characterization-effects-migrastatic-inhibitors-on-3d-tumor-spheroid

Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and ...

University of Huddersfield

TOF-LisH-PLL motifs define FOP family proteins; some members are involved in flagellum assembly. The critical role of FOP family protein FOR20 is poorly understood. Here, we report relative localisations of the four FOP family proteins in parasitic Trypanosoma brucei: TbRP2, TbOFD1 and TbFOP/FOP1-like are mature basal body proteins whereas TbFOR20 is present on pro- and mature basal bodies - on the latter it localises distal to TbRP2. We discuss how the data, together with published work for another protist Giardia intestinalis, informs on likely FOR20 function. Moreover, our localisation study provides convincing evidence that the antigen recognised by monoclonal antibody YL1/2 at trypanosome mature basal bodies is FOP family protein TbRP2, not tyrosinated α-tubulin as widely stated in the literature. Curiously, FOR20 proteins from T. brucei and closely related African trypanosomes possess short, negatively-charged N-terminal extensions absent from FOR20 in other trypanosomatids and other eukaryotes. The extension is necessary for protein targeting, but insufficient to re-direct TbRP2 to probasal bodies. Yet, FOR20 from the American trypanosome T. cruzi, which lacks any extension, localises to pro- and mature basal bodies when expressed in T. brucei. This identifies unexpected variation in FOR20 architecture that is presently unique to one clade of trypanosomatids.

Variation in Basal Body Localisation and Targeting of Trypanosome RP2 and FOR20 Proteins.

Parasitic trypanosomatids diverged from free-living kinetoplastid ancestors several hundred million years ago. These parasites are relatively well known, due in part to several unusual cell biological and molecular traits and in part to the significance of a few - pathogenic Leishmania and Trypanosoma species - as aetiological agents of serious neglected tropical diseases. However, the majority of trypanosomatid biodiversity is represented by osmotrophic monoxenous parasites of insects. In two lineages, novymonads and strigomonads, osmotrophic lifestyles are supported by cytoplasmic endosymbionts, providing hosts with macromolecular precursors and vitamins. Here we discuss the two independent origins of endosymbiosis within trypanosomatids and subsequently different evolutionary trajectories that see entrainment vs tolerance of symbiont cell divisions cycles within those of the host. With the potential to inform on the transition to obligate parasitism in the trypanosomatids, interest in the biology and ecology of free-living, phagotrophic kinetoplastids is beginning to enjoy a renaissance. Thus, we take the opportunity to additionally consider the wider relevance of endosymbiosis during kinetoplastid evolution, including the indulged lifestyle and reductive evolution of basal kinetoplastid Perkinsela.

Farming, slaving and enslavement: histories of endosymbioses during kinetoplastid evolution.

Proteins of the FGR1 oncogene partner (or FOP) family are found at microtubule organizing centres (MTOCs) including, in flagellate eukaryotes, the centriole or flagellar basal body from which the axoneme extends. We report conservation of FOP family proteins, FOPL and OFD1, in the evolutionarily divergent sleeping sickness parasite , showing (in contrast with mammalian cells, where FOP is essential for flagellum assembly) depletion of a trypanosome FOP homologue, FOPL, affects neither axoneme nor flagellum elongation. Instead, FOPL depletion causes catastrophic failure in assembly of a lineage-specific, extra-axonemal structure, the paraflagellar rod (PFR). That depletion of centriolar FOPL causes failure in PFR assembly is surprising because PFR nucleation commences approximately 2 µm distal from the basal body. When over-expressed with a C-terminal myc-epitope, FOPL was also observed at mitotic spindle poles. Little is known about bi-polar spindle assembly during closed trypanosome mitosis, but indication of a possible additional MTOC function for FOPL parallels MTOC localization of FOP-like protein TONNEAU1 in acentriolar plants. More generally, our functional analysis of FOPL emphasizes significant differences in evolutionary cell biology trajectories of FOP-family proteins. We discuss how at the molecular level FOP homologues may contribute to flagellum assembly and function in diverse flagellates.

Jane Harmer

School of Applied Sciences

University of Huddersfield

Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy

Jane Harmer

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University of Huddersfield

Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy

School of Applied Sciences