Although messenger RNA (mRNA) translation is a fundamental biological process, it has never been imaged in real time in vivo with single-molecule precision. To achieve this, we developed nascent chain tracking (NCT), a technique that uses multi-epitope tags and antibody-based fluorescent probes to quantify protein synthesis dynamics at the single-mRNA level. NCT reveals an elongation rate of ~10 amino acids per second, with initiation occurring stochastically every ~30 seconds. Polysomes contain ~1 ribosome every 200 to 900 nucleotides and are globular rather than elongated in shape. By developing multicolor probes, we showed that most polysomes act independently; however, a small fraction (~5%) form complexes in which two distinct mRNAs can be translated simultaneously. The sensitivity and versatility of NCT make it a powerful new tool for quantifying mRNA translation kinetics.

SummaryPrecise control of the attachment strength between kinetochores and spindle microtubules is essential to preserve genomic stability. Aurora B kinase has been implicated in regulating the stability of kinetochore-microtubule attachments but its relevant kinetochore targets in cells remain unclear. Here, we identify multiple serine residues within the N-terminus of the kinetochore protein Hec1 that are phosphorylated in an Aurora-B-kinase-dependent manner during mitosis. On all identified target sites, Hec1 phosphorylation at kinetochores is high in early mitosis and decreases significantly as chromosomes bi-orient. Furthermore, once dephosphorylated, Hec1 is not highly rephosphorylated in response to loss of kinetochore-microtubule attachment or tension. We find that a subpopulation of Aurora B kinase remains localized at the outer kinetochore even upon Hec1 dephosphorylation, suggesting that Hec1 phosphorylation by Aurora B might not be regulated wholly by spatial positioning of the kinase. Our results define a role for Hec1 phosphorylation in kinetochore-microtubule destabilization and error correction in early mitosis and for Hec1 dephosphorylation in maintaining stable attachments in late mitosis.

Accurate regulation of kinetochore–microtubule affinity is driven by incremental phosphorylation of an NDC80 molecular “lawn,” in which NDC80–microtubule bonds reorganize dynamically in response to the number and stability of microtubule attachments.

Precise regulation of kinetochore–microtubule attachments is essential for successful chromosome segregation. DeLuca et al. show that Aurora A kinase regulates kinetochore–microtubule dynamics of metaphase chromosomes through phosphorylation of Hec1 S69, a previously uncharacterized phosphorylation target site on the Hec1 tail.

Cryptophycin-52 (LY355703) is a new synthetic member of the cryptophycin family of antimitotic antitumor agents that is currently undergoing clinical evaluation. At high concentrations (>10 times the IC 50 ), cryptophycin-52 blocked HeLa cell proliferation at mitosis by depolymerizing spindle microtubules and disrupting chromosome organization. However, low concentrations of cryptophycin-52 inhibited cell proliferation at mitosis (IC 50 ‫؍‬ 11 pM) without significantly altering spindle microtubule mass or organization. Cryptophycin-52 appears to be the most potent suppressor of microtubule dynamics found thus far. It suppressed the dynamic instability behavior of individual microtubules in vitro (IC 50 ‫؍‬ 20 nM), reducing the rate and extent of shortening and growing without significantly reducing polymer mass or mean microtubule length. Using [ 3 H]cryptophycin-52, we found that the compound bound to microtubule ends in vitro with high affinity (K d , 47 nM, maximum of Ϸ19.5 cryptophycin-52 molecules per microtubule). By analyzing the effects of cryptophycin-52 on dynamics in relation to its binding to microtubules, we determined that Ϸ5-6 molecules of cryptophycin-52 bound to a microtubule were sufficient to decrease dynamicity by 50%. Cryptophycin-52 became concentrated in cells 730-fold, and the resulting intracellular cryptophycin-52 concentration was similar to that required to stabilize microtubule dynamics in vitro. The data suggest that cryptophycin-52 potently perturbs kinetic events at microtubule ends that are required for microtubule function during mitosis and that it acts by forming a reversible cryptophycin-52-tubulin stabilizing cap at microtubule ends.