Subcellular localization and role of Ran1 in Tetrahymena thermophila amitotic macronucleus

Abstract

Amitosis, a direct method of cell division is common in ciliated protozoan, fungi and some animal and plant cells. During amitosis, intranuclear microtubules are reorganized into specified arrays which assist in separation of nucleus, despite lack of a bipolar spindle. However, the regulation of amitosis is not understood. Here, we focused on the localization and role of mitotic spindle assembly regulator: Ran GTPase (Ran1) in macronuclear amitosis in binucleated protozoan Tetrahymena thermophila. HA-tagged Ran1 was localized in the macronucleus throughout the cell cycle of Tetrahymena during vegetative growth, and the accessory factor binding domains of Ran1 contributed to its macronuclear localization. Incomplete somatic knockout of RAN1 resulted in aberrant intramacronuclear microtubule array formation, missegregation of macronuclear chromosomes and ultimately blocked macronuclei proliferation. When the Ran1 cycle was perturbed by overexpression of Ran1T25N (GDP-bound Ran1-mimetic) or Ran1Q70L (GTP-bound Ran1-mimetic), intramacronuclear microtubule assembly was inhibited or multi-micronucleate cells formed. These results suggest that Ran GTPase pathway is involved in assembly of a specialized intramacronuclear microtubule network and coordinates amitotic progression in Tetrahymena.

Ran1 is essential for parental macronuclear import of apoptosis‐inducing factor and programmed nuclear death in Tetrahymena thermophila

“…The conversion and distribution of the two nucleotidebound Ran act as signals for the cellular processes. To determine the involvement of Ran1 in parental MAC PND in Tetrahymena, it was necessary to detect the subcellular localizations of Ran1, Ran1T25N and Ran1Q70L [37] during conjugation stage (Fig. 1A).…”

“…To further determine whether Ran1 was involved in nuclear degeneration, we observed the nuclear development in conjugating iΔRAN1 (Δ::NEO4) cells (which is an incomplete somatic knockout RAN1 Tetrahymena) [37] and mating cells between iΔRAN1 and OERAN1 (overexpression of RAN1 in Tetrahymena; Fig. 2) [37]. During the early stages of conjugation, mating iΔRAN1 cells and mating cells between iΔRAN1 and OERAN1 initiated normal nuclear events as the mating wild-type (WT) cells ( Fig.…”

“…OERAN1 (exogenous HA-Ran1 overexpression strain), OERan1T25N/Q70L (exogenous HA-Ran1 T25N/Q70L overexpression strain) and iΔRAN1 (Δ::NEO4, incomplete somatic knockout RAN1 strain) were described previously [37].…”

“…For co-transformation of Tetrahymena, pBsr vector was generated by removing the NEO4 cassette and replacing it with the Bsr cassette containing the blasticidin S resistance gene (Bsr) as a selection marker using the pNeo4 [62] as backbone vector. The N-terminal HA tagging construct for the AIF gene (HA-AIF construct) was produced with pBsr as described previously [37]. First, the 3 0 flanking homology arm was amplified by overlapping PCR in which the HA tag was inserted after the start codon of AIF.…”

“…It plays an essential role in nuclear import-export during interphase by a steep RanGTP-RanGDP gradient, which is high RanGTP in the nucleus and high RanGDP in the cytoplasm across the nuclear envelope [34][35][36]. Spatial partitioning of macronuclear (MAC) RanGTP and cytoplasmic RanGDP also significantly exists across the MAC envelop in amitosis during vegetative growth of Tetrahymena [37]. Importantly, a peculiar membranous structure encloses the parental MAC during PND [10].…”