Decreased MCM2-6 in Drosophila S2 cells does not generate significant DNA damage or cause a marked increase in sensitivity to replication interference.

A reduction in the level of some MCM proteins in human cancer cells (MCM5 in U20S cells or MCM3 in Hela cells) causes a rapid increase in the level of DNA damage under normal conditions of cell proliferation and a loss of viability when the cells are subjected to replication interference. Here we show that Drosophila S2 cells do not appear to show the same degree of sensitivity to MCM2-6 reduction. Under normal cell growth conditions a reduction of >95% in the levels of MCM3, 5, and 6 causes no significant short term alteration in the parameters of DNA replication or increase in DNA damage. MCM depleted cells challenged with HU do show a decrease in the density of replication forks compared to cells with normal levels of MCM proteins, but this produces no consistent change in the levels of DNA damage observed. In contrast a comparable reduction of MCM7 levels has marked effects on viability, replication parameters and DNA damage in the absence of HU treatment

M phase phosphoprotein 1 is a human plus-end-directed kinesin-related protein required for cytokinesis.

International audienceThe human M phase phosphoprotein 1 (MPP1), previously identified through a screening of a subset of proteins specifically phosphorylated at the G2/M transition (Matsumoto-Taniura, N., Pirollet, F., Monroe, R., Gerace, L., and Westendorf, J. M. (1996) Mol. Biol. Cell 7, 1455-1469), is characterized as a plus-end-directed kinesin-related protein. Recombinant MPP1 exhibits in vitro microtubule-binding and microtubule-bundling properties as well as microtubule-stimulated ATPase activity. In gliding experiments using polarity-marked microtubules, MPP1 is a slow molecular motor that moves toward the microtubule plus-end at a 0.07 microm/s speed. In cycling cells, MPP1 localizes mainly to the nuclei in interphase. During mitosis, MPP1 is diffuse throughout the cytoplasm in metaphase and subsequently localizes to the midzone to further concentrate on the midbody. MPP1 suppression by RNA interference induces failure of cell division late in cytokinesis. We conclude that MPP1 is a new mitotic molecular motor required for completion of cytokinesis

Etude sur l'inhibiteur de la ribonuclease: son role eventuel dans la maladie d'Alzheimer, etude d'un peptide trypsique montant une activite inhibitrice vis-a-vis de la ribonuclease A

SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : T 83934 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Single-headed mode of kinesin-5

In most organisms, kinesin-5 motors are essential for mitosis and meiosis, where they crosslink and slide apart the antiparallel microtubule half-spindles. Recently, it was shown using single-molecule optical trapping that a truncated, double-headed human kinesin-5 dimer can step processively along microtubules. However, processivity is limited (~8 steps) with little coordination between the heads, raising the possibility that kinesin-5 motors might also be able to move by a nonprocessive mechanism. To investigate this, we engineered single-headed kinesin-5 dimers. We show that a set of these single-headed Eg5 dimers drive microtubule sliding at about 90% of wild-type velocity, indicating that Eg5 can slide microtubules by a mechanism in which one head of each Eg5 head-pair is effectively redundant. On the basis of this, we propose a muscle-like model for Eg5-driven microtubule sliding in spindles in which most force-generating events are single-headed interactions and alternate-heads processivity is rare

DmRecQ4 localisation does not change after H₂O₂ or etoposide treatment.

<p>S2 cells expressing the full length DmRecQ4 protein 1 h after exposure to etoposide were subjected to detergent washes and then fixed and analysed for DNA (LHS) using DAPI or DmRecQ4 (RHS) by detection of the V5 tag on the fusion protein.</p

DmRecQ4 depleted cells show abnormal loading of key replication proteins.

<p>Chromatin was prepared from untreated S2 cells, cells that had been treated with HU for 24 h and cells which had been challenged with dsRNA corresponding to the N terminus of DmRecQ4 3 days after the challenge. Chromatin samples were analysed for the presence of PCNA and DNA polymerase alpha using the corresponding antibodies on western blots. In each case equal numbers of cells were subject to chromatin extraction and in addition the loading was confirmed using antibodies against the Orc2 protein.</p

DmRecQ4 proteins without the SLD2 domain cannot rescue either cell cycle or proliferation defects while those without the Drosophila specific domain, or containing only the first 1234aa show differential effects on these parameters.

<p>A) Diagrammatic representation to show the deletions of the SLD2 homology domain, the Drosophila specific domain (228–610) and the 1234–1579 region. B) Cells expressing full length DmRecQ4 either with (red) or without (blue) the SLD2 domain were challenged with dsRNA corresponding to the N (NRecQ4) and C (CRecQ4) terminal regions of DmRecQ4. Cell proliferation was measured by cell count on days 4, 5 and 6. C) Control cells (S2) and cells expressing full length DmRecQ4 either with (FL) or without (FΔSld2) the SLD2 domain were challenged with dsRNA corresponding to the N terminal region of DmRecQ4.The cell cycle profile of the cells was measured by FACS analysis cell on days 0, 4, 5 and 6. D) Control cells (S2), cells expressing the Drosophila specific deletion DmRecQ4 (Δ228–610) and cells expressing the first 1234aa only (Δ1234–1579) were challenged with dsRNA corresponding to the N (NRecQ4) and C (CRecQ4) terminal regions of DmRecQ4. Cell proliferation was measured by cell count on days 3, 5 and 7. E) Control cells (S2), cells expressing the Drosophila specific deletion DmRecQ4 (Δ228–610) and cells expressing the first 1234aa only (D1234–1579) were challenged with dsRNA corresponding to the N (NRecQ4) and C (CRecQ4) terminal regions of DmRecQ4. The cell cycle profile of the cells was measured by FACS analysis on days 0, 3, 5 and 7.</p

Expression of full length, N and C terminal regions in S cells.

<p>A) Diagrammatic representation of DmRecQ4 deletions constructed. B) Western blots to determine the localisation of endogenous DmRecQ4 and overexpressed full length, C terminal and N terminal fragments of DmRecQ4. Wt S2 cells or cells expressing the full length (FL), N terminal (N) or C terminal (C) DmRecQ4 were either added directly to loading buffer to make the total extract (whole) or fractionated as describe in the materials and methods to produce cytoplasmic (cyto), nuclear (NE) and pellet fractions. Western blots of these fractions were analysed with affinity purified DmRecQ4 antibody to detect the endogenous protein, V5 antibody to detect the DmRecQ4 fusion, tubulin antibody as a loading control for the whole extracts, and tubulin and Orc2 antibodies to confirm that the fractionation was successful.</p

DmRecQ4 accumulates in the nucleus soon after UV treatment in a manner dependent on the presence of the C terminus.

<p>A) Untreated (top) or UV treated (80 KJ/M²)(bottom) S2 cells stably expressing the full length DmRecQ4 protein were subjected to detergent washes and then fixed and analysed for DNA (LHS) using DAPI or DmRecQ4 (RHS) by detection of the V5 tag on the fusion protein. B) Untreated (-) or UV treated (80 KJ/M²) (+) S2 cells stably expressing the isolated N (N) or C (C)) terminus of the DmRecQ4 protein were subjected to detergent washes and then fixed and analysed for DNA (LHS) using DAPI or DmRecQ4 (RHS) by detection of the V5 tag on the fusion protein.</p

Reduction of DmRecQ4 in S2 cells interferes with cell proliferation and DNA synthesis.

<p>A) Total extracts of S2 cells which had been mock depleted (control) or depleted of DmRecQ4 by dsRNA from the N terminal (NRecQ4) or C terminal (CRecQ4) region DmRecQ4 were analysed by western blotting with affinity purified anti-DmRecQ4 antibody. In each case extracts from 2.5×10 5 cells were loaded. The DmRecQ4 specific band is shown in the top panel and the bottom panel shows the tubulin control. B) Equal numbers of cells were treated with dsRNA against 2 different regions DmRecQ4 (N terminal – NRecQ4 and C terminal – CRecQ4), control dsRNA (TTC4) or left untreated (no RNA) and the proliferation of the cells was measured by performing a cell count on days 3,4, 5 and 6. C) Untreated cells and Cells treated with dsRNA against 2 different regions DmRecQ4 (N terminal – NRecQ4 and C terminal – CRecQ4) or against a control dsRNA were analysed for their cell cycle distribution using FACS analysis on days 3, 4, 5 and 6. D) Untreated cells and cells which had been exposed to dsRNA corresponding the N terminus of DmRecQ4 or control dsRNA were analysed for BrdU incorporation by dot blot titration 3 days after addition of dsRNA as described in the materials and methods.</p