Cellular functions of myotubularins, amphiphysins, and dynamins implicated in human diseases and their related pathological mechanisms.

<p>(A) Human diseases, (B) their related pathological mechanisms. Membrane fission is necessary for vesicle formation and subsequent trafficking, while inhibition of membrane fission or membrane addition at the T-tubules in muscle may be necessary for their formation and maintenance.</p

Protein domains and disease-causing mutations in the myotubularin, amphiphysin, and dynamin families.

<p>Myotubularin contains a PH-GRAM domain that may bind lipids and a coil-coiled-PDZ binding site to form homo- and hetero-dimers with other members of the myotubularin family. Only the disease-causing missense mutations in MTM1 are represented, based on the international UMD-MTM1 database, existing currently in a local version in Strasbourg (France). MTM1 mutations identified in more than two patients are R69C(9 families), P205L(5), V227M(3), R241C(13), G378R(4), E404K(4), and Y397C(5). AMPH1 and BIN1 possess an N-BAR domain able to sense and eventually curve membrane and a C-terminal SH3 domain binding to proteins with proline-rich domains, such as dynamins <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002595#pgen.1002595-Peter1" target="_blank">[48]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002595#pgen.1002595-Owen1" target="_blank">[88]</a>. In addition some isoforms have clathrin-binding and Myc-binding domains (CBD, MBD); a phosphoinositide-binding motif is present between the BAR and MBD domains specifically in skeletal muscle. DNM2 contains a GTPase domain, a central middle (MID) domain, a Pleckstrin Homology (PH) domain, a GTPase Effector Domain (GED), and a C-terminal Proline Rich Domain (PRD). Dominant mutations in DNM2 lead to either centronuclear myopathy (above), or peripheral CMT neuropathy (below). Only coding mutations are listed for all genes.</p

Myotubularin/amphiphysin/dynamin protein functions in specific tissues, based on animal and cell models.

<p>Myotubularin/amphiphysin/dynamin protein functions in specific tissues, based on animal and cell models.</p

Phylogenetic relationships.

<p>Phylogenetic relationships within the amphiphysin (A), dynamin (B), and myotubularin (C) protein families. Sequences were collected using the eggNOG database, which groups genes into families at different taxonomic levels. A high quality multiple sequence alignment was computed for each protein family on all proteins members including, respectively, 91 myotubularin protein sequences, 23 dynamin protein sequences, and 13 amphiphysin protein sequences. For a more detailed description, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002595#pgen.1002595.s001" target="_blank">Protocol S1</a>. Scale represents the percentage of divergence.</p

Mouse models for 16p11.2 rearrangements.

<p>(A) Top: human 16p11.2 region and proximal segmental duplications (SDs) prone to generate BP4-BP5 copy number variations (CNVs) by a non-allelic homologous recombination (NAHR) mechanism. All genomic positions are given according to UCSC human genome browser GRCh38/hg38. Bottom: 16p11.2 syntenic region on mouse chromosome 7F3. Colored boxes indicate genetic intervals studied in previous publications and in the present study. All genomic positions are given according to UCSC mouse genome browser GRCm38/mm10. (B) Strategy for <i>in vivo</i> cre-mediated recombination and targeted meiotic recombination (TAMERE) crossing strategy. LoxP sites were inserted upstream of <i>Sult1a1</i> and downstream of <i>Spn</i>. The breeding strategy aimed to have trans-loxer females expressing the <i>Hprt</i>^{<i>tm1(cre)Mnn</i>} transgene and carrying the two loxP sites in a <i>trans</i> configuration. The last step consisted of mating trans-loxer females with wt males to generate progeny carrying the deletion or the duplication of the <i>Sult1a1-Spn</i> region. <i>Del/+</i> and <i>Dup/+</i> animals were crossed with wt animals to generate <i>Del/+</i> and <i>Dup/+</i> cohorts. For the <i>Del-Dup</i> cohorts, we crossed <i>Del/+</i> with <i>Dup/+</i> to generate <i>Del/+</i>, wt, <i>Dup/+</i> and <i>Del/Dup</i> animals. (C) Molecular validation. PCR products specific for the <i>Dup/+</i> and <i>Del/+</i> alleles are 500-bp and 461-bp long, respectively.</p

Behavioral map of phenotypes observed in mice carrying deletions and duplications of <i>Slx1b-Sept1</i> [21], <i>Coro1a-Spn</i> [22] and <i>Sult1a1-Spn</i> (this study) genetic intervals on hybrid and inbred genetic backgrounds.

<p>Common phenotypes were found for circadian locomotor activity and repetitive behaviors in the 3 mouse models on different genetic background. Recognition memory was not investigated for the <i>Slx1b-Sept1</i> models. Mice carrying the <i>Coro1a-Spn</i> deletion displayed an impairment of the object recognition memory (1 h retention delay) similar to <i>Sult1a1-Spn Del/+</i> mice that exhibited deficits with 30 min and 3 h retention delays. Changes in open field locomotor activity levels of the <i>Del/+</i> mice were inconsistent between the different studies. In the present study, mice carrying rearrangements for the <i>Sult1a1-Spn</i> region bred on a hybrid genetic background displayed lower levels of social interaction and mice carrying the deletion also showed an absence of social preference for the new individual in the three-chamber sociability test. Phenotype with a significant higher, or respectively lower, level in mutant compared to wt are indicated in green, or in red. When no difference in mutant and wt has been observed, the result is indicated with a grey cell while it is shown with a white box when the phenotype was not investigated. C: Climbing; Ci: Circling; H: Horizontal activity; J: Jumping; R: Rearing; TC: Time in Center; V: Vertical activity; Retention delays 0.5: 30 min; 1: 1h; 3: 3h</p

Behavioral characterization of the separated C57BL/6NxC3B <i>Del/+</i> and <i>Dup/+</i> cohorts.

<p>(A) Circadian activity test. Graphs plot the ambulatory activity (count) and the vertical activity/rears (count) during dark and light phases. (B) Open field test. Distance travelled (m), vertical activity/rears (count) and time percentage spent in the central area over 30 min of testing. (C) Repetitive behavior data. Observations of rearing, jumping, climbing and digging behaviors during 10 min of observation in a novel cage. (D) Social interaction test. Graph plots the duration of sniffing and following behaviors. (E) Novel object recognition test. Discrimination index was calculated as the ratio of time spent exploring the novel object vs the familiar object in the choice trial. (F) Motor coordination evaluation. Graphs plot the latency (s) that mice stayed on the rod before falling under acceleration speed over 3 consecutive days of test. Data are represented as the mean + SEM. *<i>P</i> < 0.05, **<i>P</i> < 0.01 and ***<i>P</i> < 0.001, significantly different from matched wt littermates, Student’s t-test.</p

Expression levels of genes within the <i>Sult1a1-Spn</i> region in brain and peripheral tissues.

<p>Expression levels for 30 probe sets within the engineered region in the hippocampus (A), the striatum (B), the cerebellum (C), and the liver (D). Vertical axis represents log2-fold change in the normalized expression ratio. Blue dots—<i>Del/+</i> animals, black circles—wt animals, orange dots—<i>Dup/+</i> animals. (E) Principal component analysis of the expression of genes from the <i>Sult1a1-Spn</i> region. The first component (horizontal axis) explaining 58.2% of the variance is mainly capturing the CNV effect, the second component (vertical axis) explaining 28.7% of the variance is mainly capturing the regional effect, especially in the cerebellum (+) versus the striatum (*) and the hippocampus (-). Blue dots—<i>Del/+</i> animals, black circles—wt mice, orange dots—<i>Dup/+</i> animals. (F) A Venn diagram representing the number of gene set enriched pathways identified using the transcriptome analysis in the three regions of the brain and the liver, showing some major impact in the striatum and the liver and 16 pathways found altered in at least two tissues.</p

Vacuolar morphologies quantification in yeast cells producing MTM1.

<p>The <i>ymr1Δ</i> cells expressing either wild-type MTM1 or the different MTM1 mutants from either pVV200 (2 µ, overexpression) or pVV204 (CEN, expression) plasmid were analyzed. For each strain, 300 to 600 cells were observed by microscopy (DIC and FM4-64) and sorted into one of the three categories: unilobar large or giant (in white), small one or two lobes (in grey) and more than two lobes or fragmented (in black) vacuoles. The main vacuolar phenotype of the non-transformed <i>ymr1Δ</i> mutant cells is fragmented vacuoles with more than two lobes. Histograms are the mean of three independent experiments and show the proportion of each category in the different transformed yeast cells.</p

Behavioral characterization of the <i>Del-Dup</i> cohort.

<p>(A) Circadian activity test. The light-dark cycle was set as 12-h light and 12-h dark (lights on at 7 am). Plots represent counts of spontaneous locomotor activity and rearing behavior during the dark and light phases. (B) Open field test results illustrate exploratory locomotor activity (distance travelled in m) and percentage of time spent in the central area over 30 min of test. (C) Repetitive behaviors are illustrated by the occurrences of rearing, jumping, climbing and digging behaviors during 10 min of observation in a novel home cage. (D) The social interaction test graph indicates the duration of social interaction behaviors (sniffing and following) between pairs of unfamiliar mice of the same genotype and equivalent body weight tested in a familiar open field area during 10 min. (E) Results of the novel object recognition test. Discrimination index reflects the ability of mice to distinguish the novel object from the familiar object after a short (30 min) and a long (3 hours) retention delay. All genotypes performed significantly above the chance level (one-sample Student's <i>t</i>-test). (F) Motor coordination evaluation. The left graph illustrates the latency (s) with which mice stayed on the rod before falling under accelerating speed (4–40 rpm in 5 min) over 3 consecutive days of testing. The right graph shows hindlimb errors during the notched bar test. Animals had to cross a notched bar and each time a hind paw went through a gap, it was counted as an error. The data are represented as the mean + s.e.m., *<i>P</i> < 0.05 vs wt and ^# <i>P</i> < 0.05 vs all other groups. Tukey's and Mann-Whitney <i>U</i>-tests were applied following a significant one-way ANOVA and Kruskal-Wallis results, respectively.</p