Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response.

NRF2 is a transcription factor responsible for antioxidant stress responses that is usually regulated in a redox-dependent manner. p62 bodies formed by liquid-liquid phase separation contain Ser349-phosphorylated p62, which participates in the redox-independent activation of NRF2. However, the regulatory mechanism and physiological significance of p62 phosphorylation remain unclear. Here, we identify ULK1 as a kinase responsible for the phosphorylation of p62. ULK1 colocalizes with p62 bodies, directly interacting with p62. ULK1-dependent phosphorylation of p62 allows KEAP1 to be retained within p62 bodies, thus activating NRF2. p62S351E/+ mice are phosphomimetic knock-in mice in which Ser351, corresponding to human Ser349, is replaced by Glu. These mice, but not their phosphodefective p62S351A/S351A counterparts, exhibit NRF2 hyperactivation and growth retardation. This retardation is caused by malnutrition and dehydration due to obstruction of the esophagus and forestomach secondary to hyperkeratosis, a phenotype also observed in systemic Keap1-knockout mice. Our results expand our understanding of the physiological importance of the redox-independent NRF2 activation pathway and provide new insights into the role of phase separation in this process.

"I'd like to let people know what we did:" values of Fukushima medical students following the Great East Japan Earthquake.

BACKGROUND: The Great East Japan Earthquake and the resulting tsunami and nuclear disaster on March 11, 2011 have had a profound and lasting effect on residents of Japan's Fukushima Prefecture, particularly among evacuees. While there continues to be extensive news coverage and academic study of Fukushima Prefecture's recovery, there has been little exploration of individual narratives. This study aims to illuminate some individual stories of medical students at Fukushima Medical University (FMU) who lived in the Prefecture at the time of the Earthquake. METHODS: A qualitative approach was taken in order to investigate individuals' experiences with the goal of adding a personal dimension to quantitative studies on the subject. 10 open-ended ethnographic interviews were conducted with medical students at FMU in years 1-5 who lived in Fukushima Prefecture at the time of the Great East Japan Earthquake. All interviews were audio recorded and transcribed. Transcriptions were reviewed using inductive thematic analysis under the lens of ethnographic anthropology. RESULTS: Three major themes emerged from these interviews: first, that the events following the Earthquake influenced not only these students' decisions to pursue careers in medicine, but the ways in which they hope to practice medicine in the future. Second, that these students were motivated to share their experiences by a want to change Fukushima Prefecture's public image. And lastly, that the students viewed the opportunity to discuss their experiences through these interviews as healing, both for themselves and for the future. CONCLUSIONS: While multiple factors undoubtably contributed these students' medical education, they cite the Earthquake as essential to their approach to their medical careers. Additionally, opportunities for the participants to discuss their experiences following the Earthquake appear to be rare but valued, as the students view their stories as their "legacies." The enduring, burdening effects of the Earthquake appear to have galvanized the participating students to act on behalf of their communities and their Prefecture. Further qualitative studies in more generalizable populations are needed to improve and deepen our understanding of the societal, cultural, and personal impacts of the Great East Japan Earthquake.

NBR1-mediated p62-liquid droplets enhance the Keap1-Nrf2 system.

p62/SQSTM1 is a multivalent protein that has the ability to cause liquid-liquid phase separation and serves as a receptor protein that participates in cargo isolation during selective autophagy. This protein is also involved in the non-canonical activation of the Keap1-Nrf2 system, a major oxidative stress response pathway. Here, we show a role of neighbor of BRCA1 gene 1 (NBR1), an autophagy receptor structurally similar to p62/SQSTM1, in p62-liquid droplet formation and Keap1-Nrf2 pathway activation. Overexpression of NBR1 blocks selective degradation of p62/SQSTM1 through autophagy and promotes the accumulation and phosphorylation of p62/SQSTM1 in liquid-like bodies, which is required for the activation of Nrf2. NBR1 is induced in response to oxidative stress, which triggers p62-mediated Nrf2 activation. Conversely, loss of Nbr1 suppresses not only the formation of p62/SQSTM1-liquid droplets, but also of p62-dependent Nrf2 activation during oxidative stress. Taken together, our results show that NBR1 mediates p62/SQSTM1-liquid droplet formation to activate the Keap1-Nrf2 pathway.

Phosphorylation of p62 activates the Keap1-Nrf2 pathway during selective autophagy.

The Keap1-Nrf2 system and autophagy are both involved in the oxidative-stress response, metabolic pathways, and innate immunity, and dysregulation of these processes is associated with pathogenic processes. However, the interplay between these two pathways remains largely unknown. Here, we show that phosphorylation of the autophagy-adaptor protein p62 markedly increases p62's binding affinity for Keap1, an adaptor of the Cul3-ubiquitin E3 ligase complex responsible for degrading Nrf2. Thus, p62 phosphorylation induces expression of cytoprotective Nrf2 targets. p62 is assembled on selective autophagic cargos such as ubiquitinated organelles and subsequently phosphorylated in an mTORC1-dependent manner, implying coupling of the Keap1-Nrf2 system to autophagy. Furthermore, persistent activation of Nrf2 through accumulation of phosphorylated p62 contributes to the growth of human hepatocellular carcinomas (HCCs). These results demonstrate that selective autophagy and the Keap1-Nrf2 pathway are interdependent, and that inhibitors of the interaction between phosphorylated p62 and Keap1 have potential as therapeutic agents against human HCC.

Biallelic Variants in UBA5 Link Dysfunctional UFM1 Ubiquitin-like Modifier Pathway to Severe Infantile-Onset Encephalopathy.

The ubiquitin fold modifier 1 (UFM1) cascade is a recently identified evolutionarily conserved ubiquitin-like modification system whose function and link to human disease have remained largely uncharacterized. By using exome sequencing in Finnish individuals with severe epileptic syndromes, we identified pathogenic compound heterozygous variants in UBA5, encoding an activating enzyme for UFM1, in two unrelated families. Two additional individuals with biallelic UBA5 variants were identified from the UK-based Deciphering Developmental Disorders study and one from the Northern Finland Intellectual Disability cohort. The affected individuals (n = 9) presented in early infancy with severe irritability, followed by dystonia and stagnation of development. Furthermore, the majority of individuals display postnatal microcephaly and epilepsy and develop spasticity. The affected individuals were compound heterozygous for a missense substitution, c.1111G>A (p.Ala371Thr; allele frequency of 0.28% in Europeans), and a nonsense variant or c.164G>A that encodes an amino acid substitution p.Arg55His, but also affects splicing by facilitating exon 2 skipping, thus also being in effect a loss-of-function allele. Using an in vitro thioester formation assay and cellular analyses, we show that the p.Ala371Thr variant is hypomorphic with attenuated ability to transfer the activated UFM1 to UFC1. Finally, we show that the CNS-specific knockout of Ufm1 in mice causes neonatal death accompanied by microcephaly and apoptosis in specific neurons, further suggesting that the UFM1 system is essential for CNS development and function. Taken together, our data imply that the combination of a hypomorphic p.Ala371Thr variant in trans with a loss-of-function allele in UBA5 underlies a severe infantile-onset encephalopathy.

Autophagy-deficient mice develop multiple liver tumors.

Autophagy is a major pathway for degradation of cytoplasmic proteins and organelles, and has been implicated in tumor suppression. Here, we report that mice with systemic mosaic deletion of Atg5 and liver-specific Atg7⁻/⁻ mice develop benign liver adenomas. These tumor cells originate autophagy-deficient hepatocytes and show mitochondrial swelling, p62 accumulation, and oxidative stress and genomic damage responses. The size of the Atg7⁻/⁻ liver tumors is reduced by simultaneous deletion of p62. These results suggest that autophagy is important for the suppression of spontaneous tumorigenesis through a cell-intrinsic mechanism, particularly in the liver, and that p62 accumulation contributes to tumor progression.

Autocrine and Paracrine Interactions between Multiple Myeloma Cells and Bone Marrow Stromal Cells by Growth Arrest-specific Gene 6 Cross-talk with Interleukin-6.

The pathogenesis of multiple myeloma (MM) has not yet been fully elucidated. Our microarray analysis and immunohistochemistry revealed significant up-regulation of growth arrest-specific gene 6 (Gas6), a vitamin K-dependent protein with a structural homology with protein S, in bone marrow (BM) cells of MM patients. ELISA showed that the serum levels of soluble Gas6 were significantly increased in the MM patients when compared with healthy controls. Gas6 was overexpressed in the human CD138-positive MM cell line RPMI-8226. Exogenous Gas6 suppressed apoptosis induced by serum deprivation and enhanced cell proliferation of the MM cells. The conditional medium from the human BM stromal cell line HS-5 induced cell proliferation and anti-apoptosis of the MM cells with extracellular signal-regulated kinase, Akt, and nuclear factor-κB phosphorylation, which were reversed by the neutralizing antibody to Gas6 or IL-6. The TAM family receptor Mer, which has been identified as a Gas6 receptor, was overexpressed in BM cells of MM patients. The knockdown of Mer by siRNA inhibited cell proliferation, anti-apoptosis, and up-regulation of intercellular cell adhesion molecule-1 (ICAM-1) in MM cells stimulated by an HS-5 cell-conditioned medium. Furthermore, the Gas6-neutralizing antibody reduced the up-regulation of IL-6 and ICAM-1 induced by a HS-5 cell-conditioned medium in MM cells. The present study provides new evidence that autocrine and paracrine stimulation of Gas6 in concert with IL-6 contributes to the pathogenesis of MM, suggesting that Gas6-Mer-related signaling pathways may be a promising novel target for treating MM.

Mon1-Ccz1 activates Rab7 only on late endosomes and dissociates from the lysosome in mammalian cells.

Rab GTPases act as molecular switches regulating various aspects of membrane trafficking. Among them, Rab5 and Rab7 play central roles in the endolysosomal network. Although many effectors downstream of Rab7 have been elucidated, our present understanding of the mechanism regulating Rab7 activity is extremely limited. It has only recently been accepted that the Mon1-Ccz1 complex is a Rab7 guanine nucleotide exchange factor, but it still remains unclear what the location where Mon1-Ccz1 works with Rab7 is. To address what kind of change or switch exists in the regulatory mechanism upstream of Rab7 during its transition from the late endosome to lysosome, we examined Rab7 activity in steady-state cells and during EGF-induced macropinocytosis using a newly developed FRET sensor. A combination of a Rab7 sensor and confocal FRET imaging techniques revealed that the activation of Rab7 on late endosomes depends on Mon1-Ccz1 and is implicated in late-endosome-lysosome fusion. In contrast, Rab7 activity on lysosomes was independent of Mon1-Ccz1 and active Rab7 played a role in perinuclear clustering of lysosomes.

Sqstm1-GFP knock-in mice reveal dynamic actions of Sqstm1 during autophagy and under stress conditions in living cells.

Sqstm1 serves as a signaling hub and receptor for selective autophagy. Consequently, dysregulation of Sqstm1 causes imbalances in signaling pathways and disrupts proteostasis, thereby contributing to the development of human diseases. Environmental stresses influence the level of Sqstm1 by altering its expression and/or autophagic degradation, and also changes the localization of Sqstm1, making it difficult to elucidate the actions and roles of this protein. In this study, we developed knock-in mice expressing Sqstm1 fused to GFP (Sqstm1-GFP(KI/+)). Using these Sqstm1-GFP(KI/+) mice, we revealed for the first time the dynamics of endogenous Sqstm1 in living cells. Sqstm1-GFP was translocated to a restricted area of LC3-positive structures, which primarily correspond to the inside of autophagosomes, and then degraded. Moreover, exposure to arsenite induced expression of Sqstm1-GFP, followed by accumulation of the fusion protein in large aggregates that were degraded by autophagy. Furthermore, suppression of autophagy in Sqstm1-GFP(KI/+) mouse livers caused accumulation of Sqstm1-GFP and formation of GFP-positive aggregate structures, leading to severe hepatic failure. These results indicate that Sqstm1-GFP(KI/+) mice are a useful tool for analyzing Sqstm1 in living cells and intact animals.

Metastasis of breast cancer cells to the bone, lung, and lymph nodes promotes resistance to ionizing radiation.

BACKGROUND: Metastasis represents the leading cause of breast cancer deaths, necessitating strategies for its treatment. Although radiotherapy is employed for both primary and metastatic breast cancers, the difference in their ionizing radiation response remains incompletely understood. This study is the first to compare the radioresponse of a breast cancer cell line with its metastatic variants and report that such metastatic variants are more radioresistant. MATERIALS AND METHODS: A luciferase expressing cell line was established from human basal-like breast adenocarcinoma MDA-MB-231 and underwent in vivo selections, whereby a cycle of inoculations into the left cardiac ventricle or the mammary fat pad of athymic nude mice, isolation of metastases to the bone, lung and lymph nodes visualized with bioluminescence imaging, and expansion of obtained cells was repeated twice or three times. The established metastatic cell lines were assessed for cell proliferation, wound healing, invasion, clonogenic survival, and apoptosis. RESULTS: The established metastatic cell lines possessed an increased proliferative potential in vivo and were more chemotactic, invasive, and resistant to X­ray-induced clonogenic inactivation and apoptosis in vitro. CONCLUSION: Breast cancer metastasis to the bone, lung, and lymph nodes promotes radioresistance.

Proteasome dysfunction activates autophagy and the Keap1-Nrf2 pathway.

The ubiquitin-proteasome system and autophagy are crucially important for proteostasis in cells. These pathways are interdependent, and dysfunction in either pathway causes accumulation of ubiquitin-positive aggregates, a hallmark of human pathological conditions. To elucidate in vivo compensatory action(s) against proteasomal dysfunction, we developed mice with reduced proteasome activity in their livers. The mutant mice exhibited severe liver damage, accompanied by formation of aggregates positive for ubiquitin and p62/Sqstm1, an adaptor protein for both selective autophagy and the anti-oxidative Keap1-Nrf2 pathway. These aggregates were selectively entrapped by autophagosomes, and pathological features of livers with impaired proteasome activity were exacerbated by simultaneous suppression of autophagy. In contrast, concomitant loss of p62/Sqstm1 had no apparent effect on the liver pathology though p62/Sqstm1 was indispensable for the aggregates formation. Furthermore, defective proteasome function led to transcriptional activation of the Nrf2, which served as a physiological adaptation. Our in vivo data suggest that cells contain networks of cellular defense mechanisms against defective proteostasis.

Uptake through glycoprotein 2 of FimH(+) bacteria by M cells initiates mucosal immune response.

The mucosal immune system forms the largest part of the entire immune system, containing about three-quarters of all lymphocytes and producing grams of secretory IgA daily to protect the mucosal surface from pathogens. To evoke the mucosal immune response, antigens on the mucosal surface must be transported across the epithelial barrier into organized lymphoid structures such as Peyer's patches. This function, called antigen transcytosis, is mediated by specialized epithelial M cells. The molecular mechanisms promoting this antigen uptake, however, are largely unknown. Here we report that glycoprotein 2 (GP2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for mucosal antigens. Recombinant GP2 protein selectively bound a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane. Consistently, these bacteria were colocalized with endogenous GP2 on the apical plasma membrane as well as in cytoplasmic vesicles in M cells. Moreover, deficiency of bacterial FimH or host GP2 led to defects in transcytosis of type-I-piliated bacteria through M cells, resulting in an attenuation of antigen-specific immune responses in Peyer's patches. GP2 is therefore a previously unrecognized transcytotic receptor on M cells for type-I-piliated bacteria and is a prerequisite for the mucosal immune response to these bacteria. Given that M cells are considered a promising target for oral vaccination against various infectious diseases, the GP2-dependent transcytotic pathway could provide a new target for the development of M-cell-targeted mucosal vaccines.

Rab11 regulates exocytosis of recycling vesicles at the plasma membrane.

Rab11 is known to associate primarily with perinuclear recycling endosomes and regulate recycling of endocytosed proteins. However, the recycling step in which Rab11 participates remains unknown. We show here that, in addition to causing tubulation of recycling endosomes, Rab11 depletion gives rise to accumulation of recycling carriers containing endocytosed transferrin and transferrin receptor beneath the plasma membrane. We also show that the carriers are transported from perinuclear recycling endosomes to the cell periphery along microtubules. Total internal reflection fluorescence microscopy of cells expressing EGFP-tagged transferrin receptor revealed that Rab11 depletion inhibits tethering and fusion of recycling carriers to the plasma membrane. Depletion of Sec15, which interacts with Rab11, or Exo70, both components of the exocyst tethering complex, leads to essentially the same phenotypes as those of Rab11 depletion. Thus, in addition to its role in recycling processes at perinuclear recycling endosomes, Rab11 is transported along microtubules to the cell periphery through association with recycling carriers, and directly regulates vesicle exocytosis at the plasma membrane in concert with the exocyst.

AP-1 clathrin adaptor and CG8538/Aftiphilin are involved in Notch signaling during eye development in Drosophila melanogaster.

Clathrin adaptor protein complex-1 (AP-1) and its accessory proteins play a role in the sorting of integral membrane proteins at the trans-Golgi network and endosomes. Their physiological functions in complex organisms, however, are not fully understood. In this study, we found that CG8538p, an uncharacterized Drosophila protein, shares significant structural and functional characteristics with Aftiphilin, a mammalian AP-1 accessory protein. The Drosophila Aftiphilin was shown to interact directly with the ear domain of γ-adaptin of Drosophila AP-1, but not with the GAE domain of Drosophila GGA. In S2 cells, Drosophila Aftiphilin and AP-1 formed a complex and colocalized at the Golgi compartment. Moreover, tissue-specific depletion of AP-1 or Aftiphilin in the developing eyes resulted in a disordered alignment of photoreceptor neurons in larval stage and roughened eyes with aberrant ommatidia in adult flies. Furthermore, AP-1-depleted photoreceptor neurons showed an intracellular accumulation of a Notch regulator, Scabrous, and downregulation of Notch by promoting its degradation in the lysosomes. These results suggest that AP-1 and Aftiphilin are cooperatively involved in the intracellular trafficking of Notch during eye development in Drosophila.

Mice deficient in ficolin, a lectin complement pathway recognition molecule, are susceptible to Streptococcus pneumoniae infection.

Mannose-binding lectin (MBL) and ficolin are complexed with MBL-associated serine proteases, key enzymes of complement activation via the lectin pathway, and act as soluble pattern recognition molecules in the innate immune system. Although numerous reports have revealed the importance of MBL in infectious diseases and autoimmune disorders, the role of ficolin is still unclear. To define the specific role of ficolin in vivo, we generated model mice deficient in ficolins. The ficolin A (FcnA)-deficient (Fcna(-/-)) and FcnA/ficolin B double-deficient (Fcna(-/-)b(-/-)) mice lacked FcnA-mediated complement activation in the sera, because of the absence of complexes comprising FcnA and MBL-associated serine proteases. When the host defense was evaluated by transnasal infection with a Streptococcus pneumoniae strain, which was recognized by ficolins, but not by MBLs, the survival rate was significantly reduced in all three ficolin-deficient (Fcna(-/-), Fcnb(-/-), and Fcna(-/-)b(-/-)) mice compared with wild-type mice. Reconstitution of the FcnA-mediated lectin pathway in vivo improved survival rate in Fcna(-/-) but not in Fcna(-/-)b(-/-) mice, suggesting that both FcnA and ficolin B are essential in defense against S. pneumoniae. These results suggest that ficolins play a crucial role in innate immunity against pneumococcal infection through the lectin complement pathway.

Endosomal protein expression of γ1-adaptin is associated with tumor growth activity and relapse-free survival in breast cancer.

BACKGROUND: γ1-Adaptin is a subunit of adaptor protein complex-1 (AP-1), which regulates intracellular transport between the trans-Golgi network (TGN) and endosomes. Since expression levels of AP-1 subunits have been reported to be associated with cell proliferation and cancer malignancy, we investigated the relationships between the immunohistochemical expression of γ1-adaptin and both clinicopathological factors and relapse-free survival (RFS) in breast cancer tissue. MATERIALS AND METHODS: SK-BR-3 cell line depleted of γ1-adaptin was used for cell proliferation, migration, and invasion assay. Intracellular localization of γ1-adaptin was examined with immunohistochemistry (IHC) using an antibody against γ1-adaptin, and with double immunohistofluorescence (IHF) microscopy using markers for the TGN and endosome. γ1-Adaptin intensities in IHC samples from 199 primary breast cancer patients were quantified and assessed in relation to clinicopathological factors and RFS. RESULTS: Cell growth, migration, and invasion of SK-BR-3 cells were significantly suppressed by the depletion of γ1-adaptin. Although the staining patterns in the cancer tissues varied among cases by IHC, double IHF demonstrated that γ1-adaptin was mainly localized in EEA1-positive endosomes, but not in the TGN. γ1-Adaptin intensity was significantly higher in the tumor regions than in non-tumor regions. It was also higher in patients with Ki-67 (high), ER (-), PgR (-), and HER2 (+). Among subtypes of breast cancer, γ1-adaptin intensity was higher in HER2 than in luminal A or luminal B. The results of the survival analysis indicated that high γ1-adaptin intensity was significantly associated with worse RFS, and this association was also observed in group with ER (+), PgR (+), HER2 (-), Ki-67 (high), or luminal B. In addition, the Cox proportional hazards model showed that high γ1-adaptin intensity was an independent prognostic factor. CONCLUSION: These results suggest that the endosomal expression of γ1-adaptin is positively correlated with breast cancer malignancy and could be a novel prognostic marker.

Mitosis-coupled, microtubule-dependent clustering of endosomal vesicles around centrosomes.

Upon cell division, not only cells themselves but also their organelles undergo drastic shape changes, although the behaviors of organelles other than the Golgi apparatus remain poorly understood. We followed the spatiotemporal changes in the localization of transferrin receptor (TfnR) and other proteins. In early mitotic phases, a population of proteins cycling through the endocytic recycling compartment (ERC) exhibits a distinct spatiotemporal change from that of Golgi proteins. In prophase/prometaphase, when the cell surface-to-volume ratio is reaching its minimum, the ERC proteins are transiently assembled around the centrated centrosome in a microtubule- and dynein-dependent manner, and soon separated polewards into two clusters concomitant with separation of duplicated centrosomes. Electron microscopic analysis revealed that endosomal vesicles containing endocytosed transferrin cluster tightly around centrosomes without fusing with one another. As cytokinesis proceeds, the clusters gradually collapse, and the ERC proteins reassemble around the furrowing equatorial region. FRAP (fluorescence recovery after photobleaching) analyses of EGFP-TfnR-expressing cells revealed minimal membrane exchange between the endosomal clusters and other cellular compartments until anaphase/telophase, when membrane traffic resumes. Our observations indicate that ERC clustering around centrosomes plays a fundamental role in restricting membrane delivery to the plasma membrane during early mitotic phases, when the cell surface-to-volume ratio reaches its minimum.

[The MD-PhD program at Fukushima Medical University].

Fukushima Medical University launched an MD-PhD program in July, 2011, to foster a research-oriented mindset among medical students. This program consists of a first phase that proceeds in parallel with the regular medical school curriculum, and a second postgraduate phase. Upon completion of the first phase, the second phase is expected to take only 3 years, rather than the usual 4 required for a PhD. Moreover, students can obtain financial support from the university. Because of such attractive benefits, 19 medical students enrolled in 2011, much more than we had expected. Here we outline our program and report on a questionnaire survey taken 8 months after the program started.

ATG9A supports Chlamydia trachomatis infection via autophagy-independent mechanisms.

Chlamydia trachomatis infection can be regulated by autophagy-related (ATG) genes. Here, we found that the depletion of ATG9A, one of the core ATG genes, in HeLa cells suppressed C. trachomatis growth in the inclusion. The growth was restored by re-expressing ATG9A or an ATG9A mutant impairing lipid scramblase activity in ATG9A-knockout (KO) cells. Moreover, the depletion of lipid transfer proteins ATG2A/B, responsible for isolation membrane expansion together with ATG9A, did not significantly alter the growth, suggesting that the non-autophagic function of ATG9A supports C. trachomatis infection. ATG9A-KO cells showed no infection-induced redistribution of the Golgi from the perinuclear region to inclusion, which was restored by re-expressing the mutant but not the ATG9A mutant lacking an N-terminal adapter protein-binding domain. Re-expression of the N-terminal deletion mutant in ATG9A-KO cells did not rescue C. trachomatis growth, suggesting the importance of this domain for its growth. Although ATG9A-KO cells showed enhanced TBK1 activation, interferon (IFN)-ß was not significantly increased, excluding the possibility that upregulation of stimulator of IFN genes (STING) signaling suppressed bacterial growth. Taken together, these findings suggest that the proper trafficking, rather than the isolation membrane expansion function, of ATG9A assists C. trachomatis growth in the inclusion. IMPORTANCE ATG9A is an autophagy-related gene that functions during the isolation membrane expansion process to form autophagosomes, but it also has other functions independent of autophagy. In this study, we employed ATG9A-deficient HeLa cells and found that the absence of ATG9A negatively impacted proliferation of Chlamydia trachomatis in inclusions. Furthermore, rescue experiments using ATG9A mutants revealed that this action was mediated not by its autophagic function but by its binding ability to clathrin adapter proteins. These findings suggest that the proper trafficking of ATG9A assists C. trachomatis growth in the inclusion.