513E PDK1 PH domains to lipid bilayers containing the PIP3 target lipid, using both single-molecule and ensemble measurements. Single-molecule analysis of the brightness of the fluorescent PH domain shows that the PIP3-bound WT PH domain on membranes is predominantly dimeric while the PIP3-bound T513E PH domain is monomeric, demonstrating that negative charge at the T513 position is sufficient to dissociate the PH domain dimer and is thus likely to play a central role in PDK1 monomerization and activation. Single-molecule analysis of two-dimensional (2D) diffusion of PH domain-PIP3 complexes reveals that the dimeric WT PH domain diffuses at the same rate as a single lipid molecule, indicating that only one of its two PIP3 binding sites is occupied and there is little penetration of the protein into the bilayer as observed for other PH domains. The 2D diffusion of T513E PH domain is slower, suggesting the negative charge disrupts local structure in a way that allows deeper insertion of the protein into the viscous bilayer, thereby increasing the diffusional friction. Ensemble measurements of PH domain affinity for PIP3 on plasma membrane-like bilayers reveal that the dimeric WT PH domain possesses a one order of magnitude higher target membrane affinity than the previously characterized monomeric PH domains, consistent with a dimerization-triggered, allosterically enhanced affinity for one PIP3 molecule (a much larger affinity enhancement would be expected for dimerization-triggered binding to two PIP3 molecules). The monomeric T513E PDK1 PH domain, like other monomeric PH domains, exhibits a PIP3 affinity and bound state lifetime that are each 1 order of magnitude lower than those of the dimeric WT PH domain, which is predicted to facilitate release of activated, monomeric PDK1 to the cytoplasm. Overall, the study yields the first molecular picture of PH domain regulation via electrostatic control of dimer-monomer conversion.

Peripheral membrane proteins bound to lipids on bilayer surfaces play central roles in a wide array of cellular processes, including many signaling pathways. These proteins diffuse in the plane of the bilayer and often undergo complex reactions involving the binding of regulatory and substrate lipids and proteins they encounter during their 2-D diffusion. Some peripheral proteins, for example pleckstrin homology (PH) domains, dock to the bilayer in a relatively shallow position with little penetration into the bilayer. Other peripheral proteins exhibit more complex bilayer contacts, for example classical protein kinase C isoforms (PKCs) bind as many as six lipids in stepwise fashion, resulting in the penetration of three PKC domains (C1A, C1B, C2) into the bilayer headgroup and hydrocarbon regions. A molecular understanding of the molecular features that control the diffusion speeds of proteins bound to supported bilayers would enable key molecular information to be extracted from experimental diffusion constants, revealing protein-lipid and protein-bilayer interactions difficult to study by other methods. The present study investigates a range of 11 different peripheral protein constructs comprised by 1 to 3 distinct domains (PH, C1A, C1B, C2, anti-lipid antibody). By combining these constructs with various combinations of target lipids, the study measures 2-D diffusion constants on supported bilayers for 17 different protein-lipid complexes. The resulting experimental diffusion constants, together with the known membrane interaction parameters of each complex, are used to analyze the molecular features correlated with diffusional slowing and bilayer friction. The findings show that both 1) individual bound lipids and 2) individual protein domains that penetrate into the hydrocarbon core make additive contributions to the friction against the bilayer, thereby defining the 2-D diffusion constant. An empirical formula is developed that accurately estimates the diffusion constant and bilayer friction of a peripheral protein in terms of its number of bound lipids and its geometry of penetration into the bilayer hydrocarbon core, yielding an excellent global best fit (R2 of 0.97) to the experimental diffusion constants. Finally, the observed additivity of the frictional contributions suggests that further development of current theory describing bilayer dynamics may be needed. The present findings provide constraints that will be useful in such theory development.

Protein complexes assembled on membrane surfaces regulate a wide array of signaling pathways and cell processes. Thus a molecular understanding of the membrane surface diffusion and regulatory events leading to the assembly of active membrane complexes is crucial to signaling biology and medicine. Here we present a novel single molecule diffusion analysis designed to detect complex formation on supported lipid bilayers. The usefulness of the method is illustrated by detection of an engineered, heterodimeric complex in which two membrane-bound pleckstrin homology (PH) domains associate stably, but reversibly, upon Ca2+-triggered binding of calmodulin (CaM) to a target peptide from myosin light chain kinase (MLCKp). Specifically, when a monomeric, fluorescent PH-CaM domain fusion protein diffusing on a supported bilayer binds a dark MLCKp-PH domain fusion protein, the heterodimeric complex is observed to diffuse nearly 2-fold more slowly than the monomer because both of its twin PH domains can simultaneously bind to the viscous bilayer. In a mixed population of monomers and heterodimers, the single molecule diffusion analysis resolves and quantitates the rapidly diffusing monomer and slowly diffusing heterodimer subpopulations. The affinity of the CaM-MLCKp interaction is measured by titrating dark MLCKp-PH construct into the system, while monitoring the changing average diffusion coefficient of the fluorescent PH-CaM population, yielding a saturating binding curve. Strikingly, the apparent affinity of the CaM-MLCKp complex is ∼102-fold greater in the membrane system than in solution, apparently due both to faster complex association and slower complex dissociation on the membrane surface. More broadly, the present findings suggest that single molecule diffusion measurements on supported bilayers will provide an important tool for analyzing the 2D diffusion and assembly reactions governing the formation of diverse membrane-bound complexes, including key complexes from critical signaling pathways. The approach may also prove useful in pharmaceutical screening for compounds that inhibit membrane complex assembly or stability.

The Protein Kinase C family of enzymes is a group of serine/threonine kinases that play central roles in cell-cycle regulation, development and cancer. A key step in the activation of PKC is translocation to membranes and binding of membrane-associated activators including diacylglycerol (DAG). Interaction of novel and conventional isotypes of PKC with DAG and phorbol esters occurs through the two C1 regulatory domains (C1A and C1B), which exhibit distinct ligand binding selectivity that likely controls enzyme activation by different co-activators. PKC has also been implicated in physiological responses to alcohol consumption and it has been proposed that PKCα (Slater et al. J Biol Chem 272(10):6167-6173, 1997; Slater et al. Biochemistry 43(23):7601-7609, 2004), PKCε (Das et al. Biochem J 421(3):405-413, 2009) and PKCδ (Das et al. J Biol Chem 279(36):37964-37972, 2004; Das et al. Protein Sci 15(9):2107-2119, 2006) contain specific alcohol-binding sites in their C1 domains. We are interested in understanding how ethanol affects signal transduction processes through its affects on the structure and function of the C1 domains of PKC. Here we present the (1)H, (15)N and (13)C NMR chemical shift assignments for the Rattus norvegicus PKCδ C1A and C1B proteins.

The leukocyte chemosensory pathway detects attractant gradients and directs cell migration to sites of inflammation, infection, tissue damage, and carcinogenesis. Previous studies have revealed that local Ca2+ and PIP3 signals at the leading edge of polarized leukocytes play central roles in positive feedback loop essential to cell polarization and chemotaxis. These prior studies showed that stimulation of the leading edge Ca2+ signal can strongly activate PI3K, thereby triggering a larger PIP3 signal, but did not elucidate the mechanistic link between Ca2+ and PIP3 signaling. A hypothesis explaining this link emerged, postulating that Ca2+-activated PKC displaces the MARCKS protein from plasma membrane PIP2, thereby releasing sequestered PIP2 to serve as the target and substrate lipid of PI3K in PIP3 production. In vitro single molecule studies of the reconstituted pathway on lipid bilayers demonstrated the feasibility of this PKC-MARCKS-PI3K regulatory module linking Ca2+ and PIP3 signals in the reconstituted system. The present study tests the model predictions in live macrophages by quantifying the effects of: (a) two pathway activators—PDGF and ATP that stimulate chemoreceptors and Ca2+ influx, respectively; and (b) three pathway inhibitors—wortmannin, EGTA, and Go6976 that inhibit PI3K, Ca2+ influx, and PKC, respectively; on (c) four leading edge activity sensors—AKT-PH-mRFP, CKAR, MARCKSp-mRFP, and leading edge area that report on PIP3 density, PKC activity, MARCKS membrane binding, and leading edge expansion/contraction, respectively. The results provide additional evidence that PKC and PI3K are both essential elements of the leading edge positive feedback loop, and strongly support the existence of a PKC-MARCKS-PI3K regulatory module linking the leading edge Ca2+ and PIP3 signals. As predicted, activators stimulate leading edge PKC activity, displacement of MARCKS from the leading edge membrane and increased leading edge PIP3 levels, while inhibitors trigger the opposite effects. Comparison of the findings for the ameboid chemotaxis of leukocytes with recently published findings for the mesenchymal chemotaxis of fibroblasts suggests that some features of the emerging leukocyte leading edge core pathway (PLC-DAG-Ca2+-PKC-MARCKS-PIP2-PI3K-PIP3) may well be shared by all chemotaxing eukaryotic cells, while other elements of the leukocyte pathway may be specialized features of these highly optimized, professional gradient-seeking cells. More broadly, the findings suggest a molecular mechanism for the strong links between phospho-MARCKS and many human cancers.

The lipid-anchored GTPase Ras is well known for its role in oncogenesis – about one quarter of all human tumors show mutations in a Ras family member. One of the best characterized Ras targets is the lipid kinase Phosphatidylinositol 3-Kinase (PI3K) which, like Ras, is highly oncogenic. PI3K phosphorylates the constitutive plasma membrane lipid phosphatidyl-inositol-(4,5)-bisphosphate (PIP2) yielding the essential signaling lipid phosphatidylinositol-(3,4,5)-bisphosphate (PIP3). The resulting PIP3 lipid signal regulates an array of cell processes including migration and growth.The underlying mechanism by which Ras activates PI3K is unclear - this project aims to better elucidate the activation mechanism using single molecule TIRF microscopy to probe the interactions and activities of single proteins on a target membrane surface. One hypothesis is that the lipid-anchored, membrane-bound Ras protein increases the surface density of PI3K on the membrane by increasing kon and/or decreasing koff. An alternative hypothesis proposes that Ras binding increases the PI3K kcat and/or affinity for substrate lipid PI(4,5)P2 (PIP2) and/or ATP. Single-molecule fluorescence methods will be used to distinguish between these possibilities by observing the number of single particle diffusion tracks, track length, diffusion speed, specific activity, kcat, and Km of PI3K in the presence and absence of Ras. Our poster will present the latest findings of these studies.

Protein kinase C-α (PKCα) is a member of the conventional family of protein kinase C isoforms (cPKCs) that regulate diverse cellular signaling pathways, share a common activation mechanism, and are linked to multiple pathologies. The cPKC domain structure is modular, consisting of an N-terminal pseudosubstrate peptide, two inhibitory domains (C1A and C1B), a targeting domain (C2), and a kinase domain. Mature, cytoplasmic cPKCs are inactive until they are switched on by a multistep activation reaction that occurs largely on the plasma membrane surface. Often, this activation begins with a cytoplasmic Ca(2+) signal that triggers C2 domain targeting to the plasma membrane where it binds phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP2). Subsequently, the appearance of the signaling lipid diacylglycerol (DAG) activates the membrane-bound enzyme by recruiting the inhibitory pseudosubstrate and one or both C1 domains away from the kinase domain. To further investigate this mechanism, this study has utilized single-molecule total internal reflection fluorescence microscopy (TIRFM) to quantitate the binding and lateral diffusion of full-length PKCα and fragments missing specific domain(s) on supported lipid bilayers. Lipid binding events, and events during which additional protein is inserted into the bilayer, were detected by their effects on the equilibrium bound particle density and the two-dimensional diffusion rate. In addition to the previously proposed activation steps, the findings reveal a major, undescribed, kinase-inactive intermediate. On bilayers containing PS or PS and PIP2, full-length PKCα first docks to the membrane via its C2 domain, and then its C1A domain embeds itself in the bilayer even before DAG appears. The resulting pre-DAG intermediate with membrane-bound C1A and C2 domains is the predominant state of PKCα while it awaits the DAG signal. The newly detected, membrane-embedded C1A domain of this pre-DAG intermediate confers multiple useful features, including enhanced membrane affinity and longer bound state lifetime. The findings also identify the key molecular step in kinase activation: because C1A is already membrane-embedded in the kinase off state, recruitment of C1B to the bilayer by DAG or phorbol ester is the key regulatory event that stabilizes the kinase on state. More broadly, this study illustrates the power of single-molecule methods in elucidating the activation mechanisms and hidden regulatory states of membrane-bound signaling proteins.

The chemotactic migration of eukaryotic ameboid cells up concentration gradients is among the most advanced forms of cellular behavior. Chemotaxis is controlled by a complex network of signaling proteins bound to specific lipids on the cytoplasmic surface of the plasma membrane at the front of the cell, or the leading edge. The central lipid players in this leading edge signaling pathway include the phosphoinositides PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3), both of which play multiple roles. The products of PI(4,5)P2 hydrolysis, diacylglycerol (DAG) and Ins(1,4,5)P3 (IP3), are also implicated as important players. Together, these leading edge phosphoinositides and their degradation products, in concert with a local Ca(2+) signal, control the recruitment and activities of many peripheral membrane proteins that are crucial to the leading edge signaling network. The present critical review summarizes the current molecular understanding of chemotactic signaling at the leading edge, including newly discovered roles of phosphoinositide lipids and Ca(2+), while highlighting key questions for future research.

Anopheles gambiae mosquitoes that transmit malaria are attracted to humans by the odor molecules that emanate from skin and sweat. Odorant binding proteins (OBPs) are the first component of the olfactory apparatus to interact with odorant molecules, and so present potential targets for preventing transmission of malaria by disrupting the normal olfactory responses of the insect. AgamOBP20 is one of a limited subset of OBPs that it is preferentially expressed in female mosquitoes and its expression is regulated by blood feeding and by the day/night light cycles that correlate with blood-feeding behavior. Analysis of AgamOBP20 in solution reveals that the apo-protein exhibits significant conformational heterogeneity but the binding of odorant molecules results in a significant conformational change, which is accompanied by a reduction in the conformational flexibility present in the protein. Crystal structures of the free and bound states reveal a novel pathway for entrance and exit of odorant molecules into the central-binding pocket, and that the conformational changes associated with ligand binding are a result of rigid body domain motions in α-helices 1, 4, and 5, which act as lids to the binding pocket. These structures provide new insights into the specific residues involved in the conformational adaptation to different odorants and have important implications in the selection and development of reagents targeted at disrupting normal OBP function.

The second messenger lipid PIP(3) (phosphatidylinositol-3,4,5-trisphosphate) is generated by the lipid kinase PI3K (phosphoinositide-3-kinase) in the inner leaflet of the plasma membrane, where it regulates a broad array of cell processes by recruiting multiple signaling proteins containing PIP(3)-specific pleckstrin homology (PH) domains to the membrane surface. Despite the broad importance of PIP(3)-specific PH domains, the membrane docking geometry of a PH domain bound to its target PIP(3) lipid on a bilayer surface has not yet been experimentally determined. The present study employs EPR site-directed spin labeling and relaxation methods to elucidate the membrane docking geometry of GRP1 PH domain bound to bilayer-embedded PIP(3). The model target bilayer contains the neutral background lipid PC and both essential targeting lipids: (i) PIP(3) target lipid that provides specificity and affinity, and (ii) PS facilitator lipid that enhances the PIP(3) on-rate via an electrostatic search mechanism. The EPR approach measures membrane depth parameters for 18 function-retaining spin labels coupled to the PH domain, and for calibration spin labels coupled to phospholipids. The resulting depth parameters, together with the known high resolution structure of the co-complex between GRP1 PH domain and the PIP(3) headgroup, provide sufficient constraints to define an optimized, self-consistent membrane docking geometry. In this optimized geometry the PH domain engulfs the PIP(3) headgroup with minimal bilayer penetration, yielding the shallowest membrane position yet described for a lipid binding domain. This binding interaction displaces the PIP(3) headgroup from its lowest energy position and orientation in the bilayer, but the headgroup remains within its energetically accessible depth and angular ranges. Finally, the optimized docking geometry explains previous biophysical findings including mutations observed to disrupt membrane binding, and the rapid lateral diffusion observed for PIP(3)-bound GRP1 PH domain on supported lipid bilayers.

Many signaling pathways are controlled by protein complexes assembled on membrane surfaces via collisions between diffusing membrane components. Subsequently, the active, membrane-bound complex often collides with other effector proteins or lipids on the membrane surface to transmit its essential downstream signal. Thus, 2D-diffusion of membrane-bound proteins, complexes and effectors plays a central role in signaling biology. We have proposed that single molecule analysis of membrane protein diffusion on supported lipid bilayers could provide a powerful approach for studies of complex formation between membrane-associated proteins (Knight, Lerner, Marcano-Velazquez, Pastor & Falke (2010) Biophys J 99:2879-87). In principle the approach could yield information about complex stoichiometry, stability, and kinetics, and about specific lipid requirements for complex formation. Here we test these ideas using a simple model system in which membrane-bound pleckstrin homology (PH) domains are forced to dimerize by the calcium-triggered association of calmodulin (CaM) and its target peptide from skeletal muscle light chain kinase (MLCKp). Two fusion protein constructs (CaM-PH) and (MLCKp-PH) have been generated and bind normally to the PH domain target lipid, PIP3. Single molecule TIRF analysis of CaM-PH diffusion reveals that in the presence of calcium and MLCKp-PH a stable CaM-PH/MLCKp-PH heterodimer is formed on the membrane surface that diffuses approximately 2-fold more slowly than the single CaM-PH molecule. Addition of excess EDTA to chelate calcium reverses the dimer back to monomers. Substoichiometric levels of MLCKp-PH yield a mixed population of monomers and dimers, and the two populations can be resolved by single molecule analysis. Overall, the findings indicate that single molecule diffusion provides a useful new window into many critical, molecular features of protein complex formation on membrane surfaces. [Supported by NIH grant GM063235.]

The master kinase PKCα is a central player in the normal function of many signaling pathways, and also plays a role in multiple pathologies. The conventional model for activation of classical PKCs, including PKCα, proposes two major steps during conversion of inactive, cytoplasmic enzyme to active, membrane-bound enzyme. First, a cytoplasmic Ca2+ signal activates C2 domain that serves to recruit the inactive enzyme from cytoplasm to the PS-rich inner leaflet of plasma membrane. Second, inactive enzyme, bound to plasma membrane via its C2 domain, is activated by appearance of the second messenger lipid diacylglycerol (DAG) that recruits the inhibitory C1A and C1B domains from the kinase domain to the membrane, thus activating the kinase. To test the predictions of this model, we have employed single molecule methods to analyze membrane binding and surface diffusion of full length PKCα, and also simpler constructs containing a subset of its domains. TIRF microscopy was used to visualize these proteins on supported lipid bilayers containing different combinations of target lipids in order to investigate the contributions of individual domains to membrane binding and lipid-induced kinase activation. Previous studies have shown that single molecule TIRF analysis of peripheral proteins on supported lipid bilayers provides extensive information on protein-lipid interactions difficult to obtain by other methods (Ziemba, Knight & Falke (2012) Biochemistry 51(8):1638-1647. Knight, Lerner, Marcano-Velazquez, Pastor & Falke (2010) Biophys J 99:2879-87). The present single molecule analysis reveals a previously unknown intermediate in the PKCα activation reaction, and indicates this is the major intermediate while the enzyme awaits the appearance of activating diacylglycerol or phorbol ester. The findings yield new insights into the PKCα activation mechanism, and show that the single molecule approach provides a new window into the activation mechanism of membrane-bound signaling enzymes.