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Fig.3.A showing Western Blotting from the publication: Tollip coordinates Parkin-dependent trafficking of mitochondrial-derived vesicles.

Fig.5.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: Vacuolar H+-ATPase Subunit V0C Regulates Aerobic Glycolysis of Esophageal Cancer Cells via PKM2 Signaling.

Fig.2.B showing Immunocytochemistry-immunofluorescence from the publication: Targeting Heparan Sulfate Proteoglycans as a Novel Therapeutic Strategy for Mucopolysaccharidoses.

Fig.1.N showing Immunocytochemistry in a Homo sapiens (Human) sample from the publication: Mitosis can drive cell cannibalism through entosis.

4 images found

VPS41 recruits biosynthetic LAMP-positive vesicles through interaction with Arl8b.

In The Journal of Cell Biology on 3 April 2025 by Sanzà, P., van der Beek, J., et al.

Vacuolar protein sorting 41 (VPS41), a component of the homotypic fusion and protein sorting (HOPS) complex for lysosomal fusion, is essential for the trafficking of lysosomal membrane proteins via lysosome-associated membrane protein (LAMP) carriers from the trans-Golgi network (TGN) to endo/lysosomes. However, the molecular mechanisms underlying this pathway and VPS41's role herein remain poorly understood. Here, we investigated the effects of ectopically localizing VPS41 to mitochondria on LAMP distribution. Using electron microscopy, we identified that mitochondrial-localized VPS41 recruited LAMP1- and LAMP2A-positive vesicles resembling LAMP carriers. The retention using selective hooks (RUSH) system further revealed that newly synthesized LAMPs were specifically recruited by mitochondrial VPS41, a function not shared by other HOPS subunits. Notably, we identified the small GTPase Arl8b as a critical factor for LAMP carrier trafficking. Arl8b was present on LAMP carriers and bound to the WD40 domain of VPS41, enabling their recruitment. These findings reveal a unique role of VPS41 in recruiting TGN-derived LAMP carriers and expand our understanding of VPS41-Arl8b interactions beyond endosome-lysosome fusion, providing new insights into lysosomal trafficking mechanisms.
© 2025 Sanzà et al.

Neutrophil extracellular traps promote tumor chemoresistance to anthracyclines

Preprint on BioRxiv : the Preprint Server for Biology on 11 November 2024 by Lin, C., Herlihy, S. E., et al.

The microenvironment plays an important role in promoting tumor cell chemoresistance, but the mechanisms responsible for this effect are not clear. Here, using models of multiple myeloma (MM) and solid cancers, we demonstrate a novel mechanism mediated by neutrophils, a major cell population in the bone marrow (BM), that protects cancer cells from chemotherapeutics. We show that in response to tumor-derived soluble factors, BM neutrophils release their DNA in the form of neutrophil extracellular traps (NETs). Cell-free DNA derived from NETs is then taken up by tumor cells via endocytosis and localizes to the cytoplasm. We found that both NETs and cell-free DNA taken up by tumor cells can bind anthracyclines, leading to tumor cell resistance to this class of chemotherapeutic agents. Targeting cell-free DNA with Pulmozyme or blocking NET formation with a PAD4 inhibitor abrogates the chemoprotective effect of neutrophils and restores sensitivity of tumor cells to anthracyclines.

NKX3-2 Induces Ovarian Cancer Cell Migration by HDAC6-Mediated Repositioning of Lysosomes and Inhibition of Autophagy.

In Cells on 4 November 2024 by Ferraresi, A., Ghezzi, I., et al.

Several soluble factors secreted by the stromal cells and cancer cells within the tumor microenvironment facilitate the progression and invasiveness of ovarian cancer. In ovarian cancer cells, lysophosphatidic acid (LPA) modulates the transcriptome profile and promotes cell invasiveness by the downregulation of autophagy. Here, we further elucidate this mechanism by focusing on the molecular and cellular events regulating autophagy. Transcriptomic and Western blotting analyses revealed NKX3-2, a transcriptional factor, to be among the genes hyperexpressed in LPA-stimulated ovarian cancer cells. Bioinformatic analyses revealed that in ovarian cancer patients, the expression of NKX3-2 positively correlates with genes involved in cell motility and migration, while it negatively correlates with macromolecular catabolic pathways. In various ovarian cancer cell lines, NKX3-2 silencing abrogated LPA-induced cell migration. Mechanistically, this effect is linked to the restoration of the HDAC6-mediated relocation of the lysosomes in the para-golgian area, and this results in an increase in autolysosome formation and the overall upregulation of autophagy. Silencing the expression of ATG7 or BECN1, two autophagy genes, rescued the migratory phenotype of the NKX3-2-silenced ovarian cancer cells. Taken together, these data reveal the mechanism by which the LPA-NKX3-2 axis promotes the invasiveness of ovarian cancer cells and supports the possibility of targeting NKX3-2 to reduce the migratory capacity of cancer cells in response to a permissive microenvironment.

Migfilin promotes autophagic flux through direct interaction with SNAP29 and Vamp8.

In The Journal of Cell Biology on 4 November 2024 by Cai, R., Bai, P., et al.

Autophagy plays a crucial role in cancer cell survival by facilitating the elimination of detrimental cellular components and the recycling of nutrients. Understanding the molecular regulation of autophagy is critical for developing interventional approaches for cancer therapy. In this study, we report that migfilin, a focal adhesion protein, plays a novel role in promoting autophagy by increasing autophagosome-lysosome fusion. We found that migfilin is associated with SNAP29 and Vamp8, thereby facilitating Stx17-SNAP29-Vamp8 SNARE complex assembly. Depletion of migfilin disrupted the formation of the SNAP29-mediated SNARE complex, which consequently blocked the autophagosome-lysosome fusion, ultimately suppressing cancer cell growth. Restoration of the SNARE complex formation rescued migfilin-deficiency-induced autophagic flux defects. Finally, we found depletion of migfilin inhibited cancer cell proliferation. SNARE complex reassembly successfully reversed migfilin-deficiency-induced inhibition of cancer cell growth. Taken together, our study uncovers a new function of migfilin as an autophagy-regulatory protein and suggests that targeting the migfilin-SNARE assembly could provide a promising therapeutic approach to alleviate cancer progression.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

IER3IP1-mutations cause microcephaly by selective inhibition of ER-Golgi transport.

In Cellular and Molecular Life Sciences : CMLS on 8 August 2024 by Anitei, M., Bruno, F., et al.

Mutations in the IER3IP1 (Immediate Early Response-3 Interacting Protein 1) gene can give rise to MEDS1 (Microcephaly with Simplified Gyral Pattern, Epilepsy, and Permanent Neonatal Diabetes Syndrome-1), a severe condition leading to early childhood mortality. The small endoplasmic reticulum (ER)-membrane protein IER3IP1 plays a non-essential role in ER-Golgi transport. Here, we employed secretome and cell-surface proteomics to demonstrate that the absence of IER3IP1 results in the mistrafficking of proteins crucial for neuronal development and survival, including FGFR3, UNC5B and SEMA4D. This phenomenon correlates with the distension of ER membranes and increased lysosomal activity. Notably, the trafficking of cargo receptor ERGIC53 and KDEL-receptor 2 are compromised, with the latter leading to the anomalous secretion of ER-localized chaperones. Our investigation extended to in-utero knock-down of Ier3ip1 in mouse embryo brains, revealing a morphological phenotype in newborn neurons. In summary, our findings provide insights into how the loss or mutation of a 10 kDa small ER-membrane protein can cause a fatal syndrome.
© 2024. The Author(s).

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Tollip coordinates Parkin-dependent trafficking of mitochondrial-derived vesicles.

In EMBO J on 2 June 2020 by Ryan, T. A., Phillips, E. O., et al.

Fig.3.A

Tollip and Parkin are in complex in a CUE domain‐dependent manner, but independent of autophagy and Tom1HeLa cells expressing mycBioID–Tollip and HA‐Parkin were either left untreated or treated with 5 μM antimycin A/10 μM oligomycin (AO) or AO/100 μM bafilomycin A (BfnA1) for 6 h in the presence of biotin. Cells were lysed and streptavidin pulldowns performed overnight to isolate biotinylated proteins. Proteins in whole‐cell extracts and pulldowns from each condition were then separated by SDS–PAGE and membranes probed with specific antibodies. Immunoblotting for Tom1 and mycBioID–Tollip was used as positive controls. Cells cultured in media lacking biotin were used as a negative control to assess background levels. Biotinylation of Parkin suggested Tollip specifically interacted with Parkin under these conditions.HeLa cells expressing mycBioID alone and HA‐Parkin were used to confirm the specificity of the Tollip–Parkin interaction.HeLa cells coexpressing GFP‐Tollip and HA‐Parkin were either left untreated or treated with AO for 2 h prior to lysis. Cell lysates were subjected to either a normal rabbit IgG IP or HA IP, and Western blot analysis was performed using antibodies against Parkin and GFP.HeLa cells expressing mycBioID–Tollip containing the CUE domain mutation (CUEmut) and HA‐Parkin were subjected to biotinylation and streptavidin pulldowns, followed by Western blot analysis.HeLa wild type (WT), Tom1 KO and ATG5 KO cells expressing mycBioID–Tollip and HA‐Parkin underwent the same conditions as those above and immunoblotted for the indicated proteins.Source data are available online for this figure. less

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Vacuolar H+-ATPase Subunit V0C Regulates Aerobic Glycolysis of Esophageal Cancer Cells via PKM2 Signaling.

In Cells on 24 September 2019 by Son, S. W., Chau, G. C., et al.

Fig.5.B

ATP6V0C increases expression of glycolytic genes by inducing nuclear translocation of PKM2. (A) TE8 cells were immunostained with PKM2 antibody (PKM2, green). The nucleus is marked with DAPI (blue). Merged images (Merge) are shown. Fluorescenceintensity was shown in linear histogram. The intensity was divided into two groups: PKM2 fluorescence intensity is plotted on the green peak, and DAPI intensity is plotted on the blue peak. The line profiles were placed across cells, and the simultaneously occurring intensity peaks were evaluated. The line profiles of PKM2 and DAPI signals were measured by ZEN 2011 software (Carl Zeiss). Scale bars = 10 μm. (B) Nuclear and cytosolic lysates were prepared from ATP6V0C-depleted TE8 cells, followed by immunoblotting. (C) Recruitment of PKM2 by ATP6V0C at the HRE site of the indicated promoter. TE8 cells were transfected with NS or si-V0C and subjected to ChIP assays. Each data point is the average of three independent measurements. Values are mean ± SEM. (Student’s t-test; * p < 0.05). (D) Frequency of copy number alterations and mutations of ATP6V0C across several tumor types. (E) Proposed model of how ATP6V0C mediates metabolic programming in ECCs. ATP6V0C associates with PKM2 to enhance its nuclear localization and increases PKM2 phosphorylation, inducing transcriptional expression of glycolytic genes and thus facilitating glycolysis and motility in ECCs. less

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Mitosis can drive cell cannibalism through entosis.

In Elife on 11 July 2017 by Durgan, J., Tseng, Y. Y., et al.

Fig.1.N

Cdc42 controls entosis in adherent epithelial cells.(a) Control and siRNA or shRNA Cdc42-depleted 16HBE cell lysates were probed for Cdc42 and GAPDH expression by western blotting. (b) Representative confocal images of control and Cdc42-depleted 16HBE monolayers stained for β-catenin (adherens junctions), ZO-1 (tight junctions) and DNA. Scale bar = 20 μm. (c) Representative images of cell-in-cell structures formed in matrix detached control and Cdc42-depleted 16HBE cells. Cell were stained for DNA (blue) and imaged by IF/confocal and DIC. Scale bar = 5 μm. (d) Quantification of suspension cell-in-cell formation in control and Cdc42-depleted cells. >200 cells were counted per sample/experiment, across three separate experiments. Error bars denote mean±SEM. ns = no significant difference, t-test. (e) Representative images of a cell-in-cell structure formed under adherent conditions in Cdc42-depleted 16HBE cells. Cells were stained for cell body (green) and DNA (blue) and imaged by IF/confocal and DIC. Scale bar = 10 μm. (f) Quantification of adherent cell-in-cell formation. >200 16HBE cells were counted per sample/experiment, across three separate experiments. Error bars denote mean±SEM. **p<0.002; ***p<0.0002; ****p<0.0001, t-test. (g) Control and shRNA Cdc42-depleted MCF7 cell lysates were probed for Cdc42 and GAPDH expression by western blotting. (h) Representative images of a cell-in-cell structure formed under adherent conditions in Cdc42-depleted MCF7 cells. Cells were stained for cell body (green) and DNA (blue) and imaged by IF/confocal and DIC. Scale bar = 10 μm. (i) Quantification of adherent cell-in-cell formation. >200 MCF7 cells were counted per sample/experiment, across three separate experiments. Error bars denote mean±SEM. *p<0.02, t-test. (j) Lysates from 16HBE cells co-depleted of Cdc42 and α-catenin (aCat) were probed for α-catenin, Cdc42 and GAPDH by western blotting. (k) Representative confocal images of 16HBE cells co-depleted of Cdc42 and siControl or α-catenin and stained for β-catenin (green) and DNA (blue). Scale bar = 20 μm. (l) Quantification of cell-in-cell structures in adherent 16HBE cells treated with siCdc42 and siControl or siα-catenin, treated −/+10 μM Y-27632 (ROCKi), for 3 days. >200 cells were scored per sample/experiment, across three separate experiments. Error bars denote mean±SEM. **p<0.002; ***p<0.0002, t-test. (m) Confocal images of a forming cell-in-cell structure in adherent, Cdc42-depleted 16HBE cells fixed and costained for pMLC2 (S19; green), β-catenin (red) and DNA (Hoechst, blue). The arrowhead indicates the tail of the internalising cell. Scale bar = 10 μm. (n) Cell-in-cell structures in Cdc42-depleted 16HBE cells were fixed and costained for LC3 (green), LAMP1 (red) and DNA (blue), and imaged by IF/confocal and DIC. The arrowhead indicates a dying internalised cell. Scale bar = 10 μm.DOI:http://dx.doi.org/10.7554/eLife.27134.00310.7554/eLife.27134.004Figure 1—source data 1.DOI:http://dx.doi.org/10.7554/eLife.27134.004DOI:http://dx.doi.org/10.7554/eLife.27134.004 less

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