A genome-wide CRISPR screen identifies the CCT chaperonin as a critical regulator of vesicle trafficking.

Vesicle trafficking is a fundamental cellular process that controls the transport of various proteins and cargos between cellular compartments in eukaryotes. Using a combination of genome-wide CRISPR screening in mammalian cells and RNAi screening in Caenorhabditis elegans, we identify chaperonin containing TCP-1 subunit 4 (CCT4) as a critical regulator of protein secretion and vesicle trafficking. In C. elegans, deficiency of cct-4 as well as other CCT subunits impairs the trafficking of endocytic markers in intestinal cells, and this defect resembles that of dyn-1 RNAi worms. Consistent with these findings, the silencing of CCT4 in human cells leads to defective endosomal trafficking, and this defect can be rescued by the dynamin activator Ryngo 1-23. These results suggest that the cytosolic chaperonin CCT may regulate vesicle trafficking by promoting the folding of dynamin in addition to its known substrate tubulin. Our findings establish an essential role for the CCT chaperonin in regulating vesicle trafficking, and provide new insights into the regulation of vesicle trafficking and the cellular function of the cytosolic chaperonin.

HRG-9 homologues regulate haem trafficking from haem-enriched compartments.

Haem is an iron-containing tetrapyrrole that is critical for a variety of cellular and physiological processes1-3. Haem binding proteins are present in almost all cellular compartments, but the molecular mechanisms that regulate the transport and use of haem within the cell remain poorly understood2,3. Here we show that haem-responsive gene 9 (HRG-9) (also known as transport and Golgi organization 2 (TANGO2)) is an evolutionarily conserved haem chaperone with a crucial role in trafficking haem out of haem storage or synthesis sites in eukaryotic cells. Loss of Caenorhabditis elegans hrg-9 and its paralogue hrg-10 results in the accumulation of haem in lysosome-related organelles, the haem storage site in worms. Similarly, deletion of the hrg-9 homologue TANGO2 in yeast and mammalian cells induces haem overload in mitochondria, the site of haem synthesis. We demonstrate that TANGO2 binds haem and transfers it from cellular membranes to apo-haemoproteins. Notably, homozygous tango2-/- zebrafish larvae develop pleiotropic symptoms including encephalopathy, cardiac arrhythmia and myopathy, and die during early development. These defects partially resemble the symptoms of human TANGO2-related metabolic encephalopathy and arrhythmias, a hereditary disease caused by mutations in TANGO24-8. Thus, the identification of HRG-9 as an intracellular haem chaperone provides a biological basis for exploring the aetiology and treatment of TANGO2-related disorders.

Caenorhabditis elegans homologue of Fam210 is required for oogenesis and reproduction.

Mitochondria are the central hub for many metabolic processes, including the citric acid cycle, oxidative phosphorylation, and fatty acid oxidation. Recent studies have identified a new mitochondrial protein family, Fam210, that regulates bone metabolism and red cell development in vertebrates. The model organism Caenorhabditis elegans has a Fam210 gene, y56a3a.22, but it lacks both bones and red blood cells. In this study, we report that Y56A3A.22 plays a crucial role in regulating mitochondrial protein homeostasis and reproduction. The nematode y56a3a.22 is expressed in various tissues, including the intestine, muscle, hypodermis, and germline, and its encoded protein is predominantly localized in mitochondria. y56a3a.22 deletion mutants are sterile owing to impaired oogenesis. Loss of Y56A3A.22 induced mitochondrial unfolded protein response (UPRmt), which is mediated through the ATFS-1-dependent pathway, in tissues such as the intestine, germline, hypodermis, and vulval muscle. We further show that infertility and UPRmt induces by Y56A3A.22 deficiency are not attributed to systemic iron deficiency. Together, our study reveals an important role of Y56A3A.22 in regulating mitochondrial protein homeostasis and oogenesis and provides a new genetic tool for exploring the mechanisms regulating mitochondrial metabolism and reproduction as well as the fundamental role of the Fam210 family.

Experimental Study on Effects of Adipose-Derived Stem Cell-Seeded Silk Fibroin Chitosan Film on Wound Healing of a Diabetic Rat Model.

BACKGROUND: Wound healing is a complex process that relies on growth factors and stimulation of angiogenesis. Tissue engineering materials composed of adipose-derived stem cells (ADSCs) and silk fibroin (SF)/chitosan (CS) may be able to solve this problem. The aim of this study was to investigate the wound-healing potential of ADSC-seeded SF/CS in streptozotocin-induced diabetic rats. MATERIALS AND METHODS: Thirty-six male Sprague-Dawley rats were purchased and randomly assigned into 3 groups: a control group (no graft), a group treated with SF/CS film graft, and a group treated with ADSC-seeded SF/CS graft. The number of animals in each group was 12. Diabetes was induced by an intraperitoneal injection of streptozotocin. A cutaneous wound was incised at the dorsal region of all the experimental animals. The ADSCs were labeled with CM-Dil fluorescent staining. Wound healing was assessed for all animal groups by observing the rate of wound closure and hematoxylin and eosin staining. The expression of epidermal growth factor, transforming growth factor-ß, and vascular endothelial growth factor at the wound sites was studied by enzyme-linked immunosorbent assay to evaluate the effect of growth factors secreted by ADSCs. The differentiation of ADSCs was analyzed by immunofluorescence staining. RESULTS: The ADSC-seeded SF/CS film treatment significantly increased the rates of wound closure in treated animals, and hence wound healing was drastically enhanced for ADSC-SF/CS treatment groups compared with control groups and SF/CS film treatment group. Histological observations showed the condition of wound healing. Enzyme-linked immunosorbent assay and immunofluorescence staining observations showed the secretion and differentiation of ADSCs, respectively. CONCLUSIONS: Our analyses clearly suggested that it is feasible and effective to enhance wound healing in a diabetic rat model with ADSC-seeded SF/CS film.

[Diagnosis and treatment of pediatric anismus].

OBJECTIVE: To explore the diagnosis and treatment methods of pediatric anismus. METHODS: Twenty-nine patients with idiopathic chronic constipation, diagnosed with anismus by colon barium contrast and anorectal manometry from Nov. 2001 to Nov. 2004 in our hospital, were investigated retrospectively. RESULTS: This group consisted of 13 men and 16 women whose mean age was (6.7+/-4.0) years. Hirschsprung diseases were excluded from the patients by colon barium contrast and rectoanal inhibitory reflex (RAIR) examination. Normal RAIR (5-10 ml elicited) was showed on 21 cases while weakened RAIR (15-30 ml elicited) was showed on 8 cases. After the diagnosis, the patients were treated by toilet training, diet regulation and laxative for 1-2 months. 4 cases were recovered, 5 cases were improved and 20 cases were relied on glycerin suppository. Four cases, relied on glycerin suppository, underwent Lynn procedure and had good results after 5-24 months follow-up. Two cases were re-examined by anorectal manometry 3 and 6 months after surgery, the resting pressure and the high pressure zone (HPZ) decreased, but the simulation defecation reflex was still abnormal. CONCLUSIONS: The diagnosis of pediatric anismus relies on history of constipation, combined with anorectal manometry and colon barium contrast. Lynn procedure could be chosen for the patients unsatisfied in toilet training and other non-operative treatment.

[Diagnosis and surgical management of adult Hirschsprung disease].

OBJECTIVE: To investigate the diagnosis and surgical treatment of adult Hirschsprung disease (AHD). METHODS: Clinical data of 10 patients with AHD undergoing operation from May 1985 to May 2005 were analyzed retrospectively. RESULTS: There were 7 males and 3 females with an age ranged from 14 to 40 years. All the cases had constipation, and were diagnosed by barium enema. Aganglionosis was located in distal sigmoid and rectum in 2 cases, in rectum in 7 cases, unknown in one case. Colostomy was performed in one, Ikeda s operation in 6, Rehbein operation in two,modified Swenson operation in one. After radical operation,7 patients had excellent continence function, one had good function, one had poor function. CONCLUSIONS: The diagnosis of adult HD mainly depends on the history of constipation, barium enema and manometry examination. The pull-through procedures are effective surgical treatments for adult HD. The operation type should be selected individually.