Summary The nucleolus and other ribonucleoprotein (RNP) bodies are membrane-less organelles that appear to assemble through phase separation of their molecular components. However, many such RNP bodies contain internal sub-compartments, and the mechanism of their formation remains unclear. Here, we combine in vivo and in vitro studies, together with computational modeling, to show that sub-compartments within the nucleolus represent distinct, coexisting liquid phases. Consistent with their in vivo immiscibility, purified nucleolar proteins phase separate into droplets containing distinct non-coalescing phases that are remarkably similar to nucleoli in vivo. This layered droplet organization is caused by differences in the biophysical properties of the phases – particularly droplet surface tension – which arises from sequence-encoded features of their macromolecular components. These results suggest that phase separation can give rise to multilayered liquids that may facilitate sequential RNA processing reactions in a variety of RNP bodies.

Dietary cholesterol consumption and intestinal cholesterol absorption contribute to plasma cholesterol levels, a risk factor for coronary heart disease. The molecular mechanism of sterol uptake from the lumen of the small intestine is poorly defined. We show that Niemann-Pick C1 Like 1(NPC1L1) protein plays a critical role in the absorption of intestinal cholesterol. NPC1L1 expression is enriched in the small intestine and is in the brush border membrane of enterocytes. Although otherwise phenotypically normal, NPC1L1-deficient mice exhibit a substantial reduction in absorbed cholesterol, which is unaffected by dietary supplementation of bile acids. Ezetimibe, a drug that inhibits cholesterol absorption, had no effect in NPC1L1 knockout mice, suggesting that NPC1L1 resides in an ezetimibe-sensitive pathway responsible for intestinal cholesterol absorption.

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Niemann-Pick C1 Like 1 (NPC1L1) is a protein localized in jejunal enterocytes that is critical for intestinal cholesterol absorption. The uptake of intestinal phytosterols and cholesterol into absorptive enterocytes in the intestine is not fully defined on a molecular level, and the role of NPC1L1 in maintaining whole body cholesterol homeostasis is not known. NPC1L1 null mice had substantially reduced intestinal uptake of cholesterol and sitosterol, with dramatically reduced plasma phytosterol levels. The NPC1L1 null mice were completely resistant to diet-induced hypercholesterolemia, with plasma lipoprotein and hepatic cholesterol profiles similar to those of wild type mice treated with the cholesterol absorption inhibitor ezetimibe. Cholesterol/cholate feeding resulted in down-regulation of intestinal NPC1L1 mRNA expression in wild type mice. NPC1L1 deficiency resulted in up-regulation of intestinal hydroxymethylglutaryl-CoA synthase mRNA and an increase in intestinal cholesterol synthesis, down-regulation of ABCA1 mRNA, and no change in ABCG5 and ABCG8 mRNA expression. NPC1L1 is required for intestinal uptake of both cholesterol and phytosterols and plays a major role in cholesterol homeostasis. Thus, NPC1L1 may be a useful drug target for the treatment of hypercholesterolemia and sitosterolemia.Cholesterol absorption of both dietary cholesterol and cholesterol cleared from the liver through biliary secretion contributes along with regulation of cholesterol biosynthesis to maintain a tight control of cholesterol homeostasis. The mechanism by which cholesterol moves from the intestinal lumen into the absorptive enterocytes lining the proximal small intestine is poorly understood. The identification of ezetimibe as a potent selective inhibitor of intestinal cholesterol uptake and absorption confirmed this mechanism as a key point of therapeutic intervention for lowering plasma cholesterol levels and indicated that this process is mediated by a specific transporter (1-4). Based on the properties of ezetimibe in animal models of cholesterol uptake, it was predicted that such a transporter would be expressed in jejunal enterocytes and localized to the brush border membrane, which forms the interface between the intestinal lumen and the intracellular compartments responsible for cholesterol esterification and packaging into chylomicrons.Through studies designed to understand the mechanism by which ezetimibe inhibits cholesterol absorption, we recently identified Niemann-Pick C1 Like 1 (NPC1L1) 1 as a critical protein for the intestinal absorption of dietary and biliary cholesterol (5). NPC1L1 was identified through a genomics-bioinformatics approach by sequencing an expression sequence tags library from rat jejunum, annotating the sequences, and searching databases for intestinal proteins with features of a cholesterol transporter (5). NPC1L1 was found to be highly expressed in the jejunum and localized on the surface of the absorptive enterocytes. Mice deficient in NPC1L1 exhibited a significant reduction in chol...

Summary Recent studies show that liquid-liquid phase separation plays a key role in the assembly of diverse intracellular structures. However, the biophysical principles by which phase separation can be precisely localized within subregions of the cell are still largely unclear, particularly for low-abundance proteins. Here we introduce an oligomerizing biomimetic system, “Corelets”, and utilize its rapid and quantitative light-controlled tunability to map full intracellular phase diagrams, which dictate the concentrations at which phase separation occurs, and the mode of phase separation. Surprisingly, both experiments and simulations show that while intracellular concentrations may be insufficient for global phase separation, sequestering protein ligands to slowly diffusing nucleation centers can move the cell into a different region of the phase diagram, resulting in localized phase separation. This diffusive capture mechanism liberates the cell from the constraints of global protein abundance and is likely exploited to pattern condensates associated with diverse biological processes.

The cell nucleus contains a large number of membrane-less bodies that play important roles in the spatiotemporal regulation of gene expression. Recent work suggests that low complexity/disordered protein motifs and repetitive binding domains drive assembly of droplets of nuclear RNA/protein by promoting nucleoplasmic phase separation. Nucleation and maturation of these structures is regulated by, and may in turn affect, factors including post-translational modifications, protein concentration, transcriptional activity, and chromatin state. Here we present a concise review of these exciting recent advances, and discuss current and future challenges in understanding the assembly, regulation, and function of nuclear RNA/protein bodies.

The three currently available male contraceptive approaches are 1) the barrier method such as the condom, 2) hormonal methods by disrupting the pituitary-testicular axis so as to impair spermatogenesis, and 3) immunological methods by preparing vaccines against male-specific antigens. We hereby describe an alternative approach in which attachments of developing germ cells onto the seminiferous epithelium are disrupted, thereby inducing their premature release into the tubular lumen. This in turn leads to infertility. A panel of analogues based on the core structure of 1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid was synthesized. These compounds were subjected to an in vivo screening assay assessing their effects in inducing the expression of testin, a testicular marker whose expression correlates with the integrity of Sertoli-germ cell junctions. An induction of testin expression in the testis signifies a disruption of Sertoli-germ cell junctions that is followed by depletion of germ cells from the seminiferous epithelium. Two compounds, namely 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) and 1-(2,4-dichlorobenzyl)-indazole-3-acrylic acid (AF-2785), were identified that caused detachment of germ cells, in particular round and elongated spermatids, from the epithelium inducing their premature release into the tubular lumen as confirmed by histological analysis. Adult rats receiving several oral doses of either one of these compounds became infertile within 3-7 wk after the epididymal sperm reserve was exhausted. Depending on the dosing of the administered compound, rats became infertile for 4-14 wk before their fertility gradually bounced back, illustrating the reversibility and efficacy of these new compounds. Also, these compounds did not appear to impair the hypothalamus-pituitary-testicular axis because the serum levels of LH, FSH, and testosterone of the treated animals did not change significantly when compared to control rats. In addition, results of serum microchemistry illustrate that liver and kidney function was not affected in animals treated with both compounds.

Transgenic mice were generated containing a 1542-base pair fragment of the kidney androgen-regulated protein (KAP) promoter fused to the human angiotensinogen (HAGT) gene with the goal of specifically targeting inducible expression of renin-angiotensin system components to the kidney. High level expression of both KAP-HAGT and endogenous KAP mRNA was evident in the kidney of male mice from two independent transgenic lines. Renal expression of the transgene in female mice was undetectable under basal conditions but could be strongly induced by administration of testosterone. Testosterone treatment did not cause a transcriptional induction in any other tissues examined. However, an analysis of six androgen target tissues in males revealed that the transgene was expressed in epididymis. No other extra-renal expression of the transgene was detected. In situ hybridization demonstrated that expression of HAGT (and KAP) mRNA in males and testosterone-treated females was restricted to proximal tubule epithelial cells in the renal cortex. Although there was no detectable human angiotensinogen protein in plasma, it was evident in the urine, consistent with a pathway of synthesis in proximal tubule cells and release into the tubular lumen. These results demonstrate that 1542 base pairs of the KAP promoter is sufficient to drive expression of a heterologous reporter gene in a tissue-specific, cell-specific, and androgen-regulated fashion in transgenic mice.The renin-angiotensin system (RAS) 1 is a classical endocrine system activated by the release of renin from the kidney and angiotensinogen (AGT) from the liver. In blood, renin proteolytically cleaves AGT to form angiotensin I (Ang-I) which is further processed by angiotensin converting enzyme to form Ang-II, a potent vasoconstrictor and antinatriuretic peptide. The RAS has been implicated in the genetic basis of hypertension and pre-eclampsia (1-4). Our understanding of the RAS in normal and pathophysiological regulation of blood pressure has been complicated by the fact that in addition to its actions as an endocrine system, certain individual tissues, such as the kidney (5-7), heart (8, 9), brain (10), and vasculature (11), contain all the components of the RAS cascade and therefore have the potential for local synthesis and action of Ang-II. In the kidney, for example, renin, AGT and ACE mRNAs, and proteins are synthesized in juxtaglomerular cells, proximal convoluted tubule (PCT) cells, and endothelial and tubular cells, respectively, and Ang-II type-1 (AT-1) and type-2 (AT-2) receptors are localized in glomeruli, collecting ducts, tubules, and vasa recta (12-18). The intrarenal RAS has been postulated to regulate various aspects of renal function including blood flow, natriuresis, and tubular-glomerular feedback, and may therefore participate in the pathogenesis of hypertension (19 -21). Our current understanding of the relative importance of the intrarenal versus systemic RAS comes largely from pharmacological studies (22) which have been limited by the specificity...