Cortical tension drug screen links mitotic spindle integrity to Rho pathway.

Mechanical force generation plays an essential role in many cellular functions, including mitosis. Actomyosin contractile forces mediate changes in cell shape in mitosis and are implicated in mitotic spindle integrity via cortical tension. An unbiased screen of 150 small molecules that impact actin organization and 32 anti-mitotic drugs identified two molecular targets, Rho kinase (ROCK) and tropomyosin 3.1/2 (Tpm3.1/2), whose inhibition has the greatest impact on mitotic cortical tension. The converse was found for compounds that depolymerize microtubules. Tpm3.1/2 forms a co-polymer with mitotic cortical actin filaments, and its inhibition prevents rescue of multipolar spindles induced by anti-microtubule chemotherapeutics. We examined the role of mitotic cortical tension in this rescue mechanism. Inhibition of ROCK and Tpm3.1/2 and knockdown (KD) of cortical nonmuscle myosin 2A (NM2A), all of which reduce cortical tension, inhibited rescue of multipolar mitotic spindles, further implicating cortical tension in the rescue mechanism. GEF-H1 released from microtubules by depolymerization increased cortical tension through the RhoA pathway, and its KD also inhibited rescue of multipolar mitotic spindles. We conclude that microtubule depolymerization by anti-cancer drugs induces cortical-tension-based rescue to ensure integrity of the mitotic bipolar spindle mediated via the RhoA pathway. Central to this mechanism is the dependence of NM2A on Tpm3.1/2 to produce the functional engagement of actin filaments responsible for cortical tension.

Spatiotemporally mapping temperature dynamics of lysosomes and mitochondria using cascade organelle-targeting upconversion nanoparticles.

The intracellular metabolism of organelles, like lysosomes and mitochondria, is highly coordinated spatiotemporally and functionally. The activities of lysosomal enzymes significantly rely on the cytoplasmic temperature, and heat is constantly released by mitochondria as the byproduct of adenosine triphosphate (ATP) generation during active metabolism. Here, we developed temperature-sensitive LysoDots and MitoDots to monitor the in situ thermal dynamics of lysosomes and mitochondria. The design is based on upconversion nanoparticles (UCNPs) with high-density surface modifications to achieve the exceptionally high sensitivity of 2.7% K-1 and low uncertainty of 0.8 K for nanothermometry to be used in living cells. We show the measurement is independent of the ion concentrations and pH values. With Ca2+ ion shock, the temperatures of both lysosomes and mitochondria increased by ∼2 to 4 °C. Intriguingly, with chloroquine (CQ) treatment, the lysosomal temperature was observed to decrease by up to ∼3 °C, while mitochondria remained relatively stable. Lastly, with oxidative phosphorylation inhibitor treatment, we observed an ∼3 to 7 °C temperature increase and a thermal transition from mitochondria to lysosomes. These observations indicate different metabolic pathways and thermal transitions between lysosomes and mitochondria inside HeLa cells. The nanothermometry probes provide a powerful tool for multimodality functional imaging of subcellular organelles and interactions with high spatial, temporal, and thermal dynamics resolutions.