Ciliary entry of the kinesin-2 motor KIF17 is regulated by importin-beta2 and RanGTP

The biogenesis, maintenance, and function of primary cilia are controlled through intraflagellar transport (IFT) driven by two kinesin-2 family members, the heterotrimeric KIF3A/KIF3B/KAP complex and the homodimeric KIF17 motor1,2. How these motors and their cargoes gain access to the ciliary compartment is poorly understood. We identify a ciliary localization signal (CLS) in the KIF17 tail domain that is necessary and sufficient for ciliary targeting. Similarities between the CLS and classic nuclear localization signals (NLS) suggests that similar mechanisms regulate nuclear and ciliary import. We hypothesize that ciliary targeting of KIF17 is regulated by a Ran-GTP gradient across the ciliary base. Consistent with this, cytoplasmic expression of GTP-locked Ran(G19V) disrupts the gradient and abolishes ciliary entry of KIF17. Furthermore, KIF17 interacts with importin-β2 in a manner dependent on the CLS and inhibited by Ran-GTP. We propose that Ran plays a global role in regulating cellular compartmentalization by controlling the shuttling of cytoplasmic proteins into nuclear and ciliary compartments

Structure and dynamics of single-isoform recombinant Neuronal Human Tubulin

Microtubules are polymers that cycle stochastically between polymerization and depolymerization i.e., they exhibit 'dynamic instability'. This behavior is crucial for cell division, motility and differentiation. While studies in the last decade have made fundamental breakthroughs in our understanding of how cellular effectors modulate microtubule dynamics, analysis of the relationship between tubulin sequence, structure and dynamics has been held back by a lack of dynamics measurements with and structural characterization of homogenous, isotypically pure, engineered tubulin. Here we report for the first time the cryo-EM structure and in vitro dynamics parameters of recombinant isotypically pure human tubulin. α1A/βIII is a purely neuronal tubulin isoform. The 4.2 Å structure of unmodified human α1A/βIII microtubules shows overall similarity to that of heterogeneous brain microtubules, but is distinguished by subtle differences at polymerization interfaces, which are hotspots for sequence divergence between tubulin isoforms. In vitro dynamics assays show that, like mosaic brain microtubules, recombinant homogenous microtubules undergo dynamic instability but they polymerize slower and catastrophe less frequently. Interestingly, we find that epitaxial growth of α1A/βIII microtubules from heterogeneous brain seeds is inefficient, but can be fully rescued by incorporating as little as 5% of brain tubulin into the homogenous α1A/βIII lattice. Our study establishes a system to examine the structure and dynamics of mammalian microtubules with well-defined tubulin species and is a first and necessary step towards uncovering how tubulin genetic and chemical diversity is exploited to modulate intrinsic microtubule dynamics

Comparing recruitment, retention, and safety reporting among geographic regions in multinational Alzheimer’s disease clinical trials

INTRODUCTION: Most Alzheimer’s disease (AD) clinical trials enroll participants multinationally. Yet, few data exist to guide investigators and sponsors regarding the types of patients enrolled in these studies and whether participant characteristics vary by region. METHODS: We used data derived from four multinational phase III trials in mild to moderate AD to examine whether regional differences exist with regard to participant demographics, safety reporting, and baseline scores on the Mini Mental State Examination (MMSE), the 11-item Alzheimer’s Disease Assessment Scale–Cognitive subscale (ADAS-cog11), the Clinical Dementia Rating scale Sum of Boxes (CDR-SB), the Alzheimer’s Disease Cooperative Study–Activities of Daily Living Inventory (ADCS-ADL), and the Neuropsychiatric Inventory (NPI). We assigned 31 participating nations to 7 geographic regions: North America, South America/Mexico, Western Europe/Israel, Eastern Europe/Russia, Australia/South Africa, Asia, and Japan. RESULTS: North America, Western Europe/Israel, and Australia/South Africa enrolled similar proportions of men, apolipoprotein E ε4 carriers, and participants with spouse study partners, whereas Asia, Eastern Europe/Russia, and South America/Mexico had lower proportions for these variables. North America and South America/Mexico enrolled older subjects, whereas Asia and South America/Mexico enrolled less-educated participants than the remaining regions. Approved AD therapy use differed among regions (range: 73% to 92%) and was highest in North America, Western Europe/Israel, and Japan. Dual therapy was most frequent in North America (48%). On the MMSE, North America, Western Europe/Israel, Japan, and Australia/South Africa had higher (better) scores, and Asia, South America/Mexico, and Eastern Europe/Russia had lower scores. Eastern Europe/Russia had more impaired ADAS-cog11 scores than all other regions. Eastern Europe/Russia and South America/Mexico had more impaired scores for the ADCS-ADL and the CDR-SB. Mean scores for the CDR-SB in Asia were milder than all regions except Japan. NPI scores were lower in Asia and Japan than in all other regions. Participants in North America and Western Europe/Israel reported more adverse events than those in Eastern Europe/Russia and Japan. CONCLUSIONS: These findings suggest that trial populations differ across geographic regions on most baseline characteristics and that multinational enrollment is associated with sample heterogeneity. The data provide initial guidance with regard to the regional differences that contribute to this heterogeneity and are important to consider when planning global trials

Induction of Ran GTP drives ciliogenesis

The small GTPase Ran and the importin proteins regulate nucleocytoplasmic transport. New evidence suggests that Ran GTP and the importins are also involved in conveying proteins into cilia. In this study, we find that Ran GTP accumulation at the basal bodies is coordinated with the initiation of ciliogenesis. The Ran-binding protein 1 (RanBP1), which indirectly accelerates Ran GTP → Ran GDP hydrolysis and promotes the dissociation of the Ran/importin complex, also localizes to basal bodies and cilia. To confirm the crucial link between Ran GTP and ciliogenesis, we manipulated the levels of RanBP1 and determined the effects on Ran GTP and primary cilia formation. We discovered that RanBP1 knockdown results in an increased concentration of Ran GTP at basal bodies, leading to ciliogenesis. In contrast, overexpression of RanBP1 antagonizes primary cilia formation. Furthermore, we demonstrate that RanBP1 knockdown disrupts the proper localization of KIF17, a kinesin-2 motor, at the distal tips of primary cilia in Madin–Darby canine kidney cells. Our studies illuminate a new function for Ran GTP in stimulating cilia formation and reinforce the notion that Ran GTP and the importins play key roles in ciliogenesis and ciliary protein transport

ともにいればいるほど、我々は幸せである：ジンバブエのメンタルヘルスにおけるピア・ミュージック より一緒に、より幸せに：ジンバブエの精神保健治療における、仲間どうしでの音楽

Ruth Verheyは、ジンバブエのHarareで、メンタルヘルスケア主導を基盤とする包括的グループ「なかまのベンチ［Friendship Bench］」の発展と運営に携わる臨床心理士である。2013年、彼女はニューヨーク市から音楽療法士のJeffrey Angellを招聘し、Harare病院において毎週行われるZeebagサポートグループを設立した。Zeebagグループはうつを患う8人の女性で構成されている。このエッセイは、Jeffreyがその経験を一人称で述べると同時に、音楽や音楽プロセスについて、またこれらがいかにしてFriendship Benchのピア・エンパワメント・モデルに統合されていったかについて報告する。Ruth Verhey is a clinical psychologist involved in developing and running the Friendship Bench, a comprehensive, group based mental healthcare initiative in Harare, Zimbabwe.  In 2013, she invited Jeffrey Angell, a music therapist from New York City, to facilitate one of weekly Zeebag support groups at Harare Hospital.  The Zeebag group is comprised of eight women who suffer from depression.  The essay provides Jeffrey's first person accounts of this experience, as well as describing the music, music process, and how these intergrate into the peer empowerment model of the Frienship Bench.Ruth Verhey est un psychologue clinicien impliqué dans le développement et l'exécution du Banc d'amitié, un initiative basée sur groupe globale de soins de santé mentale à Harare, au Zimbabwe. En 2013, elle a invité Jeffrey Angell, un musicothérapeute de New York, afin de faciliter l'un des groupes de soutien Zeebag hebdomadaires à l'hôpital de Harare. Le groupe Zeebag est composé de huit femmes qui souffrent de dépression. L'essai fournit des premiers comptes de personne de Jeffrey de cette expérience, ainsi que décrire la musique, le processus de la musique, et comment ceux-ci intergré dans le modèle d'autonomisation par les pairs du Banc Amitié

Luminal Localization of α-tubulin K40 Acetylation by Cryo-EM Analysis of Fab-Labeled Microtubules

<div><p>The αβ-tubulin subunits of microtubules can undergo a variety of evolutionarily-conserved post-translational modifications (PTMs) that provide functional specialization to subsets of cellular microtubules. Acetylation of α-tubulin residue Lysine-40 (K40) has been correlated with increased microtubule stability, intracellular transport, and ciliary assembly, yet a mechanistic understanding of how acetylation influences these events is lacking. Using the anti-acetylated tubulin antibody 6-11B-1 and electron cryo-microscopy, we demonstrate that the K40 acetylation site is located inside the microtubule lumen and thus cannot directly influence events on the microtubule surface, including kinesin-1 binding. Surprisingly, the monoclonal 6-11B-1 antibody recognizes both acetylated and deacetylated microtubules. These results suggest that acetylation induces structural changes in the K40-containing loop that could have important functional consequences on microtubule stability, bending, and subunit interactions. This work has important implications for acetylation and deacetylation reaction mechanisms as well as for interpreting experiments based on 6-11B-1 labeling.</p> </div

2D and 3D EM visualization of the 6-11B-1 Fab within the microtubule lumen.

<p><b>A-D</b>) Microtubules polymerized from A,D) untreated B) MEC-17-treated (acetylated), or C) SIRT2-treated (deacetylated) tubulins were incubated with A-C) 6-11B-1 Fab fragments or D) GST-KHC motor domain and visualized after embedding in negative stain. The insets show expanded views of the boxed areas. White arrows in D) indicate kinesin-1 motors on the microtubule surface. Scale bars, 50 nm. <b>E–G</b>) Side and minus end views of 3D helical reconstructions of vitrified microtubules. Visible density thresholds have been adjusted to levels comparable to docked αβ-tubulin. All maps have been low-pass filtered to 22Å resolution. <b>E</b>) Control microtubule without Fab labeling. <b>F</b>) Cross section of acetylated microtubule decorated with 6-11B-1 Fab (orange). The structure of the αβ-tubulin dimer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048204#pone.0048204-Maruta1" target="_blank">[30]</a> has been docked into the right side of the density map (α-tubulin is shown in teal, β-tubulin is shown in purple). <b>G</b>) Cross section of deacetylated microtubule decorated with 6-11B-1 Fab (orange).</p

Monoclonal (6-11B-1) and polyclonal (anti-acetyl-K40) antibodies differ in their ability to recognize deacetylated microtubules <i>in vitro</i>.

<p>Taxol-stabilized microtubules polymerized from acetylated or deacetylated tubulins were stained immediately (Live) or after fixation with paraformaldehyde (PFA fixed) with A) monoclonal 6-11B-1 antibody (magenta) or B) polyclonal anti-acetyl-K40 antibody (magenta). The total tubulin in each sample was detected with DM1A antibody (green). Scale bars, 20 µm.</p

Generation of acetylated and deacetylated tubulins.

<p><b>A</b>) Lysates of COS7 and PtK2 cells either untransfected (lanes 1 and 2) or expressing the acetyltransferase MEC-17 (lanes 3 and 4) were immunoblotted for K40 acetylation of α-tubulin using monoclonal 6-11B-1 and polyclonal anti-acetyl-K40 antibodies. <b>B</b>) Purified brain tubulin was untreated (lane 1) or treated with recombinant MEC-17 (lane 2) or SIRT2 (lane 3) enzymes. The total tubulin in all samples was determined in parallel by blotting with an anti-β-tubulin antibody.</p