Nilah Monnier

Mapping translation 'hot-spots' in live cells by tracking single molecules of mRNA and ribosomes

Messenger RNA localization is important for cell motility by local protein translation. However, while single mRNAs can be imaged and their movements tracked in single cells, it has not yet been possible to determine whether these mRNAs are actively translating. Therefore, we imaged single β-actin mRNAs tagged with MS2 stem loops colocalizing with labeled ribosomes to determine when polysomes formed. A dataset of tracking information consisting of thousands of trajectories per cell demonstrated that mRNAs co-moving with ribosomes have significantly different diffusion properties from non-translating mRNAs that were exposed to translation inhibitors. These data indicate that ribosome load changes mRNA movement and therefore highly translating mRNAs move slower. Importantly, β-actin mRNA near focal adhesions exhibited sub-diffusive corralled movement characteristic of increased translation. This method can identify where ribosomes become engaged for local protein production and how spatial regulation of mRNA-protein interactions mediates cell directionality. DOI: http://dx.doi.org/10.7554/eLife.10415.001

Bayesian Approach to the Analysis of Fluorescence Correlation Spectroscopy Data II: Application to Simulated and In Vitro Data

Fluorescence correlation spectroscopy (FCS) is a powerful approach to characterizing the binding and transport dynamics of macromolecules. The unbiased interpretation of FCS data relies on the evaluation of multiple competing hypotheses to describe an underlying physical process under study, which is typically unknown a priori. Bayesian inference provides a convenient framework for this evaluation based on the temporal autocorrelation function (TACF), as previously shown theoretically using model TACF curves (He, J., Guo, S., and Bathe, M. Anal. Chem. 2012, 84). Here, we apply this procedure to simulated and experimentally measured photon-count traces analyzed using a multitau correlator, which results in complex noise properties in TACF curves that cannot be modeled easily. As a critical component of our technique, we develop two means of estimating the noise in TACF curves based either on multiple independent TACF curves themselves or a single raw underlying intensity trace, including a general procedure to ensure that independent, uncorrelated samples are used in the latter approach. Using these noise definitions, we demonstrate that the Bayesian approach selects the simplest hypothesis that describes the FCS data based on sampling and signal limitations, naturally avoiding overfitting. Further, we show that model probabilities computed using the Bayesian approach provide a reliability test for the downstream interpretation of model parameter values estimated from FCS data. Our procedure is generally applicable to FCS and image correlation spectroscopy and therefore provides an important advance in the application of these methods to the quantitative biophysical investigation of complex analytical and biological systems.

UMD‐DYSF, a novel locus specific database for the compilation and interactive analysis of mutations in the dysferlin gene

Mutations in the dysferlin gene (DYSF) lead to a complete or partial absence of the dysferlin protein in skeletal muscles and are at the origin of dysferlinopathies, a heterogeneous group of rare autosomal recessive inherited neuromuscular disorders. As a step towards a better understanding of the DYSF mutational spectrum, and towards possible inclusion of patients in future therapeutic clinical trials, we set up the Universal Mutation Database for Dysferlin (UMD‐DYSF), a Locus‐Specific Database developed with the UMD® software. The main objective of UMD‐DYSF is to provide an updated compilation of mutational data and relevant interactive tools for the analysis of DYSF sequence variants, for diagnostic and research purposes. In particular, specific algorithms can facilitate the interpretation of newly identified intronic, missense‐ or isosemantic‐exonic sequence variants, a problem encountered recurrently during genetic diagnosis in dysferlinopathies. UMD‐DYSF v1.0 is freely accessible at www.umd.be/DYSF/. It contains a total of 742 mutational entries corresponding to 266 different disease‐causing mutations identified in 558 patients worldwide diagnosed with dysferlinopathy. This article presents for the first time a comprehensive analysis of the dysferlin mutational spectrum based on all compiled DYSF disease‐causing mutations reported in the literature to date, and using the main bioinformatics tools offered in UMD‐DYSF. ©2011 Wiley‐Liss, Inc. Hum Mutat 33:E2317–E2331, 2012.

4th Annual Dysferlin Conference 11-14 September 2010, Washington, USA.

The 4th Annual Dysferlin Conference, sponsored and organized by the Jain Foundation, was held from September 11–14, 2010 in Bellevue, WA, USA. Participants included 36 invited speakers from 10 countries around the world, 16 additional scientists and clinicians, 26 poster presenters, 4 dysferlinopathy patients, 2 representatives of other muscular dystrophy foundations, and 7 members of the Jain Foundation scientific team. The objective of the annual Dysferlin Conferences is to accelerate research on the dysferlinopathies, Limb Girdle Muscular Dystrophy 2B (LGMD2B) and Miyoshi Myopathy, by promoting collaboration and sharing between leading scientists and clinicians in the field. LGMD2B/MM are caused by mutations in dysferlin, and are characterized by progressive muscle wasting that begins in the late teenage years. Although dysferlin has been implicated in muscle fiber membrane repair, effective therapy development requires a more complete understanding of dysferlin’s function and how it leads to muscle pathology. This year’s meeting implemented a novel “lab-meeting” conference format that emphasized the open exchange of information by multiple researchers that converged on a specific scientific question or topic. This format allowed a detailed analysis of key questions in the field, and a venue to air potential solutions.

3rd Annual Dysferlin Conference 2–5 June 2009, Boston, Massachusetts, USA

The 3rd Annual Dysferlin Conference, held from June 2–5, 2009 in Boston, MA, USA, brought together leading scientists and clinicians to discuss recent progress towards understanding and developing a therapy for the dysferlinopathies, Limb Girdle Muscular Dystrophy 2B (LGMD2B) and Miyoshi myopathy. These diseases involve progressive muscle wasting, typically beginning in the late teenage years, and are caused by mutations in the gene encoding dysferlin [1,2]. Dysferlin is required for repair of muscle fiber membrane tears [3,4], but little is known about how this defect contributes to disease pathology or whether dysferlin is also involved in other cellular processes. The conference was sponsored and organized by the Jain Foundation, and participants included 37 invited speakers, 10 additional scientists and clinicians, 40 poster presenters, 5 dysferlinopathy patients, 3 representatives of other muscular dystrophy foundations, and 7 members of the Jain Foundation scientific team. Research was presented from 11 countries around the world. Prior to the start of the main conference, a satellite meeting on the design of a natural history study for dysferlinopathy came up with specific recommendations about issues specific to this disease, including the high clinical variability, small patient numbers, and slow disease progression.

5th Annual Dysferlin Conference 11–14 July 2011, Chicago, Illinois, USA

The fifth Annual Dysferlin Conference, sponsored and organized by the Jain Foundation, was held from July 11 to 14, 2011 in Chicago, Illinois. Participants included 34 speakers, 31 poster presenters, 14 dysferlinopathy patients and family members, 5 representatives from other muscular dystrophy foundations, and 12 members of the Jain Foundation team. The objective of the dysferlin conference is to discuss progress towards developing therapies for the dysferlinopathies, Limb Girdle Muscular Dystrophy 2B (LGMD2B) and Miyoshi Myopathy (MMD1). These diseases manifest progressive muscle loss, usually beginning in the late teenage years, and are caused by mutations in the gene encoding dysferlin. The dysferlin protein is required for effective repair of muscle fiber membranes, but little is known about how this defect leads to the muscle loss experienced by patients, or if dysferlin is involved in other cellular processes that contribute to the pathology. The meeting addressed these and other issues specific to dysferlin deficiency, including the best tools for studying dysferlin and possible causes and interventions for the disease. Four dysferlinopathy patients from the Jain Foundation’s patient registry also shared their experiences with this disease, including the risk of misdiagnosis with polymyositis and the damaging outcome of prednisone treatment.