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1.
PLoS Biol ; 22(1): e3002486, 2024 Jan.
Article in English | MEDLINE | ID: mdl-38236896

ABSTRACT

Acute gastrointestinal infection with intracellular pathogens like Salmonella Typhimurium triggers the release of the proinflammatory cytokine interleukin 1ß (IL-1ß). However, the role of IL-1ß in intestinal defense against Salmonella remains unclear. Here, we show that IL-1ß production is detrimental during Salmonella infection. Mice lacking IL-1ß (IL-1ß -/-) failed to recruit neutrophils to the gut during infection, which reduced tissue damage and prevented depletion of short-chain fatty acid (SCFA)-producing commensals. Changes in epithelial cell metabolism that typically support pathogen expansion, such as switching energy production from fatty acid oxidation to fermentation, were absent in infected IL-1ß -/- mice which inhibited Salmonella expansion. Additionally, we found that IL-1ß induces expression of complement anaphylatoxins and suppresses the complement-inactivator carboxypeptidase N (CPN1). Disrupting this process via IL-1ß loss prevented mortality in Salmonella-infected IL-1ß -/- mice. Finally, we found that IL-1ß expression correlates with expression of the complement receptor in patients suffering from sepsis, but not uninfected patients and healthy individuals. Thus, Salmonella exploits IL-1ß signaling to outcompete commensal microbes and establish gut colonization. Moreover, our findings identify the intersection of IL-1ß signaling and the complement system as key host factors involved in controlling mortality during invasive Salmonellosis.


Subject(s)
Interleukin-1beta , Salmonella Infections , Animals , Humans , Mice , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Neutrophils/metabolism , Salmonella Infections/metabolism , Salmonella typhimurium/metabolism , Virulence
2.
Transl Psychiatry ; 13(1): 305, 2023 10 02.
Article in English | MEDLINE | ID: mdl-37783686

ABSTRACT

Autism is a neurodevelopmental disorder characterized by early-onset social behavioral deficits and repetitive behaviors. Chromodomain helicase DNA-binding protein (CHD8) is among the genes most strongly associated with autism. In addition to the core behavioral symptoms of autism, affected individuals frequently present with gastrointestinal symptoms that are also common among individuals harboring mutations in the gene encoding CHD8. However, little is known regarding the mechanisms whereby CHD8 affects gut function. In addition, it remains unknown whether gastrointestinal manifestations contribute to the behavioral phenotypes of autism. The current study found that mice haploinsufficient for the large isoform of Chd8 (Chd8L) exhibited increased intestinal permeability, transcriptomic dysregulation in gut epithelial cells, reduced tuft cell and goblet cell counts in the gut, and an overall increase in microbial load. Gut epithelial cell-specific Chd8 haploinsufficiency was associated with increased anxiety-related behaviors together with a decrease in tuft cell numbers. Antibiotic treatment of Chd8L haploinsufficient mice attenuated social behavioral deficits. Together, these results suggest Chd8 as a key determinant of autism-related gastrointestinal deficits, while also laying the ground for future studies on the link between GI deficits and autism-related behaviors.


Subject(s)
Autism Spectrum Disorder , Autistic Disorder , Mice , Animals , Autistic Disorder/genetics , Brain-Gut Axis , Gene Expression Regulation, Developmental , DNA-Binding Proteins/genetics , Autism Spectrum Disorder/genetics , Epithelial Cells
3.
Trends Cell Biol ; 33(10): 817-819, 2023 10.
Article in English | MEDLINE | ID: mdl-37586983

ABSTRACT

Intestinal epithelial cells form the largest barrier in the body, separating us from the outside world. Here, we review recent findings that highlight the role of autophagy in the cell-intrinsic response of the epithelial cells to the harsh intestinal environment and how they shape host physiology.


Subject(s)
Autophagy , Epithelial Cells , Humans , Inflammation
4.
Autophagy ; 19(11): 3014-3016, 2023 11.
Article in English | MEDLINE | ID: mdl-37436421

ABSTRACT

Mucus secretion from colonic goblet cells is an important host defense mechanism against the harsh lumenal environment. Yet how mucus secretion is regulated is not well understood. We discovered that constitutive activation of macroautophagy/autophagy via BECN1 (beclin 1) relieves endoplasmic reticulum (ER) stress in goblet cells, which in turn produce a thicker and less penetrable mucus barrier. Pharmacological reduction of the ER stress or activation of the unfolded protein response (UPR) in mice, regardless of autophagy activation, lead to excess mucus secretion. This regulation of mucus secretion by ER stress is microbiota-dependent and requires the activity of the intracellular sensor NOD2 (nucleotide-binding oligomerization domain containing 2). Excess mucus production in the colon alters the gut microbiota and protects from chemical- and infection-driven inflammation. Our findings provide new insights into the mechanisms by which autophagy regulates mucus secretion and susceptibility to intestinal inflammation.Abbreviations:BECN1- Beclin 1; ER- endoplasmic reticulum; UPR - unfolded protein response; NOD2 - nucleotide-binding oligomerization domain containing 2; IBD- inflammatory bowel disease; BCL2- B cell leukemia/lymphoma 2; TUDCA- tauroursodeoxycholic acid; ATG16L1- autophagy related 16 like 1; LRRK2- leucine-rich repeat kinase 2.


Subject(s)
Autophagy , Endoplasmic Reticulum Stress , Animals , Mice , Beclin-1/metabolism , Autophagy/physiology , Inflammation/metabolism , Mucus/metabolism , Nucleotides/metabolism
5.
Cell Host Microbe ; 31(3): 433-446.e4, 2023 03 08.
Article in English | MEDLINE | ID: mdl-36738733

ABSTRACT

Colonic goblet cells are specialized epithelial cells that secrete mucus to physically separate the host and its microbiota, thus preventing bacterial invasion and inflammation. How goblet cells control the amount of mucus they secrete is unclear. We found that constitutive activation of autophagy in mice via Beclin 1 enables the production of a thicker and less penetrable mucus layer by reducing endoplasmic reticulum (ER) stress. Accordingly, genetically inhibiting Beclin 1-induced autophagy impairs mucus secretion, while pharmacologically alleviating ER stress results in excessive mucus production. This ER-stress-mediated regulation of mucus secretion is microbiota dependent and requires the Crohn's-disease-risk gene Nod2. Overproduction of mucus alters the gut microbiome, specifically expanding mucus-utilizing bacteria, such as Akkermansia muciniphila, and protects against chemical and microbial-driven intestinal inflammation. Thus, ER stress is a cell-intrinsic switch that limits mucus secretion, whereas autophagy maintains intestinal homeostasis by relieving ER stress.


Subject(s)
Goblet Cells , Inflammation , Animals , Mice , Beclin-1 , Mucus , Autophagy , Intestinal Mucosa/microbiology
6.
Cell Rep ; 41(7): 111657, 2022 11 15.
Article in English | MEDLINE | ID: mdl-36384106

ABSTRACT

Changes in microbiome composition are associated with a wide array of human diseases, turning the human microbiota into an attractive target for therapeutic intervention. Yet, clinical translation of these findings requires the establishment of causative connections between specific microbial taxa and their functional impact on host tissues. Here, we infuse gut organ cultures with longitudinal microbiota samples collected from therapy-naive patients with irritable bowel syndrome (IBS) under a low-fermentable oligo-, di-, mono-saccharides and polyols (FODMAP) diet. We show that post-diet microbiota regulates intestinal expression of inflammatory and neuro-muscular gene sets. Specifically, we identify Bifidobacterium adolescentis as a diet-sensitive pathobiont that alters tight junction integrity and disrupts gut barrier functions. Collectively, we present a pathway discovery platform for mechanistic dissection and identification of functional diet-host-microbiota modules. Our data support the hypothesis that the gut microbiota mediates the beneficial effects of a low-FODMAP diet and reinforce the potential feasibility of microbiome-based therapies in IBS.


Subject(s)
Gastrointestinal Microbiome , Irritable Bowel Syndrome , Humans , Irritable Bowel Syndrome/therapy , Diet, Carbohydrate-Restricted , Homeostasis
8.
Int J Mol Sci ; 22(7)2021 Mar 25.
Article in English | MEDLINE | ID: mdl-33806191

ABSTRACT

Metabolic plasticity is a hallmark of the ability of metastatic cancer cells to survive under stressful conditions. The intracellular Fer kinase is a selective constituent of the reprogramed mitochondria and metabolic system of cancer cells. In the current work, we deciphered the modulatory roles of Fer in the reprogrammed metabolic systems of metastatic, lung (H358), non-small cell lung cancer (NSCLC), and breast (MDA-MB-231), triple-negative breast cancer (TNBC), carcinoma cells. We show that H358 cells devoid of Fer (H358ΔFer), strictly depend on glucose for their proliferation and growth, and fail to compensate for glucose withdrawal by oxidizing and metabolizing glutamine. Furthermore, glucose deficiency caused increased reactive oxygen species (ROS) production and induction of a DNA damage response (DDR), accompanied by the onset of apoptosis and attenuated cell-cycle progression. Analysis of mitochondrial function revealed impaired respiratory and electron transport chain (ETC) complex 1 (comp. I) activity in the Fer-deficient H358ΔFer cells. This was manifested by decreased levels of NAD+ and ATP and relatively low abundance of tricarboxylic acid (TCA) cycle metabolites. Impaired electron transport chain comp. I activity and dependence on glucose were also confirmed in Fer-deficient, MDA-MB-231ΔFer cells. Although both H358ΔFer and MDA-MB-231ΔFer cells showed a decreased aspartate level, this seemed to be compensated by the predominance of pyrimidines synthesis over the urea cycle progression. Notably, absence of Fer significantly impeded the growth of H358ΔFer and MDA-MB-231ΔFer xenografts in mice provided with a carb-deficient, ketogenic diet. Thus, Fer plays a key role in the sustention of metabolic plasticity of malignant cells. In compliance with this notion, targeting Fer attenuates the progression of H358 and MDA-MB-231 tumors, an effect that is potentiated by a glucose-restrictive diet.


Subject(s)
Breast Neoplasms/metabolism , Lung Neoplasms/metabolism , Mitochondria/metabolism , Protein-Tyrosine Kinases/metabolism , Alleles , Animals , Carcinoma/metabolism , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Line, Tumor , DNA Damage , Homeostasis , Humans , Mice , Mice, Nude , Neoplasm Metastasis , Neoplasm Transplantation , Phosphorylation , Reactive Oxygen Species/metabolism
9.
Trends Immunol ; 41(12): 1054-1056, 2020 12.
Article in English | MEDLINE | ID: mdl-33158739

ABSTRACT

Lysozyme-secreting Paneth cells are abnormally present in the distal colons of patients with inflammatory bowel disease (IBD), along with high amounts of lysozyme in feces. In a recent article in Immunity, Yu et al. show that lysozyme-mediated processing of luminal bacteria in the colon triggers a proinflammatory response and predisposes mice to experimental IBD.


Subject(s)
Inflammatory Bowel Diseases , Microbiota , Animals , Colon , Expectorants , Humans , Mice , Muramidase , Paneth Cells
10.
Cell Host Microbe ; 25(6): 777-788.e8, 2019 Jun 12.
Article in English | MEDLINE | ID: mdl-31101494

ABSTRACT

Vitamin A deficiency increases susceptibility to skin infection. However, the mechanisms by which vitamin A regulates skin immunity remain unclear. Here, we show that resistin-like molecule α (RELMα), a small secreted cysteine-rich protein, is expressed by epidermal keratinocytes and sebocytes and serves as an antimicrobial protein that is required for vitamin-A-dependent resistance to skin infection. RELMα was induced by microbiota colonization of the murine skin, was bactericidal in vitro, and was protected against bacterial infection of the skin in vivo. RELMα expression required dietary vitamin A and was induced by the therapeutic vitamin A analog isotretinoin, which protected against skin infection in a RELMα-dependent manner. The RELM family member Resistin was expressed in human skin, was induced by vitamin A analogs, and killed skin bacteria, indicating a conserved function for RELM proteins in skin innate immunity. Our findings provide insight into how vitamin A promotes resistance to skin infection.


Subject(s)
Antimicrobial Cationic Peptides/metabolism , Immunologic Factors/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Skin Diseases, Bacterial/prevention & control , Skin/immunology , Vitamin A/metabolism , Animals , Cells, Cultured , Disease Models, Animal , Epithelial Cells/immunology , Epithelial Cells/metabolism , Humans , Mice , Resistin/metabolism , Skin Diseases, Bacterial/immunology , Transcriptional Activation/drug effects
11.
Autophagy ; 14(4): 719-721, 2018.
Article in English | MEDLINE | ID: mdl-29388875

ABSTRACT

Secretion of antimicrobial proteins is an important host defense mechanism against bacteria, yet how secretory cells maintain function during bacterial invasion has been unclear. We discovered that Paneth cells, specialized secretory cells in the small intestine, react to bacterial invasion by rerouting a critical secreted antibacterial protein through a macroautophagy/autophagy-based secretion system termed secretory autophagy. Mice harboring a mutation in an essential autophagy gene, a mutation which is common in Crohn disease patients, cannot reroute their antimicrobial cargo during bacterial invasion and thus have compromised innate immunity. We showed that this alternative secretion system is triggered by both a cell-intrinsic mechanism, involving the ER stress response, and a cell-extrinsic mechanism, involving subepithelial innate immune cells. Our findings uncover a new role for secretory autophagy in host defense and suggest how a mutation in an autophagy gene can predispose individuals to Crohn disease.


Subject(s)
Autophagy/physiology , Endoplasmic Reticulum Stress/genetics , Intestine, Small/microbiology , Paneth Cells/metabolism , Autophagy-Related Proteins/metabolism , Biological Transport/physiology , Carrier Proteins/metabolism , Crohn Disease/genetics , Crohn Disease/microbiology , Muramidase/metabolism , Mutation/genetics , Paneth Cells/microbiology
12.
Science ; 357(6355): 1047-1052, 2017 09 08.
Article in English | MEDLINE | ID: mdl-28751470

ABSTRACT

Intestinal Paneth cells limit bacterial invasion by secreting antimicrobial proteins, including lysozyme. However, invasive pathogens can disrupt the Golgi apparatus, interfering with secretion and compromising intestinal antimicrobial defense. Here we show that during bacterial infection, lysozyme is rerouted via secretory autophagy, an autophagy-based alternative secretion pathway. Secretory autophagy was triggered in Paneth cells by bacteria-induced endoplasmic reticulum (ER) stress, required extrinsic signals from innate lymphoid cells, and limited bacterial dissemination. Secretory autophagy was disrupted in Paneth cells of mice harboring a mutation in autophagy gene Atg16L1 that confers increased risk for Crohn's disease in humans. Our findings identify a role for secretory autophagy in intestinal defense and suggest why Crohn's disease is associated with genetic mutations that affect both the ER stress response and autophagy.


Subject(s)
Endoplasmic Reticulum Stress/immunology , Muramidase/metabolism , Paneth Cells/immunology , Paneth Cells/metabolism , Salmonella Infections/immunology , Salmonella enterica , Animals , Autophagy/genetics , Autophagy-Related Proteins , Carrier Proteins/genetics , Carrier Proteins/metabolism , Crohn Disease/genetics , Crohn Disease/immunology , Crohn Disease/microbiology , Endoplasmic Reticulum Stress/genetics , Mice , Mice, Inbred C57BL , Microtubule-Associated Proteins/metabolism , Mutation , Paneth Cells/enzymology , Salmonella Infections/genetics
13.
PLoS One ; 10(12): e0145277, 2015.
Article in English | MEDLINE | ID: mdl-26701263

ABSTRACT

TMF/ARA160 is known to be a TATA element Modulatory Factor (TMF). It was initially identified as a DNA-binding factor and a coactivator of the Androgen receptor. It was also characterized as a Golgi-associated protein, which is essential for acrosome formation during functional sperm development. However, the molecular roles of TMF in this intricate process have not been revealed. Here, we show that during spermiogenesis, TMF undergoes a dynamic change of localization throughout the Golgi apparatus. Specifically, TMF translocates from the cis-Golgi to the trans-Golgi network and to the emerging vesicles surface, as the round spermatids develop. Notably, lack of TMF led to an abnormal spatial orientation of the Golgi and to the deviation of the trans-Golgi surface away from the nucleus of the developing round spermatids. Concomitantly, pro-acrosomal vesicles derived from the TMF-/- Golgi lacked targeting properties and did not tether to the spermatid nuclear membrane thereby failing to form the acrosome anchoring scaffold, the acroplaxome, around the cell-nucleus. Absence of TMF also perturbed the positioning of microtubules, which normally lie in proximity to the Golgi and are important for maintaining Golgi spatial orientation and dynamics and for chromatoid body formation, which is impaired in TMF-/- spermatids. In-silico evaluation combined with molecular and electron microscopic analyses revealed the presence of a microtubule interacting domain (MIT) in TMF, and confirmed the association of TMF with microtubules in spermatogenic cells. Furthermore, the MIT domain in TMF, along with microtubules integrity, are required for stable association of TMF with the Golgi apparatus. Collectively, we show here for the first time that a Golgi and microtubules associated protein is crucial for maintaining proper Golgi orientation during a cell developmental process.


Subject(s)
Golgi Apparatus/metabolism , Spermatogenesis , Ubiquitin-Protein Ligases/physiology , Vesicular Transport Proteins/physiology , Animals , Cell Differentiation/genetics , DNA-Binding Proteins , Gene Deletion , Golgi Matrix Proteins , Male , Mice , Mice, Inbred ICR , Microtubules/metabolism , Microtubules/ultrastructure , NIH 3T3 Cells , Protein Structure, Tertiary , Sequence Analysis, Protein , Spermatids/metabolism , Spermatids/ultrastructure , Transcription Factors , Tubulin/metabolism , Ubiquitin-Protein Ligases/genetics , Vesicular Transport Proteins/genetics
15.
Proc Natl Acad Sci U S A ; 111(13): 4964-9, 2014 Apr 01.
Article in English | MEDLINE | ID: mdl-24639530

ABSTRACT

Tata Element Modulatory Factor (TMF/ARA160) is a multifunctional Golgi-associated protein, which accumulates in colonic enterocytes and goblet cells. Mice lacking TMF/ARA160 (TMF(-/-)) produce thick and uniform colonic mucus that resists adherent bacterial colonization and diminishes susceptibility of these mice to induced acute colitis, through a mechanism that is not fully understood. Here, we show that mucus secretion by goblet cells is altered in the colon of TMF(-/-) mice, resulting in the formation of a highly oligomerized colonic gel-forming mucin, MUC2. Microbiome analysis revealed a shift in the microbiota of TMF(-/-) mice leading to predominance of the Firmicutes phylum and a significantly higher abundance of probiotic beneficial bacterial species. Notably, this trait was transmissible, and when cohoused with wild-type animals, TMF(-/-) mice influenced the microbiota and diminished the susceptibility of wild-type mice to chemically induced dextran sulfate sodium colitis. Thus, altered mucus secretion in TMF(-/-) mouse colons is accompanied by a reprogrammed intestinal microbiota, leading to a transmissible reduced sensitivity to induced colitis.


Subject(s)
Colitis/microbiology , Colitis/pathology , Intestines/microbiology , Intestines/pathology , Microbiota , Ubiquitin-Protein Ligases/deficiency , Vesicular Transport Proteins/deficiency , Animals , Cell Shape , Colitis/chemically induced , Colon/metabolism , Colon/pathology , Colon/ultrastructure , DNA-Binding Proteins , Disease Susceptibility/microbiology , Disease Susceptibility/pathology , Feces/microbiology , Golgi Matrix Proteins , Intestines/ultrastructure , Mice , Mice, Inbred C57BL , Mice, Knockout , Mucin-2/metabolism , Mucus/metabolism , Protein Multimerization , Transcription Factors , Ubiquitin-Protein Ligases/metabolism , Vesicular Transport Proteins/metabolism
16.
Mol Cell Endocrinol ; 365(1): 52-63, 2013 Jan 05.
Article in English | MEDLINE | ID: mdl-23000399

ABSTRACT

TMF/ARA160 is a Golgi-associated protein, which is essential for spermiogenesis. In this study, we show that lack of TMF/ARA160 leads to defects in both the testis and the epididymis. In the testis, spermatid retention and extensive proliferation of Leydig cells were observed. Concomitantly, the serum levels of luteinizing hormone (LH), a stimulator of Leydig cell proliferation, were significantly increased in TMF(-/-) mice. Structural and functional defects were also seen in the epididymis. These included apoptosis of epithelial epididymal cells and sperm stasis in the cauda. Notably, the serum testosterone levels of TMF(-/-) mice were significantly lower than those of wt mice, and external testosterone administration decreased the number of apoptotic epithelial epididymal cells in TMF(-/-) animals. In summary, we show here for the first time that TMF/ARA160 participates in the control of serum testosterone levels in males, and its absence results in major testicular and epididymal defects.


Subject(s)
Epididymis/pathology , Testis/pathology , Testosterone/blood , Ubiquitin-Protein Ligases/metabolism , Vesicular Transport Proteins/metabolism , Age Factors , Animals , Apoptosis , Cell Proliferation , DNA-Binding Proteins , Epididymis/abnormalities , Epididymis/metabolism , Gene Expression Profiling , Gene Expression Regulation , Golgi Matrix Proteins , Hormone Replacement Therapy , Leydig Cells/enzymology , Leydig Cells/metabolism , Leydig Cells/pathology , Luteinizing Hormone/blood , Male , Mice , Mice, Inbred ICR , Mice, Knockout , Oligonucleotide Array Sequence Analysis , RNA, Messenger/metabolism , Spermatogenesis , Testis/abnormalities , Testis/metabolism , Testosterone/metabolism , Testosterone/therapeutic use , Transcription Factors , Ubiquitin-Protein Ligases/deficiency , Ubiquitin-Protein Ligases/genetics , Vesicular Transport Proteins/deficiency , Vesicular Transport Proteins/genetics
17.
J Biol Chem ; 287(30): 25631-9, 2012 Jul 20.
Article in English | MEDLINE | ID: mdl-22553199

ABSTRACT

TMF/ARA160 is a Golgi-associated protein with several cellular functions, among them direction of the NF-κB subunit, p65 RelA, to ubiquitination and proteasomal degradation in stressed cells. We sought to investigate the role of TMF/ARA160 under imposed stress conditions in vivo. TMF(-/-) and wild-type (WT) mice were treated with the ulcerative agent dextran sulfate sodium (DSS), and the severity of the inflicted acute colitis was determined. TMF(-/-) mice were found to be significantly less susceptible to DSS-induced colitis, with profoundly less bacterial penetration into the colonic epithelia. Surprisingly, unlike in WT mice, no bacterial colonies were visualized in colons of healthy untreated TMF(-/-) mice, indicating the constitutive resistance of TMF(-/-) colonic mucus to bacterial retention and penetration. Gene expression analysis of colon tissues from unchallenged TMF(-/-) mice revealed 5-fold elevated transcription of the muc2 gene, which encodes the major component of the colonic mucus gel, the MUC2 mucin. Accordingly, the morphology of the colonic mucus in TMF(-/-) mice was found to differ from the mucus structure in WT colons. The NF-κB subunit, p65, a well known transcription inducer of muc2, was up-regulated significantly in TMF(-/-) intestinal epithelial cells. However, this did not cause spontaneous inflammation or increased colonic crypt cell proliferation. Collectively, our findings demonstrate that absence of TMF/ARA160 renders the colonic mucus refractory to bacterial colonization and the large intestine less susceptible to the onset of colitis.


Subject(s)
Bacteria , Bacterial Translocation , Colitis , Colon , Immunity, Innate , Ubiquitin-Protein Ligases/metabolism , Vesicular Transport Proteins/metabolism , Animals , Bacterial Translocation/genetics , Bacterial Translocation/immunology , Colitis/chemically induced , Colitis/genetics , Colitis/immunology , Colitis/metabolism , Colitis/microbiology , Colon/immunology , Colon/metabolism , Colon/microbiology , Colon/pathology , DNA-Binding Proteins , Dextran Sulfate/toxicity , Golgi Matrix Proteins , Intestinal Mucosa/immunology , Intestinal Mucosa/metabolism , Intestinal Mucosa/microbiology , Intestinal Mucosa/pathology , Mice , Mice, Knockout , Mucin-2/genetics , Mucin-2/immunology , Mucin-2/metabolism , Transcription Factor RelA/genetics , Transcription Factor RelA/metabolism , Transcription Factors , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/immunology , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/immunology
18.
Dev Biol ; 348(1): 12-21, 2010 Dec 01.
Article in English | MEDLINE | ID: mdl-20691678

ABSTRACT

TMF/ARA160 is a Golgi-associated protein to which several cellular activities have been attributed. These include, trafficking of Golgi-derived vesicles and E3 ubiquitin ligase activity. Here we show that TMF/ARA160 is required for the onset of key processes which underlie the development of mature sperm in mammals. TMF/ARA160 is highly expressed in specific spermatogenic stages. While the protein is not detected in the spermatogenic progenitor cells - spermatogonia, it accumulates in the Golgi of spermatocytes and spermatids but then disappears and is absent from spermatozoa and epididymal sperm cells. Mice that are homozygous null for TMF develop normally are healthy and the females are fertile. However, the males are sterile and their spermatids suffer from several developmental defects. They lack homing of Golgi-derived proacrosomal vesicles to the perinuclear surface, resulting in spermatozoa and epididymal sperm cells which lack acrosome. In a later developmental stage, the cytoplasm is not properly removed, thus resulting in spermatids which bare the nucleus with tightly packed DNA, surrounded by a cytoplasm. Finally, the spermatozoa of TMF(-/-) mice also suffer from misshapen heads, tails coiling around the sperm heads, and lack of motility. Taken together our findings portray TMF/ARA160 as a key regulator which is essential for the onset of key events in the differentiation and maturation of mammalian sperm and whose absence severely compromises their ability to fertilize ova.


Subject(s)
Infertility, Male/physiopathology , Sperm Maturation/physiology , Spermatozoa/physiology , Ubiquitin-Protein Ligases/physiology , Vesicular Transport Proteins/physiology , Acrosome/chemistry , Acrosome/ultrastructure , Actin Cytoskeleton/ultrastructure , Animals , Cell Differentiation , Cytoplasm/metabolism , DNA-Binding Proteins , Female , Golgi Apparatus/metabolism , Golgi Matrix Proteins , Infertility, Male/genetics , Male , Mice , Mice, Knockout , Microscopy, Electron , Mitochondria/ultrastructure , Reverse Transcriptase Polymerase Chain Reaction , Sperm Head/ultrastructure , Sperm Motility , Sperm Tail/ultrastructure , Sperm-Ovum Interactions/physiology , Spermatids/metabolism , Spermatids/ultrastructure , Spermatocytes/metabolism , Spermatocytes/ultrastructure , Spermatozoa/abnormalities , Spermatozoa/ultrastructure , Transcription Factors , Ubiquitin-Protein Ligases/deficiency , Ubiquitin-Protein Ligases/genetics , Vesicular Transport Proteins/deficiency , Vesicular Transport Proteins/genetics
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