Sptpalm

The morphology of protein assemblies impacts their behaviour and contributes to beneficial and aberrant cellular responses. While single-molecule localization microscopy provides the required spatial resolution to investigate these assemblies, the lack of universal robust analytical tools to extract and quantify underlying structures limits this powerful technique. Here we present SEMORE, a semi-automatic machine learning framework for universal, system- and input-dependent, analysis of super-resolution data...

Neuronal and hormonal communication relies on the exocytic fusion of vesicles containing neurotransmitters and hormones with the plasma membrane. This process is tightly regulated by key protein-protein and protein-lipid interactions and culminates in the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation and zippering that promotes vesicular fusion. Located on both sides of the vesicle and the plasma membrane, the zippering of the SNARE complex acts to overcome the energy barrier afforded by the repulsive electrostatic force stemming from apposing two negatively charged phospholipid membranes...

Super-resolution microscopy techniques have pushed the limit of optical imaging to unprecedented spatial resolutions. However, one of the frontiers in nanoscopy is its application to intact living organisms. Here we describe the implementation and application of super-resolution single-particle tracking photoactivated localization microscopy (sptPALM) to probe single-molecule dynamics of membrane proteins in live roots of the model plant Arabidopsis thaliana. We first discuss the advantages and limitations of sptPALM for studying the diffusion properties of membrane proteins and compare this to fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS)...

The fusion of synaptic vesicles with the plasma membrane underpins neurotransmission. A number of presynaptic proteins play a critical role in overcoming the energy barrier inherent to the fusion of the negatively charged vesicular and plasma membranes. Emerging concepts suggest that this process is hierarchical and dependent on rapid and transient reorganization of proteins in and out of small nanoclusters located in the active zones of nerve terminals. Examining the nanoscale organization of presynaptic molecules requires super-resolution microscopy to overcome the limits of conventional light microscopy...

γ-Secretase is a multi-subunit enzyme whose aberrant activity is associated with Alzheimer's disease and cancer. While its structure is atomically resolved, γ-secretase localization in the membrane in situ relies mostly on biochemical data. Here, we combined fluorescent tagging of γ-secretase subunits with super-resolution microscopy in fibroblasts. Structured illumination microscopy revealed single γ-secretase complexes with a monodisperse distribution and in a 1:1 stoichiometry of PSEN1 and nicastrin subunits...

BACKGROUND: Fluorescence microscopy is a powerful tool in cell biology, especially for the study of dynamic processes. Intensive irradiation of bacteria with UV, blue and violet light has been shown to be able to kill cells, but very little information is available on the effect of blue or violet light during live-cell imaging. RESULTS: We show here that in the model bacterium Bacillus subtilis chromosome segregation and cell growth are rapidly halted by standard violet (405 nm) and blue light (CFP) (445-457 nm) excitation, whereas they are largely unaffected by green light (YFP)...

The antimicrobial activity and mechanism of silver ions (Ag+ ) have gained broad attention in recent years. However, dynamic studies are rare in this field. Here, we report our measurement of the effects of Ag+ ions on the dynamics of histone-like nucleoid structuring (H-NS) proteins in live bacteria using single-particle tracking photoactivated localization microscopy (sptPALM). It was found that treating the bacteria with Ag+ ions led to faster diffusive dynamics of H-NS proteins. Several techniques were used to understand the mechanism of the observed faster dynamics...

Green-to-red photoconvertible fluorescent proteins repeatedly enter dark states, causing interrupted tracks in single-particle-tracking localization microscopy (sptPALM). We identified a long-lived dark state in photoconverted mEos4b that results from isomerization of the chromophore and efficiently absorbs cyan light. Addition of weak 488-nm light swiftly reverts this dark state to the fluorescent state. This strategy largely eliminates slow blinking and enables the recording of longer tracks in sptPALM with minimum effort...

Primary cilia play a vital role in cellular sensing and signaling. An essential component of ciliogenesis is intraflagellar transport (IFT) involving in IFT-protein recruitment, axonemal engagement of IFT-protein complexes, etc. The mechanistic understanding of these processes at the ciliary base was largely missing, because it is challenging to observe the motion of IFT proteins in this crowded region using conventional microscopy. Here, we report short trajectory tracking of IFT proteins at the base of mammalian primary cilia by optimizing single-particle tracking photoactivated localization microscopy (sptPALM) for IFT88-mEOS4b in live human retinal pigment epithelial cells...

Lactic acid bacteria (LAB) are frequently used in food fermentation and are invaluable for the taste and nutritional value of the fermentation end-product. To gain a better understanding of underlying biochemical and microbiological mechanisms and cell-to-cell variability in LABs, single-molecule techniques such as single-particle tracking photo-activation localization microscopy (sptPALM) hold great promises but are not yet employed due to the lack of detailed protocols and suitable assays. Here, we qualitatively test various fluorescent proteins including variants that are photoactivatable and therefore suitable for sptPALM measurements in Lactococcus lactis, a key LAB for the dairy industry...

The anisotropic organization of plasma membrane constituents is indicative of mechanisms that drive the membrane away from equilibrium. However, defining these mechanisms is challenging due to the short spatiotemporal scales at which diffusion operates. Here, we use high-density single protein tracking combined with photoactivation localization microscopy (sptPALM) to monitor Cdc42 in budding yeast, a system in which Cdc42 exhibits anisotropic organization. Cdc42 exhibited reduced mobility at the cell pole, where it was organized in nanoclusters...

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane...

Patterning cells on microcontact-printed substrates is a powerful approach to control cell morphology and introduce specific mechanical cues on a cell's molecular organization. Although global changes in cellular architectures caused by micropatterns can easily be probed with diffraction-limited optical microscopy, studying molecular reorganizations at the nanoscale demands micropatterned substrates that accommodate the optical requirements of single molecule microscopy techniques. Here, we developed a simple micropatterning strategy that provides control of cellular architectures and is optimized for nanometer accuracy single molecule tracking and three-dimensional super-resolution imaging of plasma and nuclear membrane proteins in cells...

Neuronal growth cones move forward by dynamically connecting actin-based motility to substrate adhesion, but the mechanisms at the individual molecular level remain unclear. We cultured primary neurons on N-cadherin-coated micropatterned substrates, and imaged adhesion and cytoskeletal proteins at the ventral surface of growth cones using single particle tracking combined to photoactivated localization microscopy (sptPALM). We demonstrate transient interactions in the second time scale between flowing actin filaments and immobilized N-cadherin/catenin complexes, translating into a local reduction of the actin retrograde flow...

Adenosine triphosphate (ATP)-gated P2X7 receptors (P2X7Rs) are members of the purinergic receptor family that are expressed in several cell types including neurons. A high concentration of ATP is required for the channel opening of P2X7Rs compared to other members of this receptor family. Recent work suggests that ATP binding to members of the P2X receptor family determines the diffusion and localization of these receptors on the plasma membrane of neurons. Here, we employed single particle tracking photoactivated localization microscopy (sptPALM) to study the diffusion and ATP-dependence of rat P2X7Rs...

Recent developments in single-molecule localization techniques using photoactivatable fluorescent proteins have allowed the probing of single-molecule motion in a living cell with high specificity, millisecond time resolution, and nanometer spatial resolution. Analyzing the dynamics of individual molecules at high densities in this manner promises to provide new insights into the mechanisms of many biological processes, including protein heterogeneity in the plasma membrane, the dynamics of cytoskeletal flow, and clustering of receptor complexes in response to signaling cues...

Rapidly emerging techniques of super-resolution single-molecule microscopy of living cells rely on the continued development of genetically encoded photoactivatable fluorescent proteins. On the basis of monomeric TagRFP, we have developed a photoactivatable TagRFP protein that is initially dark but becomes red fluorescent after violet light irradiation. Compared to other monomeric dark-to-red photoactivatable proteins including PAmCherry, PATagRFP has substantially higher molecular brightness, better pH stability, substantially less sensitivity to blue light, and better photostability in both ensemble and single-molecule modes...

We combined photoactivated localization microscopy (PALM) with live-cell single-particle tracking to create a new method termed sptPALM. We created spatially resolved maps of single-molecule motions by imaging the membrane proteins Gag and VSVG, and obtained several orders of magnitude more trajectories per cell than traditional single-particle tracking enables. By probing distinct subsets of molecules, sptPALM can provide insight into the origins of spatial and temporal heterogeneities in membranes.