The identification and characterization of noncanonical functions within the autophagy pathway have unveiled intricate cellular processes, including LC3-associated phagocytosis (LAP) and LC3-associated endocytosis (LANDO). These phenomena play pivotal roles in the conjugation of ATG8 with single-membrane phagosomes and endosomes, shedding light on the dynamic interplay between autophagy and cellular homeostasis. Here, we present detailed protocols for both qualitative and quantitative assessment of LAP, including immunofluorescence, flow cytometry, and Western blotting of isolated LAPosomes. Additionally, the protocol for the evaluation of LANDO through immunofluorescent detection of receptor recycling is outlined. The methodologies presented herein serve as a practical guide for researchers seeking to unravel the intricacies of LAP and LANDO. By providing step-by-step instructions, accompanied by insights into potential challenges and optimization strategies, this chapter aims to empower investigators in the exploration of these noncanonical functions of autophagy proteins.

Selective retrograde transport from endosomes back to the trans-Golgi network (TGN) is important for maintaining protein homeostasis, recycling receptors, and returning molecules that were transported to the wrong compartments. Two important transmembrane proteins directed to this pathway are the Cation-Independent Mannose-6-phosphate receptor (CI-MPR) and the ATP7B copper transporter. Among CI-MPR functions is the delivery of acid hydrolases to lysosomes, while ATP7B facilitates the transport of cytosolic copper ions into organelles or the extracellular space. Precise subcellular localization of CI-MPR and ATP7B is essential for the proper functioning of these proteins. This study shows that both CI-MPR and ATP7B interact with a variant of the clathrin adaptor 1 (AP-1) complex that contains a specific isoform of the γ-adaptin subunit called γ2. Through synchronized anterograde trafficking and cell-surface uptake assays, we demonstrated that AP-1γ2 is dispensable for ATP7B and CI-MPR exit from the TGN while being critically required for ATP7B and CI-MPR retrieval from endosomes to the TGN. Moreover, AP-1γ2 depletion leads to the retention of endocytosed CI-MPR in endosomes enriched in retromer complex subunits. These data underscore the importance of AP-1γ2 as a key component in the sorting and trafficking machinery of CI-MPR and ATP7B, highlighting its essential role in the transport of proteins from endosomes.

OBJECTIVE: Breast cancer (BC) accounts for roughly 30% of new cancers diagnosed in women each year; thus, this cancer type represents a substantial burden for people and health care systems. Despite the existence of effective therapies to treat BC, drug resistance remains a problem and is a major cause of treatment failure. Therefore, new drugs and treatment regimens are urgently required to overcome resistance. Recent research indicates that inhibition of the endosomal recycling pathway, an intracellular membrane trafficking pathway that returns endocytosed proteins back to the plasma membrane, may be a promising strategy to downregulate clinically relevant cell surface proteins such as HER2 and HER3, and to overcome drug resistance.
METHODS: To investigate the molecular mechanism of action of an endosomal recycling inhibitor (ERI) called primaquine, we performed a reverse-phase protein array (RPPA) assay using a HER2-positive breast cancer cell line. The RPPA findings were confirmed by Western blot and RT-qPCR in several BC cell lines. Novel drug combinations were tested by MTT cell viability and clonogenic assays.
RESULTS: Among the signalling molecules downregulated by ERIs were estrogen receptor-alpha (ER-α) and androgen receptor. We confirmed this finding in other breast cancer cell lines and show that downregulation occurs at the transcriptional level. We also found that ERIs synergise with tamoxifen, a standard-of-care therapy for breast cancer.
CONCLUSIONS: Our data suggest that combining ERIs with hormone receptor antagonists may enhance their efficacy and reduce the emergence of drug resistance.

BACKGROUND: Apolipoprotein E4 (APOE4) is the most prevalent genetic risk factor of Alzheimer\'s disease. Several studies suggest that APOE4 binding to its receptors is associated with their internalization and accumulation in intracellular compartments. Importantly, this phenomenon also occurs with other, non-ApoE receptors. Based on these observations, we hypothesized that APOE4 pathological effects are mediated by impairment in the life cycle of distinct receptors (APOER2, LRP1, IR, VEGFR).
OBJECTIVE: To examine the effects of APOE genotype on receptors protein levels and compartmentalization.
METHODS: Primary mouse neurons were prepared from APOE3 or APOE4 targeted replacement mice, or APOE-KO mice. Specific receptors protein levels were evaluated in these neurons, utilizing immunofluorescent staining. Additionally, surface membrane protein levels of those receptors were assessed by cell surface biotinylation assay and ELISA. Receptors\' colocalization with intracellular compartments was assessed by double staining and confocal microscopy, followed by colocalization analysis. Finally, LRP1 or APOER2 were knocked-down with CRISPR/Cas9 system to examine their role in mediating APOE4 effects on the receptors.
RESULTS: Our results revealed lower receptors\' levels in APOE4, specifically on the membrane surface. Additionally, APOE4 affects the compartmentation of these receptors in two patterns: the first was observed with LRP1 and was associated with decreased receptor levels in numerous intracellular compartments. The second was obtained with the other receptors and was associated with their accumulation in early endosomes and their decrease in the late endosomes.
CONCLUSIONS: These results provide a unifying mechanism, in which APOE4 drives the down regulation of various receptors, which plays important roles in distinct APOE4 related pathological processes.

Prostate cancer is the second most frequent cancer diagnosed in men, and accounts for one-fifth of cancer associated deaths worldwide. Despite the availability of effective prostate cancer therapies, if it is not cured by radical local treatment, progression to drug resistant metastatic prostate cancer is inevitable. Therefore, new drugs and treatment regimens are urgently required to overcome resistance. We have recently published research demonstrating that targeting the endosomal recycling pathway, a membrane transport pathway that recycles internalised cell surface proteins back to the plasma membrane, may be a novel means to downregulate clinically relevant cell surface proteins and potentially overcome drug resistance. A reverse phase protein array (RPPA) assay of breast cancer cells treated with an endosomal recycling inhibitor identified the androgen receptor (AR) as one of the top downregulated proteins. We confirmed that endosomal recycling inhibitors also downregulated AR in prostate cancer cells and show that this occurs at the transcriptional level. We also found that endosomal recycling inhibitors synergise with enzalutamide, a standard-of-care therapy for prostate cancer. Our data suggest that combining recycling inhibitors with hormone receptor antagonists may enhance their efficacy and reduce the emergence of drug resistance.

Peroxisomes play important roles in fungal physiological processes. The RING-finger complex consists of peroxins Pex2, Pex10, and Pex12 and is essential for recycling of receptors responsible for peroxisomal targeting of matrix proteins. In this study, these three peroxins were functionally characterized in the entomopathogenic fungus Beauveria bassiana (Bb). These three peroxins are associated with peroxisomes, in which BbPex2 interacted with BbPex10 and BbPex12. Ablation of these peroxins did not completely block the peroxisome biogenesis, but abolish peroxisomal targeting of matrix proteins via both PTS1 and PTS2 pathways. Three disruptants displayed different phenotypic defects in growth on nutrients and under stress conditions, but have similar defects in acetyl-CoA biosynthesis, development, and virulence. Strikingly, BbPex10 played a less important role in fungal growth on tested nutrients than other two peroxins; whereas, BbPex2 performed a less important contribution to fungal growth under stresses. This investigation reinforces the peroxisomal roles in the lifecycle of entomopathogenic fungi and highlights the unequal functions of different peroxins in peroxisomal biology.

Aberrant glycosylation is a hallmark of a cancer cell. One prevalent alteration is an enrichment in α2,6-linked sialylation of N-glycosylated proteins, a modification directed by the ST6GAL1 sialyltransferase. ST6GAL1 is upregulated in many malignancies including ovarian cancer. Prior studies have shown that the addition of α2,6 sialic acid to the epidermal growth factor receptor (EGFR) activates this receptor, although the mechanism was largely unknown. To investigate the role of ST6GAL1 in EGFR activation, ST6GAL1 was overexpressed in the OV4 ovarian cancer line, which lacks endogenous ST6GAL1, or knocked-down in the OVCAR-3 and OVCAR-5 ovarian cancer lines, which have robust ST6GAL1 expression. Cells with high expression of ST6GAL1 displayed increased activation of EGFR and its downstream signaling targets, AKT and NFκB. Using biochemical and microscopy approaches, including total internal reflection fluorescence microscopy, we determined that the α2,6 sialylation of EGFR promoted its dimerization and higher order oligomerization. Additionally, ST6GAL1 activity was found to modulate EGFR trafficking dynamics following EGF-induced receptor activation. Specifically, EGFR sialylation enhanced receptor recycling to the cell surface following activation while simultaneously inhibiting lysosomal degradation. 3D widefield deconvolution microscopy confirmed that in cells with high ST6GAL1 expression, EGFR exhibited greater colocalization with Rab11 recycling endosomes and reduced colocalization with LAMP1-positive lysosomes. Collectively, our findings highlight a novel mechanism by which α2,6 sialylation promotes EGFR signaling by facilitating receptor oligomerization and recycling.

Sorting nexins (SNX) are a family of proteins containing the Phox homology domain, which shows a preferential endo-membrane association and regulates cargo sorting processes. Here, we established that SNX32, an SNX-BAR (Bin/Amphiphysin/Rvs) sub-family member associates with SNX4 via its BAR domain and the residues A226, Q259, E256, R366 of SNX32, and Y258, S448 of SNX4 that lie at the interface of these two SNX proteins mediate this association. SNX32, via its PX domain, interacts with the transferrin receptor (TfR) and Cation-Independent Mannose-6-Phosphate Receptor (CIMPR), and the conserved F131 in its PX domain is important in stabilizing these interactions. Silencing of SNX32 leads to a defect in intracellular trafficking of TfR and CIMPR. Further, using SILAC-based differential proteomics of the wild-type and the mutant SNX32, impaired in cargo binding, we identified Basigin (BSG), an immunoglobulin superfamily member, as a potential interactor of SNX32 in SHSY5Y cells. We then demonstrated that SNX32 binds to BSG through its PX domain and facilitates its trafficking to the cell surface. In neuroglial cell lines, silencing of SNX32 leads to defects in neuronal differentiation. Moreover, abrogation in lactate transport in the SNX32-depleted cells led us to propose that SNX32 may contribute to maintaining the neuroglial coordination via its role in BSG trafficking and the associated monocarboxylate transporter activity. Taken together, our study showed that SNX32 mediates the trafficking of specific cargo molecules along distinct pathways.

The Δ133p53β isoform is increased in many primary tumors and has many tumor-promoting properties that contribute to increased proliferation, migration and inflammation. Here we investigated whether Δ133p53β contributed to some of the most aggressive tumors that had metastasized to the brain. Δ133p53β mRNA expression was measured in lung, breast, melanoma, colorectal metastases and, where available, the matched primary tumor. The presence of Δ133p53β expression was associated with the time for the primary tumor to metastasize and overall survival once the tumor was detected in the brain. Δ133p53β was present in over 50% of lung, breast, melanoma and colorectal metastases to the brain. It was also increased in the brain metastases compared with the matched primary tumor. Brain metastases with Δ133p53β expressed were associated with a reduced time for the primary tumor to metastasize to the brain compared with tumors with no Δ133p53β expression. In-vitro-based analyses in Δ133p53β-expressing cells showed increased cancer-promoting proteins on the cell surface and increased downstream p-AKT and p-MAPK signaling. Δ133p53β-expressing cells also invaded more readily across a mock blood-brain barrier. Together these data suggested that Δ133p53β contributes to brain metastases by making cells more likely to invade the brain.

[This corrects the article DOI: 10.3389/fcell.2021.733688.].