The twin-arginine translocation (Tat) system transports folded proteins across the cytoplasmic membrane of most bacteria and archaea. TatA, which contains a single membrane-spanning helix, is believed to be responsible for the actual translocation. According to the prevalent model, multiple TatA subunits form a transient protein-conducting pore, which disassembles after each translocation event. An alternative model exists, in which TatA proteins locally weaken the lipid bilayer to translocate folded proteins. Here, we imaged eGFP-fused TatA expressed from the genome in live E. coli cells. Images showed TatA occuring both in highly mobile monomers or small oligomers and in large, stable complexes that do not dissociate. Single-particle tracking revealed that large TatA complexes switch between fast and slow diffusion. The fast diffusion is too fast for a transmembrane protein complex consisting of multiple TatA monomers. In line with recent data on rhomboid proteases, we propose that TatA complexes switch between a slowly diffusing transmembrane conformation and a rapidly diffusing membrane-disrupting state that enables folded proteins to cross the membrane, in accordance with the membrane-weakening model.

Single-molecule imaging in living cells can provide unique information about biological processes. Bacteria offer some particular challenges for single-molecule imaging due to their small size, only slightly larger than the diffraction limit of visible light. Here, we describe how reliable and reproducible single-molecule data can be obtained for a transmembrane protein in the Gram-negative bacterium Escherichia coli by using live-cell fluorescence microscopy. Fluorescent labeling of a protein by genetic fusion, cell culturing, sample preparation, imaging, and data analysis are discussed.

Cilia are built and maintained by intraflagellar transport (IFT), driving IFT trains back and forth along the ciliary axoneme. How IFT brings about the intricate ciliary structure and how this structure affects IFT are not well understood. We identify, using single-molecule super-resolution imaging of IFT components in living C. elegans, ciliary subdomains, enabling correlation of IFT-train dynamics to ciliary ultra-structure. In the transition zone, IFT dynamics are impaired, resulting in frequent pauses. At the ciliary base and tip, IFT trains show intriguing turnaround dynamics. Surprisingly, deletion of IFT motor kinesin-II not only affects IFT-train dynamics but also alters ciliary structure. Super-resolution imaging in these mutant animals suggests that the arrangement of IFT trains with respect to the axonemal microtubules is different than in wild-type animals. Our results reveal a complex, mutual interplay between ciliary ultrastructure and IFT-train dynamics, highlighting the importance of physical cues in the control of IFT dynamics.

Primary cilia act as cellular antennae to detect and transmit signals from the extracellular environment. They are built and maintained by continuous cycles of intraflagellar transport (IFT), which transports ciliary proteins from base to tip and back again. It is well known that defects in the IFT machinery can disrupt ciliary structure and function, but the effects of more controlled disturbances of the IFT machinery are not well understood. Here we study how IFT in the chemosensory cilia of C. elegans is affected by chemical inhibition and femtosecond laser ablation of the dendritic input. In C. elegans, anterograde IFT is driven by two cooperating kinesins, OSM-3 and kinesin-II, and retrograde IFT by IFT dynein. Using fluorescence microscopy, we visualize and quantify the real-time response of ciliary proteins to both disturbances. We find that laser ablation of the dendrite results in a three-stage response: (i) IFT motors moving at their normal velocity redistribute, followin a sudden surge of retrograde transport triggered by an unknown signalling process; (ii) the axoneme shortens and motor velocities decrease; and (iii) motors leave the cilium. We propose that such a multi-step response enables the cilium to adapt to outside changes. In another set of experiments, we applied the small-molecule cytoplasmic dynein antagonists, ciliobrevin A. Acute, low-concentration ciliobrevin treatment results in shortening of cilia and reduction of transport velocity in both retrograde and anterograde directions. Longer exposure to ciliobrevin leads to concentration-dependent motor accumulations and axonemal deformations. We find a strong correlation between IFT-dynein velocity and ciliary length. These experiments show that, in C. elegans chemosensory cilia, changes in IFT efficiency directly affect ciliary structure, highlighting that the cilium is a highly dynamic organelle connecting inside and outside of the cell.

Cilia are microtubule-based sensing hubs that rely on intraflagellar transport (IFT) for their development, maintenance, and function. Kinesin-2 motors transport IFT trains, consisting of IFT proteins and cargo, from ciliary base to tip. There, trains turn around and are transported back by IFT dynein. The mechanism of tip turnaround has remained elusive. Here, we employ single-molecule fluorescence microscopy of IFT components in the tips of phasmid cilia of living C. elegans. Analysis of the trajectories reveals that while motor proteins and IFT-A particle component CHE-11 mostly turn around immediately, the IFT-B particle component OSM-6 pauses for several seconds. Our data indicate that IFT trains disassemble into at least IFT-A, IFT-B, IFT-dynein, and OSM-3 complexes at the tip, where OSM-6 is temporarily retained or undergoes modification, prior to train reassembly and retrograde transport. The single-molecule approach used here is a valuable tool to study how directional switches occur in microtubule-based transport processes.

Assessing the strength and kinetics of molecular interactions of cells with the extracellular matrix is fundamental to understand cell adhesion processes. Given the relevance of these processes, there is a strong need for physical methods to quantitatively assess the mechanism of cell adhesion at the single-cell level, allowing discrimination of cells with different behaviors. Here we introduce single-cell acoustic force spectroscopy (scAFS), an approach that makes use of acoustic waves to exert controlled forces, up to 1 nN, to hundreds of individual cells in parallel. We demonstrate the potential of scAFS by measuring adhesion forces and kinetics of CD4(+) T lymphocytes (CD4) to fibronectin. We determined that CD4 adhesion is accelerated by interleukin-7, their main regulatory cytokine, whereas CD4 binding strength remains the same. Activation of these cells likely increases their chance to bind to the vessel wall in the blood flow to infiltrate inflamed tissues and locally coordinate the immune response.

Bidirectional transport driven by motor proteins is essential for the proper distribution of cargo, and therefore vital for many cellular processes. Cilia are polar, microtubule-based cellular sensing hubs that rely on a process called intraflagellar transport (IFT) for their development, maintenance and function in signal-transduction. IFT trains, consisting of cargo and the IFT-A and IFT-B protein complexes, are assembled at the ciliary base and driven co-operatively by kinesin-II and OSM-3 motors to the ciliary tip. The trains reverse direction at the tip and are transported back to the base by IFT dynein. The mechanism of IFT turnaround at the ciliary tip remains unknown. Here, we employ single-molecule fluorescence microscopy and single-particle tracking in the phasmid cilia of living C. elegans to probe IFT tip turnaround. Single-molecule trajectories reveal direct, pausing and diffusive turns in a sub-micrometer long turnaround region at the ciliary tip. Strikingly, while most IFT dyneins, OSM-3s and IFT-A particles turn almost instantaneously (within 600ms), IFT-B particles pause on average for 3 s before returning, with pauses lasting as long as 15 s. Further analysis reveals that IFT dynein and OSM-3 also exhibit diffusive behavior at the ciliary tip, whereas IFT-A and IFT-B are more spatially constrained. Our findings suggest that IFT trains dissociate at the tip and re-associate in retrograde moving trains. IFT-B, different than the other components, requires substantial remodeling while remaining spatially constrained at the tip, before it docks to an IFT-dynein driven retrograde train. Stochastic simulations of several tip turnaround scenarios support this model. Our data provides the first in vivo single-molecule quantification of IFT tip turnarounds, providing new insights into how bidirectional intracellular transport is organized and regulated.

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.

IFT-dynein (cytoplasmic dynein 2) co-operates with kinesin-2 motors to assemble and maintain cilia in a process called intraflagellar transport (IFT). Relatively little is known about this dynein, while the related cytoplasmic dynein 1 has received a lot of attention of late. Here, we study the in vivo IFT-dynein dynamics at the ensemble and single-molecule level. To this end, we use fluorescence microscopy to visualize labeled IFT-dynein motors, expressed at endogenous levels, in the chemosensory cilia of living C. elegans. Fluorescence movies were processed to kymographs, from which location-dependent velocities and motor numbers were obtained using custom kymograph-analysis software. To obtain insight into the behavior of individual motors, we employed photoactivation of PA-GFP-labeled IFT-dynein, which allowed, for the first time, to track individual IFT-dynein motors in vivo. The data revealed that IFT-dynein moves in trains consisting tens of motor proteins. Retrograde trains are smaller but more frequent than anterograde (kinesin-driven) trains. Anterograde and retrograde IFT-dynein flux are equal along cilia, indicating that the cilium is a closed system for dynein. While the kinesin composition of a train varies along the track, the amount of dynein per train remains relatively constant. Remarkably, this does not result in directionality changes along the track, like in reported ‘tug-of-war’ motor systems, suggesting that retrograde and anterograde motor activities are carefully orchestrated in IFT. Single IFT-dynein measurements support the ensemble findings. Trajectories show distinct motility behavior: diffusion at the ciliary base, pauses, turns and directed motion. Pauses in retrograde or anterograde trajectories are never followed by a directional switch. Moreover, retrograde-to-anterograde turn events are rare. This combined ensemble and single-molecule approach has provided new insights into IFT-dynein transport dynamics in living organisms, shedding light on dynein function in general.

The functional organization of prokaryotic cell membranes, which is essential for many cellular processes, has been challenging to analyze due to the small size and nonflat geometry of bacterial cells. Here, we use single-molecule fluorescence microscopy and three-dimensional quantitative analyses in live Escherichia coli to demonstrate that its cytoplasmic membrane contains microdomains with distinct physical properties. We show that the stability of these microdomains depends on the integrity of the MreB cytoskeletal network underneath the membrane. We explore how the interplay between cytoskeleton and membrane affects trans-membrane protein (TMP) diffusion and reveal that the mobility of the TMPs tested is subdiffusive, most likely caused by confinement of TMP mobility by the submembranous MreB network. Our findings demonstrate that the dynamic architecture of prokaryotic cell membranes is controlled by the MreB cytoskeleton and regulates the mobility of TMPs.

Force-spectroscopy has become an indispensable tool to unravel the structural and mechanochemical properties of biomolecules. Acoustic Force Spectroscopy (AFS) is a new acoustic manipulation method, which consists of a resonator integrated into a micro-fabricated fluidic chip. An acoustical pressure gradient is created homogeneously throughout the sample enabling to exert forces on DNA-tethered microspheres. By changing the amplitude of the driving voltage the pressure gradient can be altered, allowing sensitive control of the force applied to the DNA molecules. This approach allows exerting acoustic forces from sub-pN to hundreds of pN applied to thousands of biomolecules in parallel, with sub-millisecond response time and inherent stability. Here we present the next step in validating this new technology, a stand-alone commercial grade prototype, that makes this method available to the wider scientific community.

Intraflagellar transport (IFT) is an essential intracellular transport mechanism in cilia, the hair-like, microtubule-based protrusions of eukaryotic cells with sensory or motile functions. In the chemosensory cilia of the nematode C. elegans, IFT is driven by the cooperative action of IFT-dynein (responsible for transport from cilium tip to base) and two kinesin motor proteins, Kinesin-II and OSM-3, (responsible for transport in the opposite direction). Our goal was to understand why two kinesins are needed for IFT and what their respective roles are. To achieve this, we generated mutant nematodes expressing fluorescent versions of the motor proteins at endogenous levels and subjected them to in vivo fluorescence microscopy with single-molecule resolution. Images obtained were analyzed using automated kymograph and single-particle tracking analysis, providing unprecedented, quantitative insight in the role of the kinesins in IFT. We find that the two kinesins fulfill distinct roles in line with their distinct motility properties. Kinesin-II is the slower and less processive motor. In IFT its key role is to load of IFT trains, to initiate the transport of multiple, coupled motor proteins connected to cargo, and to effectively traverse the transition zone, the semi-permeable protein barrier between cilium and rest of the cell. After successful crossing of the transition zone, Kinesin-II leaves the trains and the other, faster and more processive kinesin, OSM-3 binds and drives the longer-distance transport to the cilium tip. Our results provide insight in how cells use a combination of motor proteins to drive intracellular transport and demonstrate the power of single-molecule fluorescence microscopy to unravel complex processes in the cells of living, multicellular organisms.

Cytoplasmic dyneins are the main drivers of microtubule-based retrograde transport in eukaryotic cells. Cytoplasmic dynein 1 plays a role in retrograde intracellular transport and cell division, whereas cytoplasmic dynein 2, also known as IFT-dynein, co-operates with kinesin motors to assemble and maintain cilia in a process called intraflagellar transport (IFT). While cytoplasmic dynein 1 has been the subject of many recent studies, relatively little is known about IFT-dynein. Here, we focus on the mechanism and dynamics of IFT-dynein: how does it behave in vivo at the ensemble and single-molecule level? To this end, we use fluorescence microscopy to visualize labeled IFT-dynein motors in the chemosensory cilia of living C. elegans. Transgene worms were generated using the Mos1-mediated single copy insertion (MosSCI) method to ensure endogenous motor expression levels. Time-lapse fluorescence movies showed that IFT-dynein moves in trains consisting of tens of motor proteins. The movies were processed to kymographs, from which location-dependent velocities and motor numbers were obtained using in-house developed kymograph-analysis software. This analysis revealed that IFT-dynein train velocities and motor numbers are dynamic, changing along the cilium. Double-labeled constructs allowed us to look more closely into motor co-operation, determining the dynein:kinesin ratio at different positions in the cilium. To obtain insight into the behavior of individual motors, we employed photoactivation of PA-GFP-labeled IFT-dynein, which allowed, for the first time, the tracking of individual IFT-dynein motors in vivo. Single-motor trajectories revealed distinct features of IFT-dynein motility: diffusive behavior at the ciliary base, pauses, turns, directed motion and switches between these behaviors. This combined ensemble and single-molecule approach has provided novel quantitative insight into IFT-dynein dynamics in living organisms, shedding light on the complex functioning of dynein motors in general.

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.

Membrane proteins perform vital cellular functions like respiration, signaling and nutrient uptake. For proper membrane-protein functioning, conformational dynamics, complex formation and ability to diffuse in the membrane are vital parameters. Despite a lot of work on model membranes, little is known about lateral diffusion of proteins in prokaryotic membranes. Here we use single-molecule wide-field epi-fluorescence microscopy to track, in living E. coli, the lateral mobility of seven trans-membrane proteins of different size fused to green fluorescent protein. We apply a novel method, IPODD (inverse projection of displacement distributions), to extract accurate diffusion coefficients from the 2-D projected diffusion trajectories along the 3-D curved bacterial membrane. The diffusion coefficients we find are significantly lower than those reported in in vitro studies of isolated membrane proteins in giant unilaminar vesicles. Our results indicate that crowding in the E. coli inner membrane substantially slows down trans-membrane protein mobility. Strikingly, we observe heterogeneity in the diffusive motion of all seven proteins: they all show a faster and a slower moving component. This heterogeneity does not appear to be connected to specific localization of the proteins in poles or other parts of the bacterium. Instead, our experiments indicate that the plasma membrane of E. coli contains patches with a different lipid composition than the bulk of the membrane, resulting in regions of slower and faster membrane-protein diffusion. These results show that the diffusion behavior of proteins embedded in the plasma membrane in E. coli is richer and far more complex than anticipated.

Intraflagellar transport (IFT) is indispensable for the assembly and maintenance of cilia. In the chemosensory cilia of C. elegans, two motors of the kinesin-2-family, heterotrimeric kinesin-II and homodimeric OSM-3, collaborate to drive anterograde IFT. It is known that both kinesins associate with IFT-trains that move along the middle segment, and that OSM-3 alone drives transport in the distal segment. However, many questions remain concerning how these kinesins cooperate, how this is regulated and why both motors are required. To address this problem, we have improved the fidelity of IFT-assays by using single transgenes encoding for fluorescently-labeled kinesins. In combination with ultrasensitive, quantitative fluorescence microscopy and dynamic photoactivated localization microscopy allowing observation of single motors in living nematodes, we find that the motor composition of IFT-trains is highly dynamic. Kinesin-II undocks gradually from IFT-trains close to the ciliary base, and not suddenly upon reaching the middle segment tips as previously thought, whereas OSM-3 docks gradually resulting in accelerating IFT-trains. Undocked kinesin-II is transported back to the ciliary base by dynein-driven retrograde trains. Consequently, the IFT-system ensures that kinesin-II stays close to the ciliary base, where, it is responsible for the loading of IFT-trains onto the axoneme, and that OSM-3 stays around the distal segment, where it drives fast long-distance transport. Our work on motor cooperation and dynamics provides new insight into how IFT drives the proper development and functioning of cilia and, more broadly, how kinesin motors work together to generate intracellular transport pathways within cells.

The cytoplasmic membrane forms the barrier between any cell's interior and the outside world. It contains many proteins that enable essential processes such as the transmission of signals, the uptake of nutrients, and cell division. In the case of prokaryotes, which do not contain intracellular membranes, the cytoplasmic membrane also contains proteins for respiration and protein folding. Mutual interactions and specific localization of these proteins depend on two-dimensional diffusion driven by thermal fluctuations. The experimental investigation of membrane-protein diffusion in bacteria is challenging due to their small size, only a few times larger than the resolution of an optical microscope. Here, we review fluorescence microscopy-based methods to study diffusion of membrane proteins in living bacteria. The main focus is on data-analysis tools to extract diffusion coefficients from single-particle tracking data obtained by single-molecule fluorescence microscopy. We introduce a novel approach, IPODD (inverse projection of displacement distributions), to obtain diffusion coefficients from the usually obtained 2-D projected diffusion trajectories of the highly 3-D curved bacterial membrane. This method provides, in contrast to traditional mean-squared-displacement methods, correct diffusion coefficients and allows unravelling of heterogeneously diffusing populations.

BACKGROUND:Searching the orthologs of a given protein or DNA sequence is one of the most important and most commonly used Bioinformatics methods in Biology. Programs like BLAST or the orthology search engine Inparanoid can be used to find orthologs when the similarity between two sequences is sufficiently high. They however fail when the level of conservation is low. The detection of remotely conserved proteins oftentimes involves sophisticated manual intervention that is difficult to automate.RESULTS:Here, we introduce morFeus, a search program to find remotely conserved orthologs. Based on relaxed sequence similarity searches, morFeus selects sequences based on the similarity of their alignments to the query, tests for orthology by iterative reciprocal BLAST searches and calculates a network score for the resulting network of orthologs that is a measure of orthology independent of the E-value. Detecting remotely conserved orthologs of a protein using morFeus thus requires no manual intervention. We demonstrate the performance of morFeus by comparing it to state-of-the-art orthology resources and methods. We provide an example of remotely conserved orthologs, which were experimentally shown to be functionally equivalent in the respective organisms and therefore meet the criteria of the orthology-function conjecture.CONCLUSIONS:Based on our results, we conclude that morFeus is a powerful and specific search method for detecting remotely conserved orthologs. morFeus is freely available at http://bio.biochem.mpg.de/morfeus/. Its source code is available from Sourceforge.net (https://sourceforge.net/p/morfeus/).