isolation-primary-rat-hepatocytes-with-multiparameter-perfusion

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...

National University of Singapore

The TGF-β/Smad signaling system decreases its activity through strong negative regulation. Several molecular mechanisms of negative regulation have been published, but the relative impact of each mechanism on the overall system is unknown. In this work, we used computational and experimental methods to assess multiple negative regulatory effects on Smad signaling in HaCaT cells. Previously reported negative regulatory effects were classified by time-scale: degradation of phosphorylated R-Smad and I-Smad-induced receptor degradation were slow-mode effects, and dephosphorylation of R-Smad was a fast-mode effect. We modeled combinations of these effects, but found no combination capable of explaining the observed dynamics of TGF-β/Smad signaling. We then proposed a negative feedback loop with upregulation of the phosphatase PPM1A. The resulting model was able to explain the dynamics of Smad signaling, under both short and long exposures to TGF-β. Consistent with this model, immuno-blots showed PPM1A levels to be significantly increased within 30 min after TGF-β stimulation. Lastly, our model was able to resolve an apparent contradiction in the published literature, concerning the dynamics of phosphorylated R-Smad degradation. We conclude that the dynamics of Smad negative regulation cannot be explained by the negative regulatory effects that had previously been modeled, and we provide evidence for a new negative feedback loop through PPM1A upregulation. This work shows that tight coupling of computational and experiments approaches can yield improved understanding of complex pathways.

The self-limiting dynamics of TGF-β signaling in silico and in vitro, with negative feedback through PPM1A upregulation.

Chemotaxis in shallow gradients of chemoattractants is accomplished by preferential maintenance of protrusions oriented towards the chemoattractant; however, the mechanism of preferential maintenance is not known. Here, we test the hypothesis that kinectin-dependent endoplasmic reticulum (ER) transport supports focal complex maturation to preferentially maintain correctly oriented protrusions. We knocked down kinectin expression in MDA-MB-231 cells using small interfering RNA and observed that kinectin contributes to the directional bias, but not the speed, of cell migration. Kymograph analysis revealed that the extension of protrusions oriented towards the chemoattractant was not affected by kinectin knockdown, but that their maintenance was. Immunofluorescence staining and live-cell imaging demonstrated that kinectin transports ER preferentially to protrusions oriented towards the chemoattractant. ER then promotes the maturation of focal complexes into focal adhesions to maintain these protrusions for chemotaxis. Our results show that kinectin-dependent ER distribution can be localized by chemoattractants and provide a mechanism for biased protrusion choices during chemotaxis in shallow gradients of chemoattractants.

Kinectin-dependent ER transport supports the focal complex maturation required for chemotaxis in shallow gradients.

A wide range of liver diseases manifest as biliary obstruction, or cholestasis. However, the sequence of molecular events triggered as part of the early hepatocellular homeostatic response in obstructive cholestasis is poorly elucidated. Pericanalicular actin is known to accumulate during obstructive cholestasis. Therefore, we hypothesized that the pericanalicular actin cortex undergoes significant remodeling as a regulatory response to obstructive cholestasis.

Actomyosin contractility drives bile regurgitation as an early response during obstructive cholestasis.

Liver is highly regenerative as it can restore its function and size even after 70% partial hepatectomy. During liver regeneration, the mechanical and chemical environment of liver is altered with accumulation of various growth factors and remodeling of extracellular environment. Cells can sense the changes in their cellular environment through various chemo and mechanosensors present on their surfaces. These changes are then transduced by initiation of multiple signaling pathways. Traditional view of liver regeneration describes the process as a cascade of chemical signaling pathways. In this review, we describe the role of mechanical forces and mechanosensing in regulating liver regeneration with focus on the role of altered shear and extracellular matrix environment following injury. These mechanosensing mechanisms either generate molecular signals that further activate downstream signaling pathways such as YAP or directly transduce mechanical signals by regulating actomyosin cytoskeleton. These signals travel to the decision center such as nucleus to switch cell fate and activate functions needed in liver regeneration, e.g. proliferation of various hepatic cell types, differentiation of hepatic stem cells, extracellular matrix remodeling and termination signals that regulate the regenerated liver size. Different mechanical and chemical signals coordinate intracellular chemical signaling pathways leading to robust liver regeneration.

Mechanosensing in liver regeneration.

The symmetry breaking of protein distribution and cytoskeleton organization is an essential aspect for the development of apicobasal polarity. In embryonic cells this process is largely cell autonomous, while differentiated epithelial cells collectively polarize during epithelium formation. Here, we demonstrate that the de novo polarization of mature hepatocytes does not require the synchronized development of apical poles on neighbouring cells. De novo polarization at the single-cell level by mere contact with the extracellular matrix and immobilized cadherin defining a polarizing axis. The creation of these single-cell liver hemi-canaliculi allows unprecedented imaging resolution and control and over the lumenogenesis process. We show that the density and localization of cadherins along the initial cell-cell contact act as key triggers of the reorganization from lateral to apical actin cortex. The minimal cues necessary to trigger the polarization of hepatocytes enable them to develop asymmetric lumens with ectopic epithelial cells originating from the kidney, breast or colon.

Biomimetic niches reveal the minimal cues to trigger apical lumen formation in single hepatocytes.

Drug-induced hepatocellular cholestasis leads to altered bile flow. Bile is propelled along the bile canaliculi (BC) by actomyosin contractility, triggered by increased intracellular calcium (Ca). However, the source of increased intracellular Ca and its relationship to transporter activity remains elusive. We identify the source of the intracellular Ca involved in triggering BC contractions, and we elucidate how biliary pressure regulates Ca homeostasis and associated BC contractions. Primary rat hepatocytes were cultured in collagen sandwich. Intra-canalicular Ca was measured with fluo-8; and intra-cellular Ca was measured with GCaMP. Pharmacological modulators of canonical Ca-channels were used to study the Ca-mediated regulation of BC contraction. BC contraction correlates with cyclic transfer of Ca from BC to adjacent hepatocytes, and not with endoplasmic reticulum Ca. A mechanosensitive Ca channel (MCC), Piezo-1, is preferentially localized at BC membranes. The Piezo-1 inhibitor GsMTx-4 blocks the Ca transfer, resulting in cholestatic generation of BC-derived vesicles whereas Piezo-1 hyper-activation by Yoda1 increases the frequency of Ca transfer and BC contraction cycles. Yoda1 can recover normal BC contractility in drug-induced hepatocellular cholestasis, supporting that Piezo-1 regulates BC contraction cycles. Finally, we show that hyper-activating Piezo-1 can be exploited to normalize bile flow in drug-induced hepatocellular cholestasis.

Article	Year
The self-limiting dynamics of TGF-β signaling in silico and in vitro, with negative feedback through PPM1A upregulation. PLoS computational biology\| PubMed ID: 24901250	2014
Kinectin-dependent ER transport supports the focal complex maturation required for chemotaxis in shallow gradients. Journal of cell science\| PubMed ID: 27221621	2016
Actomyosin contractility drives bile regurgitation as an early response during obstructive cholestasis. Journal of hepatology\| PubMed ID: 28189756	2017
Mechanosensing in liver regeneration. Seminars in cell & developmental biology\| PubMed ID: 28768152	2017
Biomimetic niches reveal the minimal cues to trigger apical lumen formation in single hepatocytes. Nature materials\| PubMed ID: 32341512	2020
Bile canaliculi contract autonomously by releasing calcium into hepatocytes via mechanosensitive calcium channel. Biomaterials\| PubMed ID: 32827796	2020

Inn Chuan Ng

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