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

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.

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

BACKGROUND & AIMS: 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. METHODS: In vivo investigations were performed in a bile duct-ligated mouse model. Actomyosin contractility was assessed using sandwich-cultured rat hepatocytes transfected with various fluorescently labeled proteins and pharmacological inhibitors of actomyosin contractility. RESULTS: Actomyosin contractility induces transient deformations along the canalicular membrane, a process we have termed inward blebbing. We show that these membrane intrusions are initiated by local ruptures in the pericanalicular actin cortex; and they typically retract following repair by actin polymerization and actomyosin contraction. However, above a certain osmotic pressure threshold, these inward blebs pinch away from the canalicular membrane into the hepatocyte cytoplasm as large vesicles (2-8µm). Importantly, we show that these vesicles aid in the regurgitation of bile from the bile canaliculi. CONCLUSION: Actomyosin contractility induces the formation of bile-regurgitative vesicles, thus serving as an early homeostatic mechanism against increased biliary pressure during cholestasis. LAY SUMMARY: Bile canaliculi expand and contract in response to the amount of secreted bile, and resistance from the surrounding actin bundles. Further expansion due to bile duct blockade leads to the formation of inward blebs, which carry away excess bile to prevent bile build up in the canaliculi.

Author Correction: PTEN-L is a novel protein phosphatase for ubiquitin dephosphorylation to inhibit PINK1-Parkin-mediated mitophagy.

We apologize for three errors that we just found in the paper published online on 22 June 2018.

PTEN-L is a novel protein phosphatase for ubiquitin dephosphorylation to inhibit PINK1-Parkin-mediated mitophagy.

Mitophagy is an important type of selective autophagy for specific elimination of damaged mitochondria. PTEN-induced putative kinase protein 1 (PINK1)-catalyzed phosphorylation of ubiquitin (Ub) plays a critical role in the onset of PINK1-Parkin-mediated mitophagy. Phosphatase and tensin homolog (PTEN)-long (PTEN-L) is a newly identified isoform of PTEN, with addition of 173 amino acids to its N-terminus. Here we report that PTEN-L is a novel negative regulator of mitophagy via its protein phosphatase activity against phosphorylated ubiquitin. We found that PTEN-L localizes at the outer mitochondrial membrane (OMM) and overexpression of PTEN-L inhibits, whereas deletion of PTEN-L promotes, mitophagy induced by various mitochondria-damaging agents. Mechanistically, PTEN-L is capable of effectively preventing Parkin mitochondrial translocation, reducing Parkin phosphorylation, maintaining its closed inactive conformation, and inhibiting its E3 ligase activity. More importantly, PTEN-L reduces the level of phosphorylated ubiquitin (pSer65-Ub) in vivo, and in vitro phosphatase assay confirms that PTEN-L dephosphorylates pSer65-Ub via its protein phosphatase activity, independently of its lipid phosphatase function. Taken together, our findings demonstrate a novel function of PTEN-L as a protein phosphatase for ubiquitin, which counteracts PINK1-mediated ubiquitin phosphorylation leading to blockage of the feedforward mechanisms in mitophagy induction and eventual suppression of mitophagy. Thus, understanding this novel function of PTEN-L provides a key missing piece in the molecular puzzle controlling mitophagy, a critical process in many important human diseases including neurodegenerative disorders such as Parkinson's disease.