Eh Klp5 is a divergent member of the kinesin 5 family that regulates genome content and microtubular assembly in Entamoeba histolytica

Abstract

SummaryEarlier studies have established two unusual features in the cell division cycle of Entamoeba histolytica. First, microtubules form a radial assembly instead of a bipolar mitotic spindle, and second, the genome content of E. histolytica cells varied from 1¥ to 6¥ or more. In this study, Eh Klp5 was identified as a divergent member of the BimC kinesin family that is known to regulate formation and stabilization of the mitotic spindle in other eukaryotes. In contrast to earlier studies, we show here that bipolar microtubular spindles were formed in E. histolytica but were visible only in 8-12% of the cells after treatment with taxol. The number of bipolar spindles was significantly increased in Eh Klp5 stable transformants (20-25%) whereas Eh Klp5 double-stranded RNA (dsRNA) transformants did not show any spindles (< 1%). The genome content of Eh Klp5 stable transformants was regulated between 1¥ and 2¥ unlike control cells. Binucleated cells accumulated in Eh Klp5 dsRNA transformants and after inhibition of Eh Klp5 with small molecule inhibitors in control cells, suggesting that cytokinesis was delayed in the absence of Eh Klp5. Taken together, our results indicate that Eh Klp5 regulates microtubular assembly, genome content and cell division in E. histolytica. Additionally, Eh Klp5 showed alterations in its drug-binding site compared with its human homologue, Hs Eg5 and this was reflected in its reduced sensitivity to Eg5 inhibitors -monastrol and HR22C16 analogues.

Identification of an Entamoeba histolytica Serine-, Threonine-, and Isoleucine-Rich Protein with Roles in Adhesion and Cytotoxicity

“…2). This methodology was previously used to downregulate expression of the formin homology gene EhDia1, as well as the kinesin family gene EhKlp5 (17,30). After selection with G418 at 48 g/ml, the expression levels of all EhSTIRP genes were drastically reduced as shown by reverse transcription-PCR (data not shown) and Northern blot analysis (Fig.…”

Entamoeba histolytica Encodes Unique Formins, a Subset of Which Regulates DNA Content and Cell Division

“…Subsequently, the HH-tagged Ehformin-2 gene was subcloned into pJST4. Stable transformants of the HAtagged Ehformin-1 gene, the HH-tagged Ehformin-2 gene, and an empty vector control (13) were stably maintained in the presence of G418.…”

“…For measurement of nuclear DNA contents, ethanol-fixed cells were stained with 4Ј,6Ј-diamidino-2-phenylindole (DAPI) (0.1 g/ml) for 10 min and spread on glass slides (13). Each slide was scanned for the DNA contents of individual nuclei under a 40ϫ objective of a Zeiss Axiovert 200M fluorescence microscope fitted with a MetaCyte scanning cytometer and Metafer 4 software (Zeiss, Germany).…”

“…Cells were prepared for flow cytometry according to the work of Dastidar et al (13), and data were acquired in a FACSCalibur instrument (Becton Dickinson) equipped with a single laser system (6-W Innova 90-6 argon ion laser). For measurement of DNA content, cells were excited with 488-nm light, and emission was measured through a 575-nm-wavelength DF20 filter (for propidium iodide fluorescence; FL2).…”

“…We have also observed that sequence homologs of several eukaryotic "checkpoint proteins" are not found in the amoeba genome (36). However, functional analysis of a kinesin-like protein, E. histolytica Klp5 (Klp5 Eh ), showed that increased expression of this protein, while promoting microtubular spindles, leads to homogenization of the average DNA content in growing cells (13). Thus, in the absence of conserved regulatory proteins, genome content and cell division may be regulated by other proteins in this parasite.…”