HiExM: high-throughput expansion microscopy enables scalable super-resolution imaging.

Expansion microscopy (ExM) enables nanoscale imaging using a standard confocal microscope through the physical, isotropic expansion of fixed immunolabeled specimens. ExM is widely employed to image proteins, nucleic acids, and lipid membranes in single cells at nanoscale resolution; however, current methods cannot be performed in multi-well cell culture plates which limits the number of samples that can be processed simultaneously. We developed High-throughput Expansion Microscopy (HiExM), a robust platform that enables expansion microscopy of cells cultured in a standard 96-well plate. Our method enables consistent ~4.2x expansion within individual wells, across multiple wells, and between plates processed in parallel. We also demonstrate that HiExM can be combined with high-throughput confocal imaging platforms greatly improve the ease and scalability of image acquisition. As an example, we analyzed the effects of doxorubicin, a known cardiotoxic agent, in human cardiomyocytes (CMs) based on Hoechst signal intensity. We show a dose dependent effect on nuclear chromatin that is not observed in unexpanded CMs, suggesting that HiExM improves the detection of cellular phenotypes in response to drug treatment. Our method broadens the application of ExM as a tool for scalable super-resolution imaging in biological research applications.

A dual role for H2A.Z.1 in modulating the dynamics of RNA polymerase II initiation and elongation.

RNA polymerase II (RNAPII) pausing immediately downstream of the transcription start site is a critical rate-limiting step for the expression of most metazoan genes. During pause release, RNAPII encounters a highly conserved +1 H2A.Z nucleosome, yet how this histone variant contributes to transcription is poorly understood. Here, using an inducible protein degron system combined with genomic approaches and live cell super-resolution microscopy, we show that H2A.Z.1 modulates RNAPII dynamics across most genes in murine embryonic stem cells. Our quantitative analysis shows that H2A.Z.1 slows the rate of RNAPII pause release and consequently impacts negative elongation factor dynamics as well as nascent transcription. Consequently, H2A.Z.1 also impacts re-loading of the pre-initiation complex components TFIIB and TBP. Altogether, this work provides a critical mechanistic link between H2A.Z.1 and the proper induction of mammalian gene expression programs through the regulation of RNAPII dynamics and pause release.

High-Resolution Imaging Methods to Analyze LINC Complex Function During Drosophila Muscle Development.

Using Drosophila muscle development as a model system makes possible the identification of genetic pathways, temporal regulation of development, mechanisms of cellular development, and physiological impacts in a single system. Here we describe the basic techniques for the evaluation of the cellular development of muscle in Drosophila in both embryos and in larvae. These techniques are discussed within the context of how the LINC complex contributes to muscle development.

Aplip1, the Drosophila homolog of JIP1, regulates myonuclear positioning and muscle stability.

During muscle development, myonuclei undergo a complex set of movements that result in evenly spaced nuclei throughout the muscle cell. In Drosophila, two separate pools of Kinesin and Dynein work in synchrony to drive this process. However, how these two pools are specified is not known. Here, we investigate the role of Aplip1 (the Drosophila homolog of JIP1, JIP1 is also known as MAPK8IP1), a known regulator of both Kinesin and Dynein, in myonuclear positioning. Aplip1 localizes to the myotendinous junction and has genetically separable roles in myonuclear positioning and muscle stability. In Aplip1 mutant embryos, there was an increase in the percentage of embryos that had both missing and collapsed muscles. Via a separate mechanism, we demonstrate that Aplip1 regulates both the final position of and the dynamic movements of myonuclei. Aplip1 genetically interacts with both Raps (also known as Pins) and Kinesin to position myonuclei. Furthermore, Dynein and Kinesin localization are disrupted in Aplip1 mutants suggesting that Aplip1-dependent nuclear positioning requires Dynein and Kinesin. Taken together, these data are consistent with Aplip1 having a function in the regulation of Dynein- and Kinesin-mediated pulling of nuclei from the muscle end.This article has an associated First Person interview with the first author of the paper.

Nucleus-dependent sarcomere assembly is mediated by the LINC complex.

Two defining characteristics of muscle cells are the many precisely positioned nuclei and the linearly arranged sarcomeres, yet the relationship between these two features is not known. We show that nuclear positioning precedes sarcomere formation. Furthermore, ZASP-GFP, a Z-line protein, colocalizes with F-actin in puncta at the cytoplasmic face of nuclei before sarcomere assembly. In embryos with mispositioned nuclei, ZASP-GFP is still recruited to the nuclei before its incorporation into sarcomeres. Furthermore, the first sarcomeres appear in positions close to the nuclei, regardless of nuclear position. These data suggest that the interaction between sarcomere proteins and nuclei is not dependent on properly positioned nuclei. Mechanistically, ZASP-GFP localization to the cytoplasmic face of the nucleus did require the linker of nucleoskeleton and cytoskeleton (LINC) complex. Muscle-specific depletion of klarsicht (nesprin) or klariod (SUN) blocked the recruitment of ZASP-GFP to the nucleus during the early stages of sarcomere assembly. As a result, sarcomeres were poorly formed and the general myofibril network was less stable, incomplete, and/or torn. These data suggest that the nucleus, through the LINC complex, is crucial for the proper assembly and stability of the sarcomere network.