TIA1 Mutations in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Promote Phase Separation and Alter Stress Granule Dynamics.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are age-related neurodegenerative disorders with shared genetic etiologies and overlapping clinical and pathological features. Here we studied a novel ALS/FTD family and identified the P362L mutation in the low-complexity domain (LCD) of T cell-restricted intracellular antigen-1 (TIA1). Subsequent genetic association analyses showed an increased burden of TIA1 LCD mutations in ALS patients compared to controls (p = 8.7 × 1

Drebrin-mediated microtubule-actomyosin coupling steers cerebellar granule neuron nucleokinesis and migration pathway selection

Neuronal migration from a germinal zone to a final laminar position is essential for the morphogenesis of neuronal circuits. While it is hypothesized that microtubule–actomyosin crosstalk is required for a neuron's ‘two-stroke' nucleokinesis cycle, the molecular mechanisms controlling such crosstalk are not defined. By using the drebrin microtubule–actin crosslinking protein as an entry point into the cerebellar granule neuron system in combination with super-resolution microscopy, we investigate how these cytoskeletal systems interface during migration. Lattice light-sheet and structured illumination microscopy reveal a proximal leading process nanoscale architecture wherein f-actin and drebrin intervene between microtubules and the plasma membrane. Functional perturbations of drebrin demonstrate that proximal leading process microtubule–actomyosin coupling steers the direction of centrosome and somal migration, as well as the switch from tangential to radial migration. Finally, the Siah2 E3 ubiquitin ligase antagonizes drebrin function, suggesting a model for control of the microtubule–actomyosin interfaces during neuronal differentiation

Cross-species genomic and epigenomic landscape of retinoblastoma

Genetically engineered mouse models (GEMMs) of human cancer are important for advancing our understanding of tumor initiation and progression as well as for testing novel therapeutics. Retinoblastoma is a childhood cancer of the developing retina that initiates with biallelic inactivation of the RB1 gene. GEMMs faithfully recapitulate the histopathology, molecular, cellular, morphometric, neuroanatomical and neurochemical features of human retinoblastoma. In this study, we analyzed the genomic and epigenomic landscape of murine retinoblastoma and compared them to human retinoblastomas to gain insight into shared mechanisms of tumor progression across species. Similar to human retinoblastoma, mouse tumors have low rates of single nucleotide variations. However, mouse retinoblastomas have higher rates of aneuploidy and regional and focal copy number changes that vary depending on the genetic lesions that initiate tumorigenesis in the developing murine retina. Furthermore, the epigenetic landscape in mouse retinoblastoma was significantly different from human tumors and some pathways that are candidates for molecular targeted therapy for human retinoblastoma such as SYK or MCL1 are not deregulated in GEMMs. Taken together, these data suggest there are important differences between mouse and human retinoblastomas with respect to the mechanism of tumor progression and those differences can have significant implications for translational research to test the efficacy of novel therapies for this devastating childhood cancer

Distinct TCR signaling pathways drive proliferation and cytokine production in T cells

The physiological basis and mechanistic requirements for a large number of functional immunoreceptor tyrosine-based activation motifs (ITAMs; high ITAM multiplicity) in the complex of the T cell antigen receptor (TCR) and the invariant signaling protein CD3 remain obscure. Here we found that whereas a low multiplicity of TCR-CD3 ITAMs was sufficient to engage canonical TCR-induced signaling events that led to cytokine secretion, a high multiplicity of TCR-CD3 ITAMs was required for TCR-driven proliferation. This was dependent on the formation of compact immunological synapses, interaction of the adaptor Vav1 with phosphorylated CD3 ITAMs to mediate the recruitment and activation of the oncogenic transcription factor Notch1 and, ultimately, proliferation induced by the cell-cycle regulator c-Myc. Analogous mechanistic events were also needed to drive proliferation in response to weak peptide agonists. Thus, the TCR-driven pathways that initiate cytokine secretion and proliferation are separable and are coordinated by the multiplicity of phosphorylated ITAMs in TCR-CD3. © 2013 Nature America, Inc. All rights reserved

Outer Hair Cell Lateral Wall Structure Constrains the Mobility of Plasma Membrane Proteins

<div><p>Nature’s fastest motors are the cochlear outer hair cells (OHCs). These sensory cells use a membrane protein, Slc26a5 (prestin), to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP. These mice and four other strains expressing fluorescently labeled membrane proteins were used to examine their lateral diffusion in the OHC lateral wall. All five proteins showed minimal diffusion, but did move after pharmacological disruption of membrane-associated structures with a cholesterol-depleting agent and salicylate. Thus, our results demonstrate that OHC lateral wall structure constrains the mobility of plasma membrane proteins and that the integrity of such membrane-associated structures are critical for Slc26a5’s active and structural roles. The structural constraint of membrane proteins may exemplify convergent evolution of cellular motors across species. Our findings also suggest a possible mechanism for disorders of cholesterol metabolism with hearing loss such as Niemann-Pick Type C diseases.</p></div

Loss of MAGEL2 in Prader-Willi syndrome leads to decreased secretory granule and neuropeptide production

Prader-Willi syndrome (PWS) is a developmental disorder caused by loss of maternally imprinted genes on 15q11-q13, including melanoma antigen gene family member L2 (MAGEL2). The clinical phenotypes of PWS suggest impaired hypothalamic neuroendocrine function; however, the exact cellular defects are unknown. Here, we report deficits in secretory granule (SG) abundance and bioactive neuropeptide production upon loss of MAGEL2 in humans and mice. Unbiased proteomic analysis of Magel2pΔ/m+ mice revealed a reduction in components of SG in the hypothalamus that was confirmed in 2 PWS patient–derived neuronal cell models. Mechanistically, we show that proper endosomal trafficking by the MAGEL2-regulated WASH complex is required to prevent aberrant lysosomal degradation of SG proteins and reduction of mature SG abundance. Importantly, loss of MAGEL2 in mice, NGN2-induced neurons, and human patients led to reduced neuropeptide production. Thus, MAGEL2 plays an important role in hypothalamic neuroendocrine function, and cellular defects in this pathway may contribute to PWS disease etiology. Moreover, these findings suggest unanticipated approaches for therapeutic intervention

Topology, molecular weight, and mobile fractions before and after treatment of Slc26a5 and four heterologously expressed membrane proteins.

<p>Mobile fraction for each molecule was shown either before or after co-treatment of MβCD and salicylate. Molecular weight (M. W. in kDa) was predicted based on amino acid sequences. Means and S.E.M. are expressed.</p

FRAP analysis reveals that Slc26a5 has little lateral mobility in the isolated <i>Slc26a5</i>^<i>YFP/+</i><i>(+neo)</i> OHCs.

<p>FRAP examples using the mice at P18-22 for untreated (A) and PFA-treated (B) OHCs are shown. (C) The normalized fluorescence recovery curves for Slc26a5-YFP based on analysis of the bleached spots (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005500#sec013" target="_blank">Materials and Methods</a>). White arrows (A-B) show bleached spots and the black arrow (C) indicates the time of bleaching. Error bars express S.E.M. Scale bar expresses 10 μm. Numbers (n) of OHCs used in two mice from two litters were shown.</p

B & N analysis confirms the minimal lateral mobility of Slc26a5-YFP in <i>Slc26a5</i>^<i>YFP/+</i><i>(+neo)</i> mice.

<p>(A) YFP fluorescence distribution in a temporal bone preparation from a <i>Slc26a5</i>^<i>YFP/+</i><i>(+neo)</i> mouse at P38 is shown. (B) Variance of the image series (225 frames) divided with the average brightness (σ²/μ) is shown corresponding to A. (C) YFP fluorescence distribution in Min6m9 cells expressing cytosolic YFP (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005500#sec013" target="_blank">Materials and Methods</a>) is shown. The image was taken under an identical condition with (A). B & N analysis for cytosolic YFP in Min6m9 cells (C) is shown in (D). Scale bars express 20 μm.</p