Peripheral thickening of the sarcomeres and pointed end elongation of the thin filaments are both promoted by SALS and its formin interaction partners.

During striated muscle development the first periodically repeated units appear in the premyofibrils, consisting of immature sarcomeres that must undergo a substantial growth both in length and width, to reach their final size. Here we report that, beyond its well established role in sarcomere elongation, the Sarcomere length short (SALS) protein is involved in Z-disc formation and peripheral growth of the sarcomeres. Our protein localization data and loss-of-function studies in the Drosophila indirect flight muscle strongly suggest that radial growth of the sarcomeres is initiated at the Z-disc. As to thin filament elongation, we used a powerful nanoscopy approach to reveal that SALS is subject to a major conformational change during sarcomere development, which might be critical to stop pointed end elongation in the adult muscles. In addition, we demonstrate that the roles of SALS in sarcomere elongation and radial growth are both dependent on formin type of actin assembly factors. Unexpectedly, when SALS is present in excess amounts, it promotes the formation of actin aggregates highly resembling the ones described in nemaline myopathy patients. Collectively, these findings helped to shed light on the complex mechanisms of SALS during the coordinated elongation and thickening of the sarcomeres, and resulted in the discovery of a potential nemaline myopathy model, suitable for the identification of genetic and small molecule inhibitors.

Syndecan-4 Modulates Cell Polarity and Migration by Influencing Centrosome Positioning and Intracellular Calcium Distribution.

Efficient cell migration requires cellular polarization, which is characterized by the formation of leading and trailing edges, appropriate positioning of the nucleus and reorientation of the Golgi apparatus and centrosomes toward the leading edge. Migration also requires the development of an asymmetrical front-to-rear calcium (Ca2+) gradient to regulate focal adhesion assembly and actomyosin contractility. Here we demonstrate that silencing of syndecan-4, a transmembrane heparan sulfate proteoglycan, interferes with the correct polarization of migrating mammalian myoblasts (i.e., activated satellite stem cells). In particular, syndecan-4 knockdown completely abolished the intracellular Ca2+ gradient, abrogated centrosome reorientation and thus decreased cell motility, demonstrating the role of syndecan-4 in cell polarity. Additionally, syndecan-4 exhibited a polarized distribution during migration. Syndecan-4 knockdown cells exhibited decreases in the total movement distance during directional migration, maximum and vectorial distances from the starting point, as well as average and maximum cell speeds. Super-resolution direct stochastic optical reconstruction microscopy images of syndecan-4 knockdown cells revealed nanoscale changes in the actin cytoskeletal architecture, such as decreases in the numbers of branches and individual branch lengths in the lamellipodia of the migrating cells. Given the crucial importance of myoblast migration during embryonic development and postnatal muscle regeneration, we conclude that our results could facilitate an understanding of these processes and the general role of syndecan-4 during cell migration.

Hot-Band Anti-Stokes Fluorescence Properties of Alexa Fluor 568.

Hot-band absorption and anti-Stokes emission properties of an organic fluorescent dye, Alexa Fluor 568, were characterized and compared with those of Rhodamine 101. The comparison of the properties (e.g., quantum efficiency, spectral distribution, thermal properties, and fluorescence lifetime) between the two dyes confirms that both dyes undergo the same process when excited in the red spectral region. Possible undesirable crosstalk effects and applications in dSTORM microscopy were demonstrated and discussed.

Superresolution Microscopy of Drosophila Indirect Flight Muscle Sarcomeres.

Sarcomeres are extremely highly ordered macromolecular assemblies where proper structural organization is an absolute prerequisite to the functionality of these contractile units. Despite the wealth of information collected, the exact spatial arrangement of many of the H-zone and Z-disk proteins remained unknown. Recently, we developed a powerful nanoscopic approach to localize the sarcomeric protein components with a resolution well below the diffraction limit. The ease of sample preparation and the near crystalline structure of the Drosophila flight muscle sarcomeres make them ideally suitable for single molecule localization microscopy and structure averaging. Our approach allowed us to determine the position of dozens of H-zone and Z-disk proteins with a quasi-molecular, ~5-10 nm localization precision. The protocol described below provides an easy and reproducible method to prepare individual myofibrils for dSTORM imaging. In addition, it includes an in-depth description of a custom made and freely available software toolbox to process and quantitatively analyze the raw localization data.

Nanoscopy reveals the layered organization of the sarcomeric H-zone and I-band complexes.

Sarcomeres are extremely highly ordered macromolecular assemblies where structural organization is intimately linked to their functionality as contractile units. Although the structural basis of actin and Myosin interaction is revealed at a quasiatomic resolution, much less is known about the molecular organization of the I-band and H-zone. We report the development of a powerful nanoscopic approach, combined with a structure-averaging algorithm, that allowed us to determine the position of 27 sarcomeric proteins in Drosophila melanogaster flight muscles with a quasimolecular, ∼5- to 10-nm localization precision. With this protein localization atlas and template-based protein structure modeling, we have assembled refined I-band and H-zone models with unparalleled scope and resolution. In addition, we found that actin regulatory proteins of the H-zone are organized into two distinct layers, suggesting that the major place of thin filament assembly is an M-line-centered narrow domain where short actin oligomers can form and subsequently anneal to the pointed end.

mmSTORM: Multimodal localization based super-resolution microscopy.

Super-resolution localization microscopy provides a powerful tool to study biochemical mechanisms at single molecule level. Although the lateral position of the fluorescent dye molecules can be determined routinely with high precision, measurement of other modalities such as 3D and multicolor without the degradation of the original super-resolved image is still in the focus. In this paper a dual-objective multimodal single molecule localization microscopy (SMLM) technique has been developed, optimized and tested. The proposed optical arrangement can be implemented onto a conventional inverted microscope without serious system modification. The performance of the method was tested using fluorescence beads, F-actin filaments and sarcomere structures. It was shown that the proposed imaging method does not degrade the image quality of the original SMLM 2D image but could provide information on the axial position or emission spectra of the dye molecules.

TestSTORM: Versatile simulator software for multimodal super-resolution localization fluorescence microscopy.

Optimization of sample, imaging and data processing parameters is an essential task in localization based super-resolution microscopy, where the final image quality strongly depends on the imaging of single isolated fluorescent molecules. A computational solution that uses a simulator software for the generation of test data stacks was proposed, developed and tested. The implemented advanced physical models such as scalar and vector based point spread functions, polarization sensitive detection, drift, spectral crosstalk, structured background etc., made the simulation results more realistic and helped us interpret the final super-resolved images and distinguish between real structures and imaging artefacts.

Polarization sensitive localization based super-resolution microscopy with a birefringent wedge.

A practical method has been presented for polarization sensitive localization based super-resolution microscopy using a birefringent dual wedge. The measurement of the polarization degree at the single molecule level can reveal the chemical and physical properties of the local environment of the fluorescent dye molecule and can hence provide information about the sub-diffraction sized structure of biological samples. Polarization sensitive STORM imaging of the F-Actins proved correlation between the orientation of fluorescent dipoles and the axis of the fibril.