Cellular Trafficking Research Articles

Abstract 6033: Understanding the role of Cep55 in initiation and progression of cancers

Abstract CEP55 is a cancer-testis antigen, highly expressed in a wide range of solid tumors as well as chromosomally unstable cancers. CEP55 is normally silent in most adult somatic tissues except germ cells; however, its expression is induced early during cell transformation. Numerous studies using cell line xenografts have shown that CEP55 is required for the viability and survival of a diverse range of cancers in vitro and in vivo. To decipher the mechanisms by which Cep55 promotes chromosomal instability and tumorigenesis in vivo, we developed a novel knock-in transgenic mouse model that ubiquitously overexpresses Cep55 (Cep55Tg/Tg) and a conditional knockout (cKO) mouse model of Cep55 (Cep55-/-). While overexpression of Cep55 was sufficient to cause spontaneous tumorigenesis in vivo, Cep55 loss hindered tumorigenesis. When we bred adult Cep55 cKO mice with other tumor-prone models: oncogenic Kras expression (Kras G12D) and Pten tumor-suppressor loss, Cep55 loss significantly delayed tumorigenesis and prolonged survival validating it as a promising therapeutic target for diverse cancers. This reduction of tumorigenesis is concomitant with the downregulation of AKT and ERK signaling pathways, attenuation of epithelial to mesenchymal transition, and increased cytotoxic CD4/CD8 T-cell tumor infiltration. This indicates that Cep55 can impair the establishment of the adaptive immune response through modification in the tumor microenvironment and regulation of cell signaling. Mass spectrophotometry of mouse embryo fibroblasts and derived exosomes from wild-type and KO mice revealed the pathways altered by Cep55 such as extracellular matrix remodeling pathways, integrin-related signaling and cellular trafficking mediators indicating the contributions of Cep55 to cancer are beyond what have been reported so far. Overall, CEP55 is a promising target to prevent tumorigenesis and the progression of cancer through various mechanisms. Citation Format: Behnam Rashidieh, Simon M. Tria, Kum Kum Khanna. Understanding the role of Cep55 in initiation and progression of cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6033.

The regulatory role of the Aspergillus flavus core retromer complex in aflatoxin metabolism

Aflatoxins are a series of highly toxic and carcinogenic secondary metabolites that are synthesized by Aspergillus species. The degradation of aflatoxin enzymes is an important regulatory mechanism which modulates mycotoxin producing. The retromer complex is responsible for the retrograde transport of specific biomolecules and the vacuolar fusion in the intracellular transport. Late endosomal-associated GTPase (Rab7) has been shown to be a downstream effector protein of the retromer complex. A deficiency in the retromer complex or Rab7 results in several cellular trafficking problems in yeast and humans, like protein abnormal accumulation. However, whether retromer dysfunction is involved in aflatoxin synthesis remains unclear. Here, we report that the core retromer complex, which comprises three vacuolar protein sorting-associated proteins (AflVps26-AflVps29-AflVps35), is essential for the development of dormant and resistant fungal forms such as conidia (asexual reproductive spore) and sclerotia (hardened fungal mycelium), as well as aflatoxin production and pathogenicity, in Aspergillus flavus. In particular, we show the AflVps26-AflVps29-AflVps35 complex is negatively correlated with aflatoxin exportation. Structural simulation, site-specific mutagenesis, and coimmunoprecipitation experiments showed that interactions among AflVps26, AflVps29, and AflVps35 played crucial roles in the retromer complex executing its core functions. We further found an intrinsic connection between AflRab7 and the retromer involved in vesicle-vacuole fusion, which in turn affected the accumulation of aflatoxin synthesis-associated enzymes, suggesting that they work together to regulate the production of toxins. Overall, these results provide mechanistic insights that contribute to our understanding of the regulatory role of the core retromer complex in aflatoxin metabolism.

Cellular uptake and cytotoxicity of PEGylated gold nanoparticles in C33A cervical cancer cells

Gold nanoparticles (GNPs) have served as an excellent candidate for biomedical applications. GNPs can be conjugated with carboxyl-polyethylene glycol-thiol (PEG) as a stealth coating which prolongs circulation time [Lipka J et al 2010 Biodistribution of PEG-modified gold nanoparticles following intratracheal instillation and intravenous injection. Biomaterials, 31 , 6574–6581, Janát-Amsbury M et al 2011 Geometry and surface characteristics of gold nanoparticles influence their biodistribution and uptake by macrophages. Eur. J. Pharm. Biopharm, 77 , 417–423] and increases cellular uptake.[He B et al 2017 Increased cellular uptake of peptide-modified PEGylated gold nanoparticles. Biochem. Biophys. Res. Commun., 494 , 339–345, Soenen S. J et al 2014 , The cellular interactions of PEGylated gold nanoparticles: effect of PEGylation on cellular uptake and cytotoxicity. Part. Part. Syst. Charact., 31 , 794–800, Guo J et al 2016 Bioconjugated gold nanoparticles enhance cellular uptake: A proof of concept study for siRNA delivery in prostate cancer cells. Int. J. Pharm., 509 , 16–27. Brandenberger C et al 2010 Quantitative evaluation of cellular uptake and trafficking of plain and polyethylene glycol‐coated gold nanoparticles. Small, 6 , 1669–1678. To examine the biological effects of PEG-coated GNPs, we investigated their cytotoxicity on human cervical cancer C33A cells as compared to citrate-capped GNPs. Our results indicated that PEGylated GNPs markedly induce apoptosis and necrosis causing cell shrinkage and cell membrane asymmetry. 30 nm citrate-capped GNPs were synthesized in aqueous solution using a citrate-reduction method. GNPs were functionalized with PEG (MW = 7500 g mol−1. The GNPs were characterized using scanning electron microscopy (SEM), confirming that the as-synthesized GNPs have a diameter of 30 nm. Dynamic light scattering (DLS) determined that the hydrodynamic diameter of PEGylated GNPs was 78.82 nm, and that of citrate-capped GNPs was 43.82 nm. Zeta potential measurements showed an increase in colloidal stability for PEGylated GNPs as compared to citrate GNPs, with a zeta potential of −33.33 mV observed for citrate-capped GNPs and a zeta potential of −43.38 mV observed for PEGylated GNPs. The PEGylated GNPs were found to effectively induce early and late-stage apoptosis in C33A cells with a significant reduction in total cell viability of 32.3%. Based on the apoptotic activity in C33A cells, PEGylated GNPs may serve as a promising radiosensitizer for cancer treatments.

Isoflavonoid-based therapeutics to remodel immunologically cold tumors in nasopharyngeal carcinoma.

e14534 Background: Nasopharyngeal carcinoma (NPC) is endemic in Southern China and is well-known to be heavily infiltrated by lymphocytes and constitute a unique but poorly defined tumor microenvironment. We aimed at delineating and defining the contribution of idronoxil (IDX), a synthetic flavonoid derivative, in restoring sensitivity to apoptosis and potentially modulating the immune microenvironment of NPC. Methods: Using multiplexed staining, we measured the levels of ENOX2 (IDX’s target), CD8 (cytotoxic T cell) and pan-cytokeratin (tumor marker) in 86 NPC patients’ cancerous specimens in tissue microarray format. NPC tissue bulk RNA-Seq and affymetrix microarray public datasets were gathered to examine ENOX2 significance. Interaction between ENOX2-expressing tumor cell lines (C17 and NPC43) and T cells were also studied. In vivo, antitumor effects of IDX on the growth of NPC43+ve tumors in nude mice and humanized mice were studied. Results: We performed median density as cut-off and tumors can be classified as hot or cold. 68.6% “cold”-phenotype tumors were observed in our cohort and the top 20% patients with highly positive ENOX2-expresing tumor cells showed lower number of CD8 infiltrates. Publicly dataset showed that highly expressed ENOX2 group associated with poorer prognosis (log-rank test p = 0.041). In vitro, we observed increased migration of T cells towards cancer cells when tumor cells were pre-treated with the combination of IDX and cisplatin (IDX+Cis) compared with monotherapy (IDX or cis alone) or untreated. We also observed increased susceptibility of tumor cells to T cell cytotoxicity when both IDX and Cis were added. In nude mice, IDX+Cis significantly decreased the tumor growth compared to saline-treated group. In humanized mice, upon treatment, we found 1.69-fold (p = 0.039) more CD3+ T cells in the tumor than in the blood when compared to control group. In humanized mice, the ratio between human T cell levels in tumor and blood was increased by the IDX+Cis compared to control group (1.67-fold, p = 0.01 by t-test). Conclusions: Distinct immunospatial profiles could be associated with clinicopathologic characteristics. The ability of IDX to modulate T cell populations indicates the potential of IDX to enhance the efficacy of current chemotherapy treatments in NPC by upregulating cellular trafficking toward tumor.[Table: see text]

Luxury iron uptake and storage in pennate diatoms from the equatorial Pacific Ocean

Iron is a key micronutrient for ocean phytoplankton, and the availability of iron controls primary production and community composition in large regions of the ocean. Pennate diatoms, a phytoplankton group that responds to iron additions in low-iron areas, can have highly variable iron contents, and some groups such as Pseudo-nitzschia, are known to use ferritin to store iron for later use. We quantified and mapped the intracellular accumulation of iron by a natural population of Pseudo-nitzschia from the Fe-limited equatorial Pacific Ocean. A total of 48 h after iron addition, nearly half of the accumulated iron was localized in storage bodies adjacent to chloroplasts believed to represent ferritin. Over the subsequent 48h, stored iron was distributed to the rest of the cell through subsequent growth and division, partially supporting the iron contents of the daughter cells. This study provides the first quantitative view into the cellular trafficking of iron in a globally relevant phytoplankton group and demonstrates the unique capabilities of synchrotron-based element imaging approaches.

Crowding-induced membrane remodeling: Interplay of membrane tension, polymer density, architecture

The plasma membrane hosts a wide range of biomolecules, mainly proteins and carbohydrates, that mediate cellular interactions with its environment. The crowding of such biomolecules regulates cellular morphologies and cellular trafficking. Recent discoveries have shown that the structure and density of cell surface polymers and hence the signaling machinery change with the state of the cell, especially in cancer progression. The alterations in membrane-attached glycocalyx and glycosylation of proteins and lipids are common features of cancer cells. The overexpression of glycocalyx polymers, such as mucin and hyaluronan, strongly correlates with cancer metastasis. Here, we present a mesoscale biophysics-based model that accounts for the shape regulation of membranes by crowding of membrane-attached biopolymer-glycocalyx and actin networks. Our computational model is based on the dynamically triangulated Monte Carlo model for membranes and coarse-grained representations of polymer chains. The model allows us to investigate the crowding-induced shape transformations in cell membranes in a tension- and graft polymer density-dependent manner. Our results show that the number of membrane protrusions and their shape depend on membrane tension, with higher membrane tension inducing more tubular protrusions than the vesicular shapes formed at low tension at high surface coverage of polymers. The shape transformations occur above the threshold density predicted by the polymer brush theory, but this threshold also depends on the membrane tension. Increasing the size of the polymer, either by changing the length or by adding side chains, is shown to increase the crowding-induced curvature. The effect of crowding is more prominent for flexible polymers than for semiflexible rigid polymers. We also present an extension of the model that incorporates properties of the actin-like filament networks and demonstrate how tubular structures can be generated by biopolymer crowding on the cytosolic side of cell membranes.

An Updated View of the Importance of Vesicular Trafficking and Transport and Their Role in Immune-Mediated Diseases: Potential Therapeutic Interventions.

Cellular trafficking is the set of processes of distributing different macromolecules by the cell. This process is highly regulated in cells, involving a system of organelles (endomembranous system), among which are a great variety of vesicles that can be secreted from the cell, giving rise to different types of extracellular vesicles (EVs) that can be captured by other cells to modulate their function. The cells of the immune system are especially sensitive to this cellular traffic, producing and releasing different classes of EVs, especially in disease states. There is growing interest in this field due to the therapeutic and translational possibilities it offers. Different ways of taking advantage of the understanding of cell trafficking and EVs are being investigated, and their use as biomarkers or therapeutic targets is being investigated. The objective of this review is to collect the latest results and knowledge in this area with a specific focus on immune-mediated diseases. Although some promising results have been obtained, further knowledge is still needed, at both the basic and translational levels, to understand and modulate cellular traffic and EVs for better clinical management of these patients.

Cryo-EM structure of human glucose transporter GLUT4

GLUT4 is the primary glucose transporter in adipose and skeletal muscle tissues. Its cellular trafficking is regulated by insulin signaling. Failed or reduced plasma membrane localization of GLUT4 is associated with diabetes. Here, we report the cryo-EM structures of human GLUT4 bound to a small molecule inhibitor cytochalasin B (CCB) at resolutions of 3.3 Å in both detergent micelles and lipid nanodiscs. CCB-bound GLUT4 exhibits an inward-open conformation. Despite the nearly identical conformation of the transmembrane domain to GLUT1, the cryo-EM structure reveals an extracellular glycosylation site and an intracellular helix that is invisible in the crystal structure of GLUT1. The structural study presented here lays the foundation for further mechanistic investigation of the modulation of GLUT4 trafficking. Our methods for cryo-EM analysis of GLUT4 will also facilitate structural determination of many other small size solute carriers.

Alterations of blood monocyte subset distribution and surface phenotype are linked to infection severity in COVID-19 inpatients.

Severe coronavirus disease 19 (COVID‐19) manifests with systemic immediate proinflammatory innate immune activation and altered iron turnover. Iron homeostasis, differentiation, and function of myeloid leukocytes are interconnected. Therefore, we characterized the cellularity, surface marker expression, and iron transporter phenotype of neutrophils and monocyte subsets in COVID‐19 patients within 72 h from hospital admission, and analyzed how these parameters relate to infection severity. Between March and November 2020, blood leukocyte samples from hospitalized COVID‐19 patients (n = 48) and healthy individuals (n = 7) were analyzed by flow cytometry enabling comparative analysis of 40 features. Inflammation‐driven neutrophil expansion, depletion of CD16+ nonclassical monocytes, and changes in surface expression of neutrophil and monocyte CD64 and CD86 were associated with COVID‐19 severity. By unsupervised self‐organizing map clustering, four patterns of innate myeloid response were identified and linked to varying levels of systemic inflammation, altered cellular iron trafficking and the severity of disease. These alterations of the myeloid leukocyte compartment during acute COVID‐19 may be hallmarks of inefficient viral control and immune hyperactivation and may help at risk prediction and treatment optimization.

FC016: Nephrotoxicity of Antiangiogenic Therapies: Direct Impact of Sorafenib on the Podocyte via GSK3Β

Abstract BACKGROUND AND AIMS Tumor angiogenesis is one of the therapeutic targets used in oncology, to limit cancer growth and spreading. The main angiogenesis signalling pathway is mediated by the vascular endothelial growth factor (VEGF) that binds to its tyrosine kinase receptor (VEGFR), mainly expressed on endothelial cells. The antiangiogenic therapy could target the VEGF ligand (anti-VEGF), or its receptors by inhibition of kinase activity (tyrosine kinase inhibitor, TKI) such as sorafenib. The toxicity of antiangiogenic therapies is a growing concern in clinical use. Indeed, nephrotoxicity is a critical side-effect that leads to discontinuation of therapy. Renal histological illustration of this toxicity is minimal change nephropathy/focal segmental glomerulosclerosis (MCNS/FSGS) and thrombotic micro-angiopathy (TMA), involving two distinct cell types, podocytes and endothelial cells respectively. Podocytes are the main source of VEGF and express VEGFR in the glomerulus. Mechanisms linking TKI therapy to podocyte dysfunction and nephrotic level proteinuria are still poorly understood. The working hypothesis is that nephrotoxicity of sorafenib is primarily through its effect on glomerular podocytes. METHOD Based on LC-MS/MS proteomic analysis, we have identified dysregulated cellular pathways on sorafenib-exposed podocytes. We validated these results by western blotting and immunofluorescence staining, on a cultured podocyte line exposed to sorafenib (2.5 µmol/L, 24-h). RESULTS Our results showed that sorafenib inhibits the downstream signalling pathways of VEGFR on podocytes, which induce injury by decreasing the expression of podocyte-specific markers (WT1, podocin). This is associated with podocyte morphology changes with podocyte cytoskeleton damages (actin and microtubules) and focal adhesions loss. We identified a novel podocyte target of sorafenib: the GSK3β. Indeed, sorafenib reduces significantly GSK3β inhibitory phosphorylation. Moreover, GSK3β regulates Tau, microtubule-associated proteins (MAP) and key regulator of microtubules remodelling. We show that tau phosphorylation increases significantly in sorafenib-treated cells. Furthermore, we found that sorafenib impairs the autophagic flux in podocytes, as indicated by an increase in LC3-II/LC3-I ratio in western blot, reflect of autophagosomes accumulation in podocytes. These results indicate that GSK3β overactivity induced by sorafenib participates in the disruption of the microtubules through tau phosphorylation, which interferes with cellular vesicle trafficking and blocks autophagic flux in podocytes. In addition, sorafenib leads to podocyte apoptosis through induction of endoplasmic reticulum (ER) stress (GADD153 protein induction and its nuclear translocation) and mitochondrial dysfunction. ER stress induced a decrease in podocyte protein synthesis with increased eIF2α phosphorylation. Sorafenib also causes mitochondrial membrane potential loss and mitochondrial depolarization. CONCLUSION Thus, in the current study, we show that sorafenib has a direct effect on podocytes, modulating specific signalling pathways to induce podocyte damage and ultimately glomerular lesions. However, we have to confirm the clinical relevance of our results on kidney biopsies from patients diagnosed with nephrotic syndrome following sorafenib treatment.

Effect of Rosa canina Methanol Extract on Membrane Trafficking in Different Niemann‐Pick C1 Phenotypes

Niemann Pick type C (NPC) disease is an autosomal recessive lysosomal storage disorder that is caused by mutations in Niemann‐Pick C1 (NPC1) or Niemann‐Pick C2 (NPC2) genes ultimately leading to progressive neurological deterioration. 95% of NPC cases arise from mutations in the NPC1 gene. NPC1 protein mediates the intracellular cholesterol trafficking and plays a crucial role in maintaining lipid homeostasis. Mutations in NPC1 are associated with defective cholesterol mobilization and an accumulation of unesterified cholesterol and subsequently sphingomyelin and glycosphingolipids in the late endo‐lysosomes. N‐butyldeoxynojirimycin, known as Miglustat, is one of the approved therapeutic options. It is an inhibitor of sphingolipid synthesis and is used in substrate reduction therapy. Nevertheless, the efficiency of Miglustat varies among NPC patients depending on the type of the mutation and moreover its accompanied by several side effects, such as diarrhea. The focus of this study is to find an alternative therapy that presents less side effects. Rosa canina L. methanol extract (RCME) has been shown to improve the protein trafficking and maintain cholesterol homeostasis. Here, we used skin‐derived fibroblasts from patients harbouring homozygote or compound heterozygote mutations to assess the effects of RCME on the trafficking of NPC1 and cellular cholesterol contents as compared to wild type cells. For this purpose, the cells were treated for 24 h with RCME (100 µg/ml) with and without Miglustat (100 µM) and the trafficking of NPC1 between the endoplasmic reticulum (ER) and the Golgi was examined by endoglycosidase H treatment. This treatment, which discriminates between mannose‐rich N‐linked glycosylated proteins and mature complex glycosylated forms, demonstrated a significant enhancement of the trafficking of a NPC1 mutants between the ER and the Golgi upon RCME treatment as compared to Miglustat. This trafficking behaviour was observed with those NPC1 mutants that are not entirely blocked in the ER and exit the ER at a slow rate. Markedly, the high cholesterol levels measured in the patients’ fibroblasts are substantially reduced upon RCME treatment, while almost similar cholesterol levels were maintained in the wild type cells. Impaired cellular cholesterol trafficking is associated with partial distortion of the lipid membrane composition, particularly of the cholesterol‐ and sphingolipids‐enriched membrane microdomains or lipid rafts (LRs). Our data show that RCME and also Miglustat maintained a normal LRs distribution as assessed by the LR marker flotillin 2.We conclude that RCME possess the capacity to reverse adverse effects caused by mutations in the NPC1gene including impaired NPC1 trafficking, elevated cholesterol levels and lipid raft alterations. All this together render the constituents of RCME, including quercetin, a promising agents in NPC therapy.

Progesterone Receptor Membrane Component 1 and its Accomplice: Emerging Therapeutic Targets in Lung Cancer.

Progesterone receptor membrane component 1 (PGRMC1) is a trans-membrane evolutionarily conserved protein with a cytochrome b5 like heme/steroid binding domain. PGRMC1 clinical levels are strongly suggested to correlate with poor patient survival and lung cancer prognosis. PGRMC1 has been reported to possess pleiotropic functions, such as participating in cellular and membrane trafficking, steroid hormone signaling, cholesterol metabolism and steroidogenesis, glycolysis and mitochondrial energy metabolism, heme transport and homeostasis, neuronal movement and synaptic function, autophagy, anti-apoptosis, stem cell survival and the list is still expanding. PGRMC1 mediates its pleiotropic functions through its ability to interact with multiple binding partners, such as epidermal growth factor receptor (EGFR), sterol regulatory element binding protein cleavage activating protein (SCAP), insulin induced gene-1 protein (Insig-1), heme binding proteins (hepcidin, ferrochelatase and cyp450 members), plasminogen activator inhibitor 1 RNA binding protein (PAIR-BP1). In this review, we provide a comprehensive overview of PGRMC1 and its associated pleiotropic functions that are indispensable for lung cancer promotion and progression, suggesting it as a prospective therapeutic target for intervention. Notably, we have compiled and reported various preclinical studies wherein prospective agonists and antagonists had been tested against PGRMC1 expressing cancer cell lines, suggesting it as a prospective therapeutic target for cancer intervention.

Role of the Cytoskeleton in Steroidogenesis.

Steroidogenesis in the adrenal cortex or gonads is a complicated process modulated by various elements either at the tissue or molecular level. The substrate cholesterol is first delivered to the outer membrane of mitochondria, undergoing a series of enzymatic reactions along with the material exchange between the mitochondria and the ER (endoplasmic reticulum) and ultimately yielding various steroids, such as aldosterone, cortisol, testosterone, and estrone. Several valves are set to adjust the amount of production as per the needs, e.g., StAR (steroidogenic acute regulator) controls the traffic of cholesterol from the outer membrane to the inner membrane of mitochondria which is a rate-limiting step. Moreover, the "need" is partly reflected by trophic signals, like ACTH, LH, and downstream pathways, such as the intracellular cAMP pathway, representing the endocrinal regulation of steroid synthesis. The coordinated activities of these related factors are all associated with another crucial cellular constituent, the cytoskeleton, which plays a crucial role in cellular architecture and substrate trafficking. Though considerable studies have been performed regarding steroid synthesis, details regarding the upstream signaling pathways and mechanisms of the regulation by the cytoskeleton network still remain unclear. The metabolism and interplays of the pivotal cellular organelles with cytoskeleton are worth exploring as well. This review summarizes the research of different periods, describing the roles of specific cytoskeleton elements in steroidogenesis and related signaling pathways involved in steroid synthesis. In addition, we discuss the inner cytoskeletal network involved in steroidogenic processes, such as mitochondrial movement, organelle interactions, and cholesterol trafficking.

Sterol metabolism regulates clathrin‐mediated endocytosis and intracellular trafficking within isogenic stem cell models

Clathrin‐mediated endocytosis (CME) is a critical cellular pathway for regulation of intracellular signaling, vesicular recycling, and receptor sensitization. While CME is a molecularly complex process which was previously shown to be inhibited by cholesterol depletion, the precise impact of sterol metabolism on CME, the cellular mechanisms involved, and the relevance to disease pathology remain unclear. To determine the impact of sterol homeostasis on CME and possible roles for CME within diseases impacted by cholesterol metabolism, CRISPR/Cas9 gene editing was utilized to fluorescently label clathrin light chain A and dynamin 2 in both immortalized and induced pluripotent stem cell (iPSC) models. To model how cholesterol levels and disease‐relevant sterol changes observed in cholesterol biosynthetic disorders (characterized by the substitution of cellular cholesterol for sterol intermediates) impact endocytosis, CME was analyzed under varying biochemical states. Live cell imaging demonstrated clathrin immobilization at the cell membrane and functional CME impairment occurred following either cholesterol depletion or upon accumulation of sterol species incapable of supporting ordered lipid domains. Polarized total internal reflection fluorescence microscopy demonstrated prolonged clathrin lifetimes associated with endocytic pits with variable curvature in correlation with sterol abundance and support of phase separation. To address the impact of sterol‐mediated CME on neurodevelopment, human iPSCs were analyzed at various stages of differentiation for trafficking deficits following sterol depletion or cholesterol metabolism disruption. CME deficits in iPSCs preceded loss of pluripotency, morphological changes, or aberrant differentiation events observed following cholesterol depletion or sterol intermediate accumulation. CME deficits were also cell‐stage specific, CME activity corresponded to the rate of intracellular sterol metabolism, and organellar deficits with disease relevance resulted from CME deficiencies. Ongoing studies are investigating the consequences of sterol depletion and sterol substitution on developmental signaling pathways and cellular function to provide insight into the mechanistic requirements for sterol homeostasis in CME‐mediated cellular trafficking.

Endocytosis at the Crossroad of Polarity and Signaling Regulation: Learning from Drosophila melanogaster and Beyond.

Cellular trafficking through the endosomal–lysosomal system is essential for the transport of cargo proteins, receptors and lipids from the plasma membrane inside the cells and across membranous organelles. By acting as sorting stations, vesicle compartments direct the fate of their content for degradation, recycling to the membrane or transport to the trans-Golgi network. To effectively communicate with their neighbors, cells need to regulate their compartmentation and guide their signaling machineries to cortical membranes underlying these contact sites. Endosomal trafficking is indispensable for the polarized distribution of fate determinants, adaptors and junctional proteins. Conversely, endocytic machineries cooperate with polarity and scaffolding components to internalize receptors and target them to discrete membrane domains. Depending on the cell and tissue context, receptor endocytosis can terminate signaling responses but can also activate them within endosomes that act as signaling platforms. Therefore, cell homeostasis and responses to environmental cues rely on the dynamic cooperation of endosomal–lysosomal machineries with polarity and signaling cues. This review aims to address advances and emerging concepts on the cooperative regulation of endocytosis, polarity and signaling, primarily in Drosophila melanogaster and discuss some of the open questions across the different cell and tissue types that have not yet been fully explored.

High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum dots are fluorescent nanoparticles with excellent photophysical properties imbued with bright and stable photoluminescence as well as narrow emission bands. Quantum dots are spherical in shape, and with the proper modification of the surface chemistry, can be used to conjugate biomolecules for cellular applications. These optical properties, combined with the ability to functionalize them with biomolecules, make them an excellent tool for investigating receptor-ligand interactions and cellular trafficking. Here, we present a method that uses quantum dots to track the binding and endocytosis of SARS-CoV-2 spike protein. This protocol can be used as a guide for experimentalists looking to utilize quantum dots to study protein-protein interactions and trafficking in the context of cellular physiology.

Loss of Multiple ABCB Auxin Transporters Recapitulates the Major twisted dwarf 1 Phenotypes in Arabidopsis thaliana.

FK506-BINDING PROTEIN 42/TWISTED DWARF 1 (FKBP42/TWD1) directly regulates cellular trafficking and activation of multiple ATP-BINDING CASSETTE (ABC) transporters from the ABCB and ABCC subfamilies. abcb1 abcb19 double mutants exhibit remarkable phenotypic overlap with twd1 including severe dwarfism, stamen elongation defects, and compact circinate leaves; however, twd1 mutants exhibit greater loss of polar auxin transport and additional helical twisting of roots, inflorescences, and siliques. As abcc1 abcc2 mutants do not exhibit any visible phenotypes and TWD1 does not interact with PIN or AUX1/LAX auxin transporters, loss of function of other ABCB auxin transporters is hypothesized to underly the remaining morphological phenotypes. Here, gene expression, mutant analyses, pharmacological inhibitor studies, auxin transport assays, and direct auxin quantitations were used to determine the relative contributions of loss of other reported ABCB auxin transporters (4, 6, 11, 14, 20, and 21) to twd1 phenotypes. From these analyses, the additional reduction in plant height and the twisted inflorescence, root, and silique phenotypes observed in twd1 compared to abcb1 abcb19 result from loss of ABCB6 and ABCB20 function. Additionally, abcb6 abcb20 root twisting exhibited the same sensitivity to the auxin transport inhibitor 1-napthalthalamic acid as twd1 suggesting they are the primary contributors to these auxin-dependent organ twisting phenotypes. The lack of obvious phenotypes in higher order abcb4 and abcb21 mutants suggests that the functional loss of these transporters does not contribute to twd1 root or shoot twisting. Analyses of ABCB11 and ABCB14 function revealed capacity for auxin transport; however, their activities are readily outcompeted by other substrates, suggesting alternate functions in planta, consistent with a spectrum of relative substrate affinities among ABCB transporters. Overall, the results presented here suggest that the ABCB1/19 and ABCB6/20 pairs represent the primary long-distance ABCB auxin transporters in Arabidopsis and account for all reported twd1 morphological phenotypes. Other ABCB transporters appear to participate in highly localized auxin streams or mobilize alternate transport substrates.

Chemo-Enzymatic Modification of the 5' Cap To Study mRNAs.

The central dogma of molecular biology hinges on messenger RNA (mRNA), which presents a blueprint of the genetic information encoded in the DNA and serves as a template for translation into proteins. In addition to its fundamental importance in basic research, this class of biomolecules has recently become the first approved Covid vaccine, underscoring its utility in medical applications.Eukaryotic mRNA is heavily processed, including the 5' cap as the primary hallmark. This 5' cap protects mRNA from degradation by exoribonucleases but also interacts specifically with several proteins and enzymes to ensure mRNA turnover and processing, like splicing, export from the nucleus to the cytoplasm, and initiation of translation. The absence of a 5' cap leads to a strong immune response, and the methylation status contributes to distinguishing self from non-self RNA.Non-natural modifications of the 5' cap provide an avenue to label mRNAs and make them accessible to analyses, which is important to study their cellular localization, trafficking, and binding partners. They bear potential to engineer mRNAs, e.g., more stable or immunogenic mRNAs that are still translated, by impacting select interactions in a distinct manner. The modification of the 5' cap itself is powerful as it can be applied to make long mRNAs (∼1000 nt, not directly accessible by solid-phase synthesis) by in vitro transcription.This Account describes our contribution to the field of chemo-enzymatic modification of mRNA at the 5' cap. Our approach relies on RNA methyltransferases (MTases) with promiscuous activity on analogues of their natural cosubstrate S-adenosyl-L-methionine (AdoMet). We will describe how RNA MTases in combination with non-natural cosubstrates provide access to site-specific modification of different positions of the 5' cap, namely, the N2 and N7 position of guanosine and the N6 position of adenosine as the transcription start nucleotide (TSN) and exemplify strategies to make long mRNAs with modified 5' caps.We will compare the chemical and enzymatic synthesis of the AdoMet analogues used for this purpose. We could overcome previous limitations in methionine adenosyltransferase (MAT) substrate scope by engineering variants (termed PC-MATs) with the ability to convert methionine analogues with benzylic and photocaging groups at the sulfonium ion.The final part of this Account will highlight applications of the modified mRNAs. Like in many chemo-enzymatic approaches, a versatile strategy is to install small functional groups enzymatically and use them as handles in subsequent bioorthogonal reactions. We showed fluorescent labeling of mRNAs via different types of click chemistry in vitro and in cells. In a second line of applications, we used the handles to make mRNAs amenable for analyses, most notably next-generation sequencing. In the case of extremely promiscuous enzymes, the direct installation of photo-cross-linking groups was successful also and provided a way to covalently bind protein-interaction partners. Finally, the non-natural modifications of mRNAs can also modulate the properties of mRNAs. Propargylation of Am as the transcription start nucleotide at its N6 position maintained the translation of mRNAs but increased their immunogenicity. The installation of photocaging groups provides a way to revert these effects and control interactions by light.

HCV egress - unconventional secretion of assembled viral particles.

It is believed that hepatitis C virus (HCV) particles are released through the canonical secretory route: from the endoplasmic reticulum (ER), via the Golgi, to the plasma membrane. While the Golgi is important for HCV release per se, its direct involvement in the trafficking of assembled virions has not yet been established. In fact, data from studies analyzing HCV egress are compatible with several potential pathways of HCV secretion. Here, we summarize and discuss the current knowledge related to the HCV export pathway. Apart from the prototypical anterograde transport, possible routes of HCV release include ER-to-endosomal transport, secretory autophagy, and poorly described mechanisms of unconventional protein secretion. Studying HCV egress promises to shed light on unconventional cellular trafficking and secretory routes.

Cellular Protein Trafficking: A New Player in Low-Temperature Response Pathway.

Unlike animals, plants are unable to escape unfavorable conditions, such as extremities of temperature. Among abiotic variables, the temperature is notableas it affects plants from the molecular to the organismal level. Because of global warming, understanding temperature effects on plants is salient today and should be focused not only on rising temperature but also greater variability in temperature that is now besetting the world’s natural and agricultural ecosystems. Among the temperature stresses, low-temperature stress is one of the major stresses that limits crop productivity worldwide. Over the years, although substantial progress has been made in understanding low-temperature response mechanisms in plants, the research is more focused on aerial parts of the plants rather than on the root or whole plant, and more efforts have been made in identifying and testing the major regulators of this pathway preferably in the model organism rather than in crop plants. For the low-temperature stress response mechanism, ICE-CBF regulatory pathway turned out to be the solely established pathway, and historically most of the low-temperature research is focused on this single pathway instead of exploring other alternative regulators. In this review, we tried to take an in-depth look at our current understanding of low temperature-mediated plant growth response mechanism and present the recent advancement in cell biological studies that have opened a new horizon for finding promising and potential alternative regulators of the cold stress response pathway.