Sitemap

A list of all the posts and pages found on the site. For you robots out there is an XML version available for digesting as well.

Pages

About me

Posts

Future Blog Post

less than 1 minute read

This post will show up by default. To disable scheduling of future posts, edit config.yml and set future: false.

Published:

Blog Post number 4

less than 1 minute read

This is a sample blog post. Lorem ipsum I can’t remember the rest of lorem ipsum and don’t have an internet connection right now. Testing testing testing this blog post. Blog posts are cool.

Published:

Blog Post number 3

less than 1 minute read

This is a sample blog post. Lorem ipsum I can’t remember the rest of lorem ipsum and don’t have an internet connection right now. Testing testing testing this blog post. Blog posts are cool.

Published:

Blog Post number 2

less than 1 minute read

This is a sample blog post. Lorem ipsum I can’t remember the rest of lorem ipsum and don’t have an internet connection right now. Testing testing testing this blog post. Blog posts are cool.

Published:

Blog Post number 1

less than 1 minute read

This is a sample blog post. Lorem ipsum I can’t remember the rest of lorem ipsum and don’t have an internet connection right now. Testing testing testing this blog post. Blog posts are cool.

Published:

bookchapters

collaborations

Musa Mhlanga

Note: Musa was the PhD supervisor of Ricardo Henriques.

contact

gallery

mainoutreach

maintech

news

outreach

preprints

projects

Structure of Biomimetic Materials

A large number of natural and synthetic hydrogels are currently used for tissue engineering and regenerative medicine. Over the last decade, there has been an increasing awareness of the role of material properties of the substrates in guiding cellular behaviour. This has inspired chemists to create a new generation of materials with mechanical properties closed to that of natural occurring biopolymer networks. Recently, the groups of Prof. Alan Rowan (Queens University, Australia) and Prof. Paul Kouwer (Radboud University of Nijmegen, The Netherlands) were able to develop a fully synthetic material that mimics in all aspects the gels prepared from cellular filaments. These synthetics gels are prepared from polyisocyanopeptides (PICs) grafted with oligo(ethylene glycol) chains and share structural features of biopolymers: their helical structure renders the polymer molecules relatively stiff while the interaction between the side chains enable the formation of bundles or fibrils of defined dimensions. The triethylene glycol side chains attached to the polymer backbone render the material thermo-responsive (it will gel upon heating beyond 20 °C and become liquid again upon cooling). Despite being characterized extensively in bulk, the fundamental dynamics and the relation between the macroscopic properties and the microscopic structure at cellular length scales of PIC-based hydrogels remains obscure.

Structure of the PIC polymer/monomer unit and cartoon of the polymer structure showing the helical structure.

Classically, structural characterization of materials is performed with electron microscopy or scanning probe microscopy. Despite the high spatial resolution achievable with these techniques, they are unable to measure dynamics ‘in situ’ and sample preparation can be a laborious process. In contrast, optical microscopy has the potential to unravel the dynamics in complex heterogeneous systems but has been limited to a spatial resolution of ca. 200 nm. In the past 10 years fluorescence imaging has been revolutionized by the successful development of sub-diffraction (super-resolution) microscopy modalities which can achieve resolutions down to tens of nanometers (see Molecular Organization at the Nanoscale).The various possibilities of fluorescence microscopy to probe dynamics and heterogeneities, with molecular resolution, for a wide range of time scales makes it an ideal tool to address many topics of polymer science. In this project we are using STED to image the polymer network at the nanometer scale.

STED image of PIC network.


For more information on PIC-based hydrogels:

  • Kouwer P.H.J., et al. (2013) Responsive biomimetic networks from polyisocyanopeptide hydrogels, Nature, 493, pages 651–655 (article can be found here)
  • Jasper M., et al. (2014) Ultra-responsive soft matter from strain-stiffening hydrogels, Nature Communications, 5, 5808 (article can be found here)
  • Jasper M., et al. (2016) Bundle Formation in Biomimetic Hydrogels, Macromolecules, 17(8), pages 2642–2649 (article can be found here)

Polymer reptation in 3D

Our current theoretical understanding of entangled polymer chain dynamics is based on the reptation model. First proposed by Doi and Edwards, and further expanded by de Gennes, the reptation model assumes that a polymer chain is confined by the surrounding matrix and is therefore forced to move inside an imaginary tube defined by the transient network of entangled neighboring chains. Intuitively this motion resembles that of a snake or worm. The reptation model predicts five dynamical regimes for segment diffusion, summarized in the figure below. These regimes are as follows: (0) sub-segmental processes (“glassy dynamics”) at very short times (microseconds), (I) small motion subject only to chain connectivity, (II) “local reptation”: short-distance motion within the constraints imposed by the surrounding chains (“tube”), (III) “reptation”: diffusive motion along the curvilinear tube over distances larger than the polymer size, and (IV) free diffusion.

Rheology at the micrometer scale

Due to the crucial role of physical cues in regulating cell behaviour, the mechanical properties of hydrogels are a key design parameter in tissue engineering applications. The shear elastic properties of viscoelastic materials are commonly measured by mechanical rheometers. Storage and loss moduli of a material can be measured by application of strain while measuring stress or vice versa. In contrast, recently developed optical micro-rheology techniques use nanometer- or micrometer-sized particles embedded in the material to obtain the viscoelastic response parameters. Thermal or passive micro-rheology for viscoelastic materials is based on an extension of the concepts of Brownian motion of particles in simple liquids. The movement of the embedded particles can be monitored using particle tracking. Initially developed to investigate the rheological properties of uniform complex fluids, particle tracking micro-rheology (PTM) is becoming a popular technique to analyze polymer blends and gels, as well as the deformability and elasticity within cells. However, if the beads locally modify the structure of the gel or are contained in a pore in an inhomogeneous matrix, the bulk rheological properties will not be retrieved. A solution is to use the cross-correlated thermal fluctuation of pairs of tracer particles, ‘two-point micro-rheology’. This method provides a better agreement between micro and macro-rheology, even in complex micro-structured fluids. However, technical constrains limit the wide application of this technique. One of the major limitations of two-point micro-rheology is the reduced number of trajectories that can be used for analysis. During particle tracking micro-rheology, the length of the calculated trajectories is limited by the time spent by the tracers in the field of view (x,y) and depth of focus (z). Consequently, mechanical characterization of complex polymer matrixes at the micrometer scale would benefit greatly of a new method for (fast) tracking in 3D. We are developing a new method for fast tracking of (fluorescent) beads in 3D using a multi-plane wide field microscope. This will allow a better mechanical characterization of soft materials, at the microscale.

Cellular adhesion in 3D matrices

Cells sense physical forces and the mechanical properties of the microenvironment via several distinct mechanisms and cellular components. The first step of cellular adhesion to the ECM occurs via transmembrane heterodimers of the integrin family. Once integrin molecules adhere to the ECM, they are activated and form clusters. As the number of bound molecules increases, some of the focal complexes evolve from small (0.5-1µm in diameter) transient ‘dot-like’ contacts to elongated structures (3-10µm) which couple with actin and associated proteins. The mechanical coupling between the ECM and the cell cytoskeleton is controlled by the dynamics of the focal adhesion complexes (assembly, disassembly and turnover).

Protein-Protein Interactions

Protein-protein interactions (PPIs) are intrinsic to all cellular processes, driving both metabolic and regulatory pathways. Despite the numerous techniques available, detection of transient short-lived PPIs remains challenging4. The main fluorescence microscopic techniques developed for visualizing PPIs in a cellular context are based on Föster resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC)5. Both techniques detect the interaction between a pair of labeled molecules. Although highly informative, they require fine positioning of the labels and in the majority of the applications the spatial resolution achieved is limited by the diffraction of light to about 200 nm. More information concerning the use of FRET to detect PPIs can be found at Cellular Signalling).

We have use a single molecule localization based super-resolution technique to detect and map PPIs at the cell membrane. This new variant of PAINT that enables mapping of short-lived transient interactions between cytosolic and membrane-bound proteins inside living mammalian cells, at the nanometer scale. In this method the protein of interest is labeled with a light-controllable fluorescent protein and imaged under TIRF illumination, which leads to the selective activation and subsequent detection of molecules in close proximity with the plasma membrane. Interacting molecules are discriminated using a stringent fitting of the fluorescence signal recorded for every single molecule.

Organoids: models for cell communication

Nowadays, human organoids are becoming a highly promising tool to model organ development, function and especially human diseases in vitro. In general, organoids are miniature, simplified organs that can easily propagate in vitro originating from one or a few cells, typically stem cells.

Single cell manipulation by endoscopy

Nanowire-based endoscopy has attracted interest due to its ability to manipulate cells at the single-cell level with minimal cellular perturbation. High-density, vertically aligned nanowire arrays have been used as an efficient gene delivery system. Despite the high transfection rates, culturing the cells on nanowire arrays might have other influences on the cellular behaviour. For example, stem cells cultured on silicon nanowires show significantly different adhesion, proliferation and differentiation, compared with flat silicon or other control substrates. Furthermore, such arrays are not location-specific and require optimization of the nanowire density and dimension for the different the cell types. In collaboration with the group of Prof. Hiroshi Uji-i we are developing a method to delivery genetic material using a single nanowire. In contrast to the existing methods, this approach can be applied to any cell type and is extremely specific: it can target a single cell and it can deliver the genetic material exactly at the desired position, such as inside of the nucleus, with no damage to the cell. Since gene editing is a stochastic event occurring in only a fraction of the cells, the transfer of genetic material (or proteins) is of crucial importance in genome editing methods, where the nucleases must be efficiently delivered. The duration and magnitude of the nuclease expression are critical parameters for the level of both on-target and off-target nuclease activity. Additionally, the dose of donor template DNA is important to ensure efficient homologous recombination. The proposed method offers the possibility to deliver different molecules at different times, in synchronization with the cell cycle. The lab of Prof. Uji-i is one of the first (and few) groups worldwide to have developed and optimized a novel nanoscopic technique using 1D nanowires, with a diameter of less than 100 nm, for SERS endoscopic studies. It has been already proven by us that the thin diameter and 1D structure of the NW greatly reduces the damage induced to a live cell during probe insertion. Although designed for a different purpose, this nanoprobe is ideal as a starting point to develop a new NW-based gene delivery system.

Principle of nanowire-based gene delivery system.

New Drug delivery systems

In this decade, the pharmacology field has been intensively exploring different approaches to deliver multiple drugs with a single drug nano-carrier, such as liposomes, polymer nanoparticles, and inorganic nanoparticles. The advantage of nanoparticle based drug delivery is the ability to unify pharmacokinetics by simultaneous delivery of multiple drugs to specific target cells.

Ever since first reported in 2001, mesoporous silica nanoparticles (MSNPs) have manifested themselves as highly potential candidates for targeted drug delivery. They owe their popularity to their high drug load capacity, chemical stability, biocompatibility and easy functionalization. Since the diameter of the nanoparticles (100 to 200 nm) is tunable, one can obtain a size suitable for passive targeting through the hyperpermeable tumor vasculature, thereby promoting accumulation of the nanoparticles in tumor tissue due to the enhanced permeability and retention effect (EPR). Additionally, functionalization of the nanoparticles with ligands which have a high affinity for tumor cell specific surface receptors promotes more specific internalization in cancer cells. For example, hyaluronic acid (HA) has been extensively used as a targeting ligand due to its affinity for CD44, a transmembrane glycoprotein receptor that plays a critical role in malignant cell activities and, most importantly, it is overexpressed in many solid tumor cells, in metastasis and cancer stem cells.

Correlative AFM and Fluorescence Microscopy

Biological processes are often carried out in the context of macromolecular assemblies. In addition, arrangements of these complexes can be dynamic, resulting in a heterogeneous ensemble. Single molecule techniques can resolve distinct populations in heterogeneous systems, in contrast to bulk experiments where heterogeneity is averaged out. In turn, mechanistic details of bio-macromolecular interactions can be uncovered. Atomic force microscopy (AFM) is a technique that can generate 3D reconstructions of individual biomolecules and complexes thereof in a label-free fashion, and with ~ nm resolution. To this end a very sharp tip, mounted on a flexible cantilever, scans a sample surface in a raster pattern using a piezo-scanner, while keeping the interaction force between sample and tip constant. In every pixel (x,y) of the scanned area, the z-position is recorded. Consequently, a 3D representation of the surface topography can be reconstructed. An alternative way to study single molecules is by fluorescence microscopy. The molecule of interest is labeled with a fluorescent tag providing high contrast. Emission of the tag after excitation, is detected through an optical system. Due to the wave character of light, the emitted light is spread out on the detector described by the point spread function (PSF) of the optical system. This effect limits the resolution achieved with optical microscopy, referred to as the diffraction limit. However, when the signal of a single molecule is detected, the position of this molecule can be determined by fitting of the recorded fluorescence signal with a mathematical approximation of the PSF such as a two-dimensional Gaussian function. This principle underlies single molecule localization microscopy (SMLM). AFM and SMLM are highly complementary technologies: AFM can provide insight in topographic features at a nanometer resolution while SMLM is sensitive towards specifically labelled molecules in complex samples. Integrated setups combining both technologies can therefore provide orthogonal information at the single-molecule level.

Cell signalling: probes and methods

Cell signaling involves the sensing of an extracellular signal by a cell surface receptor, which then transduces this signal to an intracellular response. Despite the numerous studies performed on signaling pathways and mechanisms, little is known about the initial steps occurring at the plasma membrane: receptor pre-assembly at the molecular level and potential reorganization after ligand activation. Traditionally crystallography is used to investigate receptor multimerization. However, the crystallized state might not represent the biochemically active form due to the harsh preparation conditions and the absence of the cellular environment. Other approaches include macroscopic biochemical or biophysical methods, such as chemical cross-linking, ion-channel gating, immunoprecipitation or binding assays. Nowadays, established fluorescence imaging and spectroscopic techniques offer a versatile toolbox to study membrane receptor organization in (living) cells.

In the lab we are using fluorescence fluctuation spectroscopy to quantify physicochemical processes (mobility, binding affinity, stoichiometry, absolute concentration) occurring on a micro-to-millisecond time scale. Fluorescence experiments down to picoseconds are also commonly possible with methods such as time-correlated single photon counting (TCSPC), that allow, e.g., measuring fluorescence lifetimes and molecular tumbling. Additionally, spatially resolved microscopy with high temporal resolution also has clear benefits. For example, combined with confocal laser scanning microscopy (LSM), TCSPC allows protein-protein interactions (PPIs) to be imaged via Förster resonance energy transfer (FRET) based fluorescence lifetime imaging microscopy (FLIM). Imaging based FCS methods such as raster (RICS), number and brightness analysis (N&B) or (spatio-) temporal image correlation spectroscopy [(S)TICS] combine the quantitative analytical power of fluctuation methods with spatial information to map, among many other things, mobility and stoichiometry inside living systems. Simultaneous dual-color fluorescence imaging is possible when fast alternating excitation (alias pulsed interleaved excitation, PIE) is employed. PIE renders analysis of dual-color point FCS experiments considerably more straightforward. The combination of PIE with fluctuation imaging (PIE-FI) allows extracting the maximum amount of molecular information (mobility, stoichiometry, interactions…) from each species present in dual-color LSM images.

PIE (a), PIE-FI (b) and subsequent analyses, based on spatial/temporal auto-/cross-correlation or fluorescence lifetimes, which allow to extract the maximum amount of information of the molecules present in the imaged structure.

For more information on these methods:

  • Hendrix J., Lamb D.C. (2014) Implementation and Application of Pulsed Interleaved Excitation for Dual-Color FCS and RICS. In: Engelborghs Y., Visser A. (eds) Fluorescence Spectroscopy and Microscopy. Methods in Molecular Biology (Methods and Protocols), vol 1076. Humana Press, Totowa, NJ (chapter can be found here)
  • Hendrix J., Schrimpf W., Höller M., Lamb D.C. (2013) Pulsed Interleaved Excitation Fluctuation Imaging, Biophysical Journal, 105(4), 848-861 (article can be found here)

Imaging single HIV virions

Viruses are simple agents exhibiting complex reproductive mechanisms. Decades of research have provided crucial basic insights, antiviral medication and moderately successful gene therapy trials. The most infectious viral particle is, however, not always the most abundant one in a population, questioning the utility of classic ensemble-averaging virology. Indeed, viral replication is often not particularly efficient, prone to errors or containing parallel routes. In collaboration with Prof. Zeger Debeyser (KU Leuven) and Prof Hendrix (UHasselt) we have applied different single-molecule sensitive fluorescence methods to investigate viruses, one-by-one. While this collaboration is still ongoing, there is already several publications that show-case the potential of imaging single virions.

publications

Regulated vesicle fusion generates signaling nanoterritories that control T cell activation at the immunological synapse

Abstract

How the vesicular traffic of signaling molecules contributes to T cell receptor (TCR) signal transduction at the immunological synapse remains poorly understood. In this study, we show that the protein tyrosine kinase Lck, the TCRζ subunit, and the adapter LAT traffic through distinct exocytic compartments, which are released at the immunological synapse in a differentially regulated manner. Lck vesicular release depends on MAL protein. Synaptic Lck, in turn, conditions the calcium- and synaptotagmin-7–dependent fusion of LAT and TCRζ containing vesicles. Fusion of vesicles containing TCRζ and LAT at the synaptic membrane determines not only the nanoscale organization of phosphorylated TCRζ, ZAP70, LAT, and SLP76 clusters but also the presence of phosphorylated LAT and SLP76 in interacting signaling nanoterritories. This mechanism is required for priming IL-2 and IFN-γ production and may contribute to fine-tuning T cell activation breadth in response to different stimulatory conditions

Published in Journal of Experimental Medicine, 2013

Download here

TNF and IL-1 exhibit distinct ubiquitin requirements for inducing NEMO–IKK supramolecular structures

Abstract

Nuclear factor κB (NF-κB) essential modulator (NEMO), a regulatory component of the IκB kinase (IKK) complex, controls NF-κB activation through its interaction with ubiquitin chains. We show here that stimulation with interleukin-1 (IL-1) and TNF induces a rapid and transient recruitment of NEMO into punctate structures that are anchored at the cell periphery. These structures are enriched in activated IKK kinases and ubiquitinated NEMO molecules, which suggests that they serve as organizing centers for the activation of NF-κB. These NEMO-containing structures colocalize with activated TNF receptors but not with activated IL-1 receptors. We investigated the involvement of nondegradative ubiquitination in the formation of these structures, using cells deficient in K63 ubiquitin chains or linear ubiquitin chain assembly complex (LUBAC)-mediated linear ubiquitination. Our results indicate that, unlike TNF, IL-1 requires K63-linked and linear ubiquitin chains to recruit NEMO into higher-order complexes. Thus, different mechanisms are involved in the recruitment of NEMO into supramolecular complexes, which appear to be essential for NF-κB activation.

Published in Journal of Cell Biology, 2014

Download here

High-content 3D multicolor super-resolution localization microscopy

Abstract

Super-resolution (SR) methodologies permit the visualization of cellular structures at near-molecular scale (1–30 nm), enabling novel mechanistic analysis of key events in cell biology not resolvable by conventional fluorescence imaging (∼300-nm resolution). When this level of detail is combined with computing power and fast and reliable analysis software, high-content screenings using SR becomes a practical option to address multiple biological questions. The importance of combining these powerful analytical techniques cannot be ignored, as they can address phenotypic changes on the molecular scale and in a statistically robust manner. In this work, we suggest an easy-to-implement protocol that can be applied to set up a high-content 3D SR experiment with user-friendly and freely available software. The protocol can be divided into two main parts: chamber and sample preparation, where a protocol to set up a direct STORM (dSTORM) sample is presented; and a second part where a protocol for image acquisition and analysis is described. We intend to take the reader step-by-step through the experimental process highlighting possible experimental bottlenecks and possible improvements based on recent developments in the field.

Published in Methods in cell biology, 2015

Download here

PALM and STORM: Into large fields and high-throughput microscopy with sCMOS detectors

Abstract

Single Molecule Localization Microscopy (SMLM) techniques such as Photo-Activation Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) enable fluorescence microscopy super-resolution: the overcoming of the resolution barrier imposed by the diffraction of light. These techniques are based on acquiring hundreds or thousands of images of single molecules, locating them and reconstructing a higher-resolution image from the high-precision localizations. These methods generally imply a considerable trade-off between imaging speed and resolution, limiting their applicability to high-throughput workflows. Recent advancements in scientific Complementary Metal-Oxide Semiconductor (sCMOS) camera sensors and localization algorithms reduce the temporal requirements for SMLM, pushing it toward high-throughput microscopy. Here we outline the decisions researchers face when considering how to adapt hardware on a new system for sCMOS sensors with high-throughput in mind.

Published in Methods, 2015

Download here

Infection counter: automated quantification of in vitro virus replication by fluorescence microscopy

Abstract

The ability to accurately and reliably quantify viral infection is essential to basic and translational virology research. Here, we describe a simple and robust automated method for using fluorescence microscopy to estimate the proportion of virally infected cells in a monolayer. We provide details of the automated analysis workflow along with a freely available open-source ImageJ plugin, Infection Counter, for performing image quantification. Using hepatitis C virus (HCV) as an example, we have experimentally verified our method, demonstrating that it is equivalent, if not better, than the established focus-forming assay. Finally, we used Infection Counter to assess the anti-HCV activity of SMBz-CsA, a non-immunosuppressive cyclosporine analogue.

Published in Viruses, 2016

Download here

Mitochondria mediate septin cage assembly to promote autophagy of Shigella

Abstract

Septins, cytoskeletal proteins with well‐characterised roles in cytokinesis, form cage‐like structures around cytosolic Shigella flexneri and promote their targeting to autophagosomes. However, the processes underlying septin cage assembly, and whether they influence S. flexneri proliferation, remain to be established. Using single‐cell analysis, we show that the septin cages inhibit S. flexneri proliferation. To study mechanisms of septin cage assembly, we used proteomics and found mitochondrial proteins associate with septins in S. flexneri‐infected cells. Strikingly, mitochondria associated with S. flexneri promote septin assembly into cages that entrap bacteria for autophagy. We demonstrate that the cytosolic GTPase dynamin‐related protein 1 (Drp1) interacts with septins to enhance mitochondrial fission. To avoid autophagy, actin‐polymerising Shigella fragment mitochondria to escape from septin caging. Our results demonstrate a role for mitochondria in anti‐Shigella autophagy and uncover a fundamental link between septin assembly and mitochondria.

Published in EMBO reports, 2016

Download here

VirusMapper: open-source nanoscale mapping of viral architecture through super-resolution microscopy

Abstract

The nanoscale molecular assembly of mammalian viruses during their infectious life cycle remains poorly understood. Their small dimensions, generally bellow the 300nm diffraction limit of light microscopes, has limited most imaging studies to electron microscopy. The recent development of super-resolution (SR) light microscopy now allows the visualisation of viral structures at resolutions of tens of nanometers. In addition, these techniques provide the added benefit of molecular specific labelling and the capacity to investigate viral structural dynamics using live-cell microscopy. However, there is a lack of robust analytical tools that allow for precise mapping of viral structure within the setting of infection. Here we present an open-source analytical framework that combines super-resolution imaging and naïve single-particle analysis to generate unbiased molecular models. This tool, VirusMapper, is a high-throughput, user-friendly, ImageJ-based software package allowing for automatic statistical mapping of conserved multi-molecular structures, such as viral substructures or intact viruses. We demonstrate the usability of VirusMapper by applying it to SIM and STED images of vaccinia virus in isolation and when engaged with host cells. VirusMapper allows for the generation of accurate, high-content, molecular specific virion models and detection of nanoscale changes in viral architecture.

Published in Scientific reports, 2016

Download here

Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations

Abstract

Despite significant progress, high-speed live-cell super-resolution studies remain limited to specialized optical setups, generally requiring intense phototoxic illumination. Here, we describe a new analytical approach, super-resolution radial fluctuations (SRRF), provided as a fast graphics processing unit-enabled ImageJ plugin. In the most challenging data sets for super-resolution, such as those obtained in low-illumination live-cell imaging with GFP, we show that SRRF is generally capable of achieving resolutions better than 150 nm. Meanwhile, for data sets similar to those obtained in PALM or STORM imaging, SRRF achieves resolutions approaching those of standard single-molecule localization analysis. The broad applicability of SRRF and its performance at low signal-to-noise ratios allows super-resolution using modern widefield, confocal or TIRF microscopes with illumination orders of magnitude lower than methods such as PALM, STORM or STED. We demonstrate this by super-resolution live-cell imaging over timescales ranging from minutes to hours.

Published in Nature communications, 2016

Download here

Super-resolution microscopy reveals a preformed NEMO lattice structure that is collapsed in incontinentia pigmenti

Abstract

The NF-κB pathway has critical roles in cancer, immunity and inflammatory responses. Understanding the mechanism(s) by which mutations in genes involved in the pathway cause disease has provided valuable insight into its regulation, yet many aspects remain unexplained. Several lines of evidence have led to the hypothesis that the regulatory/sensor protein NEMO acts as a biological binary switch. This hypothesis depends on the formation of a higher-order structure, which has yet to be identified using traditional molecular techniques. Here we use super-resolution microscopy to reveal the existence of higher-order NEMO lattice structures dependent on the presence of polyubiquitin chains before NF-κB activation. Such structures may permit proximity-based trans-autophosphorylation, leading to cooperative activation of the signalling cascade. We further show that NF-κB activation results in modification of these structures. Finally, we demonstrate that these structures are abrogated in cells derived from incontinentia pigmenti patients.

Published in Nature communications, 2016

Download here

HIV-1 Nef impairs the formation of calcium membrane territories controlling the signaling nanoarchitecture at the immunological synapse

Abstract

The ability of HIV-1 to replicate and to establish long-term reservoirs is strongly influenced by T cell activation. Through the use of membrane-tethered, genetically encoded calcium (Ca2+) indicators, we were able to detect for the first time, to our knowledge, the formation of Ca2+ territories and determine their role in coordinating the functional signaling nanostructure of the synaptic membrane. Consequently, we report a previously unknown immune subversion mechanism involving HIV-1 exploitation, through its Nef accessory protein, of the interconnectivity among three evolutionarily conserved cellular processes: vesicle traffic, signaling compartmentalization, and the second messenger Ca2+. We found that HIV-1 Nef specifically associates with the traffic regulators MAL and Rab11b compelling the vesicular accumulation of Lck. Through its association with MAL and Rab11b, Nef co-opts Lck switchlike function driving the formation Ca2+ membrane territories, which, in turn, control the fusion of LAT-transporting Rab27 and Rab37 vesicles and the formation of LAT nanoclusters at the immunological synapse. Consequently, HIV-1 Nef disengages TCR triggering from the generation of p-LAT and p-SLP nanoclusters driving TCR signal amplification and diversification. Altogether our results indicate that HIV-1 exploits the interconnectivity among vesicle traffic, Ca2+ membrane territories, and signaling nanoclusters to modulate T cell signaling and function.

Published in The Journal of Immunology, 2016

Download here

K63-linked ubiquitination targets Toxoplasma gondii for endo-lysosomal destruction in IFNγ-stimulated human cells

Abstract

Toxoplasma gondii is the most common protozoan parasitic infection in man. Gamma interferon (IFNγ) activates haematopoietic and non-haematopoietic cells to kill the parasite and mediate host resistance. IFNγ-driven host resistance pathways and parasitic virulence factors are well described in mice, but a detailed understanding of pathways that kill Toxoplasma in human cells is lacking. Here we show, that contrary to the widely held belief that the Toxoplasma vacuole is non-fusogenic, in an immune-stimulated environment, the vacuole of theme II Toxoplasma in human cells is able to fuse with the host endo-lysosomal machinery leading to parasite death by acidification. Similar to murine cells, we find that theme II, but not theme I Toxoplasma vacuoles are targeted by K63-linked ubiquitin in an IFNγ-dependent manner in non-haematopoetic primary-like human endothelial cells. Host defence proteins p62 and NDP52 are subsequently recruited to the theme II vacuole in distinct, overlapping microdomains with a loss of IFNγ-dependent restriction in p62 knocked down cells. Autophagy proteins Atg16L1, GABARAP and LC3B are recruited to <10% of parasite vacuoles and show no parasite strain preference, which is consistent with inhibition and enhancement of autophagy showing no effect on parasite replication. We demonstrate that this differs from HeLa human epithelial cells, where theme II Toxoplasma are restricted by non-canonical autophagy leading to growth stunting that is independent of lysosomal acidification. In contrast to mouse cells, human vacuoles do not break. In HUVEC, the ubiquitinated vacuoles are targeted for destruction in acidified LAMP1-positive endo-lysosomal compartments. Consequently, parasite death can be prevented by inhibiting host ubiquitination and endosomal acidification. Thus, K63-linked ubiquitin recognition leading to vacuolar endo-lysosomal fusion and acidification is an important, novel virulence-driven Toxoplasma human host defence pathway.

Published in PLoS pathogens, 2016

Download here

Open-source single-particle analysis for super-resolution microscopy with VirusMapper

Abstract

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm — VirusMapper — packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

Published in JoVE (Journal of Visualized Experiments), 2017

Download here

Standard and super-resolution bioimaging data analysis: a primer

Abstract

Super-resolution microscopy depends on steps that can contribute to the formation of image artifacts, leading to misinterpretation of biological information. We present NanoJ-SQUIRREL, an ImageJ-based analytical approach that provides quantitative assessment of super-resolution image quality. By comparing diffraction-limited images and super-resolution equivalents of the same acquisition volume, this approach generates a quantitative map of super-resolution defects and can guide researchers in optimizing imaging parameters.

Published in John Wiley & Sons, 2017

Download here

Heterogeneous localisation of membrane proteins in Staphylococcus aureus

Abstract

Super-resolution microscopy depends on steps that can contribute to the formation of image artifacts, leading to misinterpretation of biological information. We present NanoJ-SQUIRREL, an ImageJ-based analytical approach that provides quantitative assessment of super-resolution image quality. By comparing diffraction-limited images and super-resolution equivalents of the same acquisition volume, this approach generates a quantitative map of super-resolution defects and can guide researchers in optimizing imaging parameters.

Published in Scientific reports, 2018

Download here

Quantitative mapping and minimization of super-resolution optical imaging artifacts

Abstract

Super-resolution microscopy depends on steps that can contribute to the formation of image artifacts, leading to misinterpretation of biological information. We present NanoJ-SQUIRREL, an ImageJ-based analytical approach that provides quantitative assessment of super-resolution image quality. By comparing diffraction-limited images and super-resolution equivalents of the same acquisition volume, this approach generates a quantitative map of super-resolution defects and can guide researchers in optimizing imaging parameters.

Published in Nature methods, 2018

Download here

The role of mitotic cell-substrate adhesion re-modeling in animal cell division

Abstract

Animal cells undergo a dramatic series of shape changes as they divide, which depend on re-modeling of cell-substrate adhesions. Here, we show that while focal adhesion complexes are disassembled during mitotic rounding, integrins remain in place. These integrin-rich contacts connect mitotic cells to the underlying substrate throughout mitosis, guide polarized cell migration following mitotic exit, and are functionally important, since adherent cells undergo division failure when removed from the substrate. Further, the ability of cells to re-spread along pre-existing adhesive contacts is essential for division in cells compromised in their ability to construct a RhoGEF-dependent (Ect2) actomyosin ring. As a result, following Ect2 depletion, cells fail to divide on small adhesive islands but successfully divide on larger patterns, as the connection between daughter cells narrows and severs as they migrate away from one another. In this way, regulated re-modeling of cell-substrate adhesions during mitotic rounding aids division in animal cells.

Published in Developmental cell, 2018

Download here

SRRF: Universal live-cell super-resolution microscopy

Abstract

Super-resolution microscopy techniques break the diffraction limit of conventional optical microscopy to achieve resolutions approaching tens of nanometres. The major advantage of such techniques is that they provide resolutions close to those obtainable with electron microscopy while maintaining the benefits of light microscopy such as a wide palette of high specificity molecular labels, straightforward sample preparation and live-cell compatibility. Despite this, the application of super-resolution microscopy to dynamic, living samples has thus far been limited and often requires specialised, complex hardware. Here we demonstrate how a novel analytical approach, Super-Resolution Radial Fluctuations (SRRF), is able to make live-cell super-resolution microscopy accessible to a wider range of researchers. We show its applicability to live samples expressing GFP using commercial confocal as well as laser- and LED-based widefield microscopes, with the latter achieving long-term timelapse imaging with minimal photobleaching.

Published in The international journal of biochemistry & cell biology, 2018

Download here

Nanoscale colocalization of NK cell activating and inhibitory receptors controls signal integration

Abstract

NK cell responses depend on the balance of signals from inhibitory and activating receptors. However, how the integration of antagonistic signals occurs upon NK cell-target cell interaction is not fully understood. Here, we provide evidence that NK cell inhibition via the inhibitory receptor Ly49A is dependent on its relative colocalization at nanometer-scale with the activating receptor NKG2D upon immune synapse (IS) formation. In the presence of their respective cognate ligands, NKG2D and Ly49A colocalize at a nanometer scale leading to NK cell inhibition. However, increasing the size of the Ly49A ligand reduced the nanoscale colocalization with NKG2D consequently impairing Ly49A-mediated inhibition. Our results suggest the balance of NK cell signals, and NK cell responses, are determined by the relative nanoscale colocalization of activating and inhibitory receptors in the immune synapse.

Published in bioRxiv, 2018

Download here

Content-aware image restoration: pushing the limits of fluorescence microscopy

Abstract

Fluorescence microscopy is a key driver of discoveries in the life sciences, with observable phenomena being limited by the optics of the microscope, the chemistry of the fluorophores, and the maximum photon exposure tolerated by the sample. These limits necessitate trade-offs between imaging speed, spatial resolution, light exposure, and imaging depth. In this work we show how content-aware image restoration based on deep learning extends the range of biological phenomena observable by microscopy. We demonstrate on eight concrete examples how microscopy images can be restored even if 60-fold fewer photons are used during acquisition, how near isotropic resolution can be achieved with up to tenfold under-sampling along the axial direction, and how tubular and granular structures smaller than the diffraction limit can be resolved at 20-times-higher frame rates compared to state-of-the-art methods. All developed image restoration methods are freely available as open source software in Python, FIJI, and KNIME.

Published in Nature methods, 2018

Download here

Septins recognize and entrap dividing bacterial cells for delivery to lysosomes

Abstract

TMEM16F is a Ca2+ -gated ion channel that is required for Ca2+ -activated phosphatidylserine exposure on the surface of many eukaryotic cells. TMEM16F is widely expressed and has roles in platelet activation during blood clotting, bone formation and T cell activation. By combining microscopy and patch clamp recording we demonstrate that activation of TMEM16F by Ca2+ ionophores in Jurkat T cells triggers large-scale surface membrane expansion in parallel with phospholipid scrambling. With continued ionophore application,TMEM16F-expressing cells then undergo extensive shedding of ectosomes. The T cell co-receptor PD-1 is selectively incorporated into ectosomes. This selectivity depends on its transmembrane sequence. Surprisingly, cells lacking TMEM16F not only fail to expand surface membrane in response to elevated cytoplasmic Ca2+, but instead undergo rapid massive endocytosis with PD-1 internalisation. These results establish a new role for TMEM16F as a regulator of Ca2+ activated membrane trafficking.

Published in Cell host & microbe, 2018

Download here

TMEM16F activation by Ca 2+ triggers plasma membrane expansion and directs PD-1 trafficking

Abstract

TMEM16F is a Ca2+ -gated ion channel that is required for Ca2+ -activated phosphatidylserine exposure on the surface of many eukaryotic cells. TMEM16F is widely expressed and has roles in platelet activation during blood clotting, bone formation and T cell activation. By combining microscopy and patch clamp recording we demonstrate that activation of TMEM16F by Ca2+ ionophores in Jurkat T cells triggers large-scale surface membrane expansion in parallel with phospholipid scrambling. With continued ionophore application,TMEM16F-expressing cells then undergo extensive shedding of ectosomes. The T cell co-receptor PD-1 is selectively incorporated into ectosomes. This selectivity depends on its transmembrane sequence. Surprisingly, cells lacking TMEM16F not only fail to expand surface membrane in response to elevated cytoplasmic Ca2+, but instead undergo rapid massive endocytosis with PD-1 internalisation. These results establish a new role for TMEM16F as a regulator of Ca2+ activated membrane trafficking.

Published in Scientific reports, 2019

Download here

NanoJ: a high-performance open-source super-resolution microscopy toolbox

Abstract

Super-resolution microscopy (SRM) has become essential for the study of nanoscale biological processes. This theme of imaging often requires the use of specialised image analysis tools to process a large volume of recorded data and extract quantitative information. In recent years, our team has built an open-source image analysis framework for SRM designed to combine high performance and ease of use. We named it NanoJ—a reference to the popular ImageJ software it was developed for. In this paper, we highlight the current capabilities of NanoJ for several essential processing steps: spatio-temporal alignment of raw data (NanoJ-Core), super-resolution image reconstruction (NanoJ-SRRF), image quality assessment (NanoJ-SQUIRREL), structural modelling (NanoJ-VirusMapper) and control of the sample environment (NanoJ-Fluidics). We expect to expand NanoJ in the future through the development of new tools designed to improve quantitative data analysis and measure the reliability of fluorescent microscopy studies.

Published in Journal of Physics D: Applied Physics, 2019

Download here

Real time multi-modal super-resolution microscopy through Super-Resolution Radial Fluctuations (SRRF-Stream)

Abstract

Super-resolution radial fluctuations (SRRF) is a combination of temporal fluctuation analysis and localization microscopy. One of the key differences between SRRF and other super-resolution methods is its applicability to live-cell dynamics because it functions across a very wide range of fluorophore densities and excitation powers. SRRF is applied to data from imaging modes which include widefield, TIRF and confocal, where short frame bursts (e.g. 50 frames) can be processed to deliver spatial resolution enhancements similar to or better than structured illumination microscopy (SIM). On the other hand, with sparse data e.g. stochastic optical reconstruction microscopy (STORM), SRRF can deliver resolution similar to Gaussian fitting localization methods. Thus, SRRF could provide a route to super-resolution without the need for specialized optical hardware, exotic probes or very high-power densities. We present a fast GPUbased SRRF algorithm termed “SRRF-Stream” and apply it to imagery from an iXon EMCCD coupled to a multi-modal imaging platform, Dragonfly. The new implementation is <300 times faster than the standard CPU version running on an Intel Xeon 3.5GHz 4 core processor, and < 20 times faster than the NanoJ GPU implementation, while also being integrated with acquisition for real time use. In this paper we explore the image resolution and quality with EMCCD and sCMOS cameras and various fluorophores including fluorescent proteins and organic dyes.

Published in SPIE, 2019

Download here

Automating multimodal microscopy with NanoJ-Fluidics

Abstract

Combining and multiplexing microscopy approaches is crucial to understand cellular events, but requires elaborate workflows. Here, we present a robust, open-source approach for treating, labelling and imaging live or fixed cells in automated sequences. NanoJ-Fluidics is based on low-cost Lego hardware controlled by ImageJ-based software, making high-content, multimodal imaging easy to implement on any microscope with high reproducibility. We demonstrate its capacity on event-driven, super-resolved live-to-fixed and multiplexed STORM/DNA-PAINT experiments.

Published in Nature communications, 2019

Download here

Fix your membrane receptor imaging: Actin cytoskeleton and CD4 membrane organization disruption by chemical fixation

Abstract

Single-molecule localization microscopy (SMLM) techniques allow near molecular scale resolution (~ 20 nm) as well as precise and robust analysis of protein organization at different scales. SMLM hardware, analytics and probes have been the focus of a variety of studies and are now commonly used in laboratories across the world. Protocol reliability and artifact identification are increasingly seen as important aspects of super-resolution microscopy. The reliability of these approaches thus requires in-depth evaluation so that biological findings are based on solid foundations. Here we explore how different fixation approaches that disrupt or preserve the actin cytoskeleton affect membrane protein organization. Using CD4 as a model, we show that fixation-mediated disruption of the actin cytoskeleton correlates with changes in CD4 membrane organization. We highlight how these artifacts are easy to overlook and how careful sample preparation is essential for extracting meaningful results from super-resolution microscopy.

Published in Frontiers in immunology, 2019

Download here

Super-resolution fight club: assessment of 2D and 3D single-molecule localization microscopy software

Abstract

With the widespread uptake of two-dimensional (2D) and three-dimensional (3D) single-molecule localization microscopy (SMLM), a large set of different data analysis packages have been developed to generate super-resolution images. In a large community effort, we designed a competition to extensively characterize and rank the performance of 2D and 3D SMLM software packages. We generated realistic simulated datasets for popular imaging modalities—2D, astigmatic 3D, biplane 3D and double-helix 3D—and evaluated 36 participant packages against these data. This provides the first broad assessment of 3D SMLM software and provides a holistic view of how the latest 2D and 3D SMLM packages perform in realistic conditions. This resource allows researchers to identify optimal analytical software for their experiments, allows 3D SMLM software developers to benchmark new software against the current state of the art, and provides insight into the current limits of the field.

Published in Nature methods, 2019

Download here

Nanoscale polarization of the entry fusion complex of vaccinia virus drives efficient fusion

Abstract

To achieve efficient binding and subsequent fusion, most enveloped viruses encode between one and five proteins. For many viruses, the clustering of fusion proteins—and their distribution on virus particles—is crucial for fusion activity. Poxviruses, the most complex mammalian viruses, dedicate 15 proteins to binding and membrane fusion4. However, the spatial organization of these proteins and how this influences fusion activity is unknown. Here, we show that the membrane of vaccinia virus is organized into distinct functional domains that are critical for the efficiency of membrane fusion. Using super-resolution microscopy and single-particle analysis, we found that the fusion machinery of vaccinia virus resides exclusively in clusters at virion tips. Repression of individual components of the fusion complex disrupts fusion-machinery polarization, consistent with the reported loss of fusion activity. Furthermore, we show that displacement of functional fusion complexes from virion tips disrupts the formation of fusion pores and infection kinetics. Our results demonstrate how the protein architecture of poxviruses directly contributes to the efficiency of membrane fusion, and suggest that nanoscale organization may be an intrinsic property of these viruses to assure successful infection.

Published in Nature microbiology, 2019

Download here

Artificial intelligence for microscopy: what you should know

Abstract

Artificial Intelligence based on Deep Learning (DL) is opening new horizons in biomedical research and promises to revolutionize the microscopy field. It is now transitioning from the hands of experts in computer sciences to biomedical researchers. Here, we introduce recent developments in DL applied to microscopy, in a manner accessible to non-experts. We give an overview of its concepts, capabilities and limitations, presenting applications in image segmentation, classification and restoration. We discuss how DL shows an outstanding potential to push the limits of microscopy, enhancing resolution, signal and information content in acquired data. Its pitfalls are discussed, along with the future directions expected in this field.

Published in Biochemical Society Transactions, 2019

Download here

A hidden Markov model approach to characterizing the photo-switching behavior of fluorophores

Abstract

Fluorescing molecules (fluorophores) that stochastically switch between photon-emitting and dark states underpin some of the most celebrated advancements in super-resolution microscopy. While this stochastic behavior has been heavily exploited, full characterization of the underlying models can potentially drive forward further imaging methodologies. Under the assumption that fluorophores move between fluorescing and dark states as continuous time Markov processes, the goal is to use a sequence of images to select a model and estimate the transition rates. We use a hidden Markov model to relate the observed discrete time signal to the hidden continuous time process. With imaging involving several repeat exposures of the fluorophore, we show the observed signal depends on both the current and past states of the hidden process, producing emission probabilities that depend on the transition rate parameters to be estimated. To tackle this unusual coupling of the transition and emission probabilities, we conceive transmission (transition-emission) matrices that capture all dependencies of the model. We provide a scheme of computing these matrices and adapt the forward-backward algorithm to compute a likelihood which is readily optimized to provide rate estimates. When confronted with several model proposals, combining this procedure with the Bayesian Information Criterion provides accurate model selection.

Published in The annals of applied statistics, 2019

Download here

Proteasome-mediated protein degradation resets the cell division cycle and triggers ESCRT-III-mediated cytokinesis in an archaeon

Abstract

The archaeon Sulfolobus acidocaldarius is a relative of eukaryotes known to progress orderly through its cell division cycle despite lacking obvious CDK/cyclin homologues. Here, in exploring the mechanisms underpinning archaeal cell division cycle control, we show that the proteasome of S. acidocaldarius, like its eukaryotic counterpart, regulates the transition from the end of one cell division cycle to the beginning of the next. Further, we identify the archaeal ESCRT-III homologue CdvB as a key target of the proteasome, and show that state-dependent degradation of CdvB triggers archaeal cell division by allowing constriction of a CdvB1:CdvB2 ESCRT-III division ring. These findings suggest an ancient role for proteasome-mediated degradation in resetting the cell division cycle in both archaea and eukaryotes.

Published in bioRxiv, 2019

Download here

Closed mitosis requires local disassembly of the nuclear envelope

Abstract

At the end of mitosis, eukaryotic cells must segregate both copies of their replicated genome into two new nuclear compartments. They do this either by first dismantling and later reassembling the nuclear envelope in a so-called open mitosis, or by reshaping an intact nucleus and then dividing into two in a closed mitosis. However, while mitosis has been studied in a wide variety of eukaryotes for over a century, it is not known how the double membrane of the nuclear envelope is split into two at the end of a closed mitosis without compromising the impermeability of the nuclear compartment. In studying this problem in the fission yeast Schizosaccharomyces pombe, a classical model for closed mitosis, we use genetics, live cell imaging and electron tomography to show that nuclear fission is achieved via local disassembly of the nuclear envelope (NE) within the narrow bridge that links segregating daughter nuclei. In doing so, we identify a novel inner NE-localised protein Les1 that restricts the process of local NE breakdown (local NEB) to the bridge midzone and prevents the leakage of material from daughter nuclei. The mechanics of local NEB in a closed mitosis closely mirror those of NEB in open mitosis, revealing an unexpectedly deep conservation of nuclear remodelling mechanisms across diverse eukaryotes.

Published in bioRxiv, 2019

Download here

Nuclear pores as versatile reference standards for quantitative superresolution microscopy

Abstract

Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions. Here, we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag, HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use (1) as three-dimensional resolution standards for calibration and quality control, (2) to quantify absolute labeling efficiencies and (3) as precise reference standards for molecular counting. These cell lines will enable the broader community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements.

Published in Nature methods, 2019

Download here

vLUME: 3D Virtual Reality for Single-molecule Localization Microscopy

Abstract

Super-Resolution (SR) Microscopy based on 3D Single-Molecule Localization Microscopy (SMLM) is now well established and its wide-spread adoption has led to the development of more than 36 software packages, dedicated to quantitative evaluation of the spatial and temporal detection of fluorophore photoswitching. While the initial emphasis in the 3D SMLM field has clearly been on improving resolution and data quality, there is now a marked absence of 3D visualization approaches that enable the straightforward, high-fidelity exploration of this theme of data. Inspired by the horological phosphorescence points that illuminate watch-faces in the dark, we present vLUME (Visualization of the Universe in a Micro Environment, pronounced 9volume9) a free-for-academic-use immersive virtual reality-based (VR) visualization software package purposefully designed to render large 3D-SMLM data sets. vLUME enables robust visualization, segmentation and quantification of millions of fluorescence puncta from any 3D SMLM technique. vLUME has an intuitive user-interface and is compatible with all commercial VR hardware (Oculus Rift/Quest and HTC Vive). vLUME accelerates the analysis of highly complex 3D point-cloud data and the rapid identification of defects that are otherwise neglected in global quality metrics.

Published in bioRxiv, 2020

Download here

Between life and death: strategies to reduce phototoxicity in super-resolution microscopy

Abstract

Super-Resolution Microscopy enables non-invasive, molecule-specific imaging of the internal structure and dynamics of cells with sub-diffraction limit spatial resolution. One of its major limitations is the requirement for high-intensity illumination, generating considerable cellular phototoxicity. This factor considerably limits the capacity for live-cell observations, particularly for extended periods of time. Here, we overview new developments in hardware, software and probe chemistry aiming to reduce phototoxicity. Additionally, we discuss how the choice of biological model and sample environment impacts the capacity for live-cell observations.

Published in Journal of Physics D: Applied Physics, 2020

Download here

Fluctuation-Based Super-Resolution Traction Force Microscopy

Abstract

Cellular mechanics play a crucial role in tissue morphogenesis and homeostasis and are often misregulated in disease. Traction force microscopy (TFM) is one of the key methods that has enabled researchers to study fundamental aspects of mechanobiology; however, the power of TFM is limited by poor resolution and low throughput. Here, we propose a simplified protocol and imaging strategy, relying on super-resolution microscopy enabled by fluorophore fluctuation analysis, to enhance the output of TFM, by increasing both bead density as well as the accuracy of bead tracking in TFM gels. Our analysis pipeline can be used on either camera-based confocal or widefield microscopes and is fully compatible with available TFM analysis software. In addition, we demonstrate that our workflow can be used to gain biologically relevant information and is suitable for long-term live measurement of traction forces even in light-sensitive cells. Finally, we propose that our strategy could be used to considerably simplify the implementation of TFM screens. Our streamlined protocol can be performed with minimal hardware and software investment, and has the potential to standardize high-resolution TFM.

Published in Nano Letters, 2020

Download here

Super-Beacons: open-source probes with spontaneous tuneable blinking compatible with live-cell super-resolution microscopy

Abstract

Localization based super-resolution microscopy relies on the detection of individual molecules cycling between fluorescent and non-fluorescent states. These transitions are commonly regulated by high-intensity illumination, imposing constrains to imaging hardware and producing sample photodamage. Here, we propose single-molecule self-quenching as a mechanism to generate spontaneous photoswitching independent of illumination. To demonstrate this principle, we developed a new class of DNA-based open-source Super-Resolution probes named Super-Beacons, with photoswitching kinetics that can be tuned structurally, thermally and chemically. The potential of these probes for live-cell friendly Super-Resolution Microscopy without high-illumination or toxic imaging buffers is revealed by imaging Interferon Inducible Transmembrane proteins (IFITMs) at sub-100nm resolutions.

Published in Traffic, 2020

Download here

ZeroCostDL4Mic: an open platform to simplify access and use of Deep-Learning in Microscopy

Abstract

Deep Learning (DL) methods are increasingly recognised as powerful analytical tools for microscopy. Their potential to outperform conventional image processing pipelines is now well established. Despite the enthusiasm and innovations fuelled by DL technology, the need to access powerful and compatible resources, install multiple computational tools and modify code instructions to train neural networks all lead to an accessibility barrier that novice users often find difficult to cross. Here, we present ZeroCostDL4Mic, an entry-level teaching and deployment DL platform which considerably simplifies access and use of DL for microscopy. It is based on Google Colab which provides the free, cloud-based computational resources needed. ZeroCostDL4Mic allows researchers with little or no coding expertise to quickly test, train and use popular DL networks. In parallel, it guides researchers to acquire more knowledge, to experiment with optimising DL parameters and network architectures. We also highlight the limitations and requirements to use Google Colab. Altogether, ZeroCostDL4Mic accelerates the uptake of DL for new users and promotes their capacity to use increasingly complex DL networks.

Published in bioRxiv, 2020

Download here

Live Imaging of a Hyperthermophilic Archaeon Reveals Distinct Roles for Two ESCRT-III Homologs in Ensuring a Robust and Symmetric Division

Abstract

Live-cell imaging has revolutionized our understanding of dynamic cellular processes in bacteria and eukaryotes. While similar techniques have recently been applied to the study of halophilic archaea, our ability to explore the cell biology of thermophilic archaea is limited, due to the technical challenges of imaging at high temperatures. Here, we report the construction of the Sulfoscope, a heated chamber that enables live-cell imaging on an inverted fluorescent microscope. Using this system combined with thermostable fluorescent probes, we were able to image Sulfolobus cells as they divide, revealing a tight coupling between changes in DNA compaction, segregation and cytokinesis. By imaging deletion mutants, we observe important differences in the function of the two ESCRTIII proteins recently implicated in cytokinesis. The loss of CdvB1 compromises cell division, causing occasional division failures and fusion of the two daughter cells, whereas the deletion of cdvB2 leads to a profound loss of division symmetry, generating daughter cells that vary widely in size and eventually generating ghost cells. These data indicate that DNA separation and cytokinesis are coordinated in Sulfolobus, as is the case in eukaryotes, and that two contractile ESCRTIII polymers perform distinct roles to ensure that Sulfolobus cells undergo a robust and symmetrical division. Taken together, the Sulfoscope has shown to provide a controlled high temperature environment, in which cell biology of Sulfolobus can be studied in unprecedent details.

Published in Current Biology, 2020

Download here

The cell biologist’s guide to super-resolution microscopy

Abstract

Fluorescence microscopy has become a ubiquitous method to observe the location of specific molecular components within cells. However, the resolution of light microscopy is limited by the laws of diffraction to a few hundred nanometers, blurring most cellular details. Over the last two decades, several techniques – grouped under the ‘super-resolution microscopy’ moniker – have been designed to bypass this limitation, revealing the cellular organization down to the nanoscale. The number and variety of these techniques have steadily increased, to the point that it has become difficult for cell biologists and seasoned microscopists alike to identify the specific technique best suited to their needs. Available techniques include image processing strategies that generate super-resolved images, optical imaging schemes that overcome the diffraction limit and sample manipulations that expand the size of the biological sample. In this Cell Science at a Glance article and the accompanying poster, we provide key pointers to help users navigate through the various super-resolution methods by briefly summarizing the principles behind each technique, highlighting both critical strengths and weaknesses, as well as providing example images.

Published in Journal of Cell Science, 2020

Download here

FtsZ treadmilling is essential for Z-ring condensation and septal constriction initiation in bacterial cell division

Abstract

Despite the central role of division in bacterial physiology, how division proteins work together as a nanoscale machine to divide the cell remains poorly understood. Cell division by cell wall synthesis proteins is guided by the cytoskeleton protein FtsZ, which assembles at mid-cell as a dense Z-ring formed of motile treadmilling filaments. However, although FtsZ treadmilling is essential for cell division, the function of FtsZ treadmilling motility remains unclear. Here, we systematically resolve the function of FtsZ treadmilling across each stage of division in the Gram-positive model organism Bacillus subtilis using a novel combination of nanofabrication, advanced microscopy, and microfluidics to measure the division-protein dynamics in live cells with ultrahigh sensitivity. We find that FtsZ treadmilling has two essential functions: mediating condensation of diffuse FtsZ filaments into a dense Z-ring, and initiating constriction by guiding septal cell wall synthesis. After constriction initiation, FtsZ treadmilling has a dispensable function in accelerating septal constriction rate. Our results show that FtsZ treadmilling is critical for assembling and initiating the bacterial cell division machine.

Published in bioRxiv, 2020

Download here

research

Cell Division

Team members:

Collaborators:

Buzz Baum

Cell Morphogenesis Lab, University College London, UK

Ewa Paluch

Paluch Lab, Trinity College, University of Cambridge, UK

Gautam Dey

Cell Morphogenesis Lab, University College London, UK

Publications:

Live Imaging of a Hyperthermophilic Archaeon Reveals Distinct Roles for Two ESCRT-III Homologs in Ensuring a Robust and Symmetric Division
Andre Arashiro Pulschen, Delyan R Mutavchiev, *Siân Culley*, Kim Nadine Sebastian, Jacques Roubinet, Marc Roubinet, Gabriel Tarrason Risa, Marleen van Wolferen, Chantal Roubinet, Uwe Schmidt, Gautam Dey, Sonja-Verena Albers, *Ricardo Henriques*, Buzz Baum
Published in Current Biology, June 2020 (see publication or preprint)
Research themes: Cell Division, Microbiology, New Methods, Hardware
Type: Paper
Proteasome-mediated protein degradation resets the cell division cycle and triggers ESCRT-III-mediated cytokinesis in an archaeon
Gabriel Tarrason Risa, Fredrik Hurtig, Sian Bray, Anne E Hafner, Lena Harker-Kirschneck, Peter Faull, Colin Davis, Dimitra Papatziamou, Delyan R Mutavchiev, Catherine Fan, Leticia Meneguello, Andre Arashiro Pulschen, Gautam Dey, *Siân Culley*, Mairi Kilkenny, Luca Pellegrini, Robertus AM de Bruin, *Ricardo Henriques*, Ambrosius P Snijders, Anđela Šarić, Ann-Christin Lindås, Nick Robinson, Buzz Baum
Published in bioRxiv, September 2019 (see preprint)
Research themes: Cell Division
Type: Preprint

Published:

Cell Signalling

Team members:

Collaborators:

Helena Soares

Human immunobiology and Pathogenesis Lab, CEDOC, Nova Medical School, Portugal

Jason Mercer

Viral Cell Biology Lab, University of Birmingham, UK

Mark Marsh

Cellular Mechanisms of Virus Entry and Assembly Lab, University College London, UK

Musa Mhlanga

Gene Expression & Biophysics Lab, University of Cape Town, South Africa

Publications:

Published:

Host-Pathogen Interactions

Team members:

Collaborators:

Eva Frickel

Host-Toxoplasma Interaction Lab, University of Birmingham, UK

Helena Soares

Human immunobiology and Pathogenesis Lab, CEDOC, Nova Medical School, Portugal

Joe Grove

Grove Lab, University College London, UK

Jason Mercer

Viral Cell Biology Lab, University of Birmingham, UK

Mark Marsh

Cellular Mechanisms of Virus Entry and Assembly Lab, University College London, UK

Serge Mostowy

Cellular Microbiology Lab, London School of Hygiene and Tropical Medicine, UK

Publications:

Published:

Microbiology

Team members:

Collaborators:

Buzz Baum

Cell Morphogenesis Lab, University College London, UK

Eva Frickel

Host-Toxoplasma Interaction Lab, University of Birmingham, UK

Gautam Dey

Cell Morphogenesis Lab, University College London, UK

Helena Soares

Human immunobiology and Pathogenesis Lab, CEDOC, Nova Medical School, Portugal

Jason Mercer

Viral Cell Biology Lab, University of Birmingham, UK

Serge Mostowy

Cellular Microbiology Lab, London School of Hygiene and Tropical Medicine, UK

Publications:

Live Imaging of a Hyperthermophilic Archaeon Reveals Distinct Roles for Two ESCRT-III Homologs in Ensuring a Robust and Symmetric Division
Andre Arashiro Pulschen, Delyan R Mutavchiev, *Siân Culley*, Kim Nadine Sebastian, Jacques Roubinet, Marc Roubinet, Gabriel Tarrason Risa, Marleen van Wolferen, Chantal Roubinet, Uwe Schmidt, Gautam Dey, Sonja-Verena Albers, *Ricardo Henriques*, Buzz Baum
Published in Current Biology, June 2020 (see publication or preprint)
Research themes: Cell Division, Microbiology, New Methods, Hardware
Type: Paper

Published:

Structural Biology

Team members:

Collaborators:

Jason Mercer

Viral Cell Biology Lab, University of Birmingham, UK

Lucy Collinson

Electron Microscopy Science Technology Platform, Francis Crick Institute, UK

Musa Mhlanga

Gene Expression & Biophysics Lab, University of Cape Town, South Africa

Publications:

Nuclear pores as versatile reference standards for quantitative superresolution microscopy
Jervis Vermal Thevathasan, Maurice Kahnwald, Konstanty Cieśliński, Philipp Hoess, Sudheer Kumar Peneti, Manuel Reitberger, Daniel Heid, Krishna Chaitanya Kasuba, Sarah Janice Hoerner, Yiming Li, Yu-Le Wu, Markus Mund, Ulf Matti, *Pedro Matos Pereira*, *Ricardo Henriques*, Bianca Nijmeijer, Moritz Kueblbeck, Vilma Jimenez Sabinina, Jan Ellenberg, Jonas Ries
Published in Nature methods, October 2019 (see publication)
Research themes: Structural Biology, New Methods
Type: Paper

Published:

New Methods

Team members:

Collaborators:

Alan Lowe

Artificial Intelligence for Quantitative Biology Lab, University College London, UK

Buzz Baum

Cell Morphogenesis Lab, University College London, UK

Bassam Hajj

Cell Organization Imaging and Control Lab, Institut Curie, France

Ed Cohen

Cohen Group, Imperial College London, UK

Ewa Paluch

Paluch Lab, Trinity College, University of Cambridge, UK

Gautam Dey

Cell Morphogenesis Lab, University College London, UK

Joe Grove

Grove Lab, University College London, UK

Johanna Ivaska

Cell Adhesion and Cancer Adhesion Lab, University of Turku, Finland

Jason Mercer

Viral Cell Biology Lab, University of Birmingham, UK

Lucy Collinson

Electron Microscopy Science Technology Platform, Francis Crick Institute, UK

Mark Marsh

Cellular Mechanisms of Virus Entry and Assembly Lab, University College London, UK

Musa Mhlanga

Gene Expression & Biophysics Lab, University of Cape Town, South Africa

Publications:

Live Imaging of a Hyperthermophilic Archaeon Reveals Distinct Roles for Two ESCRT-III Homologs in Ensuring a Robust and Symmetric Division
Andre Arashiro Pulschen, Delyan R Mutavchiev, *Siân Culley*, Kim Nadine Sebastian, Jacques Roubinet, Marc Roubinet, Gabriel Tarrason Risa, Marleen van Wolferen, Chantal Roubinet, Uwe Schmidt, Gautam Dey, Sonja-Verena Albers, *Ricardo Henriques*, Buzz Baum
Published in Current Biology, June 2020 (see publication or preprint)
Research themes: Cell Division, Microbiology, New Methods, Hardware
Type: Paper
ZeroCostDL4Mic: an open platform to simplify access and use of Deep-Learning in Microscopy
*Lucas von Chamier*, Johanna Jukkala, *Christoph Spahn*, Martina Lerche, Sara Hernández-pérez, Pieta Mattila, Eleni Karinou, Seamus Holden, Ahmet Can Solak, Alexander Krull, Tim-Oliver Buchholz, Florian Jug, Loïc Alain Royer, Mike Heilemann, *Romain F. Laine*, Guillaume Jacquemet, *Ricardo Henriques*
Published in bioRxiv, March 2020 (see preprint)
Research themes: New Methods, Software
Type: Preprint, Corresponding author
Nuclear pores as versatile reference standards for quantitative superresolution microscopy
Jervis Vermal Thevathasan, Maurice Kahnwald, Konstanty Cieśliński, Philipp Hoess, Sudheer Kumar Peneti, Manuel Reitberger, Daniel Heid, Krishna Chaitanya Kasuba, Sarah Janice Hoerner, Yiming Li, Yu-Le Wu, Markus Mund, Ulf Matti, *Pedro Matos Pereira*, *Ricardo Henriques*, Bianca Nijmeijer, Moritz Kueblbeck, Vilma Jimenez Sabinina, Jan Ellenberg, Jonas Ries
Published in Nature methods, October 2019 (see publication)
Research themes: Structural Biology, New Methods
Type: Paper
Super-resolution fight club: assessment of 2D and 3D single-molecule localization microscopy software
Daniel Sage, Thanh-An Pham, Hazen Babcock, Tomas Lukes, Thomas Pengo, Jerry Chao, Ramraj Velmurugan, Alex Herbert, Anurag Agrawal, Silvia Colabrese, Ann Wheeler, Anna Archetti, Bernd Rieger, Raimund Ober, Guy M Hagen, Jean-Baptiste Sibarita, Jonas Ries, *Ricardo Henriques*, Michael Unser, Seamus Holden
Published in Nature methods, May 2019 (see publication)
Research themes: New Methods, Software
Type: Paper
Content-aware image restoration: pushing the limits of fluorescence microscopy
Martin Weigert, Uwe Schmidt, Tobias Boothe, Andreas Müller, Alexandr Dibrov, Akanksha Jain, Benjamin Wilhelm, Deborah Schmidt, Coleman Broaddus, *Siân Culley*, Mauricio Rocha-Martins, Fabián Segovia-Miranda, Caren Norden, *Ricardo Henriques*, Marino Zerial, Michele Solimena, Jochen Rink, Pavel Tomancak, Loic Royer, Florian Jug, Eugene W Myers
Published in Nature methods, December 2018 (see publication)
Research themes: New Methods, Software
Type: Paper

Published:

Software

Team members:

Collaborators:

Alan Lowe

Artificial Intelligence for Quantitative Biology Lab, University College London, UK

Buzz Baum

Cell Morphogenesis Lab, University College London, UK

Bassam Hajj

Cell Organization Imaging and Control Lab, Institut Curie, France

Ed Cohen

Cohen Group, Imperial College London, UK

Joe Grove

Grove Lab, University College London, UK

Johanna Ivaska

Cell Adhesion and Cancer Adhesion Lab, University of Turku, Finland

Jason Mercer

Viral Cell Biology Lab, University of Birmingham, UK

Lucy Collinson

Electron Microscopy Science Technology Platform, Francis Crick Institute, UK

Musa Mhlanga

Gene Expression & Biophysics Lab, University of Cape Town, South Africa

Publications:

ZeroCostDL4Mic: an open platform to simplify access and use of Deep-Learning in Microscopy
*Lucas von Chamier*, Johanna Jukkala, *Christoph Spahn*, Martina Lerche, Sara Hernández-pérez, Pieta Mattila, Eleni Karinou, Seamus Holden, Ahmet Can Solak, Alexander Krull, Tim-Oliver Buchholz, Florian Jug, Loïc Alain Royer, Mike Heilemann, *Romain F. Laine*, Guillaume Jacquemet, *Ricardo Henriques*
Published in bioRxiv, March 2020 (see preprint)
Research themes: New Methods, Software
Type: Preprint, Corresponding author
Super-resolution fight club: assessment of 2D and 3D single-molecule localization microscopy software
Daniel Sage, Thanh-An Pham, Hazen Babcock, Tomas Lukes, Thomas Pengo, Jerry Chao, Ramraj Velmurugan, Alex Herbert, Anurag Agrawal, Silvia Colabrese, Ann Wheeler, Anna Archetti, Bernd Rieger, Raimund Ober, Guy M Hagen, Jean-Baptiste Sibarita, Jonas Ries, *Ricardo Henriques*, Michael Unser, Seamus Holden
Published in Nature methods, May 2019 (see publication)
Research themes: New Methods, Software
Type: Paper
Content-aware image restoration: pushing the limits of fluorescence microscopy
Martin Weigert, Uwe Schmidt, Tobias Boothe, Andreas Müller, Alexandr Dibrov, Akanksha Jain, Benjamin Wilhelm, Deborah Schmidt, Coleman Broaddus, *Siân Culley*, Mauricio Rocha-Martins, Fabián Segovia-Miranda, Caren Norden, *Ricardo Henriques*, Marino Zerial, Michele Solimena, Jochen Rink, Pavel Tomancak, Loic Royer, Florian Jug, Eugene W Myers
Published in Nature methods, December 2018 (see publication)
Research themes: New Methods, Software
Type: Paper

Published:

Hardware

Team members:

Collaborators:

Alan Lowe

Artificial Intelligence for Quantitative Biology Lab, University College London, UK

Buzz Baum

Cell Morphogenesis Lab, University College London, UK

Bassam Hajj

Cell Organization Imaging and Control Lab, Institut Curie, France

Gautam Dey

Cell Morphogenesis Lab, University College London, UK

Publications:

Live Imaging of a Hyperthermophilic Archaeon Reveals Distinct Roles for Two ESCRT-III Homologs in Ensuring a Robust and Symmetric Division
Andre Arashiro Pulschen, Delyan R Mutavchiev, *Siân Culley*, Kim Nadine Sebastian, Jacques Roubinet, Marc Roubinet, Gabriel Tarrason Risa, Marleen van Wolferen, Chantal Roubinet, Uwe Schmidt, Gautam Dey, Sonja-Verena Albers, *Ricardo Henriques*, Buzz Baum
Published in Current Biology, June 2020 (see publication or preprint)
Research themes: Cell Division, Microbiology, New Methods, Hardware
Type: Paper

Published:

Probes

Team members:

Collaborators:

Mark Marsh

Cellular Mechanisms of Virus Entry and Assembly Lab, University College London, UK

Musa Mhlanga

Gene Expression & Biophysics Lab, University of Cape Town, South Africa

Publications:

Published:

resources

NanoJ

Published:

CARE

Published:

vLUME

Published:

talks

teaching

Teaching experience 1

This is a description of a teaching experience. You can use markdown like any other post.

Undergraduate course, University 1, Department, 2014

Teaching experience 2

This is a description of a teaching experience. You can use markdown like any other post.

Workshop, University 1, Department, 2015

team

Pedro Almada

PhD Thesis

Check out Dr. Pedro Almada PhD thesis here.

Research themes with our lab

Resources

Collaborators

Alan Lowe

Artificial Intelligence for Quantitative Biology Lab, University College London, UK

Buzz Baum

Cell Morphogenesis Lab, University College London, UK

Jason Mercer

Viral Cell Biology Lab, University of Birmingham, UK

Publications with our group

Published:

Pedro Matos Pereira

I am a cell biologist with extensive scientific knowledge in microbiology, host-pathogen interaction and advanced microscopy approaches. I have over 10 years experience in scientific project design/management, undergraduate and postgraduate student supervision and writing/communication of scientific information to both expert and non-expert audiences. To attest to this I am lead author in several high profile publications with over 1300 citations (Google Scholar: dQvQ0AAAAJ), have supervised multiple Master and PhD students, and have been actively involved in several outreach initiatives (In2Science, ITQB-NOVA open day). I have served in several decision boards in Universidade Nova de Lisboa, University College London and the Francis Crick Institute where I have worked with academic, industrial and political partners to define scientific and institutional vision and impact strategies. Experience that has provided me with a comprehensive view about the scientific landscape. From a research point of view I have made significant contributions in the fields of S. aureus microbiology (e.g. discovering a link between peptidoglycan and wall teichoic acids biosynthesis), host-pathogen interaction (e.g. importance of autolysins for immune evasion, or the role of septins in the recognition of intracellular pathogens) and hardware and software technological innovations for microscopy (e.g. NanoJ-Fluidics and NanoJ-SRRF).

Published:

Caron Jacobs

PhD Thesis

Check out Dr. Caron Jacobs PhD thesis here.

Research themes with our lab

Collaborators

Joe Grove

Grove Lab, University College London, UK

Jason Mercer

Viral Cell Biology Lab, University of Birmingham, UK

Mark Marsh

Cellular Mechanisms of Virus Entry and Assembly Lab, University College London, UK

Publications with our group (see more on Google Scholar)

Published:

Nils Gustafsson

PhD Thesis

Check out Dr. Nils Gustafsson PhD thesis here.

Research themes with our lab

Resources

Collaborators

Buzz Baum

Cell Morphogenesis Lab, University College London, UK

Ed Cohen

Cohen Group, Imperial College London, UK

Jason Mercer

Viral Cell Biology Lab, University of Birmingham, UK

Mark Marsh

Cellular Mechanisms of Virus Entry and Assembly Lab, University College London, UK

Musa Mhlanga

Gene Expression & Biophysics Lab, University of Cape Town, South Africa

Publications with our group

Published:

Jerzy Samolej

PhD Thesis

Check out Dr. Jerzy Samolej PhD thesis here.

Research themes with our lab

Collaborators

Jason Mercer

Viral Cell Biology Lab, University of Birmingham, UK

Publications with our group

Published:

Robert Gray

PhD Thesis

Check out Dr. Robert Gray PhD thesis here.

Research themes with our lab

Resources

Collaborators

Buzz Baum

Cell Morphogenesis Lab, University College London, UK

Jason Mercer

Viral Cell Biology Lab, University of Birmingham, UK

Publications with our group

Published:

David Albrecht

Note: David jointly did research between the laboratory of Dr. Jason Mercer and my own.

Published:

Neza Vadnjal

The cellular actin cortex offers mechanical support to animal cells. Cell shape changes are consequences of changes in the mechanical properties of the cortex, particularly in cortical tension. The aim of my project is to investigate the molecules controlling actin network architecture and their influence on cortex tension generation.

Published:

Lucas Von Chamier

Research themes with our lab

Collaborators

Publications with our group

Published:

Kalina Tosheva

My project aims to adapt single molecule localisation super-resolution techniques to light-sheet microscopy.

Published:

Yue (Julie) Yuan

Host receptor engagements is one of the first steps during HIV entry. There is still a lack of explicit knowledge on the precise stoichiometry of the virus Env-receptor interaction or of how flexible this might be for a successful fusion event, especially from the perspective of predominant viral target cells (CD4+ T cells). A better understanding of PM receptor distributions, and how this is modulated by the virus, is still required. I am aiming at characterising this dynamic process using Super-Resolution Microscopy on a single-cell level. We have successfully validated a pipeline to quantitatively analyse the topology of membrane receptors, CD4 and CCR5. Our next step is to investigate further what receptor topology makes a fusion-efficient by monitoring viral entry in living CD4+ T cells.

Published:

Romain F. Laine

Overview: I am interested in developing and applying advanced microscopy techniques to decipher functional and structural organisation of life at multiple scales. For this, I develop analytical and optical tools. You can find me on Twitter (@LaineBioImaging), in the optics lab tinkering with microscopes or on my computer developing image analyses.

Published:

Tchyn Lang Laure Ho

Dedicated to interdisciplinary research. Recently developed an interest for AI and Machine Learning, following from previous studies in Neuroscience and Dementia. Current PhD work focuses on exploring the potential of deep neural networks to elucidate cell biology mechanisms given the high complexity of live-cell imaging data.

Published:

Guillermo Herrera Sanchez

Cytotoxic T lymphocytes (CTLs) specialise in killing virus-infected and cancerous cells in our body. They do so by exposing targets cells to pro-apoptotic granzymes, whose entrance into the target is facilitated by the pore-forming protein perforin. Interestingly, while CTLs are exposed to this toxic protein during synapse formation they remain almost invariably unscathed, allowing them to kill multiple target cells in succession. This resistance stems from perforin’s unidirectional action, recently demonstrated to arise from a dynamic control of the CTL membrane lipid composition. Firstly, the CTL membrane inhibits perforin binding via its high plasma membrane order and secondly, exposed phosphatidylserine (PS) sequesters any residual perforin and inactivates it. This project’s first aim is to employ in vivo, in vitro and in silico methods to elucidate the spatio-temporal hierarchy of this two-fold defence mechanism in immunological synapses. Super-resolution (Lattice Light Sheet and Total Internal Reflection) microscopy studies will be conducted to elucidate the spatio-temporal interplay of PS exposure and enhanced membrane lipid order with events leading to synapse formation and target-cell killing. Secondly, a Brownian dynamics model will be developed to study the pathway for perforin nanopore formation. Research into these mechanisms holds unprecedented medical relevance, given how important successive killing by CTLs is to viral and cancer clearance. Finally, a better understanding of the pathway of perforin nanopore assembly could guide future therapeutic studies in locating targets to suppress or promote immune response.

Published:

Christoph Spahn

My past: I studied biology in the beautiful and romantic city Würzburg in Germany. Afterwards, I moved to Frankfurt to do my PhD in Mike Heilemann research group, investigating nucleoid architecture in E. coli cells using single-molecule localization microscopy (SMLM), a super-resolution approach that provides near molecular resolution. After figuring out how to directly label the nucleoid and how to combine different super-resolution methods (which took some time), my work resulted in several imaging approaches that allow for three-color SMLM in individual cells at spatial resolutions of ~ 20 – 40 nm. This is exactly what we require to explore the inner life of bugs at the nanoscale and understand how different processes and stimuli affect bacterial homeostasis and nucleoid organization.
My present: With the developed toolbox, I investigate how E. coli cells replicate and segregate their chromosome and how biological processes such as transcription and translation shape the nucleoid in a dynamic manner. As most of my work was performed in fixed specimen, I am very happy to join the Henriques group as an EMBO short term fellow, to perform live-cell imaging and ultimately provide a dynamic, super-resolved picture of bacterial chromosome replication.
My future: I want to use super-resolution microscopy to gain a deep understanding of how antimicrobial compounds perturb microbial cell homeostasis. This knowledge will become extremely important for fighting antimicrobial resistance and designing new drugs.

Published:

techniques