SMoLR: visualization and analysis of single-molecule localization microscopy data in R

Background: Single-molecule localization microscopy is a super-resolution microscopy technique that allows for nanoscale determination of the localization and organization of proteins in biological samples. For biological interpretation of the data it is essential to extract quantitative information from the super-resolution data sets. Due to the complexity and size of these data sets flexible and user-friendly software is required. Results: We developed SMoLR (Single Molecule Localization in R): a flexible framework that enables exploration and analysis of single-molecule localization data within the R programming environment. SMoLR is a package aimed at extracting, visualizing and analyzing quantitative information from localization data obtained by singlemolecule microscopy. SMoLR is a platform not only to visualize nanoscale subcellular structures but additionally provides means to obtain statistical information about the distribution and localization of molecules within them. This can be done for individual images or SMoLR can be used to analyze a large set of super-resolution images at once. Additionally, we describe a method using SMoLR for image feature-based particle averaging, resulting in identification of common features among nanoscale structures. Conclusions: Embedded in the extensive R programming environment, SMoLR allows scientists to study the nanoscale organization of biomolecules in cells by extracting and visualizing quantitative information and hence provides insight in a wide-variety of different biological processes at the single-molecule level

Synaptic vesicle dynamic changes in a model of fragile X

__Background:__ Fragile X syndrome (FXS) is a single-gene disorder that is the most common heritable cause of intellectual disability and the most frequent monogenic cause of autism spectrum disorders (ASD). FXS is caused by an expansion of trinucleotide repeats in the promoter region of the fragile X mental retardation gene (Fmr1). This leads to a lack of fragile X mental retardation protein (FMRP), which regulates translation of a wide range of messenger RNAs (mRNAs). The extent of expression level alterations of synaptic proteins affected by FMRP loss and their consequences on synaptic dynamics in FXS has not been fully investigated. __Methods:__ Here, we used an Fmr1 knockout (KO) mouse model to investigate the molecular mechanisms underlying FXS by monitoring protein expression changes using shotgun label-free liquid-chromatography mass spectrometry (LC-MSE) in brain tissue and synaptosome fractions. FXS-associated candidate proteins were validated using selected reaction monitoring (SRM) in synaptosome fractions for targeted protein quantification. Furthermore, functional alterations in synaptic release and dynamics were evaluated using live-cell imaging, and interpretation of synaptic dynamics differences was investigated using electron microscopy. __Results:__ Key findings relate to altered levels of proteins involved in GABA-signalling, especially in the cerebellum. Further exploration using microscopy studies found reduced synaptic vesicle unloading of hippocampal neurons and increased vesicle unloading in cerebellar neurons, which suggests a general decrease of synaptic transmission. __Conclusions:__ Our findings suggest that FMRP is a regulator of synaptic vesicle dynamics, which supports the role of FMRP in presynaptic functions. Taken together, these studies provide novel insights into the molecular changes associated with FXS

BRCA2 diffuses as oligomeric clusters with RAD51 and changes mobility after DNA damage in live cells

Genome maintenance by homologous recombination depends on coordinating many proteins in time and space to assemble at DNA break sites. To understand this process, we followed the mobility of BRCA2, a critical recombination mediator, in live cells at the single-molecule level using both single-particle tracking and fluorescence correlation spectroscopy. BRCA2-GFP and -YFP were compared to distinguish diffusion from fluorophore behavior. Diffusive behavior of fluorescent RAD51 and RAD54 was determined for comparison. All fluorescent proteins were expressed from endogenous loci. We found that nuclear BRCA2 existed in oligomeric clusters, and exhibited heterogeneous mobility. DNA damage increased BRCA2 transient binding, presumably including binding to damaged sites. Despite its very different size, RAD51 displayed mobility similar to BRCA2, which indicates physical interaction between these proteins both before and after induction of DNA damage. We propose that BRCA2-mediated sequestration of nuclear RAD51 serves to prevent inappropriate DNA interactions and that all RAD51 is delivered to DNA damage sites in association with BRCA2