Adaptin Research Articles

First person – Mitchell Leih

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Mitchell Leih is first author on ‘ Disordered hinge regions of the AP-3 adaptor complex promote vesicle budding from the late Golgi in yeast’, published in JCS. Mitchell is a PhD Student in the lab of Greg Odorizzi at the University of Colorado, Boulder, investigating adaptor protein complexes and their dynamics in membrane trafficking.

AP-1B regulates interactions of epithelial cells and intraepithelial lymphocytes in the intestine

Intraepithelial lymphocytes (IELs) reside in the epithelial layer and protect against foreign pathogens, maintaining the epithelial barrier function in the intestine. Interactions between IEL and epithelial cells are required for IELs to function effectively; however, the underlying molecular machinery remains to be elucidated. In this study, we found that intestinal epithelium-specific deficiency of the clathrin adaptor protein (AP)-1B, which regulates basolateral protein sorting, led to a massive reduction in IELs. Quantitative proteomics demonstrated that dozens of proteins, including known IEL-interacting proteins (E-cadherin, butyrophilin-like 2, and plexin B2), were decreased in the basolateral membrane of AP-1B-deficient epithelial cells. Among these proteins, CD166 interacted with CD6 on the surface of induced IEL. CD166 knockdown, using shRNA in intestinal organoid cultures, significantly inhibited IEL recruitment to the epithelial layer. These findings highlight the essential role of AP-1B-mediated basolateral sorting in IEL maintenance and survival within the epithelial layer. This study reveals a novel function of AP-1B in the intestinal immune system.

Disordered hinge regions of the AP-3 adaptor complex promote vesicle budding from the late Golgi in yeast.

Vesicles bud from maturing Golgi cisternae in a programmed sequence. Budding is mediated by adaptors that recruit cargoes and facilitate vesicle biogenesis. In Saccharomyces cerevisiae, the AP-3 adaptor complex directs cargoes from the Golgi to the lysosomal vacuole. The AP-3 core consists of small and medium subunits complexed with two non-identical large subunits, β3 and δ. The C termini of β3 and δ were thought to be flexible hinges linking the core to ear domains that bind accessory proteins involved in vesicular transport. We found by computational modeling that the yeast β3 and δ hinges are intrinsically disordered and lack folded ear domains. When either hinge is truncated, AP-3 is recruited to the Golgi, but vesicle budding is impaired, and cargoes normally sorted into the AP-3 pathway are mistargeted. This budding deficiency causes AP-3 to accumulate on ring-like Golgi structures adjacent to GGA adaptors that, in wild-type cells, bud vesicles downstream of AP-3 during Golgi maturation. Thus, each of the disordered hinges of yeast AP-3 has a crucial role in mediating transport vesicle formation at the Golgi.

CARD14 signalosome formation is associated with its endosomal relocation and mTORC1-induced keratinocyte proliferation.

Rare mutations in CARD14 promote psoriasis by inducing CARD14-BCL10-MALT1 complexes that activate NF-κB and MAP kinases. Here, the downstream signalling mechanism of the highly penetrant CARD14E138A alteration is described. In addition to BCL10 and MALT1, CARD14E138A associated with several proteins important in innate immune signalling. Interactions with M1-specific ubiquitin E3 ligase HOIP, and K63-specific ubiquitin E3 ligase TRAF6 promoted BCL10 ubiquitination and were essential for NF-κB and MAP kinase activation. In contrast, the ubiquitin binding proteins A20 and ABIN1, both genetically associated with psoriasis development, negatively regulated signalling by inducing CARD14E138A turnover. CARD14E138A localized to early endosomes and was associated with the AP2 adaptor complex. AP2 function was required for CARD14E138A activation of mTOR complex 1 (mTORC1), which stimulated keratinocyte metabolism, but not for NF-κB nor MAP kinase activation. Furthermore, rapamycin ameliorated CARD14E138A-induced keratinocyte proliferation and epidermal acanthosis in mice, suggesting that blocking mTORC1 may be therapeutically beneficial in CARD14-dependent psoriasis.

AP-1 contributes to endosomal targeting of ubiquitin ligase RNF13 via a secondary and novel non-canonical binding motif.

Cellular trafficking between organelles is typically assured by short motifs that contact carrier proteins to transport them to their destination. Ubiquitin E3 ligase RING finger protein 13 (RNF13), a regulator of proliferation, apoptosis, and protein trafficking, localizes to endolysosomal compartments through the binding of a dileucine motif to clathrin adaptor protein complex AP-3. Mutations within this motif reduce the ability of RNF13 to interact with AP-3. Here, our study shows the discovery of a glutamine-based motif that resembles a tyrosine-based motif within RNF13's C-terminal region that binds to the clathrin adaptor protein complex AP-1, notably without a functional interaction with AP-3. Using biochemical, molecular, and cellular approaches in HeLa cells, our study demonstrates that a RNF13 dileucine variant uses an AP-1-dependent pathway to be exported from the Golgi towards the endosomal compartment. Overall, this study provides mechanistic insights into the alternate route used by variant of RNF13's dileucine sorting motif.

The Epsin-like clathrin adaptor protein 4 SlECA4 improves tomato heat tolerance via clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) is one of the main pathways for plant cells to internalize the membrane proteins in response to changing environmental conditions. The Epsin-like Clathrin adaptor proteins (ECAs) play important roles in the assembly of clathrin coat; however, their involvement in plant response to heat stress remains unclear. Here we report that SlECA4 responded to heat stress, and the silencing and knockout of SlECA4 increased tomato sensitivity to heat stress, while the overexpression of SlECA4 enhanced tomato tolerance to heat stress. Meanwhile, the treatment with a CME inhibitor, ES9-17, reduced tomato heat tolerance. SlECA4 localized to the plasma membrane (PM), the trans-Golgi network/early endosomes (TGN/EE), and the prevacuolar compartment (PVC)/late endosomes. In SlECA4-KO line, both CME and recycling from the TGN/EE to the PM were inhibited. These data suggest that SlECA4 involved in CME. After heat treatment, more punctate structures of SlECA4:GFP accumualted in tobacco leaf epidermal cells by transient expression. Furthermore, compared to WT, the rate of CME was inhibited under heat stress in SlECA4-KO line. Taken together, the Epsin-like Clathrin adaptor protein SlECA4 plays a positive role in tomato tolerance to heat stress via the CME pathway.

Picalm, a novel regulator of GLUT4-trafficking in adipose tissue

ObjectivePicalm (phosphatidylinositol-binding clathrin assembly protein), a ubiquitously expressed clathrin-adapter protein, is a well-known susceptibility gene for Alzheimer's disease, but its role in white adipose tissue (WAT) function has not yet been studied. Transcriptome analysis revealed differential expression of Picalm in WAT of diabetes-prone and diabetes-resistant mice, hence we aimed to investigate the potential link between Picalm expression and glucose homeostasis, obesity-related metabolic phenotypes, and its specific role in insulin-regulated GLUT4 trafficking in adipocytes. MethodsPicalm expression and epigenetic regulation by microRNAs (miRNAs) and DNA methylation were analyzed in WAT of diabetes-resistant (DR) and diabetes-prone (DP) female New Zealand Obese (NZO) mice and in male NZO after time-restricted feeding (TRF) and alternate-day fasting (ADF). PICALM expression in human WAT was evaluated in a cross-sectional cohort and assessed before and after weight loss induced by bariatric surgery. siRNA-mediated knockdown of Picalm in 3T3-L1-cells was performed to elucidate functional outcomes on GLUT4-translocation as well as insulin signaling and adipogenesis. ResultsPicalm expression in WAT was significantly lower in DR compared to DP female mice, as well as in insulin-sensitive vs. resistant NZO males, and was also reduced in NZO males following TRF and ADF. Four miRNAs (let-7c, miR-30c, miR-335, miR-344) were identified as potential mediators of diabetes susceptibility-related differences in Picalm expression, while 11 miRNAs (including miR-23a, miR-29b, and miR-101a) were implicated in TRF and ADF effects. Human PICALM expression in adipose tissue was lower in individuals without obesity vs. with obesity and associated with weight-loss outcomes post-bariatric surgery. siRNA-mediated knockdown of Picalm in mature 3T3-L1-adipocytes resulted in amplified insulin-stimulated translocation of the endogenous glucose transporter GLUT4 to the plasma membrane and increased phosphorylation of Akt and Tbc1d4. Moreover, depleting Picalm before and during 3T3-L1 differentiation significantly suppressed adipogenesis, suggesting that Picalm may have distinct roles in the biology of pre- and mature adipocytes. ConclusionsPicalm is a novel regulator of GLUT4-translocation in WAT, with its expression modulated by both genetic predisposition to diabetes and dietary interventions. These findings suggest a potential role for Picalm in improving glucose homeostasis and highlight its relevance as a therapeutic target for metabolic disorders.

An AAGAB-to-CCDC32 handover mechanism controls the assembly of the AP2 adaptor complex

Vesicular transport relies on multimeric trafficking complexes to capture cargo and drive vesicle budding and fusion. Faithful assembly of the trafficking complexes is essential to their functions but remains largely unexplored. Assembly of AP2 adaptor, a heterotetrameric protein complex regulating clathrin-mediated endocytosis, is assisted by the chaperone AAGAB. Here, we found that AAGAB initiates AP2 assembly by stabilizing its α and σ2 subunits, but the AAGAB:α:σ2 complex cannot recruit additional AP2 subunits. We identified CCDC32 as another chaperone regulating AP2 assembly. CCDC32 recognizes the AAGAB:α:σ2 complex, and its binding leads to the formation of an α:σ2:CCDC32 ternary complex. The α:σ2:CCDC32 complex serves as a template that sequentially recruits the µ2 and β2 subunits of AP2 to complete AP2 assembly, accompanied by CCDC32 release. The AP2-regulating function of CCDC32 is disrupted by a disease-causing mutation. These findings demonstrate that AP2 is assembled by a handover mechanism switching from AAGAB-based initiation complexes to CCDC32-based template complexes. A similar mechanism may govern the assembly of other trafficking complexes exhibiting the same configuration as AP2.

The AP-1 adaptor complex is essential for intracellular trafficking of the ORF2 capsid protein and assembly of Hepatitis E virus

Although the Hepatitis E virus (HEV) is an emerging global health burden, little is known about its interaction with the host cell. HEV genome encodes three proteins including the ORF2 capsid protein that is produced in different forms, the ORF2i protein which is the structural component of viral particles, and the ORF2g/c proteins which are massively secreted but are not associated with infectious material. We recently demonstrated that the endocytic recycling compartment (ERC) is hijacked by HEV to serve as a viral factory. However, host determinants involved in the subcellular shuttling of viral proteins to viral factories are unknown. Here, we demonstrate that the AP-1 adaptor complex plays a pivotal role in the targeting of ORF2i protein to viral factories. This complex belongs to the family of adaptor proteins that are involved in vesicular transport between the trans-Golgi network and early/recycling endosomes. An interplay between the AP-1 complex and viral protein(s) has been described for several viral lifecycles. In the present study, we demonstrated that the ORF2i protein colocalizes and interacts with the AP-1 adaptor complex in HEV-producing or infected cells. We showed that silencing or drug-inhibition of the AP-1 complex prevents ORF2i protein localization in viral factories and reduces viral production in hepatocytes. Modeling of the ORF2i/AP-1 complex also revealed that the S domain of ORF2i likely interacts with the σ1 subunit of AP-1 complex. Hence, our study identified for the first time a host factor involved in addressing HEV proteins (i.e. ORF2i protein) to viral factories.

Intestinal epithelium dysfunctions cause IgA deposition in the kidney glomeruli of intestine-specific Ap1m2-deficient mice

Intestinal epithelial cells (IECs) serve as robust barriers against potentially hostile luminal antigens and commensal microbiota. Epithelial barrier dysfunction enhances intestinal permeability, leading to leaky gut syndrome (LGS) associated with autoimmune and chronic inflammatory disorders. However, a causal relationship between LGS and systemic disorders remains unclear. Ap1m2 encodes clathrin adaptor protein complex 1 subunit mu 2, which facilitates polarized protein trafficking toward the basolateral membrane and contributes to the establishment of epithelial barrier functions. We generated IEC-specific Ap1m2-deficient (Ap1m2ΔIEC) mice with low intestinal barrier integrity as an LSG model and examined the systemic impact. Ap1m2ΔIEC mice spontaneously developed IgA nephropathy (IgAN)-like features characterized by the deposition of IgA-IgG immune complexes and complement factors in the kidney glomeruli. Ap1m2 deficiency markedly enhanced aberrantly glycosylated IgA in the serum owing to downregulation and mis-sorting of polymeric immunoglobulin receptors in IECs. Furthermore, Ap1m2 deficiency caused intestinal dysbiosis by attenuating IL-22-STAT3 signaling. Intestinal dysbiosis contributed to the pathogenesis of IgAN because antibiotic treatment reduced aberrantly glycosylated IgA production and renal IgA deposition in Ap1m2ΔIEC mice. IEC barrier dysfunction and subsequent dysbiosis by AP-1B deficiency provoke IgA deposition in the mouse kidney. Our findings provide experimental evidence of a pathological link between LGS and IgAN. AMED, AMED-CREST, JSPS Grants-in-Aid for Scientific Research, JST CREST, Fuji Foundation for Protein Research, and Keio University Program for the Advancement of Next Generation Research Projects.

A fluorescence polarization assay for high-throughput screening of inhibitors against HIV-1 Nef-mediated CD4 downregulation

Therapeutic inhibition of the viral protein Nef is an intriguing direction of antiretroviral drug discovery—it may revitalize immune mechanisms to target, and potentially clear, HIV-1-infected cells. Of the many cellular functions of Nef, the most conserved is the downregulation of surface CD4, which takes place through Nef hijacking the clathrin adaptor protein complex 2 (AP2)-dependent endocytosis. Our recent crystal structure has unraveled the molecular details of the CD4-Nef-AP2 interaction. Guided by the new structural knowledge, we have developed a fluorescence polarization-based assay for inhibitor screening against Nef’s activity on CD4. In our assay, AP2 is included along with Nef to facilitate the proper formation of the CD4-binding pocket, and a fluorescently labeled CD4 cytoplasmic tail binds competently to the Nef-AP2 complex generating the desired polarization signal. The optimized assay has a good signal-to-noise ratio, excellent tolerance of DMSO and detergent, and the ability to detect competitive binding at the targeted Nef pocket, making it suitable for high-throughput screening.

A fluorescence polarization assay for high-throughput screening of inhibitors against HIV-1 Nef-mediated MHC-I downregulation

The multifunctional, HIV-1 accessory protein Nef enables infected cells to evade host immunity and thus plays a key role in viral pathogenesis. One prominent function of Nef is the downregulation of major histocompatibility complex class I (MHC-I), which disrupts antigen presentation and thereby allows the infected cells to evade immune surveillance by the cytotoxic T cells. Therapeutic inhibition of this Nef function is a promising direction of antiretroviral drug discovery as it may revitalize cytotoxic T cells to identify, and potentially clear, hidden HIV-1 infections. Guided by the crystal structure of the protein complex formed between Nef, MHC-I, and the hijacked clathrin adaptor protein complex 1 (AP1), we have developed a fluorescence polarization-based assay for inhibitor screening against Nef’s activity on MHC-I. The optimized assay has a good signal-to-noise ratio, substantial tolerance of DMSO, and excellent ability to detect competitive inhibition, indicating that it is suitable for high-throughput screening.

LRK-1/LRRK2 and AP-3 regulate trafficking of synaptic vesicle precursors through active zone protein SYD-2/Liprin-α.

Synaptic vesicle proteins (SVps) are transported by the motor UNC-104/KIF1A. We show that SVps travel in heterogeneous carriers in C. elegans neuronal processes, with some SVp carriers co-transporting lysosomal proteins (SV-lysosomes). LRK-1/LRRK2 and the clathrin adaptor protein complex AP-3 play a critical role in the sorting of SVps and lysosomal proteins away from each other at the SV-lysosomal intermediate trafficking compartment. Both SVp carriers lacking lysosomal proteins and SV-lysosomes are dependent on the motor UNC-104/KIF1A for their transport. In lrk-1 mutants, both SVp carriers and SV-lysosomes can travel in axons in the absence of UNC-104, suggesting that LRK-1 plays an important role to enable UNC-104 dependent transport of synaptic vesicle proteins. Additionally, LRK-1 acts upstream of the AP-3 complex and regulates its membrane localization. In the absence of the AP-3 complex, the SV-lysosomes become more dependent on the UNC-104-SYD-2/Liprin-α complex for their transport. Therefore, SYD-2 acts to link upstream trafficking events with the transport of SVps likely through its interaction with the motor UNC-104. We further show that the mistrafficking of SVps into the dendrite in lrk-1 and apb-3 mutants depends on SYD-2, likely by regulating the recruitment of the AP-1/UNC-101. SYD-2 acts in concert with AP complexes to ensure polarized trafficking & transport of SVps.

Abstract PO3-24-02: Molecular characterization and therapeutic approach for VISTA+ triple-negative breast cancers

Abstract Introduction: Current FDA-approved immunotherapeutics for triple-negative breast cancer are only available for a subset of patients, highlighting the need to develop improved immune-targeting strategies. VISTA (V-domain Ig suppressor of T cell activation) is a single-pass transmembrane immune checkpoint receptor that is an emerging clinical target for cancer immunotherapy. It is expressed widely on lymphoid and myeloid cells in healthy individuals, is commonly expressed on cells of the tumor microenvironment and can be abnormally upregulated on tumor cells in a wide variety of tumors. Although VISTA confers quiescence to naïve T cells, molecular mechanisms responsible for this effect remain unclear. Also, the effects of VISTA expression on tumor cells remains poorly defined. Methods: Multi-omic profiling was performed in human breast cancer cells lines to determine immune regulatory genes controlled by growth factor stimulation. Human triple-negative breast cancer tumor specimens (N=248) were examined by immunohistochemistry for expression of VISTA, PDL1, PD1 and FoxP3. VISTA expression was engineered into human and mouse triple-negative breast cancer cell lines and growth metrics were characterized in vitro and in immunocompetent (syngeneic) and immunodeficient (xenograft) mouse tumor models. Membrane receptor trafficking and localization of EGFR was performed in human TNBC cells lines expressing VISTA and VISTA mutants. Proximity biotinylation identified protein partners that interacted with VISTA’s intracellular domain. Deletion mutagenesis was performed to identify motifs required for biochemical interactions between VISTA and clathrin adaptor proteins. Clathrin adapter localization was examined in WT and VISTA knock-out cell lines. Cytokine response, clathrin adaptor localization and tumor growth kinetics were measured in TNBC cell lines expressing VISTA or VISTA mutants. Results: A class of human triple-negative breast cancers were identified with high VISTA expression, low tumor infiltrating lymphocytes and low proliferative index (8-20% of all TNBCs). Tumor models demonstrated that enforced expression of VISTA in tumor cells caused growth suppression, even in the absence of an immune system. This effect was mapped to the cytoplasmic domain of VISTA, which lacks defined signaling domains. Through biochemical approaches, the clathrin-adaptor molecules NUMB and GULP1 were found in complex with a VISTA intracellular NPGF motif. This motif sequestered NUMB at early endosomes, resulting in diminished NUMB recycling for EGFR endocytosis, thereby causing growth suppression. VISTA impaired NUMB recycling by blocking Rab11 effector complex formation. VISTA NPGF mutations did not disrupt cytokine secretion in mixed lymphocyte assays. VISTA-targeted antibodies did not disrupt NUMB sequestration. VISTA+ tumors were selectively sensitive to VISTA-blocking antibodies. Conclusions: VISTA+ TNBC have distinct features including slower proliferative index and increased sensitivity to VISTA-blocking antibodies. This work identifies mechanisms by which VISTA enforces a quiescent cellular state by sequestering clathrin adapter proteins away from sites of endocytosis in cancer cells. This is the first description of intracellular molecular mechanisms controlled by VISTA’s intracellular tail. It may inform features of the VISTA-dependent immune checkpoint that could be exploited to improve anti-tumor immunity. Citation Format: Yan Zhao, Fatima Charles, Andrew Lipchik, Yan Peng, James Ford, Melinda Telli, Song Zhang, Allison Kurian, Michael Snyder, Joshua Gruber. Molecular characterization and therapeutic approach for VISTA+ triple-negative breast cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-02.

The role of the AP-1 adaptor complex in outgoing and incoming membrane traffic.

The AP-1 adaptor complex is found in all eukaryotes, but it has been implicated in different pathways in different organisms. To look directly at AP-1 function, we generated stably transduced HeLa cells coexpressing tagged AP-1 and various tagged membrane proteins. Live cell imaging showed that AP-1 is recruited onto tubular carriers trafficking from the Golgi apparatus to the plasma membrane, as well as onto transferrin-containing early/recycling endosomes. Analysis of single AP-1 vesicles showed that they are a heterogeneous population, which starts to sequester cargo 30 min after exit from the ER. Vesicle capture showed that AP-1 vesicles contain transmembrane proteins found at the TGN and early/recycling endosomes, as well as lysosomal hydrolases, but very little of the anterograde adaptor GGA2. Together, our results support a model in which AP-1 retrieves proteins from post-Golgi compartments back to the TGN, analogous to COPI's role in the early secretory pathway. We propose that this is the function of AP-1 in all eukaryotes.

The roles of HCV core protein and its binding host factor in virus assembly and release

Hepatitis C virus (HCV) is a well-known virus that causes liver diseases such as liver cirrhosis and hepatocellular carcinoma. For several decades, numerous studies have been conducted to unravel the life cycle and molecular mechanisms of this virus with the aim of developing strategies to combat diseases caused by its infection. In this review, we summarize HCV assembly to budding, focusing on one of the structural proteins, the core, a viral capsid that binds both the viral genome and host membrane, along with the core-interacting host partners. The HCV core matures in the endoplasmic reticulum (ER), localizes at the lipid droplet (LD), and shuttles between the LD and ER to form viral particles. This process is controlled by many host factors known to binds core proteins, such as diacylglycerol acyltransferase-1 (DGAT-1), Rab18, μ subunit of the clathrin adaptor protein complex 2 (AP2M1), nuclear pore complex protein 98 (Nup98), Cortactin, group IVA phospholipase A2 (PLA2G4A) etc. Virion budding is thought to involve contributions from endosomal sorting complexes required for transport (ESCRT), similar to other envelope viruses. We delved into potential perspectives to enhance our understanding of the HCV mechanism by drawing insights from existing studies.

FACI is a novel clathrin adaptor protein 2-binding protein that facilitates low-density lipoprotein endocytosis

BackgroundCholesterol plays a vital role in multiple physiological processes. Cellular uptake of cholesterol is mediated primarily through endocytosis of low-density lipoprotein (LDL) receptor. New modifiers of this process remain to be characterized. Particularly, the role of fasting- and CREB-H-induced (FACI) protein in cholesterol homeostasis merits further investigation.MethodsInteractome profiling by proximity labeling and affinity purification − mass spectrometry was performed. Total internal reflection fluorescence microscopy and confocal immunofluorescence microscopy were used to analyze protein co-localization and interaction. Mutational analysis was carried out to define the domain and residues required for FACI localization and function. Endocytosis was traced by fluorescent cargos. LDL uptake in cultured cells and diet-induced hypercholesterolemia in mice were assessed.ResultsFACI interacted with proteins critically involved in clathrin-mediated endocytosis, vesicle trafficking, and membrane cytoskeleton. FACI localized to clathrin-coated pits (CCP) on plasma membranes. FACI contains a conserved DxxxLI motif, which mediates its binding with the adaptor protein 2 (AP2) complex. Disruption of this motif of FACI abolished its CCP localization but didn’t affect its association with plasma membrane. Cholesterol was found to facilitate FACI transport from plasma membrane to endocytic recycling compartment in a clathrin- and cytoskeleton-dependent manner. LDL endocytosis was enhanced in FACI-overexpressed AML12 cells but impaired in FACI-depleted HeLa cells. In vivo study indicated that hepatic FACI overexpression alleviated diet-induced hypercholesterolemia in mice.ConclusionsFACI facilitates LDL endocytosis through its interaction with the AP2 complex.

Abstract LB263: An in vivo unbiased screen identifies clathrin adaptor protein complex-2 as a novel tumor suppressor

Abstract Pancreatic ductal adenocarcinoma (PDAC) is almost uniformly lethal. It is thus imperative to find new therapeutic approaches to treat this disease. While whole genome CRISPR/Cas9 screens have successfully identified new vulnerabilities in PDAC cell lines, it is becoming increasingly clear that cell culture do not faithfully recapitulate the complex nature of tumorigenesis in vivo, and hence may fail to identify the full spectrum of potential therapeutic targets to be explored in this devastating disease. As previous screens we performed identified phosphatidylinositol (Ptdlns) metabolism as important for pancreatic cell growth, I generated a custom sgRNA library targeting this entire form of metabolism. To identify vulnerabilities specific to tumor growth, I screened this library in five different human PDAC cell lines grown in culture versus as xenograft tumors. I found that sgRNAs targeting two genes encoding separate subunits of the Adaptor Protein complex-2 (AP2), a regulator of clathrin mediated endocytosis, were negatively enriched in all five PDAC cell lines when grown in culture. In agreement, AP2 subunit genes are considered common essential by DepMap. Paradoxically however, the same sgRNAs were positively enriched in three of these five cell lines when grown as xenograft tumors. This begs the question how the loss of a supposably essential complex behaves like a tumor suppressor and instead enhances tumor growth when disrupted. Here, I will present data that loss of AP2 results in a substantial restructuring the plasma membrane proteome, which in cultured cells results in the loss of iron transport, one of the main functions of this complex, and cell death. Conversely, this restructuring in vivo retains receptors and adhesion proteins on the cell surface, where transcriptome and proteomic analysis indicate enhances proliferative signaling. These data support AP2 promoting cell viability of cultured cells through active iron transport, but suppresses tumor growth in vivo by endocytosing the very receptors needed for tumor growth. Citation Format: Seth Parker Zimmerman, Christopher M. Counter. An in vivo unbiased screen identifies clathrin adaptor protein complex-2 as a novel tumor suppressor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB263.

Eukaryotic Clathrin Adapter Protein and Mediator of Cholesterol Homeostasis, PICALM, Affects Trafficking to the Chlamydial Inclusion.

The obligate intracellular pathogen Chlamydia trachomatis has unique metabolic requirements as it proceeds through its biphasic developmental cycle from within the inclusion within the host cell. In our previous study, we identified a host protein, PICALM, which localizes to the chlamydial inclusion. PICALM functions in many host pathways including the recycling of receptors, specific SNARE proteins, and molecules like transferrin, and maintaining cholesterol homeostasis. Hence, we hypothesized that PICALM functions to maintain the cholesterol content and to moderate trafficking from the endosomal recycling pathway to the inclusion, which controls chlamydial access to this pathway. In uninfected cells, siRNA knockdown of PICALM resulted in increased cholesterol within the Golgi and transferrin receptor (TfR) positive vesicles (recycling endosomes). PICALM knockdown in cells infected with C. trachomatis resulted in increased levels of Golgi-derived lipid and protein, TfR, transferrin, and Rab11-FIP1 localized to inclusions and a decrease of Golgi fragmentation at and Rab11 trafficking to the inclusion. Interestingly, chlamydial infection alone also increases cholesterol in TfR and Rab11-associated vesicles, and PICALM knockdown reverses this effect. Our data suggest that PICALM functions to balance or limit chlamydial access to multiple subcellular trafficking pathways to maintain the health of the host cell during chlamydial infection.

Immunoprecipitation and Western Blot Analysis of AP-1 Clathrin-Coated Vesicles.

The function and integrity of epithelial cells depends on the polarized localization of transmembrane proteins at either apical or basolateral plasma membrane domains. To facilitate sorting to the basolateral domain, columnar epithelial cells express the tissue-specific AP-1B complex in addition to the ubiquitously expressed AP-1A. Both AP-1A and AP-1B are heterotetrameric clathrin adaptor protein complexes that are closely related. Here we describe a biochemical method to separate AP-1B from AP-1A clathrin-coated vesicles by immunoprecipitation from clathrin-coated vesicle pellets that were obtained by ultracentrifugation and analyzed by SDS-PAGE and western blot using fluorescently labeled secondary antibodies.