A fast blind zero-shot denoiser.

Image noise is a common problem in light microscopy. This is particularly true in real-time live-cell imaging applications in which long-term cell viability necessitates low-light conditions. Modern denoisers are typically trained on a representative dataset, sometimes consisting of just unpaired noisy shots. However, when data are acquired in real time to track dynamic cellular processes, it is not always practical nor economical to generate these training sets. Recently, denoisers have emerged that allow us to denoise single images without a training set or knowledge about the underlying noise. But such methods are currently too slow to be integrated into imaging pipelines that require rapid, real-time hardware feedback. Here we present Noise2Fast, which can overcome these limitations. Noise2Fast uses a novel downsampling technique we refer to as 'chequerboard downsampling'. This allows us to train on a discrete 4-image training set, while convergence can be monitored using the original noisy image. We show that Noise2Fast is faster than all similar methods with only a small drop in accuracy compared to the gold standard. We integrate Noise2Fast into real-time multi-modal imaging applications and demonstrate its broad applicability to diverse imaging and analysis pipelines.

Global cellular response to chemical perturbation of PLK4 activity and abnormal centrosome number.

Centrosomes act as the main microtubule organizing center (MTOC) in metazoans. Centrosome number is tightly regulated by limiting centriole duplication to a single round per cell cycle. This control is achieved by multiple mechanisms, including the regulation of the protein kinase PLK4, the most upstream facilitator of centriole duplication. Altered centrosome numbers in mouse and human cells cause p53-dependent growth arrest through poorly defined mechanisms. Recent work has shown that the E3 ligase TRIM37 is required for cell cycle arrest in acentrosomal cells. To gain additional insights into this process, we undertook a series of genome-wide CRISPR/Cas9 screens to identify factors important for growth arrest triggered by treatment with centrinone B, a selective PLK4 inhibitor. We found that TRIM37 is a key mediator of growth arrest after partial or full PLK4 inhibition. Interestingly, PLK4 cellular mobility decreased in a dose-dependent manner after centrinone B treatment. In contrast to recent work, we found that growth arrest after PLK4 inhibition correlated better with PLK4 activity than with mitotic length or centrosome number. These data provide insights into the global response to changes in centrosome number and PLK4 activity and extend the role for TRIM37 in regulating the abundance, localization, and function of centrosome proteins.

Terminally differentiated osteoclasts organize centrosomes into large clusters for microtubule nucleation and bone resorption.

Osteoclasts are highly specialized, multinucleated cells responsible for the selective resorption of the dense, calcified bone matrix. Microtubules (MTs) contribute to the polarization and trafficking events involved in bone resorption by osteoclasts; however, the origin of these elaborate arrays is less clear. Osteoclasts arise through cell fusion of precursor cells. Previous studies have suggested that centrosome MT nucleation is lost during this process, with the nuclear membrane and its surrounding Golgi serving as the major MT organizing centers (MTOCs) in these cells. Here we reveal that precursor cell centrosomes are maintained and functional in the multinucleated osteoclast and interestingly form large MTOC clusters, with the clusters organizing significantly more MTs compared with individual centrosomes. MTOC cluster formation requires dynamic MTs and minus-end directed MT motor activity. Inhibition of these centrosome clustering elements had a marked impact on both F-actin ring formation and bone resorption. Together these findings show that multinucleated osteoclasts employ unique centrosomal clusters to organize the extensive MTs during bone attachment and resorption.

Direct interaction between CEP85 and STIL mediates PLK4-driven directed cell migration.

PLK4 has emerged as a prime target for cancer therapeutics, and its overexpression is frequently observed in various types of human cancer. Recent studies have further revealed an unexpected oncogenic activity of PLK4 in regulating cancer cell migration and invasion. However, the molecular basis behind the role of PLK4 in these processes still remains only partly understood. Our previous work has demonstrated that an intact CEP85-STIL binding interface is necessary for robust PLK4 activation and centriole duplication. Here, we show that CEP85 and STIL are also required for directional cancer cell migration. Mutational and functional analyses reveal that the interactions between CEP85, STIL and PLK4 are essential for effective directional cell motility. Mechanistically, we show that PLK4 can drive the recruitment of CEP85 and STIL to the leading edge of cells to promote protrusive activity, and that downregulation of CEP85 and STIL leads to a reduction in ARP2 (also known as ACTR2) phosphorylation and reorganization of the actin cytoskeleton, which in turn impairs cell migration. Collectively, our studies provide molecular insight into the important role of the CEP85-STIL complex in modulating PLK4-driven cancer cell migration.This article has an associated First Person interview with the first author of the paper.

A tent pole twist on membrane ruffles.

Macropinocytosis or "cell drinking" involves the elaboration of membrane ruffles that enclose and internalize extracellular fluids. Using lattice light sheet microscopy, Condon et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201804137) reveal the presence of parallel membrane protrusions termed "tent poles" that flank and direct membrane ruffle formation.