Intraflagellar transport (IFT) particles, a multi-protein apparatus composed of complex A and B, are known to be involved in homeostasis of flagella formation. IFT particles have recently become an interesting topic in Giardia lamblia, which has 4 pairs of flagella. In this experiment, we examined the function of giardial IFT components. When 7 components (IFT121, 140, 20, 46, 52, 81, and 88) of IFT were expressed in Giardia trophozoites as a tagged form with mNeonGreen, all of them were found in both flagella pores and cytoplasmic axonemes. In addition, motor proteins for IFT particles (kinesin-13 and kinesin-2b), were localized to a median body and cytoplasmic flagella, respectively. The CRISPRi-mediated knockdown of IFT88 significantly affected the lengths of all 4 flagella compared to the control cells, Giardia expressing dead Cas9 using control guide RNA. Decreased expression of kinesin-2b also resulted in shortening of flagella, excluding the ventral flagella. Live Giardia cells expressing IFT88-mNeonGreen clearly demonstrated fluorescence in flagella pores and cytoplasmic axonemes. These results on IFT88 and kinesin-2b indicate that IFT complex plays a role in maintenance of G. lamblia flagella.

Intraflagellar transport (IFT) particles, a multi-protein apparatus composed of complex A and B, are known to be involved in homeostasis of flagella formation. IFT particles have recently become an interesting topic in Giardia lamblia, which has 4 pairs of flagella. In this experiment, we examined the function of giardial IFT components. When 7 components (IFT121, 140, 20, 46, 52, 81, and 88) of IFT were expressed in Giardia trophozoites as a tagged form with mNeonGreen, all of them were found in both flagella pores and cytoplasmic axonemes. In addition, motor proteins for IFT particles (kinesin-13 and kinesin-2b), were localized to a median body and cytoplasmic flagella, respectively. The CRISPRi-mediated knockdown of IFT88 significantly affected the lengths of all 4 flagella compared to the control cells, Giardia expressing dead Cas9 using control guide RNA. Decreased expression of kinesin-2b also resulted in shortening of flagella, excluding the ventral flagella. Live Giardia cells expressing IFT88-mNeonGreen clearly demonstrated fluorescence in flagella pores and cytoplasmic axonemes. These results on IFT88 and kinesin-2b indicate that IFT complex plays a role in maintenance of G. lamblia flagella.

Intraflagellar transport (IFT) particles, a multi-protein apparatus composed of complex A and B, are known to be involved in homeostasis of flagella formation. IFT particles have recently become an interesting topic in Giardia lamblia, which has 4 pairs of flagella. In this experiment, we examined the function of giardial IFT components. When 7 components (IFT121, 140, 20, 46, 52, 81, and 88) of IFT were expressed in Giardia trophozoites as a tagged form with mNeonGreen, all of them were found in both flagella pores and cytoplasmic axonemes. In addition, motor proteins for IFT particles (kinesin-13 and kinesin-2b), were localized to a median body and cytoplasmic flagella, respectively. The CRISPRi-mediated knockdown of IFT88 significantly affected the lengths of all 4 flagella compared to the control cells, Giardia expressing dead Cas9 using control guide RNA. Decreased expression of kinesin-2b also resulted in shortening of flagella, excluding the ventral flagella. Live Giardia cells expressing IFT88-mNeonGreen clearly demonstrated fluorescence in flagella pores and cytoplasmic axonemes. These results on IFT88 and kinesin-2b indicate that IFT complex plays a role in maintenance of G. lamblia flagella.

Intraflagellar transport (IFT) particles, a multi-protein apparatus composed of complex A and B, are known to be involved in homeostasis of flagella formation. IFT particles have recently become an interesting topic in Giardia lamblia, which has 4 pairs of flagella. In this experiment, we examined the function of giardial IFT components. When 7 components (IFT121, 140, 20, 46, 52, 81, and 88) of IFT were expressed in Giardia trophozoites as a tagged form with mNeonGreen, all of them were found in both flagella pores and cytoplasmic axonemes. In addition, motor proteins for IFT particles (kinesin-13 and kinesin-2b), were localized to a median body and cytoplasmic flagella, respectively. The CRISPRi-mediated knockdown of IFT88 significantly affected the lengths of all 4 flagella compared to the control cells, Giardia expressing dead Cas9 using control guide RNA. Decreased expression of kinesin-2b also resulted in shortening of flagella, excluding the ventral flagella. Live Giardia cells expressing IFT88-mNeonGreen clearly demonstrated fluorescence in flagella pores and cytoplasmic axonemes. These results on IFT88 and kinesin-2b indicate that IFT complex plays a role in maintenance of G. lamblia flagella.

Intraflagellar transport (IFT) particles, a multi-protein apparatus composed of complex A and B, are known to be involved in homeostasis of flagella formation. IFT particles have recently become an interesting topic in Giardia lamblia, which has 4 pairs of flagella. In this experiment, we examined the function of giardial IFT components. When 7 components (IFT121, 140, 20, 46, 52, 81, and 88) of IFT were expressed in Giardia trophozoites as a tagged form with mNeonGreen, all of them were found in both flagella pores and cytoplasmic axonemes. In addition, motor proteins for IFT particles (kinesin-13 and kinesin-2b), were localized to a median body and cytoplasmic flagella, respectively. The CRISPRi-mediated knockdown of IFT88 significantly affected the lengths of all 4 flagella compared to the control cells, Giardia expressing dead Cas9 using control guide RNA. Decreased expression of kinesin-2b also resulted in shortening of flagella, excluding the ventral flagella. Live Giardia cells expressing IFT88-mNeonGreen clearly demonstrated fluorescence in flagella pores and cytoplasmic axonemes. These results on IFT88 and kinesin-2b indicate that IFT complex plays a role in maintenance of G. lamblia flagella.

Kinesin-13 (Kin-13), a depolymerizer of microtubule (MT), has been known to affect the length of Giardia. Giardia Kin-13 (GlKin-13) was localized to axoneme, flagellar tips, and centrosomes, where phosphorylated forms of Giardia polo-like kinase (GlPLK) were distributed. We observed the interaction between GlKin-13 and GlPLK via co-immunoprecipitation using transgenic Giardia cells expressing Myc-tagged GlKin-13, hemagglutinin-tagged GlPLK, and in vitro-synthesized GlKin-13 and GlPLK proteins. In vitro-synthesized GlPLK was demonstrated to auto-phosphorylate and phosphorylate GlKin-13 upon incubation with [γ-32P]ATP. Morpholino-mediated depletion of both GlKin-13 and GlPLK caused an extension of flagella and a decreased volume of median bodies in Giardia trophozoites. Our results suggest that GlPLK plays a pertinent role in formation of flagella and median bodies by modulating MT depolymerizing activity of GlKin-13.

MYB2 protein was identified as a transcription factor that showed encystation-induced expression in Giardia lamblia. Although nuclear import is essential for the functioning of a transcription factor, an evident nuclear localization signal (NLS) of G. lamblia MYB2 (GlMYB2) has not been defined. Based on putative GlMYB2 NLSs predicted by 2 programs, a series of plasmids expressing hemagglutinin (HA)-tagged GlMYB2 from the promoter of G. lamblia glutamate dehydrogenase were constructed and transfected into Giardia trophozoites. Immunofluorescence assays using anti-HA antibodies indicated that GlMYB2 amino acid sequence #507–#530 was required for the nuclear localization of GlMYB2, and this sequence was named as NLSGlMYB2. We further verified this finding by demonstrating the nuclear location of a protein obtained by the fusion of NLSGlMYB2 and G. lamblia glyceraldehyde 3-phosphate dehydrogenase, a non-nuclear protein. Our data on GlMYB2 will expand our understanding on NLSs functioning in G. lamblia.

MYB2 protein was identified as a transcription factor that showed encystation-induced expression in Giardia lamblia. Although nuclear import is essential for the functioning of a transcription factor, an evident nuclear localization signal (NLS) of G. lamblia MYB2 (GlMYB2) has not been defined. Based on putative GlMYB2 NLSs predicted by 2 programs, a series of plasmids expressing hemagglutinin (HA)-tagged GlMYB2 from the promoter of G. lamblia glutamate dehydrogenase were constructed and transfected into Giardia trophozoites. Immunofluorescence assays using anti-HA antibodies indicated that GlMYB2 amino acid sequence #507–#530 was required for the nuclear localization of GlMYB2, and this sequence was named as NLSGlMYB2. We further verified this finding by demonstrating the nuclear location of a protein obtained by the fusion of NLSGlMYB2 and G. lamblia glyceraldehyde 3-phosphate dehydrogenase, a non-nuclear protein. Our data on GlMYB2 will expand our understanding on NLSs functioning in G. lamblia.

To identify the component(s) involved in cell cycle control in the protozoan Giardia lamblia, cells arrested at the G1/S- or G2-phase by treatment with nocodazole and aphidicolin were prepared from the synchronized cell cultures. RNA-sequencing analysis of the 2 stages of Giardia cell cycle identified several cell cycle genes that were up-regulated at the G2-phase. Transcriptome analysis of cells in 2 distinct cell cycle stages of G. lamblia confirmed previously reported components of cell cycle (PcnA, cyclin B, and CDK) and identified additional cell cycle components (NEKs, Mad2, spindle pole protein, and CDC14A). This result indicates that the cell cycle machinery operates in this protozoan, one of the earliest diverging eukaryotic lineages.
Aphidicolin ; Cell Culture Techniques ; Cell Cycle ; Cell Cycle Checkpoints ; Cyclin B ; Gene Expression Profiling ; Genes, cdc ; Giardia lamblia ; Giardia ; Nocodazole ; Spindle Poles

An adrenal incidentaloma is an adrenal mass found in an imaging study performed for other reasons unrelated to adrenal disease and often accompanied by obesity, diabetes, or hypertension. The prevalence and incidence of adrenal incidentaloma increase with age and are also expected to rise due to the rapid development of imaging technology and frequent imaging studies. The Korean Endocrine Society is promoting an appropriate practice guideline to meet the rising incidence of adrenal incidentaloma, in cooperation with the Korean Adrenal Gland and Endocrine Hypertension Study Group. In this paper, we discuss important core issues in managing the patients with adrenal incidentaloma. After evaluating core proposition, we propose the most critical 20 recommendations from the initially organized 47 recommendations by Delphi technique.
Adrenal Glands ; Delphi Technique ; Humans ; Hypertension ; Incidence ; Obesity ; Prevalence