Combining single-molecule and expansion microscopy in fission yeast to visualize protein structures at the nanostructural level.

In this work, we have developed an expansion microscopy (ExM) protocol that combines ExM with photoactivated localization microscopy (ExPALM) for yeast cell imaging, and report a robust protocol for single-molecule and expansion microscopy of fission yeast, abbreviated as SExY. Our optimized SExY protocol retains about 50% of the fluorescent protein signal, doubling the amount obtained compared to the original protein retention ExM (proExM) protocol. It allows for a fivefold, highly isotropic expansion of fission yeast cells, which we carefully controlled while optimizing protein yield. We demonstrate the SExY method on several exemplary molecular targets and explicitly introduce low-abundant protein targets (e.g. nuclear proteins such as cbp1 and mis16, and the centromere-specific histone protein cnp1). The SExY protocol optimizations increasing protein yield could be beneficial for many studies, when targeting low abundance proteins, or for studies that rely on genetic labelling for various reasons (e.g. for proteins that cannot be easily targeted by extrinsic staining or in case artefacts introduced by unspecific staining interfere with data quality).

Cytosolic sorting platform complexes shuttle type III secretion system effectors to the injectisome in Yersinia enterocolitica.

Bacteria use type III secretion injectisomes to inject effector proteins into eukaryotic target cells. Recruitment of effectors to the machinery and the resulting export hierarchy involve the sorting platform. These conserved proteins form pod structures at the cytosolic interface of the injectisome but are also mobile in the cytosol. Photoactivated localization microscopy in Yersinia enterocolitica revealed a direct interaction of the sorting platform proteins SctQ and SctL with effectors in the cytosol of live bacteria. These proteins form larger cytosolic protein complexes involving the ATPase SctN and the membrane connector SctK. The mobility and composition of these mobile pod structures are modulated in the presence of effectors and their chaperones, and upon initiation of secretion, which also increases the number of injectisomes from ~5 to ~18 per bacterium. Our quantitative data support an effector shuttling mechanism, in which sorting platform proteins bind to effectors in the cytosol and deliver the cargo to the export gate at the membrane-bound injectisome.

Temporal analysis of relative distances (TARDIS) is a robust, parameter-free alternative to single-particle tracking.

In single-particle tracking, individual particles are localized and tracked over time to probe their diffusion and molecular interactions. Temporal crossing of trajectories, blinking particles, and false-positive localizations present computational challenges that have remained difficult to overcome. Here we introduce a robust, parameter-free alternative to single-particle tracking: temporal analysis of relative distances (TARDIS). In TARDIS, an all-to-all distance analysis between localizations is performed with increasing temporal shifts. These pairwise distances represent either intraparticle distances originating from the same particle, or interparticle distances originating from unrelated particles, and are fitted analytically to obtain quantitative measures on particle dynamics. We showcase that TARDIS outperforms tracking algorithms, benchmarked on simulated and experimental data of varying complexity. We further show that TARDIS performs accurately in complex conditions characterized by high particle density, strong emitter blinking or false-positive localizations, and is in fact limited by the capabilities of localization algorithms. TARDIS' robustness enables fivefold shorter measurements without loss of information.

Unraveling the kinetochore nanostructure in Schizosaccharomyces pombe using multi-color SMLM imaging.

The key to ensuring proper chromosome segregation during mitosis is the kinetochore (KT), a tightly regulated multiprotein complex that links the centromeric chromatin to the spindle microtubules and as such leads the segregation process. Understanding its architecture, function, and regulation is therefore essential. However, due to its complexity and dynamics, only its individual subcomplexes could be studied in structural detail so far. In this study, we construct a nanometer-precise in situ map of the human-like regional KT of Schizosaccharomyces pombe using multi-color single-molecule localization microscopy. We measure each protein of interest (POI) in conjunction with two references, cnp1CENP-A at the centromere and sad1 at the spindle pole. This allows us to determine cell cycle and mitotic plane, and to visualize individual centromere regions separately. We determine protein distances within the complex using Bayesian inference, establish the stoichiometry of each POI and, consequently, build an in situ KT model with unprecedented precision, providing new insights into the architecture.

Effective or predatory funding? Evaluating the hidden costs of grant applications.

Researchers are spending an increasing fraction of their time on applying for funding; however, the current funding system has considerable deficiencies in reliably evaluating the merit of research proposals, despite extensive efforts on the sides of applicants, grant reviewers and decision committees. For some funding schemes, the systemic costs of the application process as a whole can even outweigh the granted resources-a phenomenon that could be considered as predatory funding. We present five recommendations to remedy this unsatisfactory situation.

Frequency modulation of a bacterial quorum sensing response.

In quorum sensing, bacteria secrete or release small molecules into the environment that, once they reach a certain threshold, trigger a behavioural change in the population. As the concentration of these so-called autoinducers is supposed to reflect population density, they were originally assumed to be continuously produced by all cells in a population. However, here we show that in the α-proteobacterium Sinorhizobium meliloti expression of the autoinducer synthase gene is realized in asynchronous stochastic pulses that result from scarcity and, presumably, low binding affinity of the key activator. Physiological cues modulate pulse frequency, and pulse frequency in turn modulates the velocity with which autoinducer levels in the environment reach the threshold to trigger the quorum sensing response. We therefore propose that frequency-modulated pulsing in S. meliloti represents the molecular mechanism for a collective decision-making process in which each cell's physiological state and need for behavioural adaptation is encoded in the pulse frequency with which it expresses the autoinducer synthase gene; the pulse frequencies of all members of the population are then integrated in the common pool of autoinducers, and only once this vote crosses the threshold, the response behaviour is initiated.

Mutual functional dependence of cyclase-associated protein 1 (CAP1) and cofilin1 in neuronal actin dynamics and growth cone function.

Neuron connectivity depends on growth cones that navigate axons through the developing brain. Growth cones protrude and retract actin-rich structures to sense guidance cues. These cues control local actin dynamics and steer growth cones towards attractants and away from repellents, thereby directing axon outgrowth. Hence, actin binding proteins (ABPs) moved into the focus as critical regulators of neuron connectivity. We found cyclase-associated protein 1 (CAP1), an ABP with unknown brain function, abundant in growth cones. Super-resolution microscopy and live cell imaging combined with pharmacological approaches on hippocampal neurons from gene-targeted mice revealed a crucial role for CAP1 in actin dynamics that is critical for growth cone morphology and function. Growth cone defects in CAP1 knockout (KO) neurons compromised neuron differentiation and was associated with impaired neuron connectivity in CAP1-KO brains. Mechanistically, by rescue experiments in double KO neurons lacking CAP1 and the key actin regulator cofilin1, we demonstrated that CAP1 was essential for cofilin1 function in growth cone actin dynamics and morphology and vice versa. Together, we identified CAP1 as a novel actin regulator in growth cones that was relevant for neuron connectivity, and we demonstrated functional interdependence of CAP1 and cofilin1 in neuronal actin dynamics and growth cone function.

Dynamic relocalization of cytosolic type III secretion system components prevents premature protein secretion at low external pH.

Many bacterial pathogens use a type III secretion system (T3SS) to manipulate host cells. Protein secretion by the T3SS injectisome is activated upon contact to any host cell, and it has been unclear how premature secretion is prevented during infection. Here we report that in the gastrointestinal pathogens Yersinia enterocolitica and Shigella flexneri, cytosolic injectisome components are temporarily released from the proximal interface of the injectisome at low external pH, preventing protein secretion in acidic environments, such as the stomach. We show that in Yersinia enterocolitica, low external pH is detected in the periplasm and leads to a partial dissociation of the inner membrane injectisome component SctD, which in turn causes the dissociation of the cytosolic T3SS components. This effect is reversed upon restoration of neutral pH, allowing a fast activation of the T3SS at the native target regions within the host. These findings indicate that the cytosolic components form an adaptive regulatory interface, which regulates T3SS activity in response to environmental conditions.

Raw Data to Results: A Hands-On Introduction and Overview of Computational Analysis for Single-Molecule Localization Microscopy.

Single-molecule localization microscopy (SMLM) is an advanced microscopy method that uses the blinking of fluorescent molecules to determine the position of these molecules with a resolution below the diffraction limit (∼5-40 nm). While SMLM imaging itself is becoming more popular, the computational analysis surrounding the technique is still a specialized area and often remains a "black box" for experimental researchers. Here, we provide an introduction to the required computational analysis of SMLM imaging, post-processing and typical data analysis. Importantly, user-friendly, ready-to-use and well-documented code in Python and MATLAB with exemplary data is provided as an interactive experience for the reader, as well as a starting point for further analysis. Our code is supplemented by descriptions of the computational problems and their implementation. We discuss the state of the art in computational methods and software suites used in SMLM imaging and data analysis. Finally, we give an outlook into further computational challenges in the field.

Establishing Live-Cell Single-Molecule Localization Microscopy Imaging and Single-Particle Tracking in the Archaeon Haloferax volcanii.

In recent years, fluorescence microscopy techniques for the localization and tracking of single molecules in living cells have become well-established and are indispensable tools for the investigation of cellular biology and in vivo biochemistry of many bacterial and eukaryotic organisms. Nevertheless, these techniques are still not established for imaging archaea. Their establishment as a standard tool for the study of archaea will be a decisive milestone for the exploration of this branch of life and its unique biology. Here, we have developed a reliable protocol for the study of the archaeon Haloferax volcanii. We have generated an autofluorescence-free H. volcanii strain, evaluated several fluorescent proteins for their suitability to serve as single-molecule fluorescence markers and codon-optimized them to work under optimal H. volcanii cultivation conditions. We found that two of them, Dendra2Hfx and PAmCherry1Hfx, provide state-of-the-art single-molecule imaging. Our strategy is quantitative and allows dual-color imaging of two targets in the same field of view (FOV) as well as DNA co-staining. We present the first single-molecule localization microscopy (SMLM) images of the subcellular organization and dynamics of two crucial intracellular proteins in living H. volcanii cells, FtsZ1, which shows complex structures in the cell division ring, and RNA polymerase, which localizes around the periphery of the cellular DNA. This work should provide incentive to develop SMLM strategies for other archaeal organisms in the near future.

Postmitotic expansion of cell nuclei requires nuclear actin filament bundling by α-actinin 4.

The actin cytoskeleton operates in a multitude of cellular processes including cell shape and migration, mechanoregulation, and membrane or organelle dynamics. However, its filamentous properties and functions inside the mammalian cell nucleus are less well explored. We previously described transient actin assembly at mitotic exit that promotes nuclear expansion during chromatin decondensation. Here, we identify non-muscle α-actinin 4 (ACTN4) as a critical regulator to facilitate F-actin reorganization and bundling during postmitotic nuclear expansion. ACTN4 binds to nuclear actin filament structures, and ACTN4 clusters associate with nuclear F-actin in a highly dynamic fashion. ACTN4 but not ACTN1 is required for proper postmitotic nuclear volume expansion, mediated by its actin-binding domain. Using super-resolution imaging to quantify actin filament numbers and widths in individual nuclei, we find that ACTN4 is necessary for postmitotic nuclear actin reorganization and actin filament bundling. Our findings uncover a nuclear cytoskeletal function for ACTN4 to control nuclear size and chromatin organization during mitotic cell division.

Super-resolution imaging and estimation of protein copy numbers at single synapses with DNA-point accumulation for imaging in nanoscale topography.

In the brain, the strength of each individual synapse is defined by the complement of proteins present or the "local proteome." Activity-dependent changes in synaptic strength are the result of changes in this local proteome and posttranslational protein modifications. Although most synaptic proteins have been identified, we still know little about protein copy numbers in individual synapses and variations between synapses. We use DNA-point accumulation for imaging in nanoscale topography as a single-molecule super-resolution imaging technique to visualize and quantify protein copy numbers in single synapses. The imaging technique provides near-molecular spatial resolution, is unaffected by photobleaching, enables imaging of large field of views, and provides quantitative molecular information. We demonstrate these benefits by accessing copy numbers of surface AMPA-type receptors at single synapses of rat hippocampal neurons along dendritic segments.

Visualizing the inner life of microbes: practices of multi-color single-molecule localization microscopy in microbiology.

In this review, we discuss multi-color single-molecule imaging and tracking strategies for studying microbial cell biology. We first summarize and compare the methods in a detailed literature review of published studies conducted in bacteria and fungi. We then introduce a guideline on which factors and parameters should be evaluated when designing a new experiment, from fluorophore and labeling choices to imaging routines and data analysis. Finally, we give some insight into some of the recent and promising applications and developments of these techniques and discuss the outlook for this field.

A Single-Molecule View of Archaeal Transcription.

The discovery of the archaeal domain of life is tightly connected to an in-depth analysis of the prokaryotic RNA world. In addition to Carl Woese's approach to use the sequence of the 16S rRNA gene as phylogenetic marker, the finding of Karl Stetter and Wolfram Zillig that archaeal RNA polymerases (RNAPs) were nothing like the bacterial RNAP but are more complex enzymes that resemble the eukaryotic RNAPII was one of the key findings supporting the idea that archaea constitute the third major branch on the tree of life. This breakthrough in transcriptional research 40years ago paved the way for in-depth studies of the transcription machinery in archaea. However, although the archaeal RNAP and the basal transcription factors that fine-tune the activity of the RNAP during the transcription cycle are long known, we still lack information concerning the architecture and dynamics of archaeal transcription complexes. In this context, single-molecule measurements were instrumental as they provided crucial insights into the process of transcription initiation, the architecture of the initiation complex and the dynamics of mobile elements of the RNAP. In this review, we discuss single-molecule approaches suitable to examine molecular mechanisms of transcription and highlight findings that shaped our understanding of the archaeal transcription apparatus. We furthermore explore the possibilities and challenges of next-generation single-molecule techniques, for example, super-resolution microscopy and single-molecule tracking, and ask whether these approaches will ultimately allow us to investigate archaeal transcription in vivo.

From single bacterial cell imaging towards in vivo single-molecule biochemistry studies.

Bacteria as single-cell organisms are important model systems to study cellular mechanisms and functions. In recent years and with the help of advanced fluorescence microscopy techniques, immense progress has been made in characterizing and quantifying the behavior of single bacterial cells on the basis of molecular interactions and assemblies in the complex environment of live cultures. Importantly, single-molecule imaging enables the in vivo determination of the stoichiometry and molecular architecture of subcellular structures, yielding detailed, quantitative, spatiotemporally resolved molecular maps and unraveling dynamic heterogeneities and subpopulations on the subcellular level. Nevertheless, open challenges remain. Here, we review the past and current status of the field, discuss example applications and give insights into future trends.

Live-cell single-particle tracking photoactivated localization microscopy of Cascade-mediated DNA surveillance.

Type I CRISPR-Cas systems utilize small CRISPR RNA (crRNA) molecules to scan DNA strands for target regions. Different crRNAs are bound by several CRISPR-associated (Cas) protein subunits that form the stable ribonucleoprotein complex Cascade. The Cascade-mediated DNA surveillance process requires a sufficient degree of base-complementarity between crRNA and target sequences and relies on the recognition of small DNA motifs, termed protospacer adjacent motifs. Recently, super-resolution microscopy and single-particle tracking methods have been developed to follow individual protein complexes in live cells. Here, we described how this technology can be adapted to visualize the DNA scanning process of Cascade assemblies in Escherichia coli cells. The activity of recombinant Type I-Fv Cascade complexes of Shewanella putrefaciens CN-32 serves as a model system that facilitates comparative studies for many of the diverse CRISPR-Cas systems.