Purification of functional Plasmodium falciparum tubulin allows for the identification of parasite-specific microtubule inhibitors.

Cytoskeletal proteins are essential for parasite proliferation, growth, and transmission, and therefore have the potential to serve as drug targets.1-5 While microtubules and their molecular building block αß-tubulin are established drug targets in a variety of cancers,6,7 we still lack sufficient knowledge of the biochemistry of parasite tubulins to exploit the structural divergence between parasite and human tubulins. For example, it remains to be determined whether compounds of interest can specifically target parasite microtubules without affecting the host cell cytoskeleton. Such mechanistic insights have been limited by the lack of functional parasite tubulin. In this study, we report the purification and characterization of tubulin from Plasmodium falciparum, the causative agent of malaria. We show that the highly purified tubulin is fully functional, as it efficiently assembles into microtubules with specific parameters of dynamic instability. There is a high degree of amino-acid conservation between human and P. falciparum α- and ß-tubulin, sharing approximately 83.7% and 88.5% identity, respectively. However, Plasmodium tubulin is more similar to plant than to mammalian tubulin, raising the possibility of identifying compounds that would selectively disrupt parasite microtubules without affecting the host cell cytoskeleton. As a proof of principle, we describe two compounds that exhibit selective toxicity toward parasite tubulin. Thus, the ability to specifically disrupt protozoan microtubule growth without affecting human microtubules provides an exciting opportunity for the development of novel antimalarials.

The effects of proliferation status and cell cycle phase on the responses of single cells to chemotherapy.

DNA-damaging chemotherapeutics are widely used in cancer treatments, but for solid tumors they often leave a residual tumor-cell population. Here we investigated how cellular states might affect the response of individual cells in a clonal population to cisplatin, a DNA-damaging chemotherapeutic agent. Using a live-cell reporter of cell cycle phase and long-term imaging, we monitored single-cell proliferation before, at the time of, and after treatment. We found that in response to cisplatin, cells either arrested or died, and the ratio of these outcomes depended on the dose. While we found that the cell cycle phase at the time of cisplatin addition was not predictive of outcome, the proliferative history of the cell was: highly proliferative cells were more likely to arrest than to die, whereas slowly proliferating cells showed a higher probability of death. Information theory analysis revealed that the dose of cisplatin had the greatest influence on the cells' decisions to arrest or die, and that the proliferation status interacted with the cisplatin dose to further guide this decision. These results show an unexpected effect of proliferation status in regulating responses to cisplatin and suggest that slowly proliferating cells within tumors may be acutely vulnerable to chemotherapy.

The centrosome protein AKNA regulates neurogenesis via microtubule organization.

The expansion of brain size is accompanied by a relative enlargement of the subventricular zone during development. Epithelial-like neural stem cells divide in the ventricular zone at the ventricles of the embryonic brain, self-renew and generate basal progenitors1 that delaminate and settle in the subventricular zone in enlarged brain regions2. The length of time that cells stay in the subventricular zone is essential for controlling further amplification and fate determination. Here we show that the interphase centrosome protein AKNA has a key role in this process. AKNA localizes at the subdistal appendages of the mother centriole in specific subtypes of neural stem cells, and in almost all basal progenitors. This protein is necessary and sufficient to organize centrosomal microtubules, and promote their nucleation and growth. These features of AKNA are important for mediating the delamination process in the formation of the subventricular zone. Moreover, AKNA regulates the exit from the subventricular zone, which reveals the pivotal role of centrosomal microtubule organization in enabling cells to both enter and remain in the subventricular zone. The epithelial-to-mesenchymal transition is also regulated by AKNA in other epithelial cells, demonstrating its general importance for the control of cell delamination.

GTSE1 is a microtubule plus-end tracking protein that regulates EB1-dependent cell migration.

The regulation of cell migration is a highly complex process that is often compromised when cancer cells become metastatic. The microtubule cytoskeleton is necessary for cell migration, but how microtubules and microtubule-associated proteins regulate multiple pathways promoting cell migration remains unclear. Microtubule plus-end binding proteins (+TIPs) are emerging as important players in many cellular functions, including cell migration. Here we identify a +TIP, GTSE1, that promotes cell migration. GTSE1 accumulates at growing microtubule plus ends through interaction with the EB1+TIP. The EB1-dependent +TIP activity of GTSE1 is required for cell migration, as well as for microtubule-dependent disassembly of focal adhesions. GTSE1 protein levels determine the migratory capacity of both nontransformed and breast cancer cell lines. In breast cancers, increased GTSE1 expression correlates with invasive potential, tumor stage, and time to distant metastasis, suggesting that misregulation of GTSE1 expression could be associated with increased invasive potential.