Dynamics of Mitochondrial RNA-Binding Protein Complex in Trypanosoma brucei and Its Petite Mutant under Optimized Immobilization Conditions

Huang, Zeqi; Kaltenbrunner, Sabine; Šimková, Eva; Staněk, David; Lukeš, Julius; Hashimi, Hassan

doi:10.1128/ec.00149-14

Cited by 4 publications

(4 citation statements)

References 39 publications

(74 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The advantage of live-cell imaging is that it avoids potential artefacts, which can be caused by fixation. In order to take pictures of moving trypanosomes after staining, we used a previously described technique, in which the cells are covered by a thin sheet of 1% agarose and directly observed by confocal microscopy [33]. TbUTP10 protein is localised in the nucleolus in both PS and BS cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In order to visualize TbUTP10, 5×10 6 live PS or BS cells were stained with 1 μl of 20 μM MitoTracker Red CMXRos (Molecular Probes) and two drops of Nuc Blue Live Cell Stain (Molecular Probes), incubated for 15 min at their respective cultivation conditions, spun down and resuspended in Iscove’s Modified Dulbecco’s Medium. The cells were subsequently immobilized under a sheet of 1% agarose and observed under an OlympusFluoViewFV1000 confocal microscope as described elsewhere [33].…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

TbUTP10, a protein involved in early stages of pre-18S rRNA processing in Trypanosoma brucei

Faktorová

Bär

Hashimi

et al. 2018

Molecular and Biochemical Parasitology

Self Cite

View full text Add to dashboard Cite

Ribosome biosynthesis, best studied in opisthokonts, is a highly complex process involving numerous protein and RNA factors. Yet, very little is known about the early stages of pre-18S rRNA processing even in these model organisms, let alone the conservation of this mechanism in other eukaryotes. Here we extend our knowledge of this process by identifying and characterizing the essential protein TbUTP10, a homolog of yeast U3 small nucleolar RNA-associated protein 10 - UTP10 (HEATR1 in human), in the excavate parasitic protist Trypanosoma brucei. We show that TbUTP10 localizes to the nucleolus and that its ablation by RNAi knock-down in two different T. brucei life cycle stages results in similar phenotypes: a disruption of pre-18S rRNA processing, exemplified by the accumulation of rRNA precursors, a reduction of mature 18S rRNA, and also a decrease in the level of U3 snoRNA. Moreover, polysome profiles of the RNAi-induced knock-down cells show a complete disappearance of the 40S ribosomal subunit, and a prominent accumulation of the 60S large ribosomal subunit, reflecting impaired ribosome assembly. Thus, TbUTP10 is an important protein in the processing of 18S rRNA.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

TbUTP10, a protein involved in early stages of pre-18S rRNA processing in Trypanosoma brucei

Faktorová

Bär

Hashimi

et al. 2018

Molecular and Biochemical Parasitology

Self Cite

View full text Add to dashboard Cite

show abstract

“…Equally, it is incompatible with the visualization of the free flagellum and imaging of free-swimming parasites. Agarose immobilization [37] and CyGEL immobilization [36] are also incompatible with visualization of the free flagellum.…”

Section: Discussionmentioning

confidence: 99%

Spatial confinement ofTrypanosoma bruceiin microfluidic traps provides a new tool to study free swimming parasites

Niz

Frachon

Gobaa

et al. 2023

Preprint

View full text Add to dashboard Cite

Trypanosoma brucei is the causative agent of African trypanosomiasis and is transmitted by the tsetse fly (Glossina spp.). All stages of this extracellular parasite possess a single flagellum that is attached to the cell body and confers a high degree of motility. While several stages are amenable to culture in vitro, longitudinal high-resolution imaging of free-swimming parasites has been challenging, mostly due to the rapid flagellar beating that permanently twists the cell body. Here, using microfabrication, we generated various microfluidic devices with traps of different geometrical properties. Investigation of trap topology allowed us to define the one most suitable for single T. brucei confinement within the field of view of an inverted microscope while allowing the parasite to remain motile. Chips populated with V-shaped traps allowed us to investigate various phenomena in cultured procyclic stage wild-type parasites, and to compare them with parasites whose motility was altered upon knockdown of a paraflagellar rod component. Among the properties that we investigated were trap invasion, exploratory behaviour, and the visualization of organelles labelled with fluorescent dyes. We envisage that this tool we have named Tryp-Chip will be a useful tool for the scientific community, as it could allow high-throughput, high-temporal and high-spatial resolution imaging of free-swimming T. brucei parasites.

show abstract

“…Equally, it is incompatible with the visualization of the free flagellum and imaging of free-swimming parasites. Agarose immobilization [36] and CyGEL immobilization [37] are also incompatible with visualization of the free flagellum. Optical trapping is a force nanoscopy-based method highly suitable for the study of propulsion forces generated by flagella [38].…”

Section: Technical Features: Strengths and Future Directionsmentioning

confidence: 99%

Spatial confinement of Trypanosoma brucei in microfluidic traps provides a new tool to study free swimming parasites

De Niz,

Frachon,

Gobaa

et al. 2023

PLoS ONE

View full text Add to dashboard Cite

Trypanosoma brucei is the causative agent of African trypanosomiasis and is transmitted by the tsetse fly (Glossina spp.). All stages of this extracellular parasite possess a single flagellum that is attached to the cell body and confers a high degree of motility. While several stages are amenable to culture in vitro, longitudinal high-resolution imaging of free-swimming parasites has been challenging, mostly due to the rapid flagellar beating that constantly twists the cell body. Here, using microfabrication, we generated various microfluidic devices with traps of different geometrical properties. Investigation of trap topology allowed us to define the one most suitable for single T. brucei confinement within the field of view of an inverted microscope while allowing the parasite to remain motile. Chips populated with V-shaped traps allowed us to investigate various phenomena in cultured procyclic stage wild-type parasites, and to compare them with parasites whose motility was altered upon knockdown of a paraflagellar rod component. Among the properties that we investigated were trap invasion, parasite motility, and the visualization of organelles labelled with fluorescent dyes. We envisage that this tool we have named “Tryp-Chip” will be a useful tool for the scientific community, as it could allow high-throughput, high-temporal and high-spatial resolution imaging of free-swimming T. brucei parasites.

show abstract

Dynamics of Mitochondrial RNA-Binding Protein Complex in Trypanosoma brucei and Its Petite Mutant under Optimized Immobilization Conditions

Cited by 4 publications

References 39 publications

TbUTP10, a protein involved in early stages of pre-18S rRNA processing in Trypanosoma brucei

TbUTP10, a protein involved in early stages of pre-18S rRNA processing in Trypanosoma brucei

Spatial confinement ofTrypanosoma bruceiin microfluidic traps provides a new tool to study free swimming parasites

Spatial confinement of Trypanosoma brucei in microfluidic traps provides a new tool to study free swimming parasites

Contact Info

Product

Resources

About