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Amine-reactive esters of aromatic fluorescent dyes are emerging as imaging probes for nondescript staining of cellular and tissue architectures. We characterised the differential staining patterns of 14 fluorescent dye ester species with varying physical and spectral properties in the broadly studied human cell line – HeLa. When combined with expansion microscopy (ExM), these stains reveal nanoscale features such as the nuclear proteome, membrane-bound compartments and vesicles. Among N-Hydroxysuccinimide (NHS) esters, we observe differential compartment specificity and weighting of labelling density which correlates with the hydrophobicity of the dye ester. We also observe changes in both staining density and compartment specificity for a given dye ester depending on the presence of a second dye ester species and on the timepoint of application in the ExM protocol. Our findings confirm these dye esters as a useful addition to the repertoire of biomedical stains of the cellular proteome, either on their own, or as counterstains to immunofluorescence.

Detroit is a dynamic city with a dynamic history, yet it has come to symbolize both White flight (beginning in the 1940s and accelerating in the late 1960s) and Black flight (beginning in the 1990s and reaching its apex in 2000). While Detroit’s Black population continues to decline, its White population increased by 22% between 2010 and 2015. Along with these shifting demographic trends comes shifting residential and educational landscapes that amplify the racial, economic, and spatial inequalities marking present-day Detroit. Drawing upon the literature of human geography and sociology of education, and utilizing GIS software, we overlay the mapping of demographic realities with the mapping of human stories. As a case study of how a non-profit, public charter school can be a vehicle for resisting gentrification, this paper examines the role of “place” in one school’s navigation of an increasingly gentrified Detroit and its commitment to primarily serving youth of its neighborhood. Using a multimodal and multiscalar approach, we find evidence of endogenous gentrification, intergenerational topophilia, and the school enacting resistance within a dialectic of its market-driven charter school status.

Many membraneless organelles (MLOs) have been shown to form via liquid-liquid phase separation (LLPS). Light and electron microscopy techniques have been indispensable in the identification and characterization of LLPS MLOs. However, for complex MLOs such as the perinuclear germ granule in C. elegans, our understanding of how the organelle as a whole is regulated is hampered by 1) the technical limitations in confocal fluorescence imaging in which only a few granule protein markers can be examined at a time and 2) the inaccessibility of electron microscopy. In this study, we take advantage of the newly developed super-resolution method of expansion microscopy (ExM) and in-situ staining of the whole proteome to examine the C. elegans germ granule, the P granule. We show that in small RNA pathway mutants, the P granule is smaller compared with wild type animals. Furthermore, we investigate the relationship between the P granule and two other germ granules, mutator foci and Z granule, and show that they are located within the same protein-dense regions while occupying distinct subdomains within this ultrastructure. The experimental workflow developed here will serve as an important tool in our understanding of germ granule biology as well as the biological role of LLPS.

Light and electron microscopy techniques have been indispensable in the identification and characterization of liquid–liquid phase separation membraneless organelles. However, for complex membraneless organelles such as the perinuclear germ granule inC. elegans, our understanding of how the intact organelle is regulated is hampered by (1) technical limitations in confocal fluorescence imaging for the simultaneous examination of multiple granule protein markers and (2) inaccessibility of electron microscopy. We take advantage of the newly developed super resolution method of expansion microscopy (ExM) and in situ staining of the whole proteome to examine theC. elegansgerm granule, the P granule. We show that in small RNA pathway mutants, the P granule is smaller compared with WT animals. Furthermore, we investigate the relationship between the P granule and two other germ granules, Mutator foci and Z granule, and show that they are located within the same protein-dense regions while occupying distinct subdomains within this ultrastructure. This study will serve as an important tool in our understanding of germ granule biology and the biological role of liquid–liquid phase separation.

Cancer treatment has been considerably advanced by the relatively recent development of small molecules capable of inhibiting oncogenic kinase signaling, but exogenous enhancement of tumor suppressive signaling remains elusive. Here, we sought to design small molecules capable of activating the tumor suppressor kinase LKB1. Designing compounds capable of increasing kinase activity has been more structurally challenging than those aimed at inhibiting them: apart from the ATP-binding pocket, there is rarely a site that would be conducive to binding a small molecule. Unlike most protein kinases, LKB1 signals as part of an obligate trimer consisting of itself, a scaffolding protein (MO25), and a pseudokinase (STRAD). As part of its mechanism of activation, LKB1 must bind to an ATP-bound STRAD to take on its active kinase conformation. Targeting STRAD provides a unique opportunity to allosterically stabilize and enhance LKB1 kinase activity. Using a structure-based drug design, we developed compounds that are able to selectively bind STRAD's ATP-binding pocket. We confirmed that our STRAD targeting small molecules are able to enhance the activity of recombinant LKB1 in a kinase assay. The observed increase in LKB1's kinase activity corresponds to increased association of complex components after drug treatment, as suggested by immunoprecipitation of the trimer. To understand the clinical contexts in which our compounds might be most effective, we performed a viability screen across multiple histologies to determine which cancer cells could be sensitive to LKB1 activation. Sensitive cells were used to investigate the mechanism of action of exogenous LKB1 stimulation. LKB1 signals through members of the AMPK-related kinase family to carry out its tumor suppressive functions in the cell. Thus, we used western blot analysis of LKB1 proximal and distal mediators to assess changes in downstream signaling. We found that activation of multiple LKB1 effectors was dose dependent and occurred rapidly, with signal enhancement seen in more tumor-relevant low adherence cell culture conditions. Real-time microscopy confirmed that our compounds slowed cell proliferation in a dose-dependent manner. This work demonstrates that targeting a pseudokinase with a small molecule to allosterically activate of a tumor suppressor kinase is possible, therapeutically effective in vitro, and triggers multiple downstream signaling pathways to decrease cancer cell proliferation. Citation Format: Dominique C. Mitchell, Jin Liu, Zheng Wang, Changliang He, Luo Ding, Marc Adler, LeeAnn L. Wang, Aidan Keith, Y. Christina Hwang, Tsz Kin M. Tsui, Roopa Ramamoorthi, Sarah Lively, Robert A. Drakas, Vijay Ramani, Kliment A. Verba, Tingting Qing, Richard T. Beresis, John D. Gordan. Small molecule activation of the LKB1 tumor suppressor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5228.

In this paper, we present a system identification (SI) procedure that enables building linear time-dependent fractional-order differential equation (FDE) models able to accurately describe time-dependent behavior of complex systems. The parameters in the models are the order of the equation, the coefficients in it, and, when necessary, the initial conditions. The Caputo definition of the fractional derivative, and the Mittag-Leffler function, is used to obtain the corresponding solutions. Since the set of parameters for the model and its initial conditions are nonunique, and there are small but significant differences in the predictions from the possible models thus obtained, the SI operation is carried out via global regression of an error-cost function by a simulated annealing optimization algorithm. The SI approach is assessed by considering previously published experimental data from a shell-and-tube heat exchanger and a recently constructed multiroom building test bed. The results show that the proposed model is reliable within the interpolation domain but cannot be used with confidence for predictions outside this region. However, the proposed system identification methodology is robust and can be used to derive accurate and compact models from experimental data. In addition, given a functional form of a fractional-order differential equation model, as new data become available, the SI technique can be used to expand the region of reliability of the resulting model.

The Lim1 gene has essential functions during several stages of kidney development. In particular, a tissue-specific knockout in the early metanephric mesenchyme results in the formation of the earliest nephron precursor, the renal vesicle, but failure of this structure to progress to the next stage, the comma-shaped body. To better understand the molecular nature of this developmental arrest, we used a laser capture microdissection-microarray strategy to examine the perturbed gene expression pattern of the mutant renal vesicles. Among the genes found differently expressed were Chrdl2, an inhibitor of BMP signaling, the proapoptotic factor Bmf, as well as myob5, an atypical myosin that modulates chemokine signaling, and pdgfrl, which is important in epithelial folding. Of particular interest, the microarray data indicated that the Dkk1 gene, which encodes an inhibitor of Wnt signaling, was downregulated ninefold in mutants. This was confirmed by in situ hybridizations. It is interesting to note that Lim1 and Dkk1 mutant mice have striking similarities in phenoytpe. These results suggest that the Dkk1 gene might be a key downstream effector of Lim1 function.

The adaptor protein growth factor receptor-bound protein 2 (Grb2) is ubiquitously expressed in eukaryotic cells and involved in a multitude of intracellular protein interactions. Grb2 plays a pivotal role in tyrosine kinase-mediated signal transduction including linking receptor tyrosine kinases to the Ras/mitogen-activated protein (MAP) kinase pathway, which is implicated in oncogenic outcome. Grb2 exists in a constitutive equilibrium between monomeric and dimeric states. Here we show that only monomeric Grb2 is capable of binding to SOS and upregulating MAP kinase signalling and that the dimeric state is inhibitory to this process. Phosphorylation of tyrosine 160 (Y160) on Grb2, or binding of a tyrosylphosphate-containing ligand to the SH2 domain of Grb2, results in dimer dissociation. Phosphorylation of Y160 on Grb2 is readily detectable in the malignant forms of human prostate, colon and breast cancers. The self-association/dissociation of Grb2 represents a switch that regulates MAP kinase activity and hence controls cancer progression.

The goal of the study was to determine whether defects in intracellular Ca2+ signaling contribute to cardiomyopathy in streptozotocin (STZ)-induced diabetic rats. Depression in cardiac systolic and diastolic function was traced from live diabetic rats to isolated individual myocytes. The depression in contraction and relaxation in myocytes was found in parallel with depression in the rise and decline of intracellular free Ca2+ concentration ([Ca2+]i). The sarcoplasmic reticulum (SR) Ca2+ store and rates of Ca2+ release and resequestration into SR were depressed in diabetic rat myocytes. The rate of Ca2+ efflux via sarcolemmal Na+/Ca2+ exchanger was also depressed. However, there was no change in the voltage-dependent L-type Ca2+ channel current that triggers Ca2+ release from the SR. The depression in SR function was associated with decreased SR Ca2+-ATPase and ryanodine receptor proteins and increased total and nonphosphorylated phospholamban proteins. The depression of Na+/Ca2+ exchanger activity was associated with a decrease in its protein level. Thus it is concluded that defects in intracellular Ca2+ signaling caused by alteration of expression and function of the proteins that regulate [Ca2+]i contribute to cardiomyopathy in STZ-induced diabetic rats. The increase in phospholamban, decrease in Na+/Ca2+ exchanger, and unchanged L-type Ca2+ channel activity in this model of diabetic cardiomyopathy are distinct from other types of cardiomyopathy.

Rictor is an essential component of mTOR (mammalian target of rapamycin) complex 2 (mTORC2), a kinase complex that phosphorylates Akt at Ser473 upon activation of phosphatidylinositol 3-kinase (PI-3 kinase). Since little is known about the role of either rictor or mTORC2 in PI-3 kinase-mediated physiological processes in adult animals, we generated muscle-specific rictor knockout mice. Muscle from male rictor knockout mice exhibited decreased insulin-stimulated glucose uptake, and the mice showed glucose intolerance. In muscle lacking rictor, the phosphorylation of Akt at Ser473 was reduced dramatically in response to insulin. Furthermore, insulin-stimulated phosphorylation of the Akt substrate AS160 at Thr642 was reduced in rictor knockout muscle, indicating a defect in insulin signaling to stimulate glucose transport. However, the phosphorylation of Akt at Thr308 was normal and sufficient to mediate the phosphorylation of glycogen synthase kinase 3 (GSK-3). Basal glycogen synthase activity in muscle lacking rictor was increased to that of insulin-stimulated controls. Consistent with this, we observed a decrease in basal levels of phosphorylated glycogen synthase at a GSK-3/protein phosphatase 1 (PP1)-regulated site in rictor knockout muscle. This change in glycogen synthase phosphorylation was associated with an increase in the catalytic activity of glycogen-associated PP1 but not increased GSK-3 inactivation. Thus, rictor in muscle tissue contributes to glucose homeostasis by positively regulating insulin-stimulated glucose uptake and negatively regulating basal glycogen synthase activity.

The mammalian target of rapamycin (mTOR) is a Ser/Thr (S/T) protein kinase, which controls mRNA translation initiation by modulating phosphorylation of the translational regulators PHAS-I and p70(S6K). Here we show that in vitro mTOR is able to phosphorylate these two regulators at comparable rates. Both (S/T)P sites, such as Thr36, Thr45, and Thr69 in PHAS-I and the h(S/T)h site (where h is a hydrophobic amino acid) Thr389 in p70(S6K), were phosphorylated. Rapamycin-FKBP12 inhibited mTOR activity. Surprisingly, the extent of inhibition depended on the substrate. Moreover, mutating Ser2035 in the rapamycin-binding domain (FRB) not only decreased rapamycin sensitivity as expected but also dramatically affected the sites phosphorylated by mTOR. The results demonstrate that mutations in Ser2035 are not silent with respect to mTOR activity and implicate the FRB in substrate recognition. The findings also impose new limitations on interpreting results from experiments in which rapamycin and/or rapamycin-resistant forms of mTOR are used to investigate mTOR function in cells.

Ruthenium red is a well known inhibitor of Ca2+ uptake into mitochondria in vitro. However, its utility as an inhibitor of Ca2+ uptake into mitochondria in vivo or in situ in intact cells is limited because of its inhibitory effects on sarcoplasmic reticulum Ca2+ release channel and other cellular processes. We have synthesized a ruthenium derivative and found it to be an oxygen-bridged dinuclear ruthenium amine complex. It has the same chemical structure as Ru360 reported previously (Emerson, J., Clarke, M. J., Ying, W-L., and Sanadi, D. R. (1993) J. Am. Chem. Soc. 115, 11799-11805). Ru360 has been shown to be a potent inhibitor of Ca2+-stimulated respiration of liver mitochondria in vitro. However, the specificity of Ru360 on Ca2+ uptake into mitochondria in vitro or in intact cells has not been determined. The present study reports in detail the potency, the effectiveness, and the mechanism of inhibition of mitochondrial Ca2+ uptake by Ru360 and its specificity in vitro in isolated mitochondria and in situ in isolated cardiac myocytes. Ru360 was more potent (IC50 = 0.184 nM) than ruthenium red (IC50 = 6.85 nM) in inhibiting Ca2+ uptake into mitochondria. 103Ru360 was found to bind to isolated mitochondria with high affinity (Kd = 0.34 nM, Bmax = 80 fmol/mg of mitochondrial protein). The IC50 of 103Ru360 for the inhibition of Ca2+ uptake into mitochondria was also 0.2 nM, indicating that saturation of a specific binding site is responsible for the inhibition of Ca2+ uptake. Ru360, as high as 10 microM, produced no effect on sarcoplasmic reticulum Ca2+ uptake or release, sarcolemmal Na+/Ca2+ exchange, actomyosin ATPase activity, L-type Ca2+ channel current, cytosolic Ca2+ transients, or cell shortening. 103Ru360 was taken up by isolated myocytes in a time-dependent biphasic manner. Ru360 (10 microM) applied outside intact voltage-clamped ventricular myocytes prevented Ca2+ uptake into mitochondria in situ where the cells were progressively loaded with Ca2+ via sarcolemmal Na+/Ca2+ exchange by depolarization to +110 mV. We conclude that Ru360 specifically blocks Ca2+ uptake into mitochondria and can be used in intact cells.

Skeletal muscle is susceptible to oxidative deterioration due to a combination of lipid oxidation catalysts and membrane lipid systems that are high in unsaturated fatty acids. To prevent or delay oxidation reactions, several endogenous antioxidant systems are found in muscle tissue. These include alpha-tocopherol, histidine-containing dipeptides, and antioxidant enzymes such as glutathione peroxidase, superoxide dismutase, and catalase. The contribution of alpha-tocopherol to the oxidative stability of skeletal muscle is largely influenced by diet. Dietary supplementation of tocopherol has been shown to increase muscle alpha-tocopherol concentrations and inhibit both lipid oxidation and color deterioration. Dietary selenium supplementation has also been shown to increase the oxidative stability of muscle presumably by increasing the activity of glutathione peroxidase. The oxidative stability of skeletal muscle is also influenced by the histidine-containing dipeptides, carnosine and anserine. Whereas carnosine and anserine are affected by diet less than alpha-tocopherol and glutathione peroxidase, their concentrations vary widely with species and muscle type. In pigs, beef, and turkey muscle, carnosine concentrations are greater than anserine, while the opposite is true in rabbit, salmon, and chicken muscle. Anserine and carnosine are found in greater concentrations in muscle high in white fibers, with chicken white muscle containing over fivefold more anserine and carnosine than red muscle. Anserine and carnosine are thought to inhibit lipid oxidation by a combination of free radical scavenging and metal chelation.

Benzohydrazides, benzothiohydrazides, and benzamidrazones have been converted to 1,3,4(2H)-oxadiazolenones, 1,3,4(2H)-thiadiazolenenones, and 1,2,4(5H)-triazolenones respectively by reaction with ethyl chloroformate, with ethyl thiolchloroformate, and with phenyl isocyanate. In some reactions, a carbamate or urea has been isolated as intermediate.

The fine-structural features of pinealocytes of the white-footed mouse, Peromyscus leucopus, were examined. A single population of pinealocytes was observed in both superficial and deep components of the gland. Cells in both locations are characterized by the presence of an indented nucleus exhibiting a prominent nucleolus. The usual organelles in the perikaryon are the Golgi complexes, mitochondria, endoplasmic reticulum, polysomes, microtubules and dense-core vesicles. In addition the perikaryonal cytoplasm often contains a single myeloid body. These bodies are usually lenticular in shape, are formed of an array of flattened membranous cisternae, and are not bounded by a limiting membrane. This organelle usually lies in the vicinity of the nucleus and is infrequently associated with lipid bodies. Complex forms were also observed. The cisternae are continuous with elements of the endoplasmic reticulum at points along their expanded rims. The outer surface of the cisternal membrane exhibits a granularity or fuzziness. The cisternae may be fenestrated. Pinealocyte processes with an abundance of clear vesicles, and some dense-core vesicles and vesicle-crowned rodlets are present in the parenchyma.