Intravital Dynamic and Correlative Imaging of Mouse Livers Reveals Diffusion-Dominated Canalicular and Flow-Augmented Ductular Bile Flux

Background and Aims Small‐molecule flux in tissue microdomains is essential for organ function, but knowledge of this process is scant due to the lack of suitable methods. We developed two independent techniques that allow the quantification of advection (flow) and diffusion in individual bile canaliculi and in interlobular bile ducts of intact livers in living mice, namely fluorescence loss after photoactivation and intravital arbitrary region image correlation spectroscopy. Approach and Results The results challenge the prevailing “mechano‐osmotic” theory of canalicular bile flow. After active transport across hepatocyte membranes, bile acids are transported in the canaliculi primarily by diffusion. Only in the interlobular ducts is diffusion augmented by regulatable advection. Photoactivation of fluorescein bis‐(5‐carboxymethoxy‐2‐nitrobenzyl)‐ether in entire lobules demonstrated the establishment of diffusive gradients in the bile canalicular network and the sink function of interlobular ducts. In contrast to the bile canalicular network, vectorial transport was detected and quantified in the mesh of interlobular bile ducts. Conclusions The liver consists of a diffusion‐dominated canalicular domain, where hepatocytes secrete small molecules and generate a concentration gradient and a flow‐augmented ductular domain, where regulated water influx creates unidirectional advection that augments the diffusive flux

High susceptibility to fatty liver disease in two-pore channel 2-deficient mice

Endolysosomal organelles play a key role in trafficking, breakdown and receptor-mediated recycling of different macromolecules such as low-density lipoprotein (LDL)-cholesterol, epithelial growth factor (EGF) or transferrin. Here we examine the role of two-pore channel (TPC) 2, an endolysosomal cation channel, in these processes. Embryonic mouse fibroblasts and hepatocytes lacking TPC2 display a profound impairment of LDL-cholesterol and EGF/EGF-receptor trafficking. Mechanistically, both defects can be attributed to a dysfunction of the endolysosomal degradation pathway most likely on the level of late endosome to lysosome fusion. Importantly, endolysosomal acidification or lysosomal enzyme function are normal in TPC2-deficient cells. TPC2-deficient mice are highly susceptible to hepatic cholesterol overload and liver damage consistent with non-alcoholic fatty liver hepatitis. These findings indicate reduced metabolic reserve of hepatic cholesterol handling. Our results suggest that TPC2 plays a crucial role in trafficking in the endolysosomal degradation pathway and, thus, is potentially involved in the homoeostatic control of many macromolecules and cell metabolites

Genetic Inactivation of Trpml3 Does Not Lead to Hearing and Vestibular Impairment in Mice

TRPML3, a member of the transient receptor potential (TRP) family, is an inwardly rectifying, non-selective Ca2+-permeable cation channel that is regulated by extracytosolic Na+ and H+ and can be activated by a variety of small molecules. The severe auditory and vestibular phenotype of the TRPML3(A419P) varitint-waddler mutation made this protein particularly interesting for inner ear biology. To elucidate the physiological role of murine TRPML3, we conditionally inactivated Trpml3 in mice. Surprisingly, lack of functional TRPML3 did not lead to circling behavior, balance impairment or hearing loss

Sublethal necroptosis signaling promotes inflammation and liver cancer

It is currently not well known how necroptosis and necroptosis responses manifest in vivo. Here, we uncovered a molecular switch facilitating reprogramming between two alternative modes of necroptosis signaling in hepatocytes, fundamentally affecting immune responses and hepatocarcinogenesis. Concomitant necrosome and NF-κB activation in hepatocytes, which physiologically express low concentrations of receptor-interacting kinase 3 (RIPK3), did not lead to immediate cell death but forced them into a prolonged "sublethal" state with leaky membranes, functioning as secretory cells that released specific chemokines including CCL20 and MCP-1. This triggered hepatic cell proliferation as well as activation of procarcinogenic monocyte-derived macrophage cell clusters, contributing to hepatocarcinogenesis. In contrast, necrosome activation in hepatocytes with inactive NF-κB-signaling caused an accelerated execution of necroptosis, limiting alarmin release, and thereby preventing inflammation and hepatocarcinogenesis. Consistently, intratumoral NF-κB-necroptosis signatures were associated with poor prognosis in human hepatocarcinogenesis. Therefore, pharmacological reprogramming between these distinct forms of necroptosis may represent a promising strategy against hepatocellular carcinoma

Non-canonical Wnt signalling regulates scarring in biliary disease via the planar cell polarity receptors

The number of patients diagnosed with chronic bile duct disease is increasing and in most cases these diseases result in chronic ductular scarring, necessitating liver transplantation. The formation of ductular scaring affects liver function; however, scar-generating portal fibroblasts also provide important instructive signals to promote the proliferation and differentiation of biliary epithelial cells. Therefore, understanding whether we can reduce scar formation while maintaining a pro-regenerative microenvironment will be essential in developing treatments for biliary disease. Here, we describe how regenerating biliary epithelial cells express Wnt-Planar Cell Polarity signalling components following bile duct injury and promote the formation of ductular scars by upregulating pro-fibrogenic cytokines and positively regulating collagen-deposition. Inhibiting the production of Wnt-ligands reduces the amount of scar formed around the bile duct, without reducing the development of the pro-regenerative microenvironment required for ductular regeneration, demonstrating that scarring and regeneration can be uncoupled in adult biliary disease and regeneration

Funktionelle und pharmakologische Charakterisierung des TRP-Kationenkanals TRPML3

TRPML3 belongs to the mucolipin subfamily of the transient receptor potential (TRP) channels. The mouse Trpml3 gene was identified in a positional cloning project to identify the gene responsible for the varitint-waddler (Va) mouse phenotype, which displays severe auditory, vestibular and pigmentation defects. A single A419P amino acid substitution within the predicted 5th transmembrane domain of TRPML3 was identified to cause this Va phenotype. The A419P mutation renders TRPML3 constitutively active, resulting in highly elevated [Ca2+]i and apoptotic cell death in cells that natively express TRPML3, such as melanocytes and sensory hair cells. However, it is remarkable that sensory hair cells of Va mice survive for several postnatal weeks. Presented here are in vitro and in vivo data supporting the hypothesis that the survival of Va hair cells is linked to their ability to deal with Ca2+ loads due to the abundance of plasma membrane calcium ATPases (PMCAs). The rescue effect of PMCA2 is due to the decrease in [Ca2+]i. Ca2+-buffering and Ca2+ extrusion abilities of hair cells are powerful enough to prevent cell death for weeks, even in the presence of constitutively active TRPML3(A419P), which is able to induce rapid apoptosis in other cells. To gain more insight into the function of the wild-type TRPML3 channel, a high-throughput small molecule activator screen was conducted. In this screen 53 compounds were identified that selectively activate TRPML3. Cheminformatics analyses of compounds revealed 9 different chemical scaffolds and 20 singletons. However, testing compounds on sensory hair cells revealed the absence of activator- responsive channels. Melanocytes showed weak or no responses to the compounds, except for SN-2, which when used at a relatively high concentration was able to elicit a significant increase of the intracellular Ca2+ concentration. The lack of substantial responses to identified activators suggested that TRPML3 in native cells is either present only in limited numbers in the plasma membrane or that the protein heteromerizes with other proteins, leading to channels that are not or only weakly responsive to the compounds. To elucidate the physiological role of murine TRPML3, a conditional knockout of Trpml3 was generated. Despite the strong Va phenotype, no overt inner ear phenotype was detectable in mice with ubiquitous Trpml3 inactivation or when the gene was inactivated from P0-P3 onwards. Both mouse models revealed that TRPML3 as a homomer is not essential for detectable inner ear function and developmet in vivo. Although these results do not necessarily exclude TRPML3 from playing a role in mechanotransduction, they do reveal that if the transduction channel complex utilizes TRPML3, TRPML3 may function as a dispensable subunit.TRPML3 gehört zu der Mukolipin Subfamilie der TRP (transient receptor potential) Kanäle. Das Trpml3 Gen wurde in einem positionalen Klonierungsprojekt identifiziert, um das verantwortliche Gen für den varitint- waddler (Va) Phänotyp mit schwerwiegenden auditorischen, vestibulären und Pigmentierungsdefekten zu bestimmen. Ein Aminosäureaustausch (A419P) innerhalb des 5. Transmembransegments von TRPML3 ist verantwortlich für den Va Phänotyp. Der A419P Austausch macht TRPML3 konstitutiv aktiv, was zu erhöhtem [Ca2+]i und zu programmierten Zelltod in Zellen mit nativem TRPML3, wie z.B. Melanozyten und sensorische Haarzellen, führt. Bemerkenswert ist, dass sensorische Haarzellen von Va Mäusen für mehrere postnatale Wochen überleben. In der vorliegenden Arbeit wurde in vivo und in vitro untersucht, inwiefern das Überleben der Va-Haarzellen und deren Fähigkeit mit der Ca2+-Belastung umzugehen, gekoppelt ist an die gleichzeitige Präsenz von Plasmamembranständigen Ca2+-ATPasen (PMCAs). Es wurde gezeigt, dass die Fähigkeit der sensorischen Haarzellen Ca2+ zu puffern und zu extrudieren ist ausreichend, um den Zelltod für mehrere Wochen hinauszuzögern, auch in Anwesenheit der konstitutiv aktiven TRPML3(A419P)-Variante, welche in anderen Zelltypen fähig ist einen schnellen Zelltod zu induzieren. Der Rettungeffekt ist die Folge der Reduzierung des [Ca2+]i. Um einen Einblick in die Funktion von Wildtyp-TRPML3 zu erlangen, wurde in einem Hochdurchsatzverfahren nach chemischen Aktivatoren gesucht. Es wurden 53 Verbindungen identifiziert, die TRPML3 selektiv aktivieren. Ein Test dieser Verbindungen auf TRPML3 exprimierende Kochleapräparate zeigte jedoch, dass auditorische Haarzellen nicht auf diese Aktivatoren reagieren. Hautmelanozyten zeigten nur schwache oder ebenfalls keine Antworten auf die Aktivatoren, mit Ausnahme der Substanz SN-2, welche einen signifikanten Anstieg der intrazellulären Ca2+-Konzentration hervorrief. Das Fehlen einer verbesserten Antwort auf die TRPML3 Aktivatoren kann dadurch erklärt werden, dass TRPML3 in nativen Zellen in der Plasmamembran nur in limitierender Menge vorkommt oder dass TRPML3 mit anderen Proteinen heteromerisiert, was zu multimeren Kanalkomplexen führt, die nicht oder nur schwach auf die Verbindungen reagieren. Um die physiologische Rolle des TRPML3’s im inneren Ohr aufzuklären, wurde eine konditionaler Trpml3 Inaktivierung generiert. Trotz des schwerwiegenden Va Phänotyps konnte kein Innenohrphänotyp in Mäusen mit ubiquitärer Trpml3 Inaktivierung oder wenn das Gen ab P0-P3 inaktiviert wurde, gezeigt werden. Beide Mausmodelle zeigten, dass TRPML3 als Homomer nicht essenziell für den Hörprozess ist. Die Ergebnisse schliessen TRPML3 nicht unbedingt von einer Rolle im Prozess der auditorischen Mechanotransduktion aus. Sie verraten jedoch, falls TRPML3 zum Mechanotransduktionskomplex gehört, dass das TRPML3-Protein als eine ersetzbare Untereinheit fungieren könnte

Information behavior of private investors in the age of the internet

The internet is increasingly used by corporations for investor relation purposes and by investors as source of information. This chapter outlines how corporations incorporate this technology in their financial reporting strategies, and reviews relevant literature in regard to the online behavior of private investors. Specifically, the chapter outlines the development of the internet from its beginning in the 1960s to a major source of information for private investors, and highlights the different types of online information disseminated by corporations. It identifies that prior research focused on the internet financial reporting practices of companies, and largely ignored information needs of investors. This chapter suggests that future research about the effectiveness of internet financial reporting should incorporate a user-perspective in their research design. For this purpose, it introduces Wilson's model of information (seeking) behavior, which may be useful in guiding future research examining the information behavior of investors. Specifically, this model depicts information seeking behavior as an iterative process which is influenced by several factors such as the context of information needs and the socio-cultural environment. Prior research focused largely on a description of internet financial reporting based on content analysis. Such research can be enhanced using experimental and qualitative research approaches derived from Wilson's model. This would be useful because it clearly shows that information seeking behavior is mainly driven by behavioral aspects such as human relationships.30 page(s

Targeting strategy for disruption of the <i>Trpml3</i> gene.

<p>A, Transmembrane-spanning domains are depicted as grey bars and numbered from 1–6. The orange frame indicates the part of the TRPML3 protein that is encoded by exon 11, which will be deleted (pore loop and TM6). Exons are shown as black and orange bars on the schematic genomic map below. B, Shown are the targeting vector and the targeted allele after homologous recombination. The blue bar represents the PGK promoter-driven <i>neo^R</i> expression cassette, which was used for positive selection. The DTA cassette, used for negative selection, is shown in green. The black and red arrowheads symbolize position and orientation of <i>FRT</i> and <i>loxP</i> sites. C, Targeted allele after Flp site-specific recombination in ES cells, resulting in excision of <i>neo^R</i> cassette, and leaving one FRT site behind. D, Excision of exon 11 using Cre site-specific recombinase, resulting in disruption of <i>Trpml3</i> gene.</p

Rotarod experiments.

<p>The average latencies to fall ± SEM (in sec) are shown for 3-month-old <i>Hprt^Cre/+;Trpml3^+/+</i> (n = 8) and <i>Hprt^Cre/+;Trpml3 ^Δ/Δ</i> (n = 8) mice. The experiment was executed over a time range of 6 days. The day 6 experiment was performed in the dark to exclude compensation via visual cues. The difference between genotypes was not statistically significant at any given time point (p>0.05, one-way ANOVA, followed by Tukey's post test).</p

Molecular and functional assessment of mutant TRPML3(Δ exon11) channels.

<p>A, Shown are representative cells expressing the respective murine (m) constructs of wild-type TRPML3 or mutant TRPML3(Δ exon11), C-terminally fused to yellow fluorescent protein, 24 h after transfection. Scale bar = 10 µm. B, Ca²⁺ imaging results showing relative [Ca²⁺]_i increases after application of TRPML3 activator SN-2 in HEK293 cells expressing wild-type TRPML3 or mutant TRPML3(Δ exon11). Shown are mean values ± SEM (numbers in parentheses are the numbers of independent experiments with 10–20 cells each). Statistical comparisons of means were made using Student's <i>t</i> test (unpaired); ***p<0.0001.</p