The zonula adherens matura redefines the apical junction of intestinal epithelia.

Cell-cell apical junctions of epithelia consist of multiprotein complexes that organize as belts regulating cell-cell adhesion, permeability, and mechanical tension: the tight junction (zonula occludens), the zonula adherens (ZA), and the macula adherens. The prevailing dogma is that at the ZA, E-cadherin and catenins are lined with F-actin bundles that support and transmit mechanical tension between cells. Using super-resolution microscopy on human intestinal biopsies and Caco-2 cells, we show that two distinct multiprotein belts are basal of the tight junctions as the intestinal epithelia mature. The most apical is populated with nectins/afadin and lined with F-actin; the second is populated with E-cad/catenins. We name this dual-belt architecture the zonula adherens matura. We find that the apical contraction apparatus and the dual-belt organization rely on afadin expression. Our study provides a revised description of epithelial cell-cell junctions and identifies a module regulating the mechanics of epithelia.

Probing ribosomal protein-RNA interactions with an external force.

Ribosomal (r-) RNA adopts a well-defined structure within the ribosome, but the role of r-proteins in stabilizing this structure is poorly understood. To address this issue, we use optical tweezers to unfold RNA fragments in the presence or absence of r-proteins. Here, we focus on Escherichia coli r-protein L20, whose globular C-terminal domain (L20C) recognizes an irregular stem in domain II of 23S rRNA. L20C also binds its own mRNA and represses its translation; binding occurs at two different sites--i.e., a pseudoknot and an irregular stem. We find that L20C makes rRNA and mRNA fragments encompassing its binding sites more resistant to mechanical unfolding. The regions of increased resistance correspond within two base pairs to the binding sites identified by conventional methods. While stabilizing specific RNA structures, L20C does not accelerate their formation from alternate conformations--i.e., it acts as a clamp but not as a chaperone. In the ribosome, L20C contacts only one side of its target stem but interacts with both strands, explaining its clamping effect. Other r-proteins bind rRNA similarly, suggesting that several rRNA structures are stabilized by "one-side" clamping.

PatternJ: an ImageJ toolset for the automated and quantitative analysis of regular spatial patterns found in sarcomeres, axons, somites, and more.

Regular spatial patterns are ubiquitous forms of organization in nature. In animals, regular patterns can be found from the cellular scale to the tissue scale, and from early stages of development to adulthood. To understand the formation of these patterns, how they assemble and mature, and how they are affected by perturbations, a precise quantitative description of the patterns is essential. However, accessible tools that offer in-depth analysis without the need for computational skills are lacking for biologists. Here, we present PatternJ, a novel toolset to analyze regular one-dimensional patterns precisely and automatically. This toolset, to be used with the popular imaging processing program ImageJ/Fiji, facilitates the extraction of key geometric features within and between pattern repeats in static images and time-lapse series. We validate PatternJ with simulated data and test it on images of sarcomeres from insect muscles and contracting cardiomyocytes, actin rings in neurons, and somites from zebrafish embryos obtained using confocal fluorescence microscopy, STORM, electron microscopy, and brightfield imaging. We show that the toolset delivers subpixel feature extraction reliably even with images of low signal-to-noise ratio. PatternJ's straightforward use and functionalities make it valuable for various scientific fields requiring quantitative one-dimensional pattern analysis, including the sarcomere biology of muscles or the patterning of mammalian axons, speeding up discoveries with the bonus of high reproducibility.

A nanobody toolbox to investigate localisation and dynamics of Drosophila titins and other key sarcomeric proteins.

Measuring the positions and dynamics of proteins in intact tissues or whole animals is key to understanding protein function. However, to date, this is challenging, as the accessibility of large antibodies to dense tissues is often limited, and fluorescent proteins inserted close to a domain of interest may affect protein function. These complications apply in particular to muscle sarcomeres, arguably one of the most protein-dense assemblies in nature, which complicates studying sarcomere morphogenesis at molecular resolution. Here, we introduce a toolbox of nanobodies recognising various domains of the two Drosophila titin homologs, Sallimus and Projectin, as well as the key sarcomeric proteins Obscurin, α-Actinin, and Zasp52. We verified the superior labelling qualities of our nanobodies in muscle tissue as compared to antibodies. By applying our toolbox to larval muscles, we found a gigantic Sallimus isoform stretching more than 2 µm to bridge the sarcomeric I-band, while Projectin covers almost the entire myosin filaments in a polar orientation. Transgenic expression of tagged nanobodies confirmed their high affinity-binding without affecting target protein function. Finally, adding a degradation signal to anti-Sallimus nanobodies suggested that it is difficult to fully degrade Sallimus in mature sarcomeres; however, expression of these nanobodies caused developmental lethality. These results may inspire the generation of similar toolboxes for other large protein complexes in Drosophila or mammals.

Our muscles are not just for lifting weights. They also keep us alive. For example, our heartbeat is powered by the muscles in the heart wall. Just like other organs in the body, muscles are made up of cells called muscle fibres. Each muscle fibre is divided into many smaller units, or 'sarcomeres', which contain specialised proteins that pull on each other to produce muscle contractions. Although the structure of mature muscles is rather well understood, we know much less about how muscles develop or how they are maintained throughout adult life. Understanding this is especially important in the case of the heart, because its muscle cells are not replaced throughout our lives. Instead, the heart muscle cells we are born with are maintained as we age while working continuously. This means that the proteins within the heart muscle sarcomeres are continuously under mechanical stress and may need to be repaired. How this repair might happen is not well understood. Nanobodies are very small versions of antibodies that recognise and bind to specific protein targets. In biological research, they are used as a tool to observe proteins of interest within cells. This is done by labelling nanobodies, for example, with chemical fluorophores or fluorescent proteins; once labelled, the nanobody binds to its target protein, and scientists can monitor its location and behaviour within the cell. Cells, and even flies, can also be genetically manipulated to produce labelled nanobodies themselves, which has the advantage of visualising the dynamic behaviour of the target protein in the living cell or organism. To better study the proteins in muscle cells, scientists from two different research groups developed a nanobody 'toolbox' that specifically targets sarcomere proteins. First, Loreau et al. made a 'library' of labelled nanobodies targeting different sarcomere proteins in Drosophila melanogaster fruit flies. Second, they used this library of nanobodies to locate several sarcomere proteins in the mature sarcomeres of different fly muscles. Third, using flies that had been genetically altered to produce the labelled nanobodies in their muscle cells, Loreau et al. were able to observe the behaviour of the target proteins in the living muscle. Together, these experiments showed that one protein in Drosophila that is similar to the human sarcomere protein titin has a similar size to the human version, whereas a second Drosophila titin-like protein is shorter and located at a different place in the sarcomere. Both of these proteins work together to stabilise muscle fibres, which is also the role of human titin. The nanobodies generated here are a significant contribution to the tools available to study muscle development and maintenance. Loreau et al. hope that they will help reveal how sarcomere proteins like titin are maintained, especially in the heart, and ultimately how the heart muscle manages to continue working throughout our lives.

Nanobodies combined with DNA-PAINT super-resolution reveal a staggered titin nanoarchitecture in flight muscles.

Sarcomeres are the force-producing units of all striated muscles. Their nanoarchitecture critically depends on the large titin protein, which in vertebrates spans from the sarcomeric Z-disc to the M-band and hence links actin and myosin filaments stably together. This ensures sarcomeric integrity and determines the length of vertebrate sarcomeres. However, the instructive role of titins for sarcomeric architecture outside of vertebrates is not as well understood. Here, we used a series of nanobodies, the Drosophila titin nanobody toolbox, recognising specific domains of the two Drosophila titin homologs Sallimus and Projectin to determine their precise location in intact flight muscles. By combining nanobodies with DNA-PAINT super-resolution microscopy, we found that, similar to vertebrate titin, Sallimus bridges across the flight muscle I-band, whereas Projectin is located at the beginning of the A-band. Interestingly, the ends of both proteins overlap at the I-band/A-band border, revealing a staggered organisation of the two Drosophila titin homologs. This architecture may help to stably anchor Sallimus at the myosin filament and hence ensure efficient force transduction during flight.

From ants to humans, the muscles that set an organism in motion are formed of bundles of fiber-like cells which can shorten and lengthen at will. At the microscopic level, changes in muscle cell lengths are underpinned by contractile filaments formed of multiple repeats of a basic unit, known as the sarcomere. Each unit is bookended by intricate 'Z-discs' and features an 'M-band' in its center. Three protein types give a sarcomere its ability to shorten and expand at will: two types of filaments (myosin and actin), which can slide on one another; and a spring-like molecule known as titin, which ensures that the unit does not fall apart by mechanically connecting myosin and actin. More specifically, actin filaments are anchored to the Z-discs and extend towards the M-band, while myosin filaments are centered around the M-band and extend towards the Z-discs. As myosin and actin slide alongside each other, the overlap between the two types of filaments increases or decreases and the whole unit changes its length. In vertebrates, one gigantic molecule of titin spans from the Z-disc to the M-band, linking together actin and myosin filaments and determining the length of the sarcomere. In insects and other invertebrates, however, this single molecule is replaced by two titin proteins known as Projectin and Sallimus. Understanding how these titins work together remains unclear and difficult to study. Traditional approaches are unable to precisely label titin in an environment teaming with other molecules, and they cannot offer the nanometer resolution required to dissect sarcomere organization. As a response, Schueder, Mangeol et al. combined super-resolution microscopy and a new toolbox of labelling molecules known as nanobodies to track the position of Sallimus and Projectin in the flight muscles of fruit flies. These experiments revealed that the two proteins are arranged in tandem along the length of the sarcomere, forming a structure that measures about 350 nm. Sallimus is anchored in the Z-disc and it runs alongside actin until it reaches the end of a myosin filament; there, it overlaps with Projectin for about 10 nm. Projectin then stretches for 250 nm along the length of the beginning myosin filament. These findings confirm the importance of titin in dictating the length of a sarcomere; they suggest that, in invertebrates, this role is split between two proteins, each possibly ruling over a section of the sarcomere. In addition, the work by Schueder, Mangeol et al. demonstrate the value of combining nanobodies and super-resolution microscopy to study complex structures in tissues.

Super-resolution imaging uncovers the nanoscopic segregation of polarity proteins in epithelia.

Epithelial tissues acquire their integrity and function through the apico-basal polarization of their constituent cells. Proteins of the PAR and Crumbs complexes are pivotal to epithelial polarization, but the mechanistic understanding of polarization is challenging to reach, largely because numerous potential interactions between these proteins and others have been found, without a clear hierarchy in importance. We identify the regionalized and segregated organization of members of the PAR and Crumbs complexes at epithelial apical junctions by imaging endogenous proteins using stimulated-emission-depletion microscopy on Caco-2 cells, and human and murine intestinal samples. Proteins organize in submicrometric clusters, with PAR3 overlapping with the tight junction (TJ) while PALS1-PATJ and aPKC-PAR6ß form segregated clusters that are apical of the TJ and present in an alternated pattern related to actin organization. CRB3A is also apical of the TJ and partially overlaps with other polarity proteins. Of the numerous potential interactions identified between polarity proteins, only PALS1-PATJ and aPKC-PAR6ß are spatially relevant in the junctional area of mature epithelial cells, simplifying our view of how polarity proteins could cooperate to drive and maintain cell polarity.

Many of our organs, including the lungs and the intestine, are lined with a single layer of cells that separate the inside of the organ from the surrounding environment inside the body. These so-called epithelial cells form a tightly packed barrier and have a very characteristic organization. The apical surface faces the outside world, while the basal surface faces the inner tissues. These different interfaces are reflected in the organization of the cells themselves. The shape, composition, and role of the apical cell surface are distinct from those of the basal surface, and they also contain different proteins. In some epithelial cells, the apical surface specializes and forms protruding structures called microvilli. Thus, epithelial cells are said to be polarized along this apical­basal axis. Over the last 30 years, many labs have identified and studied which proteins help epithelial cells become and stay polarized. Previous biochemical experiments showed that these so-called polarity proteins interact with each other in many different ways. But it remains unclear whether some of these interactions are more important than others, and where exactly in the apical or basal membranes these interactions take place. Mangeol et al. used super-resolution microscopy to observe the polarity of proteins at the apical membranes of both human and mouse cells from the small intestine to answer these questions. They focused on areas called tight junctions, where the intestinal cells connect with each other to form the barrier between the outside and the inside. First, all the polarity proteins clustered together in various formations, they were not distributed uniformly. For example, one protein called PAR3 was at the level of the tight junctions, whereas other proteins were closer to the apical surface and the outside world. Only two pairs of proteins ­ PAR6 and aPKC, and PALS1 and PATJ ­ formed stable clusters with each other. This finding was unexpected because previous biochemical experiments had predicted multiple interactions. Third, the PALS1/PATJ complexes stayed at the bottom of the microvilli protrusions, whereas PAR6/aPKC were inside the protrusions. Taken together, these experiments reveal a detailed snapshot of how the polarity proteins themselves are organized at the apical surface of epithelial cells. Future work will be able to address how these protein complexes behave over time.

RNA Unzipping and Force Measurements with a Dual Optical Trap.

In order to mechanically unfold a single RNA molecule, an RNA/DNA hybrid construction is prepared which allows specific attachment to two micrometer-sized beads. A dual-beam optical trap thus holding the construct in solution captures the beads separately. Unfolding of a molecule is obtained by increasing the distance between the traps, one trap being slowly moved while the other is held fixed. Force is measured to sub-piconewton precision by back focal plane interferometry of the bead in the fixed trap. The experiment allows us to measure structure and base-sequence-dependent force signals. In this chapter, important technical aspects of this type of single-molecule force measurements are considered.

Ensemble and single-molecule dynamics of IFT dynein in Caenorhabditis elegans cilia.

Cytoplasmic dyneins drive microtubule-based, minus-end directed transport in eukaryotic cells. Whereas cytoplasmic dynein 1 has been widely studied, IFT dynein has received far less attention. Here, we use fluorescence microscopy of labelled motors in living Caenorhabditis elegans to investigate IFT-dynein motility at the ensemble and single-molecule level. We find that while the kinesin composition of motor ensembles varies along the track, the amount of dynein remains relatively constant. Remarkably, this does not result in directionality changes of cargo along the track, as has been reported for other opposite-polarity, tug-of-war motility systems. At the single-molecule level, IFT-dynein trajectories reveal unexpected dynamics, including diffusion at the base, and pausing and directional switches along the cilium. Stochastic simulations show that the ensemble IFT-dynein distribution depends upon the probability of single-motor directional switches. Our results provide quantitative insight into IFT-dynein dynamics in vivo, shedding light on the complex functioning of dynein motors in general.

High-resolution real-time dual-view imaging with multiple point of view microscopy.

Most methods to observe three-dimensional processes in living samples are based on imaging a single plane that is sequentially scanned through the sample. Sequential scanning is inherently slow, which can make it difficult to capture objects moving quickly in three dimensions. Here we present a novel method, multiple point-of-view microscopy (MPoVM), that allows simultaneous capturing of the front and side views of a sample with high resolution. MPoVM can be implemented in most fluorescence microscopes, offering new opportunities in the study of dynamic biological processes in three dimensions.

KymographClear and KymographDirect: two tools for the automated quantitative analysis of molecular and cellular dynamics using kymographs.

Dynamic processes are ubiquitous and essential in living cells. To properly understand these processes, it is imperative to measure them in a time-dependent way and analyze the resulting data quantitatively, preferably with automated tools. Kymographs are single images that represent the motion of dynamic processes and are widely used in live-cell imaging. Although they contain the full range of dynamics, it is not straightforward to extract this quantitative information in a reliable way. Here we present two complementary, publicly available software tools, KymographClear and KymographDirect, that have the power to reveal detailed insight in dynamic processes. KymographClear is a macro toolset for ImageJ to generate kymographs that provides automatic color coding of the different directions of movement. KymographDirect is a stand-alone tool to extract quantitative information from kymographs obtained from a wide range of dynamic processes in an automated way, with high accuracy and reliability. We discuss the concepts behind these software tools, validate them using simulated data, and test them on experimental data. We show that these tools can be used to extract motility parameters from a diverse set of cell-biological experiments in an automated and user-friendly way.

Functional differentiation of cooperating kinesin-2 motors orchestrates cargo import and transport in C. elegans cilia.

Intracellular transport depends on cooperation between distinct motor proteins. Two anterograde intraflagellar transport (IFT) motors, heterotrimeric kinesin-II and homodimeric OSM-3, cooperate to move cargo along Caenorhabditis elegans cilia. Here, using quantitative fluorescence microscopy, with single-molecule sensitivity, of IFT in living strains containing single-copy transgenes encoding fluorescent IFT proteins, we show that kinesin-II transports IFT trains through the ciliary base and transition zone to a 'handover zone' on the proximal axoneme. There, OSM-3 gradually replaces kinesin-II, yielding velocity profiles inconsistent with in vitro motility assays, and then drives transport to the ciliary tip. Dissociated kinesin-II motors undergo rapid turnaround and recycling to the ciliary base, whereas OSM-3 is recycled mainly to the handover zone. This reveals a functional differentiation in which the slower, less processive kinesin-II imports IFT trains into the cilium and OSM-3 drives their long-range transport, thereby optimizing cargo delivery.

DNA unzipping and force measurements with a dual optical trap.

In order to open the DNA double helix mechanically, a molecular construction is prepared which allows specific attachment of the two complementary strands of an individual molecule to two different µm-sized beads. The beads are separately captured by a dual optical trap, thus holding the DNA construction in solution. The opening of a molecule is obtained by increasing the distance between the traps, one trap being slowly moved while the other is held fixed. Force is measured to sub-piconewton precision by back focal plane interferometry of the bead in the fixed trap. The experiment allows us to measure base sequence-dependent force signal. In this chapter, important technical aspects of this type of single-molecule force measurements are considered.

Interference and crosstalk in double optical tweezers using a single laser source.

Experimental studies of single molecule mechanics require high force sensitivity and low drift, which can be achieved with optical tweezers. We built an optical tweezer setup for force measurements in a two bead assay. A cw infrared laser beam is split by polarization and focused by a high numerical aperture objective to create two traps. The same laser is used to form both traps and to measure the force by back focal plane interferometry. We show that although the two beams entering the microscope are designed to exhibit orthogonal polarization, interference and a significant parasitic force signal occur. Comparing the experimental results with a ray optics model, we show that the interference patterns are caused by the rotation of polarization on microscope lens surfaces and slides. The model qualitatively describes the pattern and the dependence of the parasitic force signal on the experimental parameters. We present two different approaches to experimentally reduce the crosstalk, namely, polarization rectification and frequency shifting.