query: Increased microtubule acetylation exacerbates LRRK2 Roc-COR domain mutation induced locomotor deficits.

title: Increasing microtubule acetylation rescues axonal transport and locomotor deficits caused by LRRK2 Roc-COR domain mutations

text: Leucine-rich repeat kinase 2 (LRRK2) mutations are the most common genetic cause of Parkinson's disease. LRRK2 is a multifunctional protein affecting many cellular processes and has been described to bind microtubules. Defective microtubule-based axonal transport is hypothesized to contribute to Parkinson's disease, but whether LRRK2 mutations affect this process to mediate pathogenesis is not known. Here we find that LRRK2 containing pathogenic Roc-COR domain mutations (R1441C, Y1699C) preferentially associates with deacetylated microtubules, and inhibits axonal transport in primary neurons and in Drosophila, causing locomotor deficits in vivo. In vitro, increasing microtubule acetylation using deacetylase inhibitors or the tubulin acetylase αTAT1 prevents association of mutant LRRK2 with microtubules, and the deacetylase inhibitor trichostatin A (TSA) restores axonal transport. In vivo knockdown of the deacetylases HDAC6 and Sirt2, or administration of TSA rescues both axonal transport and locomotor behavior. Thus, this study reveals a pathogenic mechanism and a potential intervention for Parkinson's disease.

title: Structural basis for the activation of microtubule assembly by the EB1 and p150Glued complex.

text: Plus-end tracking proteins, such as EB1 and the dynein/dynactin complex, regulate microtubule dynamics. These proteins are thought to stabilize microtubules by forming a plus-end complex at microtubule growing ends with ill-defined mechanisms. Here we report the crystal structure of two plus-end complex components, the carboxy-terminal dimerization domain of EB1 and the microtubule binding (CAP-Gly) domain of the dynactin subunit p150Glued. Each molecule of the EB1 dimer contains two helices forming a conserved four-helix bundle, while also providing p150Glued binding sites in its flexible tail region. Combining crystallography, NMR, and mutational analyses, our studies reveal the critical interacting elements of both EB1 and p150Glued, whose mutation alters microtubule polymerization activity. Moreover, removal of the key flexible tail from EB1 activates microtubule assembly by EB1 alone, suggesting that the flexible tail negatively regulates EB1 activity. We, therefore, propose that EB1 possesses an auto-inhibited conformation, which is relieved by p150Glued as an allosteric activator.

title: Posttranslational Acetylation of α-Tubulin Constrains Protofilament Number in Native Microtubules

text:
BACKGROUND Microtubules are built from linear polymers of α-β tubulin dimers (protofilaments) that form a tubular quinary structure. Microtubules assembled from purified tubulin in vitro contain between 10 and 16 protofilaments; however, such structural polymorphisms are not found in cells. This discrepancy implies that factors other than tubulin constrain microtubule protofilament number, but the nature of these constraints is unknown.
 RESULTS Here, we show that acetylation of MEC-12 α-tubulin constrains protofilament number in C. elegans touch receptor neurons (TRNs). Whereas the sensory dendrite of wild-type TRNs is packed with a cross-linked bundle of long, 15-protofilament microtubules, mec-17;atat-2 mutants lacking α-tubulin acetyltransferase activity have short microtubules, rampant lattice defects, and variable protofilament number both between and within microtubules. All-atom molecular dynamics simulations suggest a model in which acetylation of lysine 40 promotes the formation of interprotofilament salt bridges, stabilizing lateral interactions between protofilaments and constraining quinary structure to produce stable, structurally uniform microtubules in vivo.
 CONCLUSIONS Acetylation of α-tubulin is an essential constraint on protofilament number in vivo. We propose a structural model in which this posttranslational modification promotes the formation of lateral salt bridges that fine-tune the association between adjacent protofilaments and enable the formation of uniform microtubule populations in vivo.

title: Distinct functions of nucleotide-binding/hydrolysis sites in the four AAA modules of cytoplasmic dynein.

text: Cytoplasmic dynein is a microtubule-based motor protein that is responsible for most intracellular retrograde transports along microtubule filaments. The motor domain of dynein contains six tandemly linked AAA (ATPases associated with diverse cellular activities) modules, with the first four containing predicted nucleotide-binding/hydrolysis sites (P1-P4). To dissect the functions of these multiple nucleotide-binding/hydrolysis sites, we expressed and purified Dictyostelium dynein motor domains in which mutations were introduced to block nucleotide binding at each of the four AAA modules, and then examined their detailed biochemical properties. The P1 mutant was trapped in a strong-binding state even in the presence of ATP and lost its motile activity. The P3 mutant also showed a high affinity for microtubules in the presence of ATP and lost most of the microtubule-activated ATPase activity, but retained microtubule sliding activity, although the sliding velocity of the mutant was more than 20-fold slower than that of the wild type. In contrast, mutation in the P2 or P4 site did not affect the apparent binding affinity of the mutant for microtubules in the presence of ATP, but reduced ATPase and microtubule sliding activities. These results indicate that ATP binding and its hydrolysis only at the P1 site are essential for the motor activities of cytoplasmic dynein, and suggest that the other nucleotide-binding/hydrolysis sites regulate the motor activities. Among them, nucleotide binding at the P3 site is not essential but is critical for microtubule-activated ATPase and motile activities of cytoplasmic dynein.

title: Acetylation controls Notch3 stability and function in T-cell leukemia

text: Post-translational modifications of Notch3 and their functional role with respect to Notch3 overexpression in T-cell leukemia are still poorly understood. We identify here a specific novel property of Notch3 that is acetylated and deacetylated at lysines 1692 and 1731 by p300 and HDAC1, respectively, a balance impaired by HDAC inhibitors (HDACi) that favor hyperacetylation. By using HDACi and a non-acetylatable Notch3 mutant carrying K/R1692−1731 mutations in the intracellular domain, we show that Notch3 acetylation primes ubiquitination and proteasomal-mediated degradation of the protein. As a consequence, Notch3 protein expression and its transcriptional activity are decreased both in vitro and in vivo in Notch3 transgenic (tg) mice, thus impairing downstream signaling upon target genes. Consistently, Notch3-induced T-cell proliferation is inhibited by HDACi, whereas it is enhanced by the non-acetylatable Notch3-K/R1692−1731 mutant. Finally, HDACi-induced Notch3 hyperacetylation prevents in vivo growth of T-cell leukemia/lymphoma in Notch3 tg mice. Together, our findings suggest a novel level of Notch signaling control in which Notch3 acetylation/deacetylation process represents a key regulatory switch, thus representing a suitable druggable target for Notch3-sustained T-cell acute lymphoblastic leukemia therapy.

title: A method of comparing the areas under receiver operating characteristic curves derived from the same cases.

text: Receiver operating characteristic (ROC) curves are used to describe and compare the performance of diagnostic technology and diagnostic algorithms. This paper refines the statistical comparison of the areas under two ROC curves derived from the same set of patients by taking into account the correlation between the areas that is induced by the paired nature of the data. The correspondence between the area under an ROC curve and the Wilcoxon statistic is used and underlying Gaussian distributions (binormal) are assumed to provide a table that converts the observed correlations in paired ratings of images into a correlation between the two ROC areas. This between-area correlation can be used to reduce the standard error (uncertainty) about the observed difference in areas. This correction for pairing, analogous to that used in the paired t-test, can produce a considerable increase in the statistical sensitivity (power) of the comparison. For studies involving multiple readers, this method provides a measure of a component of the sampling variation that is otherwise difficult to obtain.

title: Mitosis-specific anchoring of gamma tubulin complexes by pericentrin controls spindle organization and mitotic entry.

text: Microtubule nucleation is the best known function of centrosomes. Centrosomal microtubule nucleation is mediated primarily by gamma tubulin ring complexes (gamma TuRCs). However, little is known about the molecules that anchor these complexes to centrosomes. In this study, we show that the centrosomal coiled-coil protein pericentrin anchors gamma TuRCs at spindle poles through an interaction with gamma tubulin complex proteins 2 and 3 (GCP2/3). Pericentrin silencing by small interfering RNAs in somatic cells disrupted gamma tubulin localization and spindle organization in mitosis but had no effect on gamma tubulin localization or microtubule organization in interphase cells. Similarly, overexpression of the GCP2/3 binding domain of pericentrin disrupted the endogenous pericentrin-gamma TuRC interaction and perturbed astral microtubules and spindle bipolarity. When added to Xenopus mitotic extracts, this domain uncoupled gamma TuRCs from centrosomes, inhibited microtubule aster assembly, and induced rapid disassembly of preassembled asters. All phenotypes were significantly reduced in a pericentrin mutant with diminished GCP2/3 binding and were specific for mitotic centrosomal asters as we observed little effect on interphase asters or on asters assembled by the Ran-mediated centrosome-independent pathway. Additionally, pericentrin silencing or overexpression induced G2/antephase arrest followed by apoptosis in many but not all cell types. We conclude that pericentrin anchoring of gamma tubulin complexes at centrosomes in mitotic cells is required for proper spindle organization and that loss of this anchoring mechanism elicits a checkpoint response that prevents mitotic entry and triggers apoptotic cell death.

title: The coordination of cyclic microtubule association/dissociation and tail swing of cytoplasmic dynein.

text: The dynein motor domain is composed of a tail, head, and stalk and is thought to generate a force to microtubules by swinging the tail against the head during its ATPase cycle. For this "power stroke," dynein has to coordinate the tail swing with microtubule association/dissociation at the tip of the stalk. Although a detailed picture of the former process is emerging, the latter process remains to be elucidated. By using the single-headed recombinant motor domain of Dictyostelium cytoplasmic dynein, we address the questions of how the interaction of the motor domain with a microtubule is modulated by ATPase steps, how the two mechanical cycles (the microtubule association/dissociation and tail swing) are coordinated, and which ATPase site among the multiple sites in the motor domain regulates the coordination. Based on steady-state and pre-steady-state measurements, we demonstrate that the two mechanical cycles proceed synchronously at most of the intermediate states in the ATPase cycle: the motor domain in the poststroke state binds strongly to the microtubule with a K(d) of approximately 0.2 microM, whereas most of the motor domains in the prestroke state bind weakly to the microtubule with a K(d) of >10 microM. However, our results suggest that the timings of the microtubule affinity change and tail swing are staggered at the recovery stroke step in which the tail swings from the poststroke to the prestroke position. The ATPase site in the AAA1 module of the motor domain was found to be responsible for the coordination of these two mechanical processes.

title: Mutant dynactin in motor neuron disease

text: Impaired axonal transport in motor neurons has been proposed as a mechanism for neuronal degeneration in motor neuron disease. Here we show linkage of a lower motor neuron disease to a region of 4 Mb at chromosome 2p13. Mutation analysis of a gene in this interval that encodes the largest subunit of the axonal transport protein dynactin showed a single base-pair change resulting in an amino-acid substitution that is predicted to distort the folding of dynactin's microtubule-binding domain. Binding assays show decreased binding of the mutant protein to microtubules. Our results show that dysfunction of dynactin-mediated transport can lead to human motor neuron disease.

title: A forkhead-domain gene is mutated in a severe speech and language disorder

text: Individuals affected with developmental disorders of speech and language have substantial difficulty acquiring expressive and/or receptive language in the absence of any profound sensory or neurological impairment and despite adequate intelligence and opportunity1. Although studies of twins consistently indicate that a significant genetic component is involved1,2,3, most families segregating speech and language deficits show complex patterns of inheritance, and a gene that predisposes individuals to such disorders has not been identified. We have studied a unique three-generation pedigree, KE, in which a severe speech and language disorder is transmitted as an autosomal-dominant monogenic trait4. Our previous work mapped the locus responsible, SPCH1, to a 5.6-cM interval of region 7q31 on chromosome 7 (ref. 5). We also identified an unrelated individual, CS, in whom speech and language impairment is associated with a chromosomal translocation involving the SPCH1 interval6. Here we show that the gene FOXP2, which encodes a putative transcription factor containing a polyglutamine tract and a forkhead DNA-binding domain, is directly disrupted by the translocation breakpoint in CS. In addition, we identify a point mutation in affected members of the KE family that alters an invariant amino-acid residue in the forkhead domain. Our findings suggest that FOXP2 is involved in the developmental process that culminates in speech and language.

title: The flip side of perception-action coupling: locomotor experience and the ontogeny of visual-postural coupling.

text: The possible role of motor development on psychological function is once again a topic of great theoretical and practical importance. The revival of this issue has stemmed from a different approach to the topic, away from Gesell's interest in the long-term prediction of psychological functions from early motoric assessments, toward an attempt to understand how the acquisition of motor skills orchestrates psychological changes. This paper describes how the acquisition of one motor skill, prone locomotion, has been linked to developmental changes in an infant's ability to regulate posture based on information available in patterns of optic flow. It is argued that the onset of prone locomotion presses the infant to differentiate spatially delimited regions of optic flow to effectively and efficiently control the important subtasks nested within the larger task of locomotion, namely, steering, attending to the surface of support, and maintaining postural control. Following this argument, a research program is described that aims to determine if locomotor experience is causally linked to improvements in the ability to functionalize peripheral optic flow for postural control or whether locomotor experience is merely a maturational forecaster of such improvements. Finally, a hypothesis is put forward that links the emergence of wariness of heights to infants' ability to regulate posture on the basis of peripheral optic flow. The paper's overarching theoretical point is the principle of probabilistic epigenesis, which states that one developmental acquisition produces experiences that bring about a host of new developmental changes in the same and different domains.

title: NuA4 lysine acetyltransferase Esa1 is targeted to coding regions and stimulates transcription elongation with Gcn5.

text: NuA4, the major H4 lysine acetyltransferase (KAT) complex in Saccharomyces cerevisiae, is recruited to promoters and stimulates transcription initiation. NuA4 subunits contain domains that bind methylated histones, suggesting that histone methylation should target NuA4 to coding sequences during transcription elongation. We show that NuA4 is cotranscriptionally recruited, dependent on its physical association with elongating polymerase II (Pol II) phosphorylated on the C-terminal domain by cyclin-dependent kinase 7/Kin28, but independently of subunits (Eaf1 and Tra1) required for NuA4 recruitment to promoters. Whereas histone methylation by Set1 and Set2 is dispensable for NuA4's interaction with Pol II and targeting to some coding regions, it stimulates NuA4-histone interaction and H4 acetylation in vivo. The NuA4 KAT, Esa1, mediates increased H4 acetylation and enhanced RSC occupancy and histone eviction in coding sequences and stimulates the rate of transcription elongation. Esa1 cooperates with the H3 KAT in SAGA, Gcn5, to enhance these functions. Our findings delineate a pathway for acetylation-mediated nucleosome remodeling and eviction in coding sequences that stimulates transcription elongation by Pol II in vivo.

title: Acetylation of apurinic/apyrimidinic endonuclease-1 regulates Helicobacter pylori-mediated gastric epithelial cell apoptosis.

text:
BACKGROUND & AIMS Helicobacter pylori-induced gastric epithelial cell (GEC) apoptosis is a complex process that includes activation of the tumor suppressor p53. p53-mediated apoptosis involves p53 activation, bax transcription, and cytochrome c release from mitochondria. Apurinic/apyrimidinic endonuclease-1 (APE-1) regulates transcriptional activity of p53, and H pylori induce APE-1 expression in human GECs. H pylori infection increases intracellular calcium ion concentration [Ca2+]i of GECs, which induces APE-1 acetylation. We investigated the effects of H pylori infection and APE-1 acetylation on GEC apoptosis.
 METHODS AGS cells (wild-type or with suppressed APE-1), KATO III cells, and cells isolated from gastric biopsy specimens were infected with H pylori. Effects were examined by immunoblotting, real-time reverse-transcription polymerase chain reaction, immunoprecipitation, immunofluorescence microscopy, chromatin immunoprecipitation, mobility shift, DNA binding, and luciferase assays.
 RESULTS H pylori infection increased [Ca2+]i and acetylation of APE-1 in GECs, but the acetylation status of APE-1 did not affect the transcriptional activity of p53. In GECs, expression of a form of APE-1 that could not be acetylated increased total and mitochondrial levels of Bax and induced release of cytochrome c and fragmentation of DNA; expression of wild-type APE-1 reduced these apoptotic events. We identified a negative calcium response element in the human bax promoter and found that poly (adenosine diphosphate-ribose) polymerase 1 recruited the acetylated APE-1/histone deacetylase-1 repressor complex to bax nCaRE.
 CONCLUSIONS H pylori-mediated acetylation of APE-1 suppresses Bax expression; this prevents p53-mediated apoptosis when H pylori infect GECs.

title: The NLRP12 Sensor Negatively Regulates Autoinflammatory Disease by Modulating Interleukin-4 Production in T Cells.

text: Missense mutations in the nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing family of gene 12 (Nlrp12) are associated with periodic fever syndromes and atopic dermatitis in humans. Here, we have demonstrated a crucial role for NLRP12 in negatively regulating pathogenic T cell responses. Nlrp12(-/-) mice responded to antigen immunization with hyperinflammatory T cell responses. Furthermore, transfer of CD4(+)CD45RB(hi)Nlrp12(-/-) T cells into immunodeficient mice led to more severe colitis and atopic dermatitis. NLRP12 deficiency did not, however, cause exacerbated ascending paralysis during experimental autoimmune encephalomyelitis (EAE); instead, Nlrp12(-/-) mice developed atypical neuroinflammatory symptoms that were characterized by ataxia and loss of balance. Enhanced T-cell-mediated interleukin-4 (IL-4) production promotes the development of atypical EAE disease in Nlrp12(-/-) mice. These results define an unexpected role for NLRP12 as an intrinsic negative regulator of T-cell-mediated immunity and identify altered NF-κB regulation and IL-4 production as key mediators of NLRP12-associated disease.

title: Dynein pulls microtubules without rotating its stalk.

text: Dynein is a microtubule motor that powers motility of cilia and flagella. There is evidence that the relative sliding of the doublet microtubules is due to a conformational change in the motor domain that moves a microtubule bound to the end of an extension known as the stalk. A predominant model for the movement involves a rotation of the head domain, with its stalk, toward the microtubule plus end. However, stalks bound to microtubules have been difficult to observe. Here, we present the clearest views so far of stalks in action, by observing sea urchin, outer arm dynein molecules bound to microtubules, with a new method, "cryo-positive stain" electron microscopy. The dynein molecules in the complex were shown to be active in in vitro motility assays. Analysis of the electron micrographs shows that the stalk angles relative to microtubules do not change significantly between the ADP.vanadate and no-nucleotide states, but the heads, together with their stalks, shift with respect to their A-tubule attachments. Our results disagree with models in which the stalk acts as a lever arm to amplify structural changes. The observed movement of the head and stalk relative to the tail indicates a new plausible mechanism, in which dynein uses its stalk as a grappling hook, catching a tubulin subunit 8 nm ahead and pulling on it by retracting a part of the tail (linker).

title: ESCRT-III recognition by VPS4 ATPases

text: The ESCRT (endosomal sorting complex required for transport) pathway is required for terminal membrane fission events in several important biological processes, including endosomal intraluminal vesicle formation, HIV budding and cytokinesis. VPS4 ATPases perform a key function in this pathway by recognizing membrane-associated ESCRT-III assemblies and catalysing their disassembly, possibly in conjunction with membrane fission. Here we show that the microtubule interacting and transport (MIT) domains of human VPS4A and VPS4B bind conserved sequence motifs located at the carboxy termini of the CHMP1–3 class of ESCRT-III proteins. Structures of VPS4A MIT–CHMP1A and VPS4B MIT–CHMP2B complexes reveal that the C-terminal CHMP motif forms an amphipathic helix that binds in a groove between the last two helices of the tetratricopeptide-like repeat (TPR) of the VPS4 MIT domain, but in the opposite orientation to that of a canonical TPR interaction. Distinct pockets in the MIT domain bind three conserved leucine residues of the CHMP motif, and mutations that inhibit these interactions block VPS4 recruitment, impair endosomal protein sorting and relieve dominant-negative VPS4 inhibition of HIV budding. Thus, our studies reveal how the VPS4 ATPases recognize their CHMP substrates to facilitate the membrane fission events required for the release of viruses, endosomal vesicles and daughter cells.

title: CHD4 Is a RanGTP-Dependent MAP that Stabilizes Microtubules and Regulates Bipolar Spindle Formation

text:
BACKGROUND Production of the GTP-bound form of the Ran GTPase (RanGTP) around chromosomes induces spindle assembly by activating nuclear localization signal (NLS)-containing proteins. Several NLS proteins have been identified as spindle assembly factors, but the complexity of the process led us to search for additional proteins with distinct roles in spindle assembly.
 RESULTS We identify a chromatin-remodeling ATPase, CHD4, as a RanGTP-dependent microtubule (MT)-associated protein (MAP). MT binding occurs via the region containing an NLS and chromatin-binding domains. In Xenopus egg extracts and cultured cells, CHD4 largely dissociates from mitotic chromosomes and partially localizes to the spindle. Immunodepletion of CHD4 from egg extracts significantly reduces the quantity of MTs produced around chromatin and prevents spindle assembly. CHD4 RNAi in both HeLa and Drosophila S2 cells induces defects in spindle assembly and chromosome alignment in early mitosis, leading to chromosome missegregation. Further analysis in egg extracts and in HeLa cells reveals that CHD4 is a RanGTP-dependent MT stabilizer. Moreover, the CHD4-containing NuRD complex promotes organization of MTs into bipolar spindles in egg extracts. Importantly, this function of CHD4 is independent of chromatin remodeling.
 CONCLUSIONS Our results uncover a new role for CHD4 as a MAP required for MT stabilization and involved in generating spindle bipolarity.

title: The RNA Polymerase “Switch Region” Is a Target for Inhibitors

text: The alpha-pyrone antibiotic myxopyronin (Myx) inhibits bacterial RNA polymerase (RNAP). Here, through a combination of genetic, biochemical, and structural approaches, we show that Myx interacts with the RNAP "switch region"--the hinge that mediates opening and closing of the RNAP active center cleft--to prevent interaction of RNAP with promoter DNA. We define the contacts between Myx and RNAP and the effects of Myx on RNAP conformation and propose that Myx functions by interfering with opening of the RNAP active-center cleft during transcription initiation. We further show that the structurally related alpha-pyrone antibiotic corallopyronin (Cor) and the structurally unrelated macrocyclic-lactone antibiotic ripostatin (Rip) function analogously to Myx. The RNAP switch region is distant from targets of previously characterized RNAP inhibitors, and, correspondingly, Myx, Cor, and Rip do not exhibit crossresistance with previously characterized RNAP inhibitors. The RNAP switch region is an attractive target for identification of new broad-spectrum antibacterial therapeutic agents.

title: The ALS-associated proteins FUS and TDP-43 function together to affect Drosophila locomotion and life span.

text: The fatal adult motor neuron disease amyotrophic lateral sclerosis (ALS) shares some clinical and pathological overlap with frontotemporal dementia (FTD), an early-onset neurodegenerative disorder. The RNA/DNA-binding proteins fused in sarcoma (FUS; also known as TLS) and TAR DNA binding protein-43 (TDP-43) have recently been shown to be genetically and pathologically associated with familial forms of ALS and FTD. It is currently unknown whether perturbation of these proteins results in disease through mechanisms that are independent of normal protein function or via the pathophysiological disruption of molecular processes in which they are both critical. Here, we report that Drosophila mutants in which the homolog of FUS is disrupted exhibit decreased adult viability, diminished locomotor speed, and reduced life span compared with controls. These phenotypes were fully rescued by wild-type human FUS, but not ALS-associated mutant FUS proteins. A mutant of the Drosophila homolog of TDP-43 had similar, but more severe, deficits. Through cross-rescue analysis, we demonstrated that FUS acted together with and downstream of TDP-43 in a common genetic pathway in neurons. Furthermore, we found that these proteins associated with each other in an RNA-dependent complex. Our results establish that FUS and TDP-43 function together in vivo and suggest that molecular pathways requiring the combined activities of both of these proteins may be disrupted in ALS and FTD.

title: Caveolin-1 deletion exacerbates cardiac interstitial fibrosis by promoting M2 macrophage activation in mice after myocardial infarction.

text: Adverse remodeling following myocardial infarction (MI) leading to heart failure is driven by an imbalanced resolution of inflammation. The macrophage cell is an important control of post-MI inflammation, as macrophage subtypes secrete mediators to either promote inflammation and extend injury (M1 phenotype) or suppress inflammation and promote scar formation (M2 phenotype). We have previously shown that the absence of caveolin-1 (Cav1), a membrane scaffolding protein, is associated with adverse cardiac remodeling in mice, but the mechanisms responsible remain to be elucidated. We explore here the role of Cav1 in the activation of macrophages using wild type C57BL6/J (WT) and Cav1(tm1Mls/J) (Cav1(-/-)) mice. By echocardiography, cardiac function was comparable between WT and Cav1(-/-) mice at 3days post-MI. In the absence of Cav1, there were a surprisingly higher percentage of M2 macrophages (arginase-1 positive) detected in the infarcted zone. Conversely, restoring Cav1 function after MI in WT mice by adding back the Cav1 scaffolding domain reduced the M2 activation profile. Further, adoptive transfer of Cav1 null macrophages into WT mice on d3 post-MI exacerbated adverse cardiac remodeling at d14 post-MI. In vitro studies revealed that Cav1 null macrophages had a more pronounced M2 profile activation in response to IL-4 stimulation. In conclusion, Cav1 deletion promotes an array of maladaptive repair processes after MI, including increased TGF-β signaling, increased M2 macrophage infiltration and dysregulation of the M1/M2 balance. Our data also suggest that cardiac remodeling can be improved by therapeutic intervention regulating Cav1 function during the inflammatory response phase.


query: Increased microtubule acetylation repairs interference of axonal transport caused by LRRK2 Roc-COR domain mutations.

title: Increasing microtubule acetylation rescues axonal transport and locomotor deficits caused by LRRK2 Roc-COR domain mutations

text: Leucine-rich repeat kinase 2 (LRRK2) mutations are the most common genetic cause of Parkinson's disease. LRRK2 is a multifunctional protein affecting many cellular processes and has been described to bind microtubules. Defective microtubule-based axonal transport is hypothesized to contribute to Parkinson's disease, but whether LRRK2 mutations affect this process to mediate pathogenesis is not known. Here we find that LRRK2 containing pathogenic Roc-COR domain mutations (R1441C, Y1699C) preferentially associates with deacetylated microtubules, and inhibits axonal transport in primary neurons and in Drosophila, causing locomotor deficits in vivo. In vitro, increasing microtubule acetylation using deacetylase inhibitors or the tubulin acetylase αTAT1 prevents association of mutant LRRK2 with microtubules, and the deacetylase inhibitor trichostatin A (TSA) restores axonal transport. In vivo knockdown of the deacetylases HDAC6 and Sirt2, or administration of TSA rescues both axonal transport and locomotor behavior. Thus, this study reveals a pathogenic mechanism and a potential intervention for Parkinson's disease.

title: Mutant dynactin in motor neuron disease

title: Preferential binding of a kinesin-1 motor to GTP-tubulin–rich microtubules underlies polarized vesicle transport

text: Polarized transport in neurons is fundamental for the formation of neuronal circuitry. A motor domain-containing truncated KIF5 (a kinesin-1) recognizes axonal microtubules, which are enriched in EB1 binding sites, and selectively accumulates at the tips of axons. However, it remains unknown what cue KIF5 recognizes to result in this selective accumulation. We found that axonal microtubules were preferentially stained by the anti-GTP-tubulin antibody hMB11. Super-resolution microscopy combined with EM immunocytochemistry revealed that hMB11 was localized at KIF5 attachment sites. In addition, EB1, which binds preferentially to guanylyl-methylene-diphosphate (GMPCPP) microtubules in vitro, recognized hMB11 binding sites on axonal microtubules. Further, expression of hMB11 antibody in neurons disrupted the selective accumulation of truncated KIF5 in the axon tips. In vitro studies revealed approximately threefold stronger binding of KIF5 motor head to GMPCPP microtubules than to GDP microtubules. Collectively, these data suggest that the abundance of GTP-tubulin in axonal microtubules may underlie selective KIF5 localization and polarized axonal vesicular transport.

title: Microtubule stabilization reduces scarring and causes axon regeneration after spinal cord injury.

text: Hypertrophic scarring and poor intrinsic axon growth capacity constitute major obstacles for spinal cord repair. These processes are tightly regulated by microtubule dynamics. Here, moderate microtubule stabilization decreased scar formation after spinal cord injury in rodents through various cellular mechanisms, including dampening of transforming growth factor-β signaling. It prevented accumulation of chondroitin sulfate proteoglycans and rendered the lesion site permissive for axon regeneration of growth-competent sensory neurons. Microtubule stabilization also promoted growth of central nervous system axons of the Raphe-spinal tract and led to functional improvement. Thus, microtubule stabilization reduces fibrotic scarring and enhances the capacity of axons to grow.

title: Dynein is required for polarized dendritic transport and uniform microtubule orientation in axons

text: Axons and dendrites differ in both microtubule organization and in the organelles and proteins they contain. Here we show that the microtubule motor dynein has a crucial role in polarized transport and in controlling the orientation of axonal microtubules in Drosophila melanogaster dendritic arborization (da) neurons. Changes in organelle distribution within the dendritic arbors of dynein mutant neurons correlate with a proximal shift in dendritic branch position. Dynein is also necessary for the dendrite-specific localization of Golgi outposts and the ion channel Pickpocket. Axonal microtubules are normally oriented uniformly plus-end-distal; however, without dynein, axons contain both plus- and minus-end distal microtubules. These data suggest that dynein is required for the distinguishing properties of the axon and dendrites: without dynein, dendritic organelles and proteins enter the axon and the axonal microtubules are no longer uniform in polarity.

title: Vesicular trafficking of semaphorin 3A is activity-dependent and differs between axons and dendrites.

text: Secreted semaphorins act as guidance cues in the developing nervous system and may have additional functions in mature neurons. How semaphorins are transported and secreted by neurons is poorly understood. We find that endogenous semaphorin 3A (Sema3A) displays a punctate distribution in axons and dendrites of cultured cortical neurons. GFP-Sema3A shows a similar distribution and co-localizes with secretory vesicle cargo proteins. Live-cell imaging reveals highly dynamic trafficking of GFP-Sema3A vesicles with distinct properties in axons and dendrites regarding directionality, velocity, mobility and pausing time. In axons, most GFP-Sema3A vesicles move fast without interruption, almost exclusively in the anterograde direction, while in dendrites many GFP-Sema3A vesicles are stationary and move equally frequent in both directions. Disruption of microtubules, but not of actin filaments, significantly impairs GFP-Sema3A transport. Interestingly, depolarization induces a reversible arrest of axonal transport of GFP-Sema3A vesicles but has little effect on dendritic transport. Conversely, action potential blockade using tetrodotoxin (TTX) accelerates axonal transport, but not dendritic transport. These data indicate that axons and dendrites regulate trafficking of Sema3A and probably other secretory vesicles in distinct ways, with axons specializing in fast, uninterrupted, anterograde transport. Furthermore, neuronal activity regulates secretory vesicle trafficking in axons by a depolarization-evoked trafficking arrest.

text: Cytoplasmic dynein is a microtubule-based motor protein that is responsible for most intracellular retrograde transports along microtubule filaments. The motor domain of dynein contains six tandemly linked AAA (ATPases associated with diverse cellular activities) modules, with the first four containing predicted nucleotide-binding/hydrolysis sites (P1-P4). To dissect the functions of these multiple nucleotide-binding/hydrolysis sites, we expressed and purified Dictyostelium dynein motor domains in which mutations were introduced to block nucleotide binding at each of the four AAA modules, and then examined their detailed biochemical properties. The P1 mutant was trapped in a strong-binding state even in the presence of ATP and lost its motile activity. The P3 mutant also showed a high affinity for microtubules in the presence of ATP and lost most of the microtubule-activated ATPase activity, but retained microtubule sliding activity, although the sliding velocity of the mutant was more than 20-fold slower than that of the wild type. In contrast, mutation in the P2 or P4 site did not affect the apparent binding affinity of the mutant for microtubules in the presence of ATP, but reduced ATPase and microtubule sliding activities. These results indicate that ATP binding and its hydrolysis only at the P1 site are essential for the motor activities of cytoplasmic dynein, and suggest that the other nucleotide-binding/hydrolysis sites regulate the motor activities. Among them, nucleotide binding at the P3 site is not essential but is critical for microtubule-activated ATPase and motile activities of cytoplasmic dynein.

title: The Mammalian-Specific Protein Armcx1 Regulates Mitochondrial Transport during Axon Regeneration

text: Mitochondrial transport is crucial for neuronal and axonal physiology. However, whether and how it impacts neuronal injury responses, such as neuronal survival and axon regeneration, remain largely unknown. In an established mouse model with robust axon regeneration, we show that Armcx1, a mammalian-specific gene encoding a mitochondria-localized protein, is upregulated after axotomy in this high regeneration condition. Armcx1 overexpression enhances mitochondrial transport in adult retinal ganglion cells (RGCs). Importantly, Armcx1 also promotes both neuronal survival and axon regeneration after injury, and these effects depend on its mitochondrial localization. Furthermore, Armcx1 knockdown undermines both neuronal survival and axon regeneration in the high regenerative capacity model, further supporting a key role of Armcx1 in regulating neuronal injury responses in the adult central nervous system (CNS). Our findings suggest that Armcx1 controls mitochondrial transport during neuronal repair.

title: ESCRT-III recognition by VPS4 ATPases

title: EMSY overexpression disrupts the BRCA2/RAD51 pathway in the DNA-damage response: implications for chromosomal instability/recombination syndromes as checkpoint diseases

text: EMSY links the BRCA2 pathway to sporadic breast/ovarian cancer. It encodes a nuclear protein that binds to the BRCA2 N-terminal domain implicated in chromatin/transcription regulation, but when sporadically amplified/overexpressed, increased EMSY level represses BRCA2 transactivation potential and induces chromosomal instability, mimicking the activity of BRCA2 mutations in the development of hereditary breast/ovarian cancer. In addition to chromatin/transcription regulation, EMSY may also play a role in the DNA-damage response, suggested by its ability to localize at chromatin sites of DNA damage/repair. This implies that EMSY overexpression may also repress BRCA2 in DNA-damage replication/checkpoint and recombination/repair, coordinated processes that also require its interacting proteins: PALB2, the partner and localizer of BRCA2; RPA, replication/checkpoint protein A; and RAD51, the inseparable recombination/repair enzyme. Here, using a well-characterized recombination/repair assay system, we demonstrate that a slight increase in EMSY level can indeed repress these two processes independently of transcriptional interference/repression. Since EMSY, RPA and PALB2 all bind to the same BRCA2 region, these findings further support a scenario wherein: (a) EMSY amplification may mimic BRCA2 deficiency, at least by overriding RPA and PALB2, crippling the BRCA2/RAD51 complex at DNA-damage and replication/transcription sites; and (b) BRCA2/RAD51 may coordinate these processes by employing at least EMSY, PALB2 and RPA. We extensively discuss the molecular details of how this can happen to ascertain its implications for a novel recombination mechanism apparently conceived as checkpoint rather than a DNA repair system for cell division, survival, death, and human diseases, including the tissue specificity of cancer predisposition, which may renew our thinking about targeted therapy and prevention.

title: Structural basis for the activation of microtubule assembly by the EB1 and p150Glued complex.

text: Plus-end tracking proteins, such as EB1 and the dynein/dynactin complex, regulate microtubule dynamics. These proteins are thought to stabilize microtubules by forming a plus-end complex at microtubule growing ends with ill-defined mechanisms. Here we report the crystal structure of two plus-end complex components, the carboxy-terminal dimerization domain of EB1 and the microtubule binding (CAP-Gly) domain of the dynactin subunit p150Glued. Each molecule of the EB1 dimer contains two helices forming a conserved four-helix bundle, while also providing p150Glued binding sites in its flexible tail region. Combining crystallography, NMR, and mutational analyses, our studies reveal the critical interacting elements of both EB1 and p150Glued, whose mutation alters microtubule polymerization activity. Moreover, removal of the key flexible tail from EB1 activates microtubule assembly by EB1 alone, suggesting that the flexible tail negatively regulates EB1 activity. We, therefore, propose that EB1 possesses an auto-inhibited conformation, which is relieved by p150Glued as an allosteric activator.

title: 6-Sulphated Chondroitins Have a Positive Influence on Axonal Regeneration

text: Chondroitin sulphate proteoglycans (CSPGs) upregulated in the glial scar inhibit axon regeneration via their sulphated glycosaminoglycans (GAGs). Chondroitin 6-sulphotransferase-1 (C6ST-1) is upregulated after injury leading to an increase in 6-sulphated GAG. In this study, we ask if this increase in 6-sulphated GAG is responsible for the increased inhibition within the glial scar, or whether it represents a partial reversion to the permissive embryonic state dominated by 6-sulphated glycosaminoglycans (GAGs). Using C6ST-1 knockout mice (KO), we studied post-injury changes in chondroitin sulphotransferase (CSST) expression and the effect of chondroitin 6-sulphates on both central and peripheral axon regeneration. After CNS injury, wild-type animals (WT) showed an increase in mRNA for C6ST-1, C6ST-2 and C4ST-1, but KO did not upregulate any CSSTs. After PNS injury, while WT upregulated C6ST-1, KO showed an upregulation of C6ST-2. We examined regeneration of nigrostriatal axons, which demonstrate mild spontaneous axon regeneration in the WT. KO showed many fewer regenerating axons and more axonal retraction than WT. However, in the PNS, repair of the median and ulnar nerves led to similar and normal levels of axon regeneration in both WT and KO. Functional tests on plasticity after the repair also showed no evidence of enhanced plasticity in the KO. Our results suggest that the upregulation of 6-sulphated GAG after injury makes the extracellular matrix more permissive for axon regeneration, and that the balance of different CSs in the microenvironment around the lesion site is an important factor in determining the outcome of nervous system injury.

title: Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes

text: Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.

title: KIF3A/B: a heterodimeric kinesin superfamily protein that works as a microtubule plus end-directed motor for membrane organelle transport

text: We cloned a new member of the murine brain kinesin superfamily, KIF3B, and found that its amino acid sequence is highly homologous but not identical to KIF3A, which we previously cloned and named KIF3 (47% identical). KIF3B is localized in various organ tissues and developing neurons of mice and accumulates with anterogradely moving membranous organelles after ligation of nerve axons. Immunoprecipitation assay of the brain revealed that KIF3B forms a complex with KIF3A and three other high molecular weight (approximately 100 kD)-associated polypeptides, called the kinesin superfamily-associated protein 3 (KAP3). In vitro reconstruction using baculovirus expression systems showed that KIF3A and KIF3B directly bind with each other in the absence of KAP3. The recombinant KIF3A/B complex (approximately 50-nm rod with two globular heads and a single globular tail) demonstrated plus end-directed microtubule sliding activity in vitro. In addition, we showed that KIF3B itself has motor activity in vitro, by making a complex of wild-type KIF3B and a chimeric motor protein (KIF3B head and KIF3A rod tail). Subcellular fractionation of mouse brain homogenates showed a considerable amount of the native KIF3 complex to be associated with membrane fractions other than synaptic vesicles. Immunoprecipitation by anti-KIF3B antibody-conjugated beads and its electron microscopic study also revealed that KIF3 is associated with membranous organelles. Moreover, we found that the composition of KAP3 is different in the brain and testis. Our findings suggest that KIF3B forms a heterodimer with KIF3A and functions as a new microtubule-based anterograde translocator for membranous organelles, and that KAP3 may determine functional diversity of the KIF3 complex in various kinds of cells in vivo.

text:
BACKGROUND Microtubules are built from linear polymers of α-β tubulin dimers (protofilaments) that form a tubular quinary structure. Microtubules assembled from purified tubulin in vitro contain between 10 and 16 protofilaments; however, such structural polymorphisms are not found in cells. This discrepancy implies that factors other than tubulin constrain microtubule protofilament number, but the nature of these constraints is unknown.
 RESULTS Here, we show that acetylation of MEC-12 α-tubulin constrains protofilament number in C. elegans touch receptor neurons (TRNs). Whereas the sensory dendrite of wild-type TRNs is packed with a cross-linked bundle of long, 15-protofilament microtubules, mec-17;atat-2 mutants lacking α-tubulin acetyltransferase activity have short microtubules, rampant lattice defects, and variable protofilament number both between and within microtubules. All-atom molecular dynamics simulations suggest a model in which acetylation of lysine 40 promotes the formation of interprotofilament salt bridges, stabilizing lateral interactions between protofilaments and constraining quinary structure to produce stable, structurally uniform microtubules in vivo.
 CONCLUSIONS Acetylation of α-tubulin is an essential constraint on protofilament number in vivo. We propose a structural model in which this posttranslational modification promotes the formation of lateral salt bridges that fine-tune the association between adjacent protofilaments and enable the formation of uniform microtubule populations in vivo.

title: Increased dendritic complexity and axonal length in cultured mouse cortical neurons overexpressing methyl-CpG-binding protein MeCP2

text: Rett syndrome is caused by loss-of-function mutations in the gene encoding the methyl DNA-binding factor MeCP2. As brain mass and neuronal complexity tend to be diminished in Rett patients, we tested whether MeCP2 directly influences the morphological complexity of developing neurons. Our results show that cultured mouse neurons overexpressing MeCP2beta (MECP2A) develop more complex morphologies, having longer axonal and dendritic processes, and an increased number of axonal and dendritic terminal endings. We then tested whether overexpressing a mutant form of MeCP2beta lacking its carboxyl terminus would elicit the same effects. Interestingly, while neurons overexpressing this mutant failed to enhance axonal and dendritic process elongation, the complexity of their axonal and dendritic processes remained significantly elevated. Taken together, these data support the hypothesis that MeCP2 directly regulates neuronal maturation and/or synaptogenesis, and provides evidence that MeCP2 may influence neuritic elongation and process branching through different mechanisms.

title: No Pasaran! Role of the axon initial segment in the regulation of protein transport and the maintenance of axonal identity.

text: The transmission of information in the brain depends on the highly polarized architecture of neurons. A number of cellular transport processes support this organization, including active targeting of proteins and passive corralling between compartments. The axon initial segment (AIS), which separates the somatodendritic and axonal compartments, has a key role in neuronal physiology, as both the initiation site of action potentials and the gatekeeper of the axonal arborization. Over the years, the AIS main components and their interactions have been progressively unraveled, as well as their role in the AIS assembly and maintenance. Two mechanisms have been shown to contribute to the regulation of protein transport at the AIS: a surface diffusion barrier and an intracellular traffic filter. However, a molecular understanding of these processes is still lacking. In the view of recent results on the AIS cytoskeleton structure, we will discuss how a better knowledge of the AIS architecture can help understanding its role in the regulation of protein transport and the maintenance of axonal identity.

title: Vesicular Glycolysis Provides On-Board Energy for Fast Axonal Transport

text: Fast axonal transport (FAT) requires consistent energy over long distances to fuel the molecular motors that transport vesicles. We demonstrate that glycolysis provides ATP for the FAT of vesicles. Although inhibiting ATP production from mitochondria did not affect vesicles motility, pharmacological or genetic inhibition of the glycolytic enzyme GAPDH reduced transport in cultured neurons and in Drosophila larvae. GAPDH localizes on vesicles via a huntingtin-dependent mechanism and is transported on fast-moving vesicles within axons. Purified motile vesicles showed GAPDH enzymatic activity and produced ATP. Finally, we show that vesicular GAPDH is necessary and sufficient to provide on-board energy for fast vesicular transport. Although detaching GAPDH from vesicles reduced transport, targeting GAPDH to vesicles was sufficient to promote FAT in GAPDH deficient neurons. This specifically localized glycolytic machinery may supply constant energy, independent of mitochondria, for the processive movement of vesicles over long distances in axons.

title: A Microtubule Interactome: Complexes with Roles in Cell Cycle and Mitosis

text: The microtubule (MT) cytoskeleton is required for many aspects of cell function, including the transport of intracellular materials, the maintenance of cell polarity, and the regulation of mitosis. These functions are coordinated by MT-associated proteins (MAPs), which work in concert with each other, binding MTs and altering their properties. We have used a MT cosedimentation assay, combined with 1D and 2D PAGE and mass spectrometry, to identify over 250 MAPs from early Drosophila embryos. We have taken two complementary approaches to analyse the cellular function of novel MAPs isolated using this approach. First, we have carried out an RNA interference (RNAi) screen, identifying 21 previously uncharacterised genes involved in MT organisation. Second, we have undertaken a bioinformatics analysis based on binary protein interaction data to produce putative interaction networks of MAPs. By combining both approaches, we have identified and validated MAP complexes with potentially important roles in cell cycle regulation and mitosis. This study therefore demonstrates that biologically relevant data can be harvested using such a multidisciplinary approach, and identifies new MAPs, many of which appear to be important in cell division.

title: The potential role of miRNAs 21 and 199-a in early diagnosis of hepatocellular carcinoma.

text:
BACKGROUND Hepatocellular carcinoma (HCC) is regarded as one of the most common malignancies and among the leading causes of cancer death among the whole world. The most urgent needs are to find sensitive markers for early diagnosis for HCC. MicroRNAs (miRNAs) are reported as a group of small non-coding RNAs that can function as endogenous RNA interference to regulate expression of the targeted genes. This study was conducted to detect the serum and tissue expression of miR 21 and miR 199-a to be applied as early detectors for HCC.
 METHODS A total of 40 serum and tissue samples (17 samples from chronic hepatitis and 23 samples from HCC patients) were collected. The levels of the two mature miRNAs (miR-21 and miR-199-a) were detected by real time quantitative reverse-transcriptase PCR (RT-qPCR) in sera and tissues of chronic hepatitis and HCC patients. Besides, miR-21 and miR-199-a levels in relation to clinical and pathological factors were explored.
 RESULTS We found that the expression of serum miR-21 was distinctly increased in HCC compared with chronic hepatitis (P<0.001). miR 199-a was distinctly decreased in HCC compared with chronic hepatitis (P<0.001). In addition, median of miR 21 was increased in malignant when compared to adjacent non-malignant tissues without significant differences (P=0.191) while miR 199-a was significantly decreased in malignant when compared to adjacent nonmalignant tissues (P<0.001). ROC analysis showed that miR-21 and miR-199-a might be potential biomarkers for HCC.
 CONCLUSIONS In conclusion, the expression of miR-21 was significantly up-regulated and miR-199-a was significantly down regulated in serum of patients with HCC. Due to their reasonable sensitivity and specificity for disease progression, miR-21 and miR-199-a could be used as potential circulating biomarkers for HCC.


query: Increased microtubule acetylation worsens interference of axonal transport caused by LRRK2 Roc-COR domain mutations.

text: Leucine-rich repeat kinase 2 (LRRK2) mutations are the most common genetic cause of Parkinson's disease. LRRK2 is a multifunctional protein affecting many cellular processes and has been described to bind microtubules. Defective microtubule-based axonal transport is hypothesized to contribute to Parkinson's disease, but whether LRRK2 mutations affect this process to mediate pathogenesis is not known. Here we find that LRRK2 containing pathogenic Roc-COR domain mutations (R1441C, Y1699C) preferentially associates with deacetylated microtubules, and inhibits axonal transport in primary neurons and in Drosophila, causing locomotor deficits in vivo. In vitro, increasing microtubule acetylation using deacetylase inhibitors or the tubulin acetylase αTAT1 prevents association of mutant LRRK2 with microtubules, and the deacetylase inhibitor trichostatin A (TSA) restores axonal transport. In vivo knockdown of the deacetylases HDAC6 and Sirt2, or administration of TSA rescues both axonal transport and locomotor behavior. Thus, this study reveals a pathogenic mechanism and a potential intervention for Parkinson's disease.

title: Mutant dynactin in motor neuron disease

text: Impaired axonal transport in motor neurons has been proposed as a mechanism for neuronal degeneration in motor neuron disease. Here we show linkage of a lower motor neuron disease to a region of 4 Mb at chromosome 2p13. Mutation analysis of a gene in this interval that encodes the largest subunit of the axonal transport protein dynactin showed a single base-pair change resulting in an amino-acid substitution that is predicted to distort the folding of dynactin's microtubule-binding domain. Binding assays show decreased binding of the mutant protein to microtubules. Our results show that dysfunction of dynactin-mediated transport can lead to human motor neuron disease.

text: Polarized transport in neurons is fundamental for the formation of neuronal circuitry. A motor domain-containing truncated KIF5 (a kinesin-1) recognizes axonal microtubules, which are enriched in EB1 binding sites, and selectively accumulates at the tips of axons. However, it remains unknown what cue KIF5 recognizes to result in this selective accumulation. We found that axonal microtubules were preferentially stained by the anti-GTP-tubulin antibody hMB11. Super-resolution microscopy combined with EM immunocytochemistry revealed that hMB11 was localized at KIF5 attachment sites. In addition, EB1, which binds preferentially to guanylyl-methylene-diphosphate (GMPCPP) microtubules in vitro, recognized hMB11 binding sites on axonal microtubules. Further, expression of hMB11 antibody in neurons disrupted the selective accumulation of truncated KIF5 in the axon tips. In vitro studies revealed approximately threefold stronger binding of KIF5 motor head to GMPCPP microtubules than to GDP microtubules. Collectively, these data suggest that the abundance of GTP-tubulin in axonal microtubules may underlie selective KIF5 localization and polarized axonal vesicular transport.

title: Distinct functions of nucleotide-binding/hydrolysis sites in the four AAA modules of cytoplasmic dynein.

title: ESCRT-III recognition by VPS4 ATPases

text: The ESCRT (endosomal sorting complex required for transport) pathway is required for terminal membrane fission events in several important biological processes, including endosomal intraluminal vesicle formation, HIV budding and cytokinesis. VPS4 ATPases perform a key function in this pathway by recognizing membrane-associated ESCRT-III assemblies and catalysing their disassembly, possibly in conjunction with membrane fission. Here we show that the microtubule interacting and transport (MIT) domains of human VPS4A and VPS4B bind conserved sequence motifs located at the carboxy termini of the CHMP1–3 class of ESCRT-III proteins. Structures of VPS4A MIT–CHMP1A and VPS4B MIT–CHMP2B complexes reveal that the C-terminal CHMP motif forms an amphipathic helix that binds in a groove between the last two helices of the tetratricopeptide-like repeat (TPR) of the VPS4 MIT domain, but in the opposite orientation to that of a canonical TPR interaction. Distinct pockets in the MIT domain bind three conserved leucine residues of the CHMP motif, and mutations that inhibit these interactions block VPS4 recruitment, impair endosomal protein sorting and relieve dominant-negative VPS4 inhibition of HIV budding. Thus, our studies reveal how the VPS4 ATPases recognize their CHMP substrates to facilitate the membrane fission events required for the release of viruses, endosomal vesicles and daughter cells.

title: Posttranslational Acetylation of α-Tubulin Constrains Protofilament Number in Native Microtubules

title: Kinetics of early T cell receptor signaling regulate the pathway of lytic granule delivery to the secretory domain.

text: Cytolytic granules mediate killing of virus-infected cells by cytotoxic T lymphocytes. We show here that the granules can take long or short paths to the secretory domain. Both paths utilized the same intracellular molecular events, which have different spatial and temporal arrangements and are regulated by the kinetics of Ca(2+)-mediated signaling. Rapid signaling caused swift granule concentration near the microtubule-organizing center (MTOC) and subsequent delivery by the polarized MTOC directly to the secretory domain-the shortest path. Indolent signaling led to late recruitment of granules that moved along microtubules to the periphery of the synapse and then moved tangentially to fuse at the outer edge of the secretory domain-a longer path. The short pathway is associated with faster granule release and more efficient killing than the long pathway. Thus, the kinetics of early signaling regulates the quality of the T cell cytolytic response.

title: Acetylation controls Notch3 stability and function in T-cell leukemia

text: Post-translational modifications of Notch3 and their functional role with respect to Notch3 overexpression in T-cell leukemia are still poorly understood. We identify here a specific novel property of Notch3 that is acetylated and deacetylated at lysines 1692 and 1731 by p300 and HDAC1, respectively, a balance impaired by HDAC inhibitors (HDACi) that favor hyperacetylation. By using HDACi and a non-acetylatable Notch3 mutant carrying K/R1692−1731 mutations in the intracellular domain, we show that Notch3 acetylation primes ubiquitination and proteasomal-mediated degradation of the protein. As a consequence, Notch3 protein expression and its transcriptional activity are decreased both in vitro and in vivo in Notch3 transgenic (tg) mice, thus impairing downstream signaling upon target genes. Consistently, Notch3-induced T-cell proliferation is inhibited by HDACi, whereas it is enhanced by the non-acetylatable Notch3-K/R1692−1731 mutant. Finally, HDACi-induced Notch3 hyperacetylation prevents in vivo growth of T-cell leukemia/lymphoma in Notch3 tg mice. Together, our findings suggest a novel level of Notch signaling control in which Notch3 acetylation/deacetylation process represents a key regulatory switch, thus representing a suitable druggable target for Notch3-sustained T-cell acute lymphoblastic leukemia therapy.

title: Drosophila MICAL regulates myofilament organization and synaptic structure

text: The overall size and structure of a synaptic terminal is an important determinant of its function. In a large-scale mutagenesis screen, designed to identify Drosophila mutants with abnormally structured neuromuscular junctions (NMJs), we discovered mutations in Drosophila mical, a conserved gene encoding a multi-domain protein with a N-terminal monooxygenase domain. In mical mutants, synaptic boutons do not sprout normally over the muscle surface and tend to form clusters along synaptic branches and at nerve entry sites. Consistent with high expression of MICAL in somatic muscles, immunohistochemical stainings reveal that the subcellular localization and architecture of contractile muscle filaments are dramatically disturbed in mical mutants. Instead of being integrated into a regular sarcomeric pattern, actin and myosin filaments are disorganized and accumulate beneath the plasmamembrane. Whereas contractile elements are strongly deranged, the proposed organizer of sarcomeric structure, D-Titin, is much less affected. Transgenic expression of interfering RNA molecules demonstrates that MICAL is required in muscles for the higher order arrangement of myofilaments. Ultrastructural analysis confirms that myosin-rich thick filaments enter submembranous regions and interfere with synaptic development, indicating that the disorganized myofilaments may cause the synaptic growth phenotype. As a model, we suggest that the filamentous network around synaptic boutons restrains the spreading of synaptic branches.