Basic structure and cytocompatibility of giant membrane vesicles derived from paraformaldehyde-exposed human cells.

Exposure of cultured mammalian cells to paraformaldehyde (PFA) is an effective approach to induce membrane blebs, which is followed by their detachment from the cellular cortex to yield giant membrane vesicles in extracellular spaces. Although PFA-induced giant vesicles have attracted significant interest in the field of cell membrane dynamics, their biochemical components and cytocompatibility remain largely unknown. In this report, we exposed human cervical cancer HeLa cells to PFA under metal-free buffer conditions to produce giant vesicles.

A fluorescence method to visualize the nuclear boundary by the lipophilic dye DiI.

Unlabelled: Here, we describe a procedure to fluorescently contrast the nuclear boundary using the lipophilic carbocyanine dye DiI in cultured human cells. Our procedure is simple and is applicable to detect nuclear boundary defects, which may be relevant to studies on nuclear envelope dynamics, micronuclei formation and cancer biology.

Abbreviations: DiI: 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; DiO: 3,3'-dioctadecyloxacarbocyanine perchlorate; NE: nuclear envelope; RanBP2: Ran-binding protein 2/Nucleoporin 358.

Chemical manipulations to facilitate membrane blebbing and vesicle shedding on the cellular cortex.

Objectives: Most attention has been focused on physiologically generated membrane blebs on the cellular cortex, whereas artificial membrane blebs induced by chemicals are studied to a lesser extent.

Results: We found that exposure of HeLa human cervical cancer cells to paraformaldehyde (PFA), followed by incubation in phosphate-buffered saline (PBS) efficiently induced large membrane blebs on the cellular cortex. Intriguingly, sequential exposure of the PFA-treated cells to PBS containing dimethyl sulfoxide (DMSO) facilitated shedding of the blebs from the cellular cortex, yielding a high quantity of large extracellular vesicles in the supernatant, which was applicable to assess the potentials of compounds and proteins as membrane influencers.

Nanosecond pulsed electric fields induce extracellular release of chromosomal DNA and histone citrullination in neutrophil-differentiated HL-60 cells.

Nanosecond pulsed electric fields (nsPEFs) have gained attention as a novel physical stimulus for life sciences. Although cancer therapy is currently their promising application, nsPEFs have further potential owing to their ability to elicit various cellular responses. This study aimed to explore stimulatory actions of nsPEFs, and we used HL-60 cells that were differentiated into neutrophils under cultured conditions.

Linkage between lipid droplet formation and nuclear deformation in HeLa human cervical cancer cells.

Because lipid droplets (LDs) and the nucleus are cellular organelles that regulate seemingly very different biochemical processes, very little attention has been focused on their possible interplay. Here, we report a correlation between nuclear morphology and cytoplasmic LD formation in HeLa human cervical cells. When the cells were treated with oleic acid (OA), LDs were formed in the cytoplasm, but not in the nucleoplasm.

Wash-free instant detection of giant plasma membrane vesicles.

Giant plasma membrane vesicles (GPMVs) are large extracellular vesicles produced by the exposure of cells to paraformaldehyde or other stresses, providing an experimental system to elucidate cell surface dynamics. Here we show that addition of the membrane permeable fluorescent RNA-indicators, acridine orange and thioflavin T, to GPMV-containing solutions prepared from cultured HeLa cells was sufficient for the fluorescent visualization of seemingly all GPMVs. Our findings provide a wash-free instant method using non-lipid-type fluorescent dyes for GPMV detection, which should be useful for researchers interested in studying cell membrane dynamics and biochemistry using GPMVs.

A genetic screen to discover SUMOylated proteins in living mammalian cells.

Post-translational modification by the Small Ubiquitin-related Modifier (SUMO) is indispensable for diverse biological mechanisms. Although various attempts have been made to discover novel SUMO substrate proteins to unveil the roles of SUMOylation, the reversibility of SUMOylation, and the differences in the SUMOylation level still makes it difficult to explore infrequently-SUMOylated proteins in mammalian cells. Here, we developed a method to screen for mammalian SUMOylated proteins using the reconstitution of split fluorescent protein fragments in living mammalian cells.

Aclarubicin, an anthracycline anti-cancer drug, fluorescently contrasts mitochondria and reduces the oxygen consumption rate in living human cells.

Aclarubicin (Acla), an effective anthracycline chemotherapeutic agent for hematologic cancers and solid tumors, is documented to perturb chromatin function via histone eviction and DNA topoisomerase inhibition in the nucleus, but much less attention has been paid to cytotoxic function in the cytoplasm. Here, we showed that Acla emitted fluorescence and that human cervical cancer HeLa cells exposed to Acla exhibited bright fluorescence signals in the cytoplasm when fluorescence microscopy was performed using the red filter (excitation 530-550nm/emission 575nm). Intriguingly, most of the signals appeared to be partitioned and enriched in entangled tubule-like structures; moreover, these signals merged with the mitochondria-specific MitoTracker signals.

Thymine DNA glycosylase modulates DNA damage response and gene expression by base excision repair-dependent and independent mechanisms.

Thymine DNA glycosylase (TDG) is a base excision repair (BER) enzyme, which is implicated in correction of deamination-induced DNA mismatches, the DNA demethylation process and regulation of gene expression. Because of these pivotal roles associated, it is crucial to elucidate how the TDG functions are appropriately regulated in vivo. Here, we present evidence that the TDG protein undergoes degradation upon various types of DNA damage, including ultraviolet light (UV).

HDAC9 regulates the alternative lengthening of telomere (ALT) pathway via the formation of ALT-associated PML bodies.

Cancer cells overcome cellular senescence by activating the telomere maintenance mechanism, which can be either through telomerase or the alternative lengthening of telomeres (ALT). Being exclusive to cancer cells, targeting ALT is a more promising route for the development of drugs against cancer. The histone deacetylase (HDAC) family plays significant roles in various cellular processes.

In Situ SUMOylation and DeSUMOylation Assays: Fluorescent Methods to Visualize SUMOylation and DeSUMOylation in Permeabilized Cells.

This chapter deals with the fluorescence detection of SUMOylation and deSUMOylation in semi-intact cultured human cells, the so-called "in situ SUMOylation assay" and the "in situ deSUMOylation assay," respectively. In the in situ SUMOylation assay, the recombinant green-fluorescence protein fused to the SUMO1 (GFP-SUMO1) protein is used to visualize the nuclear rim, nucleolus, and nuclear bodies. These GFP signals represent cellular regions where SUMOylation efficiently takes place.

Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition.

We have previously reported the co-localization of O-propargyl-puromycin (OP-Puro) with SUMO-2/3 and ubiquitin at promyelocytic leukemia-nuclear bodies (PML-NBs) in the presence of the proteasome inhibitor MG132, implying a role for the ubiquitin family in sequestering OP-puromycylated immature polypeptides to the nucleus during impaired proteasome activity. Here, we found that as expected puromycin induced SUMO-1/2/3 accumulation with ubiquitin at multiple nuclear foci in HeLa cells when co-exposed to MG132. Co-administration of puromycin and MG132 also facilitated redistribution of PML and the SUMO-targeted ubiquitin ligase RNF4 concurrently with SUMO-2/3.

Detection of O-propargyl-puromycin with SUMO and ubiquitin by click chemistry at PML-nuclear bodies during abortive proteasome activities.

The amino-nucleoside antibiotic, puromycin, acts by covalently linking to elongating polypeptide chains on ribosomes to generate prematurely terminated immature polypeptides. The trafficking of puromycin-conjugated (puromycylated) immature polypeptides within cell has, however, remained elusive. In this study, using O-propargyl-puromycin (OP-Puro), the distribution of puromycylated polypeptides was assessed in HeLa cells by click chemistry.

A Convenient Technique to Fix Suspension Cells on a Coverslip for Microscopy.

Human myeloid HL-60 cells are usually cultured in suspension in medium containing 5% to 10% fetal bovine serum (FBS) and thus are often difficult to adhere to a coverslip. In this unit, we describe how removal of FBS from the culture medium facilitates adhesion of HL-60 cells to coverslips. Importantly, HL-60 cells that adhere to the coverslips immersed in FBS-free medium can be immobilized in situ by conventional chemical fixatives and thus permeabilized for probing cellular structures using specific dyes and/or reagents, followed by microscopic observation.

SUMOylation of xeroderma pigmentosum group C protein regulates DNA damage recognition during nucleotide excision repair.

The xeroderma pigmentosum group C (XPC) protein complex is a key factor that detects DNA damage and initiates nucleotide excision repair (NER) in mammalian cells. Although biochemical and structural studies have elucidated the interaction of XPC with damaged DNA, the mechanism of its regulation in vivo remains to be understood in more details. Here, we show that the XPC protein undergoes modification by small ubiquitin-related modifier (SUMO) proteins and the lack of this modification compromises the repair of UV-induced DNA photolesions.

Tunicamycin-induced neutrophil extracellular trap (NET)-like structures in cultured human myeloid cell lines.

The mechanism of neutrophil extracellular trap cell death (NETosis), a regulated cell death pathway relevant to infection, autoimmunity and sepsis, is not completely known. The reason for this, at least in part, is the lack of an in vitro system that recapitulates the NETosis pathway using established human cell lines. We show that exposure of a human promyelocytic leukemia cell line HL-60 to the glycosyltransferase inhibitor tunicamycin (TM) resulted in extrusion of decompacted genomic DNAs to extracellular space, morphologically similar to NETs.

Adhesion of suspension cells on a coverslip in serum-free conditions.

Here, we present a rapid and damage-free fixation protocol for human cells cultured in suspension. Our results demonstrated that serum-free incubation of myeloid suspension cell lines HL-60, U937, and THP-1 for 10 min resulted in cell adhesion to coverslips, allowing simple and efficient fixation for microscopy. The fixed cells exhibited an intact morphology and were suitable for immunostaining.

SUMO-modification and elimination of the active DNA demethylation enzyme TDG in cultured human cells.

Thymine DNA glycosylase (TDG) is a base excision repair enzyme that interacts with the small ubiquitin-related modifier (SUMO)-targeted ubiquitin E3 ligase RNF4 and functions in the active DNA demethylation pathway. Here we showed that both SUMOylated and non-modified forms of endogenous TDG fluctuated during the cell cycle and in response to drugs that perturbed cell cycle progression, including hydroxyurea and nocodazole. Additionally, we detected a SUMOylation-independent association between TDG and RNF4 in vitro as well as in vivo, and observed that both forms of TDG were efficiently degraded in RNF4-depleted cells when arrested at S phase.

The SUMO-targeted ubiquitin ligase RNF4 localizes to etoposide-exposed mitotic chromosomes: implication for a novel DNA damage response during mitosis.

RNF4, a SUMO-targeted ubiquitin ligase (STUbL), localizes to the nucleus and functions in the DNA damage response during interphase of the cell cycle. RNF4 also exists in cells undergoing mitosis, where its regulation and function remain poorly understood. Here we showed that administration of etoposide, an anticancer DNA topoisomerase II poison, to mitotic human cervical cancer HeLa cells induced SUMO-2/3-dependent localization of RNF4 to chromosomes.

Construction of a mouse Aos1-Uba2 chimeric SUMO-E1 enzyme, mAU, and its expression in baculovirus-insect cells.

Small ubiquitin-related modifier (SUMO) is a highly conserved protein that is covalently attached to target proteins. This posttranslational modification, designated SUMOylation, is a major protein-conjugation-driven strategy designed to regulate structure and function of cellular proteins. SUMOylation consists of an enzymatic cascade involving the E1-activating enzyme and the E2-conjugating enzyme.

Assay methods for small ubiquitin-like modifier (SUMO)-SUMO-interacting motif (SIM) interactions in vivo and in vitro using a split-luciferase complementation system.

SUMOylation is a posttranslational process that attaches a small ubiquitin-like modifier (SUMO) to its target proteins covalently. SUMOylation controls multiple cellular processes through the recognition of SUMO by a SUMO-interacting motif (SIM). In this study, we developed assay systems for detecting noncovalent interactions between SUMO and SIM in cells using split-luciferase complementation.

Spectomycin B1 as a novel SUMOylation inhibitor that directly binds to SUMO E2.

Conjugation of small ubiquitin-like modifier (SUMO) to protein (SUMOylation) regulates multiple biological systems by changing the functions and fates of a large number of proteins. Consequently, abnormalities in SUMOylation have been linked to multiple diseases, including breast cancer. Using an in situ cell-based screening system, we have identified spectomycin B1 and related natural products as novel SUMOylation inhibitors.