Functional proteoliposome-like structure derived from simultaneous evisceration and enucleation of T-lymphoblastoid A3R5.7 cells: A top-down story.

Structurally-reduced cells and cell-derived structures are powerful tools for membrane studies. Using this approach, we probed whether a cell, without its nucleus and cytoplasm, is still capable of undergoing CD4-mediated membrane fusion. For this, we needed a cell-derived structure, akin to a giant liposome functionalised with CD4 and chemokine receptors. We present a method for the simultaneous removal of cytoplasmic and nuclear material from cells presenting CD4, CCR5, and CXCR4, using Colcemid treatment followed by hypotonic cytolysis, and then enriched using preparative flow cytometry. We show that the resultant cell membrane remains intact, retains presentation of CD4, CCR5, and CXCR4, and is still capable of CD4-mediated membrane fusion with a target cell. Finally, we detail how this protocol was developed, as well as how such samples should be handled for storage and assays. We envision the use of such systems for host-pathogen interaction studies, and the development of targeted delivery vehicles.

Nanoscale characterization of drug-induced microtubule filament dysfunction using super-resolution microscopy.

BACKGROUND: The integrity of microtubule filament networks is essential for the roles in diverse cellular functions, and disruption of its structure or dynamics has been explored as a therapeutic approach to tackle diseases such as cancer. Microtubule-interacting drugs, sometimes referred to as antimitotics, are used in cancer therapy to target and disrupt microtubules. However, due to associated side effects on healthy cells, there is a need to develop safer drug regimens that still retain clinical efficacy. Currently, many questions remain open regarding the extent of effects on cellular physiology of microtubule-interacting drugs at clinically relevant and low doses. Here, we use super-resolution microscopies (single-molecule localization and optical fluctuation based) to reveal the initial microtubule dysfunctions caused by nanomolar concentrations of colcemid. RESULTS: We identify previously undetected microtubule (MT) damage caused by clinically relevant doses of colcemid. Short exposure to 30-80 nM colcemid results in aberrant microtubule curvature, with a trend of increased curvature associated to increased doses, and curvatures greater than 2 rad/µm, a value associated with MT breakage. Microtubule fragmentation was detected upon treatment with ≥ 100 nM colcemid. Remarkably, lower doses (< 20 nM after 5 h) led to subtle but significant microtubule architecture remodelling characterized by increased curvature and suppression of microtubule dynamics. CONCLUSIONS: Our results support the emerging hypothesis that microtubule-interacting drugs induce non-mitotic effects in cells, and establish a multi-modal imaging assay for detecting and measuring nanoscale microtubule dysfunction. The sub-diffraction visualization of these less severe precursor perturbations compared to the established antimitotic effects of microtubule-interacting drugs offers potential for improved understanding and design of anticancer agents.

A Basic and Simple Chromosome Preparation Protocol.

Chromosome preparation for chromosome analysis is basic and indispensable for wide area of biology such as genetics, medicine, molecular biology, or other many fields. However, it seems that the many artisans in that fields feel hard to get start chromosome preparation technique, as it is something technically demanding or troublesome, or it needs something secret manner to prepare good quality of chromosome spreads. Actually many technical variations among individual laboratories exist. The aims of this chapter is to describe concise and minimal fundamental protocols specifically focused on human chromosome preparation, as step-by-step guiding with instructing essential points.

Treatment of CHO cells with Taxol and reversine improves micronucleation and microcell-mediated chromosome transfer efficiency.

Microcell-mediated chromosome transfer is an attractive technique for transferring chromosomes from donor cells to recipient cells and has enabled the generation of cell lines and humanized animal models that contain megabase-sized gene(s). However, improvements in chromosomal transfer efficiency are still needed to accelerate the production of these cells and animals. The chromosomal transfer protocol consists of micronucleation, microcell formation, and fusion of donor cells with recipient cells. We found that the combination of Taxol (paclitaxel) and reversine rather than the conventional reagent colcemid resulted in highly efficient micronucleation and substantially improved chromosomal transfer efficiency from Chinese hamster ovary donor cells to HT1080 and NIH3T3 recipient cells by up to 18.3- and 4.9-fold, respectively. Furthermore, chromosome transfer efficiency to human induced pluripotent stem cells, which rarely occurred with colcemid, was also clearly improved after Taxol and reversine treatment. These results might be related to Taxol increasing the number of spindle poles, leading to multinucleation and delaying mitosis, and reversine inducing mitotic slippage and decreasing the duration of mitosis. Here, we demonstrated that an alternative optimized protocol improved chromosome transfer efficiency into various cell lines. These data advance chromosomal engineering technology and the use of human artificial chromosomes in genetic and regenerative medical research.

Investigation of sphingosine kinase 1 inhibitory potential of cinchonine and colcemid targeting anticancer therapy.

Sphingosine kinase 1 (SphK1) and sphingosine-1-phosphate (S1P) signaling regulates numerous diseases such as cancer, diabetes, and inflammation-related ailments, rheumatoid arthritis, atherosclerosis, and multiple sclerosis. The importance of SphK1 in chemo-resistance has been extensively explored in breast, lung, colon, and hepatocellular carcinomas. SphK1 is considered an attractive drug target for the development of anticancer therapy. New drug molecules targeting the S1P signaling are required owing to its pleiotropic nature and association with multiple downstream targets. Here, we have investigated the binding affinity and SphK1 inhibitory potential of cinchonine and colcemid using a combined molecular docking and simulation studies followed by experimental analysis. These compounds bind to SphK1 with a significantly high affinity and subsequently inhibit kinase activity (IC50 7-9 µM). Further, MD simulation studies revealed that both cinchonine and colcemid bind to the residues at the active site pocket of SphK1 with several non-covalent interactions, which may be responsible for inhibiting its kinase activity. Besides, the binding of cinchonine and colcemid causes substantial conformational changes in the structure of SphK1. Taken together, cinchonine and colcemid may be implicated in designing potential drug molecules with improved affinity and specificity for SphK1 targeting anticancer therapy.Communicated by Ramaswamy H. Sarma.

Genetic Control of Kinetochore-Driven Microtubule Growth in Drosophila Mitosis.

Centrosome-containing cells assemble their spindles exploiting three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs generated near the chromosomes/kinetochores, and MTs nucleated within the spindle by the augmin-dependent pathway. Mammalian and Drosophila cells lacking the centrosomes generate MTs at kinetochores and eventually form functional bipolar spindles. However, the mechanisms underlying kinetochore-driven MT formation are poorly understood. One of the ways to elucidate these mechanisms is the analysis of spindle reassembly following MT depolymerization. Here, we used an RNA interference (RNAi)-based reverse genetics approach to dissect the process of kinetochore-driven MT regrowth (KDMTR) after colcemid-induced MT depolymerization. This MT depolymerization procedure allows a clear assessment of KDMTR, as colcemid disrupts centrosome-driven MT regrowth but not KDMTR. We examined KDMTR in normal Drosophila S2 cells and in S2 cells subjected to RNAi against conserved genes involved in mitotic spindle assembly: mast/orbit/chb (CLASP1), mei-38 (TPX2), mars (HURP), dgt6 (HAUS6), Eb1 (MAPRE1/EB1), Patronin (CAMSAP2), asp (ASPM), and Klp10A (KIF2A). RNAi-mediated depletion of Mast/Orbit, Mei-38, Mars, Dgt6, and Eb1 caused a significant delay in KDMTR, while loss of Patronin had a milder negative effect on this process. In contrast, Asp or Klp10A deficiency increased the rate of KDMTR. These results coupled with the analysis of GFP-tagged proteins (Mast/Orbit, Mei-38, Mars, Eb1, Patronin, and Asp) localization during KDMTR suggested a model for kinetochore-dependent spindle reassembly. We propose that kinetochores capture the plus ends of MTs nucleated in their vicinity and that these MTs elongate at kinetochores through the action of Mast/Orbit. The Asp protein binds the MT minus ends since the beginning of KDMTR, preventing excessive and disorganized MT regrowth. Mei-38, Mars, Dgt6, Eb1, and Patronin positively regulate polymerization, bundling, and stabilization of regrowing MTs until a bipolar spindle is reformed.

Non-linear dynamics of chromosome condensation induced by colcemid

This study investigated the dynamical process of chromosome condensation after colcemid treatment. Two pairs of human chromosomes, #2 and #3, were highlighted for the accurate identification by fluorescence in situ hybridization (FISH). A computerized image analysis system was used to measure the lengths of the two pairs of chromosomes averaged over 50 metaphases of different cultures with colcemid (0.5 µg/mL) added either at 3 or 48 h of a total 72 h culture period. For determining whether the process of chromosome condensation was chaotic or random, the algorithm of Detrended Fluctuation Analysis (DFA) was used. In order to evaluate the power of the method, the data were shuffled and DFA was performed again. It was found that colcemid prolonged treatment induced a significantly greater chromosome condensation (p<0.05), and the dynamics of this process was determined by the DFA and showed to be chaotic, with scaling exponents with range values 0.5< α<1.0. When the data were shuffled, the scaling exponent αreduced around to 0.5, which was characteristic of random events. These findings reinforced the idea that colcemid could interfere in some manner with the structure of chromosomes and the dynamics of chromosome condensation was non-linear.