PIKfyve inhibition reveals a novel role for Inpp4b in the regulation of PtdIns(3)P and lysosome dynamics

Lysosome membranes contain diverse phosphoinositide (PtdIns) lipids that co-ordinate lysosome function and dynamics. The PtdIns repertoire on lysosomes is tightly regulated by the action of diverse PtdIns kinases and phosphatases. Specific roles for PtdIns in lysosomal function and dynamics are currently unclear and require further investigation. PIKfyve, a lipid kinase which synthesizes PtdIns(3,5)P2 from PtdIns(3)P, controls lysosome fusion-fission cycles, autophagosome turnover and endocytic cargo delivery. We have recently characterized a role for INPP4B, a PtdIns phosphatase which hydrolyses PtdIns(3,4)P2 to form PtdIns(3)P, in the regulation of lysosomal biogenesis and function. To gain a better understanding of PtdIns homeostasis on lysosomes, we investigated the consequence of disrupting PIKfyve in Inpp4b-deficient mouse embryonic fibroblasts. Surprisingly, simultaneous inhibition of Inpp4b and PIKfyve functions impair lysosome fission and exacerbate lysosome enlargement and inhibit autophagic flux. Further examination into the underlying processes that may explain exaggerated lysosome enlargement revealed elevated levels of lysosome-associated PtdIns(3)P as contributing factors that control lysosome morphology in cells where Inpp4b and PIKfyve are disrupted. Overall, our study suggests that lysosomal functions are regulated by Inpp4b, through a paradoxical role in suppressing the induction of PtdIns(3)P production.

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S6K1 Is Indispensible for Stress-Induced Microtubule Acetylation and Autophagic Flux

Cells ◽

10.3390/cells10040929 ◽

2021 ◽

Vol 10 (4) ◽

pp. 929

Author(s):

Aleksandra Hać ◽

Karolina Pierzynowska ◽

Anna Herman-Antosiewicz

Keyword(s):

Degradation Process ◽

Underlying Mechanism ◽

Stress Conditions ◽

Autophagic Flux ◽

Ratiometric Fluorescence ◽

Mouse Embryonic Fibroblasts ◽

Double Knockout ◽

Embryonic Fibroblasts ◽

Microtubule Network ◽

Tubulin Acetylation

Autophagy is a specific macromolecule and organelle degradation process. The target macromolecule or organelle is first enclosed in an autophagosome, and then delivered along acetylated microtubules to the lysosome. Autophagy is triggered by stress and largely contributes to cell survival. We have previously shown that S6K1 kinase is essential for autophagic flux under stress conditions. Here, we aimed to elucidate the underlying mechanism of S6K1 involvement in autophagy. We stimulated autophagy in S6K1/2 double-knockout mouse embryonic fibroblasts by exposing them to different stress conditions. Transient gene overexpression or silencing, immunoblotting, immunofluorescence, flow cytometry, and ratiometric fluorescence analyses revealed that the perturbation of autophagic flux in S6K1-deficient cells did not stem from impaired lysosomal function. Instead, the absence of S6K1 abolished stress-induced tubulin acetylation and disrupted the acetylated microtubule network, in turn impairing the autophagosome-lysosome fusion. S6K1 overexpression restored tubulin acetylation and autophagic flux in stressed S6K1/2-deficient cells. Similar effect of S6K1 status was observed in prostate cancer cells. Furthermore, overexpression of an acetylation-mimicking, but not acetylation-resistant, tubulin variant effectively restored autophagic flux in stressed S6K1/2-deficient cells. Collectively, S6K1 controls tubulin acetylation, hence contributing to the autophagic flux induced by different stress conditions and in different cells.

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The Polyphenolic Compound Curcumin Conjugation with an Alkyne Moiety in the Process of Autophagy

The American Journal of Chinese Medicine ◽

10.1142/s0192415x18500350 ◽

2018 ◽

Vol 46 (03) ◽

pp. 673-687 ◽

Cited By ~ 5

Author(s):

Peiyi Yan ◽

Xin Sun ◽

Xiaochen Chen ◽

Yun Chen ◽

Xiao Wang ◽

...

Keyword(s):

Therapeutic Agent ◽

Mammalian Target Of Rapamycin ◽

Autophagic Flux ◽

Cell Growth Inhibition ◽

Polyphenolic Compound ◽

Mouse Embryonic Fibroblasts ◽

Curcumin Treatment ◽

Embryonic Fibroblasts ◽

Evolutionarily Conserved ◽

Related Proteins

Curcumin is a hydrophobic polyphenol derived from turmeric: the rhizome of the herb Curcumalonga. Autophagy is an evolutionarily conserved process, in which cellular proteins and organelles are engulfed in autophagosome and then fuses with lysosome for degradation. Our previous study showed that Curcumin activates lysosome and induce autophagy through inhibition of AKT (protein kinase K, PKB)-mammalian target of rapamycin (mTOR) pathway. But whether Curucmin affects the fusion of autophagosome-lysosome is still not clear. Here, we used Curcumin-probe conjugation with an alkyne moiety to label mouse embryonic fibroblasts (MEFs) and found that Curcumin targets autophagy-related proteins, enhances autophagic flux and activates lysosome in cells. Moreover, Curcumin treatment promotes the fusion of autophasosome-lysosome in MEFs. Second, the enhanced fusion of autophagosome-lysosome is attributed to mTOR suppression. Third, blockage of the autophagosome-lysosome fusion leads to cell growth inhibition by Curcumin. Taken together, data from our study indicates the importance of the fusion of autophagosome-lysosome in Curcumin-induced autophagy, which may facilitate the development of Curcumin as a potential therapeutic agent for oxidative stress-related diseases.

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The ATG5 Interactome Links Clathrin Vesicular Trafficking With The ATG8 Lipidation Machinery For Autophagosome Assembly

10.1101/769059 ◽

2019 ◽

Cited By ~ 2

Author(s):

Kiren Baines ◽

Jon D. Lane

Keyword(s):

Membrane Trafficking ◽

Mode Of Delivery ◽

Surface Expression ◽

Vesicular Trafficking ◽

Light Chains ◽

Autophagic Flux ◽

Autophagosome Formation ◽

Mouse Embryonic Fibroblasts ◽

Embryonic Fibroblasts ◽

Phosphoinositide 3 Kinase

ABSTRACTAutophagosome formation involves the sequential actions of conserved ATG family proteins that regulate the lipidation of the ubiquitin-like modifier ATG8 at the nascent isolation membrane. Although the molecular steps driving this process are well understood, the source of membranes supplied for the expanding autophagosome and their mode of delivery remain uncertain. Here, we have used quantitative SILAC-based proteomics to identify proteins that associate with the ATG12∼ATG5 conjugate that is crucial for ATG8 lipidation. Our datasets reveal a strong enrichment of regulators of clathrin-mediated vesicular trafficking, including clathrin heavy and light chains, and several clathrin adaptors. Also identified were PIK3C2A (a phosphoinositide 3-kinase involved in clathrin-mediated endocytosis) and HIP1R (a component of clathrin vesicles), and the absence of either of these proteins caused defects in autophagic flux in cell-based starvation assays. To determine whether the ATG12∼ATG5 conjugate reciprocally influences trafficking within the endocytic compartment, we captured the cell surface proteomes of autophagy-competent and autophagy-incompetent mouse embryonic fibroblasts under fed and starved conditions. Proteins whose surface expression increased contingent on autophagic capability included EPHB2, SLC12A4, and JAG1. Those whose surface expression was decreased included CASK, SLC27A4 and LAMP1. These data provide evidence for direct regulatory coupling between the ATG12∼ATG5 conjugate and the clathrin membrane trafficking system, and suggest candidate membrane proteins whose trafficking within the cell may be modulated by the autophagy machinery.

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Stress Response of Mouse Embryonic Fibroblasts Exposed to Polystyrene Nanoplastics

International Journal of Molecular Sciences ◽

10.3390/ijms22042094 ◽

2021 ◽

Vol 22 (4) ◽

pp. 2094

Author(s):

Seung-Woo Han ◽

Jinhee Choi ◽

Kwon-Yul Ryu

Keyword(s):

Stress Response ◽

Neuronal Cells ◽

Antioxidant Response ◽

Endosomal Escape ◽

Autophagic Flux ◽

Cellular Functions ◽

Mouse Embryonic Fibroblasts ◽

Embryonic Fibroblasts ◽

Inflammatory Stress ◽

Prolonged Culture

Polystyrene (PS) nanoplastic exposure has been shown to affect the viability of neuronal cells isolated from mouse embryonic brains. However, the viability of mouse embryonic fibroblasts (MEFs) was not affected although PS nanoplastics accumulated in the cytoplasm. It is currently unknown whether MEFs do not respond to PS nanoplastics or their cellular functions are altered without compromising viability. Here, we found that PS nanoplastics entered the cells via endocytosis and were then released into the cytoplasm, probably by endosomal escape, or otherwise remained in the endosome. Oxidative and inflammatory stress caused by intracellular PS nanoplastics induced the antioxidant response pathway and activated the autophagic pathway. However, colocalization of the autophagic marker LC3B and PS nanoplastics suggested that PS nanoplastics in the cytoplasm might interfere with normal autophagic function. Furthermore, autophagic flux could be impaired, probably due to accumulation of PS nanoplastic-containing lysosomes or autolysosomes. Intriguingly, the level of accumulated PS nanoplastics decreased during prolonged culture when MEFs were no longer exposed to PS nanoplastics. These results indicate that accumulated PS nanoplastics are removed or exported out of the cells. Therefore, PS nanoplastics in the cytoplasm affect cellular functions, but it is temporal and MEFs can overcome the stress caused by PS nanoplastic exposure.

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Caveolin-1 is required for fatty acid translocase (FAT/CD36) localization and function at the plasma membrane of mouse embryonic fibroblasts

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids ◽

10.1016/j.bbalip.2006.03.016 ◽

2006 ◽

Vol 1761 (4) ◽

pp. 416-423 ◽

Cited By ~ 91

Author(s):

Axel Ring ◽

Soazig Le Lay ◽

Juergen Pohl ◽

Paul Verkade ◽

Wolfgang Stremmel

Keyword(s):

Plasma Membrane ◽

Fatty Acid ◽

Mouse Embryonic Fibroblasts ◽

Embryonic Fibroblasts ◽

Fatty Acid Translocase ◽

Caveolin 1 ◽

And Function

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Identification of biomarkers and pathways of mouse embryonic fibroblasts with the dysfunction of mitochondrial DNA

10.1101/2021.04.05.438453 ◽

2021 ◽

Author(s):

Hanming Gu

Keyword(s):

Immune Responses ◽

Metabolic Diseases ◽

Biological Function ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Mitochondrial Diseases ◽

Mouse Embryonic Fibroblasts ◽

Embryonic Fibroblasts ◽

Multiple Systems ◽

And Function

Mitochondrial diseases are clinically heterogeneous which involve multiple systems such as organs that are highly dependent on metabolism. Dysfunction of mtDNA is the main cause of mitochondrial diseases that trigger inflammation and immune responses. Here, we aim to identify the biological function and pathways of MEFs with the dysfunction of mtDNA through deletion of YME1L. The gene expression profiles of GSE161735 dataset were originally created by the Illumina NovaSeq 6000 (Mus musculus) for gene biogenesis and function panel. The biological and functional pathways were analyzed by the Kyoto Encyclopedia of Genes and Genomes pathway (KEGG), Gene Ontology (GO), and Reactom visual map. KEGG and GO results showed the metabolism and immune responses were mostly affected by the loss of mtDNA. Moreover, we discovered several interacting genes including POLR2F, HIST1H2BJ, PPP1CC, HOXB4, ARG1, APITD1, BUB1B, POLR2K, HOXC4, and HOXB3 were involved in the regulation of metabolic or cancer diseases. Further, we predicted several regulators that had the ability to affect mitochondria during the dysfunction of mtDNA by L1000fwd analysis. Thus, this study provides further insights into the mechanism of mtDNA in metabolic diseases.

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c-FLIP regulates autophagy by interacting with Beclin-1 and influencing its stability

Cell Death and Disease ◽

10.1038/s41419-021-03957-5 ◽

2021 ◽

Vol 12 (7) ◽

Author(s):

Luana Tomaipitinca ◽

Simonetta Petrungaro ◽

Pasquale D’Acunzo ◽

Angelo Facchiano ◽

Amit Dubey ◽

...

Keyword(s):

Molecular Mechanism ◽

Coiled Coil ◽

Multiple Roles ◽

Beclin 1 ◽

Autophagic Flux ◽

Mouse Embryonic Fibroblasts ◽

Inhibitory Protein ◽

Embryonic Fibroblasts ◽

Interacting Protein

Abstractc-FLIP (cellular FLICE-like inhibitory protein) protein is mostly known as an apoptosis modulator. However, increasing data underline that c-FLIP plays multiple roles in cellular homoeostasis, influencing differently the same pathways depending on its expression level and isoform predominance. Few and controversial data are available regarding c-FLIP function in autophagy. Here we show that autophagic flux is less effective in c-FLIP−/− than in WT MEFs (mouse embryonic fibroblasts). Indeed, we show that the absence of c-FLIP compromises the expression levels of pivotal factors in the generation of autophagosomes. In line with the role of c-FLIP as a scaffold protein, we found that c-FLIPL interacts with Beclin-1 (BECN1: coiled-coil, moesin-like BCL2-interacting protein), which is required for autophagosome nucleation. By a combination of bioinformatics tools and biochemistry assays, we demonstrate that c-FLIPL interaction with Beclin-1 is important to prevent Beclin-1 ubiquitination and degradation through the proteasomal pathway. Taken together, our data describe a novel molecular mechanism through which c-FLIPL positively regulates autophagy, by enhancing Beclin-1 protein stability.

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