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Lysosome-associated membrane proteins (LAMPs) play a critical role in various cellular processes, including phagocytosis, lipid transport, neoangiogenesis, and tissue remodeling. Recent discussions have focused on their involvement in autophagy related to tumor progression. Emerging studies have underscored the connection between tumorigenesis and autophagy, highlighting that dysregulation of this process is associated with the resistance of tumor cells to conventional chemotherapy across numerous malignancies, including colorectal cancer (CRC). Currently, there is a notable absence of reliable prognostic biomarkers for effectively stratifying and predicting treatment responses in CRC patients. Our study examines the expression of LAMP1 and LAMP2 proteins and genes alongside key autophagy markers such as BECLIN1 and LC3B, investigating their relationships with CRC invasiveness. We present original data illustrating the association of LAMP molecules with standard autophagy markers and tumor budding. Our findings provide novel insights into the significance of these markers in CRC invasiveness, their relationship with survival rates, and their potential role as prognostic biomarkers.

Upregulation of autophagy in acute myeloid leukemia (AML) cells contributes to the development of resistance to cytarabine (AraC). LAPTM5 is mainly expressed in hematopoietic and immune cells, and has been associated with the progression of multiple cancers; however, its role in AML drug resistance remains uncharacterized. Here, we reanalyzed publicly available single-cell RNA sequencing (scRNA-seq) data from AML patients and found distinct gene expression profiles between AraC-resistant AML cells and untreated controls. Differentially expressed genes were significantly enriched in lysosome-related pathways, with LAPTM5 being highly expressed in drug-resistant cells, suggesting that it may be a key mediator of AraC resistance in AML. Mechanistically, AraC-resistant cells exhibited enhanced autophagic flux supported by LAPTM5-mediated upregulation of LAMP1 and LAMP2. Conversely, LAPTM5 knockdown impaired autophagolysosome formation by disrupting lysosomal biogenesis, thereby sensitizing resistant cells to AraC. These findings indicate that targeting LAPTM5 could enhance AraC sensitivity in AML by modulating autophagy. In vivo experiments further confirmed that the depletion of LAPTM5 inhibited tumor growth and synergistically suppressed AML progression with AraC. Collectively, our study identifies LAPTM5 as a critical regulator of AraC resistance via autophagy modulation, highlighting its potential as a therapeutic target for AML. In AML, AraC treatment induces LAPTM5 upregulation, which promotes LAMP1/2 transcription and lysosomal biogenesis. This facilitates autophagolysosome formation and enhances autophagic flux to reduce AraC-induced apoptosis, resulting in drug resistance. Targeting LAPTM5 represents a promising strategy to overcome this autophagy-mediated resistance. Highlights Single-cell analysis identified LAPTM5 as a lysosomal transmembrane protein that drives intrinsic AraC resistance in AML. LAPTM5 promotes lysosome-dependent autophagy, thereby conferring AraC resistance in AML cells. Targeting LAPTM5 disrupts autophagy and acts synergistically with AraC to suppress AML progression.

Lysosome‐associated membrane proteins 1 and 2 (LAMP‐1 and LAMP‐2) are delivered to phagosomes during the maturation process. We used cells from LAMP‐deficient mice to analyze the role of these proteins in phagosome maturation. Macrophages from LAMP‐1‐ or LAMP‐2‐deficient mice displayed normal fusion of lysosomes with phagosomes. Because ablation of both the lamp‐1 and lamp‐2 genes yields an embryonic‐lethal phenotype, we were unable to study macrophages from double knockouts. Instead, we reconstituted phagocytosis in murine embryonic fibroblasts (MEFs) by transfection of FcγIIA receptors. Phagosomes formed by FcγIIA‐transfected MEFs obtained from LAMP‐1‐ or LAMP‐2‐ deficient mice acquired lysosomal markers. Remarkably, although FcγIIA‐transfected MEFs from double‐deficient mice ingested particles normally, phagosomal maturation was arrested. LAMP‐1 and LAMP‐2 double‐deficient phagosomes acquired Rab5 and accumulated phosphatidylinositol 3‐phosphate, but failed to recruit Rab7 and did not fuse with lysosomes. We attribute the deficiency to impaired organellar motility along microtubules. Time‐lapse cinematography revealed that late endosomes/lysosomes as well as phagosomes lacking LAMP‐1 and LAMP‐2 had reduced ability to move toward the microtubule‐organizing center, likely precluding their interaction with each other.

Toll-like receptors (TLR) are essential components of the innate immune system. Several accessory proteins, such as UNC93B1, are required for transport and activation of nucleic acid sensing Toll-like receptors in endosomes. Here, we show that BAD-LAMP (LAMP5) controls TLR9 trafficking to LAMP1+ late endosomes in human plasmacytoid dendritic cells (pDC), leading to NF-κB activation and TNF production upon DNA detection. An inducible VAMP3+/LAMP2+/LAMP1- endolysosome compartment exists in pDCs from which TLR9 activation triggers type I interferon expression. BAD-LAMP-silencing enhances TLR9 retention in this compartment and consequent downstream signalling events. Conversely, sustained BAD-LAMP expression in pDCs contributes to their lack of type I interferon production after exposure to a TGF-β-positive microenvironment or isolation from human breast tumours. Hence, BAD-LAMP limits interferon expression in pDCs indirectly, by promoting TLR9 sorting to late endosome compartments at steady state and in response to immunomodulatory cues.TLR9 is highly expressed by plasmacytoid dendritic cells and detects nucleic acids, but to discriminate between host and microbial nucleic acids TLR9 is sorted into different endosomal compartments. Here the authors show that BAD-LAMP limits type 1 interferon responses by sorting TLR9 to late endosomal compartments.

Cellular organelles and proteins are degraded and recycled through autophagy, a process during which vesicles known as autophagosomes fuse with lysosomes. Altered autophagy occurs in various diseases, but its role in preterm labor (PTL) is unknown. We investigated the role of autophagic flux in two mouse models of PTL compared to controls: 1) inflammation-induced PTL (IPTL), induced by toll-like receptor agonists; and 2) non-inflammation (hormonally)-induced PTL (NIPTL). We demonstrate that the autophagy related genes Atg4c and Atg7 (involved in the lipidation of microtubule-associated protein 1 light chain 3 (LC3) B-I to the autophagosome-associated form, LC3B-II) decrease significantly in uterus and placenta during IPTL but not NIPTL. Autophagic flux is altered in IPTL, as shown by the accumulation of LC3B paralogues and diminishment of lysosome associated membrane protein (LAMP)-1, LAMP-2 and the a2 isoform of V-ATPase (a2V, an enzyme involved in lysosome acidification). These alterations in autophagy are associated with increased activation of NF-κB and proinflammatory cytokines/chemokines in both uterus and placenta. Similar changes are seen in macrophages exposed to TLR ligands and are enhanced with blockade of a2V. These novel findings represent the first evidence of an association between altered autophagic flux and hyper-inflammation and labor in IPTL.

Neonatal interstitial lung diseases due to abnormal surfactant biogenesis are rare in humans and have never been reported as a spontaneous disorder in animals. We describe here a novel lung disorder in Airedale Terrier (AT) dogs with clinical symptoms and pathology similar to the most severe neonatal forms of human surfactant deficiency. Lethal hypoxic respiratory distress and failure occurred within the first days or weeks of life in the affected puppies. Transmission electron microscopy of the affected lungs revealed maturation arrest in the formation of lamellar bodies (LBs) in the alveolar epithelial type II (AECII) cells. The secretory organelles were small and contained fewer lamellae, often in combination with small vesicles surrounded by an occasionally disrupted common limiting membrane. A combined approach of genome-wide association study and whole exome sequencing identified a recessive variant, c.1159G>A, p.(E387K), in LAMP3, a limiting membrane protein of the cytoplasmic surfactant organelles in AECII cells. The substitution resides in the LAMP domain adjacent to a conserved disulfide bond. In summary, this study describes a novel interstitial lung disease in dogs, identifies a new candidate gene for human surfactant dysfunction and brings important insights into the essential role of LAMP3 in the process of the LB formation.

Summary The last step of Leishmania intracellular life cycle is the egress of amastigotes from the host cell and their uptake by adjacent cells. Using multidimensional live imaging of long‐term‐infected macrophage cultures we observed that Leishmania amazonensis amastigotes were transferred from cell to cell when the donor host macrophage delivers warning signs of imminent apoptosis. They were extruded from the macrophage within zeiotic structures (membrane blebs, an apoptotic feature) rich in phagolysosomal membrane components. The extrusions containing amastigotes were selectively internalized by vicinal macrophages and the rescued amastigotes remain viable in recipient macrophages. Host cell apoptosis induced by micro‐irradiation of infected macrophage nuclei promoted amastigotes extrusion, which were rescued by non‐irradiated vicinal macrophages. Using amastigotes isolated from LAMP1/LAMP2 knockout fibroblasts, we observed that the presence of these lysosomal components on amastigotes increases interleukin 10 production. Enclosed within host cell membranes, amastigotes can be transferred from cell to cell without full exposure to the extracellular milieu, what represents an important strategy developed by the parasite to evade host immune system.

Self-labeling protein tags such as HaloTag are powerful tools that can label fusion proteins with synthetic fluorophores for use in fluorescence microscopy. Here we introduce HaloTag variants with either increased or decreased brightness and fluorescence lifetime compared with HaloTag7 when labeled with rhodamines. Combining these HaloTag variants enabled live-cell fluorescence lifetime multiplexing of three cellular targets in one spectral channel using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. Additionally, the brightest HaloTag variant showed up to 40% higher brightness in live-cell imaging applications. HaloTag variants offer distinct brightness and fluorescence lifetimes compared with HaloTag7 when labeled with rhodamines. These variants were used for multiplexed imaging with a single fluorophore and to create lifetime-based cell cycle indicators.

The intracellular transport of cholesterol is subject to tight regulation. The structure of the lysosomal integral membrane protein type 2 (LIMP-2, also known as SCARB2) reveals a large cavity that traverses the molecule and resembles the cavity in SR-B1 that mediates lipid transfer. The detection of cholesterol within the LIMP-2 structure and the formation of cholesterol − like inclusions in LIMP-2 knockout mice suggested the possibility that LIMP2 transports cholesterol in lysosomes. We present results of molecular modeling, crosslinking studies, microscale thermophoresis and cell-based assays that support a role of LIMP-2 in cholesterol transport. We show that the cavity in the luminal domain of LIMP-2 can bind and deliver exogenous cholesterol to the lysosomal membrane and later to lipid droplets. Depletion of LIMP-2 alters SREBP-2-mediated cholesterol regulation, as well as LDL-receptor levels. Our data indicate that LIMP-2 operates in parallel with Niemann Pick (NPC)-proteins, mediating a slower mode of lysosomal cholesterol export. Cholesterol transport is tightly regulated in the cell and in lysosomes is regulated by NPC1/2. Here, Heybrock et al. use molecular modeling, knockout mice and cell based studies to show that LIMP-2 also mediates lysosomal cholesterol transport.

Trypanosoma cruzi enters host cells by subverting the mechanism of cell membrane repair. In this process, the parasite induces small injuries in the host cell membrane leading to calcium entry and lysosomal exocytosis, which are followed by compensatory endocytosis events that drive parasites into host cells. We have previously shown that absence of both LAMP-1 and 2, major components of lysosomal membranes, decreases invasion of T. cruzi into host cells, but the mechanism by which they interfere with parasite invasion has not been described. Here we investigated the role of these proteins in parasitophorous vacuole morphology, host cell lysosomal exocytosis, and membrane repair ability. First, we showed that cells lacking only LAMP-2 present the same invasion phenotype as LAMP1/2-/- cells, indicating that LAMP-2 is an important player during T. cruzi invasion process. Second, neither vacuole morphology nor lysosomal exocytosis was altered in LAMP-2 lacking cells (LAMP2-/- and LAMP1/2-/- cells). We then investigated the ability of LAMP-2 deficient cells to perform compensatory endocytosis upon lysosomal secretion, the mechanism by which cells repair their membrane and T. cruzi ultimately enters cells. We observed that these cells perform less endocytosis upon injury when compared to WT cells. This was a consequence of impaired cholesterol traffic in cells lacking LAMP-2 and its influence in the distribution of caveolin-1 at the cell plasma membrane, which is crucial for plasma membrane repair. The results presented here show the major role of LAMP-2 in caveolin traffic and membrane repair and consequently in T. cruzi invasion.