Novel Cytoskeleton-Associated Proteins in Trypanosoma brucei Are Essential for Cell Morphogenesis and Cytokinesis

Trypanosome brucei, the causative agent of African sleeping sickness, harbours a highly ordered, subpellicular microtubule cytoskeleton that defines many aspects of morphology, motility and virulence. This array of microtubules is associated with a large number of proteins involved in its regulation. Employing proximity-dependent biotinylation assay (BioID) using the well characterised cytoskeleton-associated protein CAP5.5 as a probe, we identified CAP50 (Tb927.11.2610). This protein colocalises with the subpellicular cytoskeleton microtubules but not with the flagellum. Depletion by RNAi results in defects in cytokinesis, morphology and partial disorganisation of microtubule arrays. Published proteomics data indicate a possible association of CAP50 with two other, yet uncharacterised, cytoskeletal proteins, CAP52 (Tb927.6.5070) and CAP42 (Tb927.4.1300), which were therefore included in our analysis. We show that their depletion causes phenotypes similar to those described for CAP50 and that they are essential for cellular integrity.

Exposure to low intensity ultrasound removes paclitaxel cytotoxicity in breast and ovarian cancer cells

Background Paclitaxel (Taxol) is a microtubule-stabilizing drug used to treat several solid tumors, including ovarian, breast, non-small cell lung, and pancreatic cancers. The current treatment of ovarian cancer is chemotherapy using paclitaxel in combination with carboplatin as a frontline agent, and paclitaxel is also used in salvage treatment as a second line drug with a dose intensive regimen following recurrence. More recently, a dose dense approach for paclitaxel has been used to treat metastatic breast cancer with success. Paclitaxel binds to beta tubulin with high affinity and stabilizes microtubule bundles. As a consequence of targeting microtubules, paclitaxel kills cancer cells through inhibition of mitosis, causing mitotic catastrophes, and by additional, not yet well defined non-mitotic mechanism(s). Results In exploring methods to modulate activity of paclitaxel in causing cancer cell death, we unexpectedly found that a brief exposure of paclitaxel-treated cells in culture to low intensity ultrasound waves prevented the paclitaxel-induced cytotoxicity and death of the cancer cells. The treatment with ultrasound shock waves was found to transiently disrupt the microtubule cytoskeleton and to eliminate paclitaxel-induced rigid microtubule bundles. When cellular microtubules were labelled with a fluorescent paclitaxel analog, exposure to ultrasound waves led to the disassembly of the labeled microtubules and localization of the signals to perinuclear compartments, which were determined to be lysosomes. Conclusions We suggest that ultrasound disrupts the paclitaxel-induced rigid microtubule cytoskeleton, generating paclitaxel bound fragments that undergo degradation. A new microtubule network forms from tubulins that are not bound by paclitaxel. Hence, ultrasound shock waves are able to abolish paclitaxel impact on microtubules. Thus, our results demonstrate that a brief exposure to low intensity ultrasound can reduce and/or eliminate cytotoxicity associated with paclitaxel treatment of cancer cells in cultures.

The SON RNA splicing factor is required for intracellular trafficking structures that promote centriole assembly and ciliogenesis

Control of centrosome assembly is critical for cell division, intracellular trafficking and cilia. Regulation of centrosome number occurs through the precise duplication of centrioles that reside in centrosomes. Here we explored transcriptional control of centriole assembly and find that the RNA splicing factor SON is specifically required for completing procentriole assembly. Whole genome mRNA sequencing identified genes whose splicing and expression are affected by the reduction of SON, with an enrichment in genes involved in the microtubule cytoskeleton, centrosome and centriolar satellites. SON is required for the proper splicing and expression of CEP131 which encodes a major centriolar satellite protein and is required to organize the trafficking and microtubule network around the centrosomes. This study highlights the importance of the distinct microtubule trafficking network that is intimately associated with nascent centrioles and is responsible for procentriole development and efficient ciliogenesis. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Functional roles of α 1 -, α 2 -, β 1 -, β 2 -tubulin in vegetative growth, microtubule assembly and sexual reproduction of Fusarium graminearum .

The plant pathogen Fusarium graminearum contains two α-tubulin (α 1 and α 2 ) isotypes and two β-tubulin isotypes (β 1 and β 2 ). The functional roles of these tubulins in microtubule assembly are not clear. Previous studies showed that α 1 - and β 2 -tubulin deletion mutants showed severe growth defects and hypersensitivity to carbendazim, which have not been well explained. Here, we investigated the interaction between α- and β-tubulin of F. graminearum . Co-localization experiments demonstrated that β 1 - and β 2 -tubulin are co-localized. Co-immunoprecipitation experiment suggested that β 1 -tubulin binds to both α 1 - and α 2 -tubulin and β 2 -tubulin can also bind to α 1 - or α 2 -tubulin. Interestingly, deletion of α 1 -tubulin increased the interaction between β 2 -tubulin and α 2 -tubulin. Microtubule observation assays showed that deletion of α 1 -tubulin completely disrupted β 1 -tubulin-containing microtubules and significantly decreased β 2 -tubulin-containing microtubules. Deletion of α 2 -, β 1 - or β 2 -tubulin respectively had no obvious effect on the microtubule cytoskeleton. However, microtubules in α 1 - and β 2 -tubulin deletion mutants were easily depolymerized in the presence of carbendazim. The sexual reproduction assay indicates that α 1 - and β 1 -tubulin deletion mutants could not produce asci and ascospores. These results implied that α 1 -tubulin may be essential for the microtubule cytoskeleton. However, our Δα 1 -2×α 2 mutant (α 1 -tubulin deletion mutant containing two copies of α 2 -tubulin) exhibited a normal microtubule network, growth and sexual reproduction. Interestingly, the Δα 1 -2×α 2 mutant was still hypersensitive to carbendazim. In addition, both β 1 -tubulin and β 2 -tubulin were found to bind the mitochondrial outer membrane voltage-dependent anion channel (VDAC), indicating they could regulate the function of VDAC. Importance: In this study, we found that F. graminearum contains four different α-/β-tubulin heterodimers (α 1 -β 1 , α 1 -β 2 , α 2 -β 1 and α 2 -β 2 ) and they assemble together into a single microtubule. Moreover, α 1 -, α 2 -tubulins are functionally interchangeable in microtubule assembly, vegetative growth and sexual reproduction. These results provide more insights into functional roles of different tubulins of F. graminearum which could be helpful for purification of tubulin heterodimers and developing new tubulin-binding agents.

The Microtubule Minus-End Binding Protein Patronin Is Required for the Epithelial Remodeling in the Drosophila Abdomen

In the developing Drosophila abdomen, the epithelial tissue displays extensive cytoskeletal remodeling. In stark contrast to the spatio-temporal control of the actin cytoskeleton, the regulation of microtubule architecture during epithelial morphogenesis has remained opaque. In particular, its role in cell motility remains unclear. Here, we show that minus-end binding protein Patronin is required for organizing microtubule arrays in histoblast cells that form the Drosophila abdomen. Loss of Patronin results in a dorsal cleft, indicating the compromised function of histoblasts. We further show that Patronin is polarized in these cells and is required for the formation of highly dynamic non-centrosomal microtubules in the migrating histoblasts. Thus, our study demonstrates that regulation of microtubule cytoskeleton through Patronin mediates epithelium remodeling.