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Semiconducting polymer nanoparticles (SPNs) are explored for applications in cancer theranostics because of their high absorption coefficients, photostability, and biocompatibility. However, SPNs are susceptible to aggregation and protein fouling in physiological conditions, which can be detrimental for in vivo applications. Here, a method for achieving colloidally stable and low-fouling SPNs is described by grafting poly(ethylene glycol) (PEG) onto the backbone of the fluorescent semiconducting polymer, poly(9,9'-dioctylfluorene-5-fluoro-2,1,3-benzothiadiazole), in a simple one-step substitution reaction, postpolymerization. Further, by utilizing azide-functionalized PEG, anti-human epidermal growth factor receptor 2 (HER2) antibodies, antibody fragments, or affibodies are site-specifically "clicked" onto the SPN surface, which allows the functionalized SPNs to specifically target HER2-positive cancer cells. In vivo, the PEGylated SPNs are found to have excellent circulation efficiencies in zebrafish embryos for up to seven days postinjection. SPNs functionalized with affibodies are then shown to be able to target HER2 expressing cancer cells in a zebrafish xenograft model. The covalent PEGylated SPN system described herein shows great potential for cancer theranostics., (© 2023 The Authors. Advanced Materials published by Wiley‐VCH GmbH.)

The ability of cells to move and migrate is required during development, but also in the adult in processes such as wound healing and immune responses. In addition, cancer cells exploit the cells' ability to migrate and invade to spread into nearby tissue and eventually metastasize. The majority of cancer deaths are caused by metastasis and the process of cell migration is therefore intensively studied. A common way to study cell migration is to observe cells through an optical microscope and record their movements over time. However, segmenting and tracking moving cells in phase contrast time-lapse video sequences is a challenging task. Several tools to track the velocity of migrating cells have been developed. Unfortunately, most of the automated tools are made for fluorescence images even though unlabelled cells are often preferred to avoid phototoxicity. Consequently, researchers are constrained with laborious manual tracking tools using ImageJ or similar software. We have therefore developed a freely available, user-friendly, automated tracking tool called CellTraxx. This software makes it easy to measure the velocity and directness of migrating cells in phase contrast images. Here, we demonstrate that our tool efficiently recognizes and tracks unlabelled cells of different morphologies and sizes (HeLa, RPE1, MDA-MB-231, HT1080, U2OS, PC-3) in several types of cell migration assays (random migration, wound healing and cells embedded in collagen). We also provide a detailed protocol and download instructions for CellTraxx., (© 2023. The Author(s).)

Cells use noncanonical autophagy, also called conjugation of ATG8 to single membranes (CASM), to label damaged intracellular compartments with ubiquitin-like ATG8 family proteins in order to signal danger caused by pathogens or toxic compounds. CASM relies on E3 complexes to sense membrane damage, but so far, only the mechanism to activate ATG16L1-containing E3 complexes, associated with proton gradient loss, has been described. Here, we show that TECPR1-containing E3 complexes are key mediators of CASM in cells treated with a variety of pharmacological drugs, including clinically relevant nanoparticles, transfection reagents, antihistamines, lysosomotropic compounds, and detergents. Interestingly, TECPR1 retains E3 activity when ATG16L1 CASM activity is obstructed by the Salmonella Typhimurium pathogenicity factor SopF. Mechanistically, TECPR1 is recruited by damage-induced sphingomyelin (SM) exposure using two DysF domains, resulting in its activation and ATG8 lipidation. In vitro assays using purified human TECPR1-ATG5-ATG12 complex show direct activation of its E3 activity by SM, whereas SM has no effect on ATG16L1-ATG5-ATG12. We conclude that TECPR1 is a key activator of CASM downstream of SM exposure., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Mitophagy is the selective degradation of mitochondria by autophagy. It promotes the turnover of mitochondria and prevents the accumulation of dysfunctional mitochondria, which can lead to cellular degeneration. Mitophagy is known to be altered in several pathological conditions, especially in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). We recently demonstrated an increase in autophagy flux in lymphoblasts from ALS patients bearing a mutation in SOD1. Thus, the identification of mitophagy inhibitors may be a therapeutic option to recover mitochondrial homeostasis. Here, using a phenotypic mitophagy assay, we identified a new mitophagy inhibitor, the small molecule named IGS2.7 from the MBC library. Interestingly, the treatment of different cellular and in vivo models of ALS with mutations on SOD1 and TARDBP with this inhibitor restores autophagy to control levels. These results point mitophagy inhibitors, especially IGS2.7, to a new therapeutic approach for familial ALS patients.

Cell migration is a complex process underlying physiological and pathological processes such as brain development and cancer metastasis. The autophagy-linked FYVE protein (ALFY; also known as WDFY3), an autophagy adaptor protein known to promote clearance of protein aggregates, has been implicated in brain development and neural migration during cerebral cortical neurogenesis in mice. However, a specific role of ALFY in cell motility and extracellular matrix adhesion during migration has not been investigated. Here, we reveal a novel role for ALFY in the endocytic pathway and in cell migration. We show that ALFY localizes to RAB5- and EEA1-positive early endosomes in a PtdIns(3)P-dependent manner and is highly enriched in cellular protrusions at the leading and lagging edge of migrating cells. We find that cells lacking ALFY have reduced attachment and altered protein levels and glycosylation of integrins, resulting in the inability to form a proper leading edge and loss of directional cell motility., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The removal of mitochondria in a programmed or stress-induced manner is essential for maintaining cellular homeostasis. To date, much research has focused upon stress-induced mitophagy that is largely regulated by the E3 ligase PRKN, with limited insight into the mechanisms regulating basal "housekeeping" mitophagy levels in different model organisms. Using iron chelation as an inducer of PRKN-independent mitophagy, we recently screened an siRNA library of lipid-binding proteins and determined that two kinases, GAK and PRKCD, act as positive regulators of PRKN-independent mitophagy. We demonstrate that PRKCD is localized to mitochondria and regulates recruitment of ULK1-ATG13 upon induction of mitophagy. GAK activity, by contrast, modifies the mitochondrial network and lysosomal morphology that compromise efficient transport of mitochondria for degradation. Impairment of either kinase in vivo blocks basal mitophagy, demonstrating the biological relevance of our findings. Abbreviations: CCCP: carbonyl cyanide-m-chlorophenyl hydrazone; DFP: deferiprone; GAK: cyclin G associated kinase; HIF1A: hypoxia inducible factor 1 subunit alpha; PRKC/PKC: protein kinase C; PRKCD: protein kinase C delta; PRKN: parkin RBR E3 ubiquitin protein ligase.

Mitophagy, the selective degradation of mitochondria by autophagy, involved in important physiological processes and defects in pathways has been reported in pathological conditions, such as neurodegeneration. Thus, mitophagy is an interesting target for drug discovery programs. In this investigation, we used robust phenotypic assay to screen a set of 50 small heterocyclic compounds to identify inducers of mitophagy. We identified two compounds, VP07 and JAR1.39, that induce Parkin-dependent mitophagy. Based on structure-activity relationship studies, we proposed the ability of the compounds to act as light chain 3 (LC3) interactors, similar to cardiolipin or ceramide, triggering mitophagy via Pink1/Parkin. Finally, we show promising therapeutic applicability in a cellular model of Parkinson's disease.

The mechanisms involved in programmed or damage-induced removal of mitochondria by mitophagy remains elusive. Here, we have screened for regulators of PRKN-independent mitophagy using an siRNA library targeting 197 proteins containing lipid interacting domains. We identify Cyclin G-associated kinase (GAK) and Protein Kinase C Delta (PRKCD) as regulators of PRKN-independent mitophagy, with both being dispensable for PRKN-dependent mitophagy and starvation-induced autophagy. We demonstrate that the kinase activity of both GAK and PRKCD are required for efficient mitophagy in vitro, that PRKCD is present on mitochondria, and that PRKCD facilitates recruitment of ULK1/ATG13 to early autophagic structures. Importantly, we demonstrate in vivo relevance for both kinases in the regulation of basal mitophagy. Knockdown of GAK homologue (gakh-1) in C. elegans or knockout of PRKCD homologues in zebrafish led to significant inhibition of basal mitophagy, highlighting the evolutionary relevance of these kinases in mitophagy regulation., (© 2021. The Author(s).)

Eukaryotic cells have the capacity to degrade intracellular components through a lysosomal degradation pathway called macroautophagy (henceforth referred to as autophagy) in which superfluous or damaged cytosolic entities are engulfed and separated from the rest of the cell constituents into double membraned vesicles known as autophagosomes. Autophagosomes then fuse with endosomes and lysosomes, where cargo is broken down into basic building blocks that are released to the cytoplasm for the cell to reuse. Autophagic degradation can target either cytoplasmic material in bulk (non-selective autophagy) or particular cargo in what is called selective autophagy. Proper autophagic turnover requires the orchestrated participation of several players that need to be tightly and temporally coordinated. Whereas a large number of autophagy-related (ATG) proteins have been identified and their functions and regulation are starting to be understood, there is substantially less knowledge regarding the specific lipids constituting the autophagic membranes as well as their role in initiating, enabling or regulating the autophagic process. This review focuses on lipids and their corresponding binding proteins that are crucial in the process of selective autophagy., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The centrosome is the master orchestrator of mitotic spindle formation and chromosome segregation in animal cells. Centrosome abnormalities are frequently observed in cancer, but little is known of their origin and about pathways affecting centrosome homeostasis. Here we show that autophagy preserves centrosome organization and stability through selective turnover of centriolar satellite components, a process we termed doryphagy. Autophagy targets the satellite organizer PCM1 by interacting with GABARAPs via a C-terminal LIR motif. Accordingly, autophagy deficiency results in accumulation of large abnormal centriolar satellites and a resultant dysregulation of centrosome composition. These alterations have critical impact on centrosome stability and lead to mitotic centrosome fragmentation and unbalanced chromosome segregation. Our findings identify doryphagy as an important centrosome-regulating pathway and bring mechanistic insights to the link between autophagy dysfunction and chromosomal instability. In addition, we highlight the vital role of centriolar satellites in maintaining centrosome integrity.

Covalent modification of LC3 and GABARAP proteins to phosphatidylethanolamine in the double-membrane phagophore is a key event in the early phase of macroautophagy, but can also occur on single-membrane structures. In both cases this involves transfer of LC3/GABARAP from ATG3 to phosphatidylethanolamine at the target membrane. Here we have purified the full-length human ATG12-5-ATG16L1 complex and show its essential role in LC3B/GABARAP lipidation in vitro. We have identified two functionally distinct membrane-binding regions in ATG16L1. An N-terminal membrane-binding amphipathic helix is required for LC3B lipidation under all conditions tested. By contrast, the C-terminal membrane-binding region is dispensable for canonical autophagy but essential for VPS34-independent LC3B lipidation at perturbed endosomes. We further show that the ATG16L1 C-terminus can compensate for WIPI2 depletion to sustain lipidation during starvation. This C-terminal membrane-binding region is present only in the β-isoform of ATG16L1, showing that ATG16L1 isoforms mechanistically distinguish between different LC3B lipidation mechanisms under different cellular conditions.

Mechanical and metabolic cues independently contribute to the regulation of cell and tissue homeostasis. However, how they cross-regulate each other during this process remains largely unknown. Here, we show that cellular metabolism can regulate integrin rigidity-sensing via the sphingolipid metabolic pathway controlled by the amino acid transporter and integrin coreceptor CD98hc (SLC3A2). Genetic invalidation of CD98hc in dermal cells and tissue impairs rigidity sensing and mechanical signaling downstream of integrins, including RhoA activation, resulting in aberrant tissue mechanical homeostasis. Unexpectedly, we found that this regulation does not occur directly through regulation of integrins by CD98hc but indirectly, via the regulation of sphingolipid synthesis and the delta-4-desaturase DES2. Loss of CD98hc decreases sphingolipid availability preventing proper membrane recruitment, shuttling and activation of upstream regulators of RhoA including Src kinases and GEF-H1. Altogether, our results unravel a novel cross-talk regulation between integrin mechanosensing and cellular metabolism which may constitute an important new regulatory framework contributing to mechanical homeostasis.

The molecular mechanisms responsible for the pathophysiological traits of type 2 diabetes are incompletely understood. Here we have performed transcriptomic analysis in skeletal muscle, and plasma metabolomics from subjects with classical and early-onset forms of type 2 diabetes (T2D). Focused studies were also performed in tissues from ob/ob and db/db mice. We document that T2D, both early and late onset, are characterized by reduced muscle expression of genes involved in branched-chain amino acids (BCAA) metabolism. Weighted Co-expression Networks Analysis provided support to idea that the BCAA genes are relevant in the pathophysiology of type 2 diabetes, and that mitochondrial BCAA management is impaired in skeletal muscle from T2D patients. In diabetic mice model we detected alterations in skeletal muscle proteins involved in BCAA metabolism but not in obese mice. Metabolomic analysis revealed increased levels of branched-chain keto acids (BCKA), and BCAA in plasma of T2D patients, which may result from the disruption of muscle BCAA management. Our data support the view that inhibition of genes involved in BCAA handling in skeletal muscle takes place as part of the pathophysiology of type 2 diabetes, and this occurs both in early-onset and in classical type 2 diabetes.

The CD98/LAT1 complex is overexpressed in aggressive human cancers and is thereby described as a potential therapeutic target. This complex promotes tumorigenesis with CD98 (4F2hc) engaging β-integrin signaling while LAT1 (SLC7A5) imports essential amino acids (EAA) and promotes mTORC1 activity. However, it is unclear as to which member of the heterodimer carries the most prevalent protumoral action. To answer this question, we explored the tumoral potential of each member by gene disruption of CD98, LAT1, or both and by inhibition of LAT1 with the selective inhibitor (JPH203) in six human cancer cell lines from colon, lung, and kidney. Each knockout respectively ablated 90% (CD98 KO: ) and 100% (LAT1 KO: ) of Na(+)-independent leucine transport activity. LAT1 KO: or JPH203-treated cells presented an amino acid stress response with ATF4, GCN2 activation, mTORC1 inhibition, and severe in vitro and in vivo tumor growth arrest. We show that this severe growth phenotype is independent of the level of expression of CD98 in the six tumor cell lines. Surprisingly, CD98 KO: cells with only 10% EAA transport activity displayed a normal growth phenotype, with mTORC1 activity and tumor growth rate undistinguishable from wild-type cells. However, CD98 KO: cells became extremely sensitive to inhibition or genetic disruption of LAT1 (CD98 KO: /LAT1 KO: ). This finding demonstrates that the tumoral potential of CD98 KO: cells is due to residual LAT1 transport activity. Therefore, these findings clearly establish that LAT1 transport activity is the key growth-limiting step of the heterodimer and advocate the pharmacology development of LAT1 transporter inhibitors as a very promising anticancer target. Cancer Res; 76(15); 4481-92. ©2016 AACR., (©2016 American Association for Cancer Research.)

CD98hc functions as an amino acid (AA) transporter (together with another subunit) and integrin signaling enhancer. It is overexpressed in highly proliferative cells in both physiological and pathological conditions. CD98hc deletion induces strong impairment of cell proliferation in vivo and in vitro Here, we investigate CD98hc-associated AA transport in cell survival and proliferation. By using chimeric versions of CD98hc, the two functions of the protein can be uncoupled. Although recovering the CD98hc AA transport capacity restores the in vivo and in vitro proliferation of CD98hc-null cells, reconstitution of the integrin signaling function of CD98hc is unable to restore in vitro proliferation of those cells. CD98hc-associated transporters (i.e. xCT, LAT1, and y(+)LAT2 in wild-type cells) are crucial to control reactive oxygen species and intracellular AA levels, thus sustaining cell survival and proliferation. Moreover, in CD98hc-null cells the deficiency of CD98hc/xCT cannot be compensated, leading to cell death by ferroptosis. Supplementation of culture media with β-mercaptoethanol rescues CD98hc-deficient cell survival. Under such conditions null cells show oxidative stress and intracellular AA imbalance and, consequently, limited proliferation. CD98hc-null cells also present reduced intracellular levels of branched-chain and aromatic amino acids (BCAAs and ARO AAs, respectively) and induced expression of peptide transporter 1 (PEPT1). Interestingly, external supply of dipeptides containing BCAAs and ARO AAs rescues cell proliferation and compensates for impaired uptake of CD98hc/LAT1 and CD98hc/y(+)LAT2. Our data establish CD98hc as a master protective gene at the cross-road of redox control and AA availability, making it a relevant therapeutic target in cancer., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

4F2hc (CD98hc) is a multifunctional type II membrane glycoprotein involved in amino acid transport and cell fusion, adhesion, and transformation. The structure of the ectodomain of human 4F2hc has been solved using monoclinic (Protein Data Bank code 2DH2) and orthorhombic (Protein Data Bank code 2DH3) crystal forms at 2.1 and 2.8 A, respectively. It is composed of a (betaalpha)(8) barrel and an antiparallel beta(8) sandwich related to bacterial alpha-glycosidases, although lacking key catalytic residues and consequently catalytic activity. 2DH3 is a dimer with Zn(2+) coordination at the interface. Human 4F2hc expressed in several cell types resulted in cell surface and Cys(109) disulfide bridge-linked homodimers with major architectural features of the crystal dimer, as demonstrated by cross-linking experiments. 4F2hc has no significant hydrophobic patches at the surface. Monomer and homodimer have a polarized charged surface. The N terminus of the solved structure, including the position of Cys(109) residue located four residues apart from the transmembrane domain, is adjacent to the positive face of the ectodomain. This location of the N terminus and the Cys(109)-intervening disulfide bridge imposes space restrictions sufficient to support a model for electrostatic interaction of the 4F2hc ectodomain with membrane phospholipids. These results provide the first crystal structure of heteromeric amino acid transporters and suggest a dynamic interaction of the 4F2hc ectodomain with the plasma membrane.

The matrix metalloproteinases (MMPs) play a key role in normal and pathological angiogenesis by mediating extracellular matrix degradation and/or controlling the biological activity of growth factors, chemokines, and/or cytokines. Specific functions of individual MMPs as anti- or proangiogenic mediators remain to be elucidated. In the present study, we assessed the impact of single or combined MMP deficiencies in in vivo and in vitro models of angiogenesis (malignant keratinocyte transplantation and the aortic ring assay, respectively). MMP-9 was predominantly expressed by neutrophils in tumor transplants, whereas MMP-2 and MMP-3 were stromal. Neither the single deficiency of MMP-2, MMP-3, or MMP-9, nor the combined absence of MMP-9 and MMP-3 did impair tumor invasion and vascularization in vivo. However, there was a striking cooperative effect in double MMP-2:MMP-9-deficient mice as demonstrated by the absence of tumor vascularization and invasion. In contrast, the combined lack of MMP-2 and MMP-9 did not impair the in vitro capillary outgrowth from aortic rings. These results point to the importance of a cross talk between several host cells for the in vivo tumor promoting and angiogenic effects of MMP-2 and MMP-9. Our data demonstrate for the first time in an experimental model that MMP-2 and MMP-9 cooperate in promoting the in vivo invasive and angiogenic phenotype of malignant keratinocytes.