Template-free 13-protofilament microtubule–MAP assembly visualized at 8 A resolution

Microtubule-associated proteins (MAPs) are essential for regulating and organizing cellular microtubules (MTs). However, our mechanistic understanding of MAP function is limited by a lack of detailed structural information. Using cryo-electron microscopy and single particle algorithms, we solved the 8 Å structure of doublecortin (DCX)-stabilized MTs. Because of DCX’s unusual ability to specifically nucleate and stabilize 13-protofilament MTs, our reconstruction provides unprecedented insight into the structure of MTs with an in vivo architecture, and in the absence of a stabilizing drug. DCX specifically recognizes the corner of four tubulin dimers, a binding mode ideally suited to stabilizing both lateral and longitudinal lattice contacts. A striking consequence of this is that DCX does not bind the MT seam. DCX binding on the MT surface indirectly stabilizes conserved tubulin–tubulin lateral contacts in the MT lumen, operating independently of the nucleotide bound to tubulin. DCX’s exquisite binding selectivity uncovers important insights into regulation of cellular MTs

PCNA and DNA Polymerase δ Catalytic Subunit fromSchizosaccharomyces pombeDo Not Interact Directly

International audienceDNA polymerase delta (pol delta) is constituted of at least two subunits: the catalytic subunit of about 125 kDa (p125), and a subunit of approximately 50 kDa (p50) of unknown function. Processivity of pol delta is dependent on its auxiliary protein PCNA (proliferating cell nuclear antigen). Contradictory data were reported regarding a direct interaction between p125 and PCNA. We investigated this matter further using the baculovirus system to overexpress p125 and PCNA from S. pombe. We show that the recombinant p125 is active for basal DNA polymerase activity and for 3'-->5' exonuclease activity but is not stimulated by PCNA. Interaction between p125 and PCNA was tested by: (i) co-immunoprecipitation assay using antibodies specific for one or other polypeptides after co-expression in insect cells, and (ii) a two-hybrid assay. In both cases, no direct interaction between the two proteins was detected. Taken together, our data show that p125 and PCNA do not interact directly

Isolation of Basic FGF Receptors from Adult Bovine Brain Membranes

International audienc

Human RTEL1 deficiency causes Hoyeraal- Hreidarsson syndrome with short telomeres and genome instability

International audienceHoyeraal-Hreidarsson syndrome (HHS), a severe variant of dyskeratosis congenita (DC), is characterized by early onset bone marrowfailure, immunodeficiency and developmental defects. Several factors involved in telomere length maintenance and/or protection are defective in HHS/DC, underlining the relationship between telomeredysfunctionandthese diseases.Bycombining whole-genomelinkage analysisandexomesequencing,we identified compound heterozygous RTEL1 (regulator of telomere elongation helicase 1) mutations in three patients with HHS from two unrelated families. RTEL1 is a DNA helicase that participates in DNA replication, DNA repair and telomere integrity. We show that, in addition to short telomeres, RTEL1-deficient cells from patients exhibit hallmarks of genome instability, including spontaneous DNA damage, anaphase bridges and telomeric aberrations. Collectively, these results identify RTEL1 as a novel HHS-causing gene and highlight its role as a genomic caretaker in humans

Genome-wide Control of Heterochromatin Replication by the Telomere Capping Protein TRF2

International audienc

Structural basis for ARF1-mediated recruitment of ARHGAP21 to Golgi membranes

ARHGAP21 is a Rho family GTPase-activating protein (RhoGAP) that controls the Arp2/3 complex and F-actin dynamics at the Golgi complex by regulating the activity of the small GTPase Cdc42. ARHGAP21 is recruited to the Golgi by binding to another small GTPase, ARF1. Here, we present the crystal structure of the activated GTP-bound form of ARF1 in a complex with the Arf-binding domain (ArfBD) of ARHGAP21 at 2.1 Å resolution. We show that ArfBD comprises a PH domain adjoining a C-terminal α helix, and that ARF1 interacts with both of these structural motifs through its switch regions and triggers structural rearrangement of the PH domain. We used site-directed mutagenesis to confirm that both the PH domain and the helical motif are essential for the binding of ArfBD to ARF1 and for its recruitment to the Golgi. Our data demonstrate that two well-known small GTPase-binding motifs, the PH domain and the α helical motif, can combine to create a novel mode of binding to Arfs

Distinct roles of doublecortin modulating the microtubule cytoskeleton

Doublecortin is a neuronal microtubule-stabilising protein, mutations of which cause mental retardation and epilepsy in humans. How doublecortin influences microtubule dynamics, and thereby brain development, is unclear. We show here by video microscopy that purified doublecortin has no effect on the growth rate of microtubules. However, it is a potent anti-catastrophe factor that stabilises microtubules by linking adjacent protofilaments and counteracting their outward bending in depolymerising microtubules. We show that doublecortin-stabilised microtubules are substrates for kinesin translocase motors and for depolymerase kinesins. In addition, doublecortin does not itself oligomerise and does not bind to tubulin heterodimers but does nucleate microtubules. In cells, doublecortin is enriched at the distal ends of neuronal processes and our data raise the possibility that the function of doublecortin in neurons is to drive assembly and stabilisation of non-centrosomal microtubules in these doublecortin-enriched distal zones. These distinct properties combine to give doublecortin a unique function in microtubule regulation, a role that cannot be compensated for by other microtubule-stabilising proteins and nucleating factors