Colchicine-induced degeneration of the micronucleus during conjugation in Tetrahymena

Abstract

One of the most dramatic examples of nuclear morphogenesis occurs during conjugation in Tetrahymena when the micronucleus elongates to a size longer than the cell itself. After contraction to a spherical shape, the nucleus moves directly to chromosome separation in the first meiotic division. Here we investigate the consequences of interrupting the elongation process. Colchicine, a microtubule inhibitor, caused retraction of elongated structures. With time, cells began to lose their micronuclei, and by five hours more than half of the paired cells had at least one cell missing a micronucleus. After reversing the colchicine block, existing micronuclei did not undergo elongation again, nor did meiosis occur. These observations indicate that micronuclear elongation is critical to subsequent meiotic division. Further, nuclear elimination occurs, which could be due to meiotic failure or possibly a problem downstream from meiosis. An analysis of the process of colchicine-induced micronuclear degeneration indicated that it was regulated by a caspase-dependent mechanism, characteristic of apoptosis, and then resorbed by a lysosome-dependent autophagic mechanism. Amicronucleate cells failed to grow when returned to nutrient medium, likely because of a lesion in the post-conjugation reconstruction of a functioning oral apparatus. The ease by which a large number of nuclei are induced to “self-destruct” may make this system useful in investigating the link between colchicine treatment and nuclear death in Tetrahymena, and in investigating how nuclear death could be regulated in living cells more generally. Finally, we note that this phenomenon might relate to the evolution of amicronucleate species of Tetrahymena.

A Novel 3D Scaffold for Cell Growth to Assess Electroporation Efficacy

“…The 3D cell culture was stained with Hoechst 33342 (ThermoFisher, Waltham, MA, USA), Acridine Orange (ThermoFisher, Waltham, MA, USA) and Propidium Iodide (Sigma Aldrich, Saint Louis, MO, USA) solutions in order to dye the nucleus, identify alive and apoptotic cells, identify the necrotic cells, respectively. The nucleus of the cell is in blue due to Hoechst 33342 (5 µg/mL in PBS) [46,47]. Propidium Iodide (100 µg/mL in deionized water) dies selectively in red the nucleus of necrotic cells and is discarded by alive or apoptotic cells [48,49,50], while Acridine Orange (10 µg/mL in deionized water) dies the cell cytoplasm and nucleus in green if the cells are alive [46,47,51].…”

“…The nucleus of the cell is in blue due to Hoechst 33342 (5 µg/mL in PBS) [46,47]. Propidium Iodide (100 µg/mL in deionized water) dies selectively in red the nucleus of necrotic cells and is discarded by alive or apoptotic cells [48,49,50], while Acridine Orange (10 µg/mL in deionized water) dies the cell cytoplasm and nucleus in green if the cells are alive [46,47,51]. In acridine orange staining, apoptotic cells are characterized by disaggregated DNA, while alive cells possess a well distinguishable nucleus [52,53,54,55].…”