Biology subjects

Shafaq-Zadah, M.

Publications and source records attributed to Shafaq-Zadah, M..

6 recordsLinked to original sources

bioRxiv2026

Targeting GL-Lect driven endocytosis to suppress cell plasticity in breast cancer

Aberrant endocytosis has long been associated with epithelial plasticity and tumorigenesis, but direct in vivo evidence of its causal role in tumor progression and metastasis has been lacking. Here, we identify and molecularly characterize a previously unrecognized form of E-cadherin (ECAD) internalization in mammary epithelial cells. This process is mediated by the endocytic adaptor Epsin 3 (EPN3) through glycolipid-lectin (GL-Lect) driven endocytosis requiring galectin-3 and Eps15-family adaptors. Leveraging an EPN3 knock-in mouse model, we show that dysregulation of GL-Lect driven endocytosis disrupts mammary gland morphogenesis and activates epithelial-to-mesenchymal plasticity (EMP), synergizing with the ERBB2/Neu breast oncogene to drive metastasis. Pharmacologic inhibition of the GL-Lect mechanism suppresses morphogenetic and invasive phenotypes ex vivo, providing proof-of-concept for therapeutic targeting. These findings establish the GL-Lect mechanism as a driver of metastatic plasticity and uncover a tractable vulnerability in BC.

cancer biology↗

bioRxiv2024

Clathrin-independent endocytosis and retrograde transport in cancer cells promote cytotoxic CD8 T cell activation

Endophilin A3-mediated clathrin-independent endocytosis (EndoA3-mediated CIE) contributes to the internalization of immunoglobulin-like proteins, including key immune synapse components. Here, we identify ICAM1 as a novel EndoA3-dependent cargo, alongside ALCAM. We demonstrate that both proteins subsequently follow retromer-dependent retrograde transport to the trans-Golgi network (TGN) in cancer cells. From there, we propose that they undergo polarized redistribution to the plasma membrane, where they contribute to immune synapse formation between cancer cells and cytotoxic CD8 T cells. Disruption of EndoA3 or retromer components significantly affects the response of autologous cytotoxic CD8 T cells, as evidenced by reduced cytokine production and secretion, but increased lytic activity, while proliferation and later activation marker expression remain intact. This is accompanied by diminished ICAM1 density at the immune synapse, where we observe it arriving via polarized vesicular transport, indicating altered synapse organization. Indeed, cancer cells lacking EndoA3-mediated CIE or retromer form enlarged immune synapses that fail to sustain full T cell cytokine secretion, suggesting a compensatory attempt by T cells to overcome the defective synapse, while likely promoting more transient contacts that potentially favor serial killing. Together, these findings reveal that EndoA3-mediated CIE and retrograde transport act in concert in cancer cells to relocate immune synapse components via the Golgi, thereby fine-tuning the balance between cytotoxic T cell cytokine secretion and lytic activity. These insights contribute to a better understanding of the mechanisms governing immune synapse formation and organization, providing a necessary foundation for the long-term identification of new strategies to enhance T cell-mediated anti-tumor immunity. Significance StatementThis study uncovers a novel mechanism by which clathrin-independent endocytosis (CIE) and retrograde transport collaborate to regulate immune synapse dynamics in cancer cells. We identify ICAM1 as a new cargo of Endophilin A3-mediated CIE, highlighting its role in the polarized redistribution of immune synapse components critical for cytotoxic CD8 T cell activation. By linking a specific CIE mechanism and retrograde transport to immune synapse function, our findings provide new insights into cancer-immunity interactions and suggest potential therapeutic strategies to enhance immune responses by targeting protein trafficking pathways.

cell biology↗

bioRxiv2023

Spatial N-glycan rearrangement on α5β1 integrin nucleates galectin-3 oligomers to determine endocytic fate α

Membrane glycoproteins frequently adopt different conformations when altering between active and inactive states. Here, we discover a molecular switch that exploits dynamic spatial rearrangements of N-glycans during such conformational transitions to control protein function. For the conformationally switchable cell adhesion glycoprotein 5{beta}1 integrin, we find that only the bent-closed state arranges N-glycans to nucleate the formation of up to tetrameric oligomers of the glycan-binding protein galectin-3. We propose a structural model of how these galectin-3 oligomers are assembled and how they clamp the bent-closed state to prime it for endocytic uptake and subsequent retrograde trafficking to the Golgi for polarized distribution in cells. Our findings highlight an unexpectedly dynamic regulation of the glycan landscape at the cell surface to achieve oligomerization of galectin-3. Galectin-3 oligomers are thereby identified as decoders of defined spatial patterns of N-glycans and as functional extracellular interactors of specifically the bent- closed conformational state of 5{beta}1 integrin and possibly other family members.

cell biology↗

bioRxiv2023

Growth factor-induced desialylation for the fast control of endocytosis

It is commonly assumed that the glycan makeup of glycoproteins that reach the cell surface is final and static. Here, we challenge this notion by the discovery of a molecular switch that induces acute and reversible changes of glycans on the plasma membrane. We demonstrate that within minutes, the epidermal growth factor triggers the galectin-driven endocytosis of cell surface glycoproteins, such as integrins, that are key regulators of cell adhesion and migration. The onset of this process, mediated by the Na+/H+ antiporter NHE-1 and the neuraminidases Neu1/3, requires the pH-triggered enzymatic removal of sialic acids whose presence otherwise prevents galectin binding. Desialylated glycoproteins are then retrogradely transported to the Golgi apparatus where their glycan makeup is reset, and their function is repurposed to regulate EGF-dependent invasive cell migration. Glycosylation at the cell surface thereby emerges as a dynamic and reversible regulatory post-translational modification that controls a highly adaptable trafficking pathway.

cell biology↗

bioRxiv2022

SLC3A2 N-glycosylation and alternate evolutionary trajectories for amino acid metabolism

SLC3A2 (4F2hc, CD98) is an adaptor to the SLC7A exchangers and has undergone extensive repositioning of N-glycosylation sites with vertebrate evolution, presumably in synchrony with the species-specific demands of metabolism. The SLC3A2*SLC7A5 heterodimer imports essential amino acids (AA) and thereby stimulates mTOR signaling, while SLC3A2*SLC7A11 imports cystine required for glutathione synthesis and mitigation of oxidative stress. Analysis of SLC3A2 N-glycans revealed stable site-specific profiles of Golgi remodeling, apart from the conserved N365 site where branching and poly-N-acetylglucosamine content were sensitive to the insertion of lost ancestral sites and to metabolism. N-glycans at N381 and N365 stabilized SLC3A2 in the galectin lattice and opposed endocytosis, while N365 which is nearest the membrane, also promoted down-regulation by galectin-driven clathrin-independent endocytosis (glycolipid-lectin GL-Lect). This is the first report of both positive and negative regulation by galectin binding to N-glycans that are strategically positioned in the same membrane glycoproteins. Proteomics analysis in SLC3A2 mutant HeLa cells with induced re-expression of SLC3A2 as bait revealed the canonical non-N-glycosylated interactors, SLC7A5 and SLC7A11 exchangers, but also AA transporters that were dependent on SLC3A2 N-glycosylation, and are themselves, N-glycosylated AA/Na+ symporters (SLC7A1, SLC38A1, SLC38A2, SLC1A4, SLC1A5). The results suggest that the N-glycans on SLC3A2 regulate clustering of SLC7A exchangers with AA/Na+ symporters, thereby promoting Gln/Glu export-driven import of essential AA and cystine, with the potential to adversely impact redox balance. The evolution of modern birds (Neoaves) led to improved control of bioenergetics with the loss of genes including SLC3A2, SLC7A-5, -7, -8, -10, BCAT2, KEAP1, as well as duplications of SLC7A9, SLC7A11 and the Golgi branching enzymes MGAT4B and MGAT4C known to enhance affinities for galectins. Analyzing the fate of these and other genes in the down-sized genomes of birds, spanning [~]10,000 species and >100 Myr of evolution, may reveal the mystery of their longevity with prolonged vitality. Key PointsO_LIGolgi N-glycan remodeling at each site on SLC3A2 differs with the microenvironment. C_LIO_LIThe galectin lattice and GL-Lect mediated endocytosis act as opposing forces on trafficking, controlled by N-glycans at the distal N381 and membrane proximal N365 sites, respectively. C_LIO_LIMutation at N381 or N365 decreased SLC3A2 association with SLC7A5, SLC7A11 and N-glycosylated AA/Na+ symporters as well as the capacity to mitigate stress. C_LIO_LIClustering of SLC3A2*SLC7A exchangers, with AA/Na+ symporter and ATPase Na+/K+ exchanger promotes growth but continuously consumes ATP in non-proliferating cells. C_LIO_LIBird evolution has improved bioenergetics with the deletion of SLC3A2 and associated transporters; - replaced by transporters of keto acids and a re-enforced galectin lattice. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/516651v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1cb7958org.highwire.dtl.DTLVardef@18ba32dorg.highwire.dtl.DTLVardef@861269org.highwire.dtl.DTLVardef@22df0e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2020

Retrograde And Anterograde Transport Of LAT-Vesicles During The Immunological Synapse Formation: Defining The Finely-Tuned Mechanism

LAT is an important player of the signaling cascade induced by TCR activation. This adapter molecule is present at the plasma membrane of T lymphocytes and more abundantly in intracellular compartments. Upon T-cell activation the intracellular pool of LAT is recruited to the immune synapse (IS). We previously described two pathways controlling LAT trafficking: retrograde transport from endosomes to the TGN, and anterograde traffic from the Golgi to the IS. We address the specific role of 4 proteins, the GTPase Rab6, the t-SNARE syntaxin-16, the v-SNARE VAMP7 and the golgin GMAP210, in each pathway. Using different methods (endocytosis and Golgi trap assays, confocal and TIRF microscopy, TCR-signalosome pull down) we show that syntaxin-16 is regulating the retrograde transport of LAT whereas VAMP7 is regulating the anterograde transport. Moreover, GMAP210 and Rab6, known to contribute in both pathways, are in our cellular context specifically and respectively involved in anterograde and retrograde transport of LAT. Altogether, our data describe how retrograde and anterograde pathways coordinate LAT enrichment at the IS and point the Golgi as a central hub for the polarized recruitment of LAT to the IS. The role that this finely-tuned transport of signaling molecules plays in T-cell activation is discussed.

cell biology↗