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Coordination of IFT20 With Other IFT Components Is Required for Ciliogenesis

Journal of Clinical Laboratory Analysis 2025年第9期39卷 e70000页

作者： Wang, Weishu Shan, Ying Liu, Ruming Li, Dengwen Zhou, Jun Lu, Quanlong Zhao, Huijie Department of Genetics and Cell Biology State Key Laboratory of Medicinal Chemical Biology Haihe Laboratory of Cell Ecosystem College of Life Sciences Nankai University Tianjin China Center for Cell Structure and Function Shandong Provincial Key Laboratory of Animal Resistance Biology College of Life Sciences Shandong Normal University Jinan China

Background: Primary cilia are organelles formed on the cell surface. They can act as cellular antennae to sense signals and play important roles in various biological processes. Abnormalities in primary cilia lead to a variety of diseases collectively known as ciliopathies. Intraflagellar transport protein 20 (IFT20) has been implicated in ciliogenesis. Methods: IFT20 knockout cell lines were established using the CRISPR-Cas9 gene editing technology. The GFP-IFT20 plasmid was constructed with the Gateway cloning system. Protein levels were detected via immunoblotting, and the localization of IFT20, acetylated α-tubulin, ARL13B, CP110, MKS3, IFT88, and IFT140 in wild-type and IFT20 knockout cells was examined by immunofluorescence microscopy. The fluorescence intensities were analyzed using ImageJ. Data quantifications and mass spectrometry results were analyzed using GraphPad Prism and Metascape. Results: The IFT20 deficiency impaired ciliogenesis and reduced cilium length. IFT20 depletion did not affect the removal of centriolar coiled-coil protein 110 (CP110) from the mother centriole or the recruitment of Meckel–Gruber syndrome type 3 (MKS3) to the transition zone. Mass spectrometry analysis revealed that proteins interacting with IFT20 were mainly IFT components. IFT20 knockout decreased the levels of both IFT88 and IFT140, and abrogated IFT88 localization at the basal body and ciliary axoneme. IFT20 knockout also impaired IFT140 localization at the ciliary axoneme but did not affect its localization at the basal body. Conclusions: IFT20 is involved in ciliogenesis by regulating the level and localization of other IFT proteins and may have important implications in ciliopathies and related diseases. © 2025 The Author(s). Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.

关键词： axoneme basal body ciliogenesis cilium IFT20

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UFMylation Modulates OFIP Stability and Centrosomal Localization

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Journal of Clinical Laboratory Analysis 2025年第6期39卷 e70004页

作者： Yang, Mulin Zhao, Zihe Di, Jie Dong, Dan Li, Dengwen Ran, Jie Department of Genetics and Cell Biology College of Life Sciences State Key Laboratory of Medicinal Chemical Biology Nankai University Tianjin China Center for Cell Structure and Function Shandong Provincial Key Laboratory of Animal Resistance Biology College of Life Sciences Shandong Normal University Jinan China

Background: OFIP, also known as KIAA0753, is a centrosomal and pericentriolar satellite protein implicated in ciliogenesis, centriolar duplication, and microtubule stability. In humans, genetic mutations affecting OFIP have been implicated in the pathogenesis of Oral-Facial-Digital (OFD) Syndrome and Joubert Syndrome. Ubiquitin-fold Modifier 1 (UFM1), the most recently identified ubiquitin-like protein, is covalently transferred to its substrates, in a process known as UFMylation. This modification has recently emerged as a key regulator of various biological processes by altering their stability, activity, or localization. Methods: The interaction between UFL1 and OFIP, as well as the UFMylation of OFIP, were assessed through immunoprecipitation and immunoblotting analyses. The mRNA levels of OFIP were examined using reverse transcription quantitative PCR (RT-qPCR). Immunofluorescence microscopy was employed to examine the localization and distribution patterns of OFIP. Results: Our findings demonstrate that UFL1 interacts with OFIP both in vivo and in vitro. We also found that OFIP undergoes UFMylation, and UFL1 promotes the OFIP UFMylation. Mechanistic studies demonstrate that OFIP UFMylation inhibits its protein stability and maintains its proper centrosomal localization. However, the efficacy of these regulatory mechanisms varies significantly between different cell types, being notably pronounced in HeLa cells but markedly reduced in RPE1 cells. Conclusions: OFIP is identified as a novel substrate for UFMylation. UFL1-mediated OFIP UFMylation is essential for its stability and centrosomal localization in HeLa cells. However, these effects are not observed in RPE1 cells, highlighting cell type-specific heterogeneity in the role of OFIP UFMylation. © 2025 The Author(s). Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.

关键词： centrosome OFIP protein stability UFL1 UFMylation

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PROTEIN FACTOR(S) FROM MITOTIC CHO cellS INDUCE MEIOTIC MATURATION IN XENOPUS-LAEVIS OOCYTES

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FEBS LETTERS 1980年第2期109卷 233-238页

作者： NELKIN, B NICHOLS, C VOGELSTEIN, B Cell Structure and Function Laboratory The Johns Hopkins Oncology Center Baltimore MD 21205 USA

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HYPOMETHYLATION DISTINGUISHES GENES OF SOME HUMAN CANCERS FROM THEIR NORMAL COUNTERPARTS

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NATURE 1983年第5895期301卷 89-92页

作者： FEINBERG, AP VOGELSTEIN, B Cell Structure and Function Laboratory The Oncology Center Johns Hopkins University School of Medicine Baltimore USA

It has been suggested that cancer represents an alteration in DNA, heritable by progeny cells, that leads to abnormally regulated expression of normal cellular genes; DNA alterations such as mutations1,2, rearrangements3–5and changes in methylation6–8have been proposed to have such a role. Because of increasing evidence that DNA methylation is important in gene expression (for review see refs 7, 9–11), several investigators have studied DNA methylation in animal tumours, transformed cells and leukaemia cells in culture8,12–30. The results of these studies have varied; depending on the techniques and systems used, an increase12–19, decrease20–24, or no change25–29in the degree of methylation has been reported. To our knowledge, however, primary human tumour tissues have not been used in such studies. We have now examined DNA methylation in human cancer with three considerations in mind: (1) the methylation pattern of specific genes, rather than total levels of methylation, was determined; (2) human cancers and adjacent analogous normal tissues, unconditioned by culture media, were analysed; and (3) the cancers were taken from patients who had received neither radiation nor chemotherapy. In four of five patients studied, representing two histological types of cancer, substantial hypomethylation was found in genes of cancer cells compared with their normal counterparts. This hypomethylation was progressive in a metastasis from one of the patients.

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SUPERCOILED LOOPS AND EUKARYOTIC DNA-REPLICATION

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cell 1980年第1期22卷 79-85页

作者： VOGELSTEIN, B PARDOLL, DM COFFEY, DS Cell Structure and Function Laboratory The Oncology Center The Johns Hopkins University School of Medicine Baltimore Maryland 21205 USA

Recent studies have indicated that the DNA of the eukaryotic nucleus is organized in the form of supercoiled loops. After depleting interphase nuclei of histones and other nuclear proteins by treatment with nonionic detergent and high salt, intact (unnicked) loops of DNA can be visualized as a halo surrounding a nuclear skeleton or matrix. This halo of DNA loops can be reversibly wound using various concentrations of ethidium bromide and is irreversibly unwound when the DNA is gently nicked with DNase I or exposure to UV light. These structures, comprising a halo of unwound DNA loops anchored to a nuclear matrix, provided a way in which to examine the relationship of DNA replication to the DNA loops. Mouse fibroblast 3T3 cells were pulse-labeled with 3H-thymidine for various periods and autoradiography was performed on the matrix-halo structures. After a 1 min pulse, > 80% of the autoradiographic grains representing newly replicated DNA were found within the nuclear matrix even though the surrounding halo region contained 80% of the total nuclear DNA. With longer pulse times, the autoradiographic grains could be seen to move progressively outward from the nuclear matrix into the halo region. Over 70% of the autoradiographic grains could be chased out of the central matrix into the surrounding halo if the 1 min pulse was followed by a 1 h incubation with excess unlabeled thymidine. DNA replication occurs at fixed sites at the base of the loops. The replicating DNA loops may be motile with respect to their nuclear matrix anchorage sites.

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A TECHNIQUE FOR RADIOLABELING DNA RESTRICTION ENDONUCLEASE FRAGMENTS TO HIGH SPECIFIC ACTIVITY

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ANALYTICAL BIOCHEMISTRY 1983年第1期132卷 6-13页

作者： FEINBERG, AP VOGELSTEIN, B Cell Structure and Function Laboratory The Oncology Center Johns Hopkins University School of Medicine Baltimore Maryland 21205 USA

A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA and specific activities of over 109 dpm/.mu.g of DNA can be obtained using relatively small amounts of precursor. These oligolabeled DNA fragments serve as efficient probes in filter hybridization experiments. [Escherichia coli was used.].

关键词： radiolabeling DNA fragments hybridization Southern transfers agarose gels cloned DNA

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HYPOMETHYLATION OF RAS ONCOGENES IN PRIMARY HUMAN CANCERS

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 1983年第1期111卷 47-54页

作者： FEINBERG, AP VOGELSTEIN, B Cell Structure and Function Laboratory The Oncology Center Johns Hopkins University School of Medicine Baltimore Maryland 21205 USA

The methylation status of 2 cellular oncogenes, c-Ha-ras and c-Ki-ras, in primary human carcinomas and the adjacent analogous normal tissues from which the tumors derived. The c-Ha-ras gene was hypomethylated in 6 of 8 carcinomas, including 5 colonic adenocarcinomas and 1 small cell lung carcinoma, when compared to adjacent normal tissues. The c-ki-ras gene was hypomethylated to a lesser extent in 2 colonic adenocarcinomas. This is the 1st demonstration of alterations in methylation of cellular oncogenes in human cancer.

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Reproductive aging:biological pathways and potential interventive strategies

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Journal of Genetics and Genomics 2023年第3期50卷 141-150页

作者： Yuanyuan Liu Jinmin Gao Center for Cell Structure and Function College of Life SciencesKey Laboratory of Animal Resistance Biology of Shandong ProvinceShandong Normal UniversityJinanShandong 250014China

Reproductive aging is a natural process conserved across species and is well-known in *** shows age-related follicle depletion and reduction of oocyte quality,eventually causing reproductive senescence and *** reproductive aging in males is not well noticed as in females,it also causes infertility and has deleterious consequences on the *** factors have been suggested to contribute to reproductive aging,including oxidative stress,mitochondrial defects,telomere shortening,meiotic chromosome segregation errors and genetic *** the increasing trend of pregnancy age,it is particularly crucial to find interventions to preserve or extend human *** in humans and model organisms have provided insights into the biological pathways associated with reproductive aging,and a series of potential interventive strategies have been ***,we review factors affecting reproductive aging in females and males and summarize interventive strategies that may help delay or rescue the aging phenotypes of reproduction.

关键词： Reproductive aging Infertility Oocytes Sperm Gamete quality

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ASSOCIATION OF NEWLY REPLICATED DNA WITH THE NUCLEAR MATRIX OF PHYSARUM-POLYCEPHALUM

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NUCLEIC ACIDS RESEARCH 1981年第2期9卷 349-363页

作者： HUNT, BF VOGELSTEIN, B Cell Structure and Function Laboratory The Johns Hopkins Hospital Oncology Center Room 1-127600 North Wolfe Street Baltimore MD 21205 USA

We have studied the role of the nuclear matrix in DNA replication in a naturally synchronized eucaryote, Physarum polycephalum. When P. polycephalum. When P. polycephalum macroplasmodia were pulse labeled with 3H-thymidine, the DNA remaining tightly associated with the matrix was highly enriched in newly synthesized DNA. This enrichment was found both in nuclei that had just initiated DNA replication as well as in nuclei isolated later during S phase. Pulse chase experiments showed that the association of newly replicated DNA with the matrix is transient, since most of the newly replicated DNA could be chased from the matrix by incubating pulse labeled macroplasmodia in media containing unlabeled thymidine. Studies measuring the size distribution of the matrix DNA supported the hypothesis that replication forks are attached to the nuclear matrix. Reconstitution controls indicated that these results were unlikely to be due to preferential, nonspecific binding of nascent DNA to the matrix during the extraction procedures. These results with P. polycephalum in combination with previous studies in non-synchronized rodent cells, suggest that the association of newly replicated DNA with the nuclear matrix may be a general feature of eucaryotic DNA replication.

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Bovine β1->4-galactosyltransferase: Two sets of mRNA transcripts encode two forms of the protein with different amino-terminal domains: In vitro translation experiments demonstrate that both the short and the long forms of the enzyme are type II membrane-bound glycoproteins

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Journal of Biological Chemistry 1990年第6期265卷 3324-3331页

作者： Russo, Ruth N. Shaper, Nancy L. Shaper, Joel H. Cell Structure/Function Lab. Oncology Center Johns Hopkins University 600 N. Wolfe Street Baltimore MD 21205 United States

We have used S1 and primer extension analysis to demonstrate that the gene for bovine β1->4-galactosyltransferase specifies two sets of mRNA transcripts of different lengths. The longer mRNA transcripts initiate upstream of two in-frame ATG codons and encode a protein of 402 amino acids (long form). The shorter mRNA transcripts initiate between the two inframe ATG codons and encode a protein of 389 amino acids (short form). These two related forms of β1->4-galactosyltransferase have an identical large (358 amino acids), potentially glycosylated, COOH-terminal catalytic domain, and an identical single transmembrane domain. The only difference in primary structure between the two forms is that the long form contains an NH2-terminal extension of 13 amino acids. Thus, bovine β1->4-galactosyltransferase fits the pattern established for murine β1->4-galactosyltransferase (Shaper, N. L., Hollis, G. L., Douglas, J. G., Kirsch, I. R., and Shaper, J. H. (1988) J. Biol. Chem. 263, 10420-10428). Inspection of the NH2-terminal domain suggests that the long form of the bovine enzyme, like its murine counterpart, has a functional cleavable signal sequence which would dictate that the two forms of the membrane-bound enzyme are oriented in opposite directions. We have tested this hypothesis by in vitro translation in the absence or presence of dog pancreas microsomes. In vitro translation of RNA transcripts for the long and short form of β1->4-galactosyltransferase in the absence of microsomes results in the synthesis of polypeptides with apparent Mr of 44,500 and 43,000, respectively. In vitro translation of each transcript in the presence of microsomes results in the synthesis of two glycosylated, endoglycosidase H-sensitive proteins with apparent Mr of 47,500 and 46,000. These experiments and additional protease protection experiments demonstrate that the COOH-terminal domain of both the short and the long form of bovine β1->4-galactosyltransferase are translocated into the micro

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