Blt1 and Mid1 Provide Overlapping Membrane Anchors To Position the Division Plane in Fission Yeast

Spatial control of cytokinesis is essential for proper cell division. The molecular mechanisms that anchor the dynamic assembly and constriction of the cytokinetic ring at the plasma membrane remain unclear. In the fission yeast Schizosaccharomyces pombe, the cytokinetic ring is assembled in the cell middle from cortical node precursors that are positioned by the anillin-like protein Mid1. During mitotic entry, cortical nodes mature and then compact into a contractile ring positioned in the cell middle. The molecular link between Mid1 and medial cortical nodes remains poorly defined. Here we show that Blt1, a previously enig- matic cortical node protein, promotes the robust association of Mid1 with cortical nodes. Blt1 interacts with Mid1 through the RhoGEF Gef2 to stabilize nodes at the cell cortex during the early stages of contractile ring assembly. The Blt1 N terminus is re- quired for localization and function, while the Blt1 C terminus promotes cortical localization by interacting with phospholipids. In cells lacking membrane binding by both Mid1 and Blt1, nodes detach from the cell cortex and generate aberrant cytokinetic rings. We conclude that Blt1 acts as a scaffolding protein for precursors of the cytokinetic ring and that Blt1 and Mid1 provide overlapping membrane anchors for proper division plane positioning

Spatial Control of Cytokinesis by Cdr2 Kinase and Mid1/Anillin Nuclear Export

International audienc

Spindle pole cohesion requires glycosylation-mediated localization of NuMA

Glycosylation is critical for the regulation of several cellular processes. One glycosylation pathway, the unusual O-linked β-N-acetylglucosamine glycosylation (O-GlcNAcylation) has been shown to be required for proper mitosis, likely through a subset of proteins that are O-GlcNAcylated during metaphase. As lectins bind glycosylated proteins, we asked if specific lectins interact with mitotic O-GlcNAcylated proteins during metaphase to ensure correct cell division. Galectin-3, a small soluble lectin of the Galectin family, is an excellent candidate, as it has been previously described as a transient centrosomal component in interphase and mitotic epithelial cells. In addition, it has recently been shown to associate with basal bodies in motile cilia, where it stabilizes the microtubule-organizing center (MTOC). Using an experimental mouse model of chronic kidney disease and human epithelial cell lines, we investigate the role of Galectin-3 in dividing epithelial cells. Here we find that Galectin-3 is essential for metaphase where it associates with NuMA in an O-GlcNAcylation-dependent manner. We provide evidence that the NuMA-Galectin-3 interaction is important for mitotic spindle cohesion and for stable NuMA localization to the spindle pole, thus revealing that Galectin-3 is a novel contributor to epithelial mitotic progress

Temporal Control of Contractile Ring Assembly by Plo1 Regulation of Myosin II Recruitment by Mid1/Anillin

International audienc

A Small Nucleolar RNA Linked to Diamond-Blackfan Anemia

Résumé de congrè

C11ORF24 Is a Novel Type I Membrane Protein That Cycles between the Golgi Apparatus and the Plasma Membrane in Rab6-Positive Vesicles

<div><p>The Golgi apparatus is an intracellular compartment necessary for post-translational modification, sorting and transport of proteins. It plays a key role in mitotic entry through the Golgi mitotic checkpoint. In order to identify new proteins involved in the Golgi mitotic checkpoint, we combine the results of a knockdown screen for mitotic phenotypes and a localization screen. Using this approach, we identify a new Golgi protein C11ORF24 (NP_071733.1). We show that C11ORF24 has a signal peptide at the N-terminus and a transmembrane domain in the C-terminal region. C11ORF24 is localized on the Golgi apparatus and on the <i>trans-</i>Golgi network. A large part of the protein is present in the lumen of the Golgi apparatus whereas only a short tail extends into the cytosol. This cytosolic tail is well conserved in evolution. By FRAP experiments we show that the dynamics of C11ORF24 in the Golgi membrane are coherent with the presence of a transmembrane domain in the protein. C11ORF24 is not only present on the Golgi apparatus but also cycles to the plasma membrane <i>via</i> endosomes in a pH sensitive manner. Moreover, via video-microscopy studies we show that C11ORF24 is found on transport intermediates and is colocalized with the small GTPase RAB6, a GTPase involved in anterograde transport from the Golgi to the plasma membrane. Knocking down C11ORF24 does not lead to a mitotic phenotype or an intracellular transport defect in our hands. All together, these data suggest that C11ORF24 is present on the Golgi apparatus, transported to the plasma membrane and cycles back through the endosomes by way of RAB6 positive carriers.</p> </div

C11ORF24 is not necessary for the formation of RAB6 positive transport carriers.

<p>(A) After a 48 hours knockdown by shRNA HeLa cells were transfected with GFP-RAB6 and the next day they were imaged every second for 30 seconds by spinning disk microscopy. Snap shots from the Movies S1 and S2 are presented for the control shRNA (upper lines) compared to the C11ORF24 shRNA (lower lines). (B) The number of RAB6 positive transport carrier per cell and the speed of theses carriers was then quantified for both treatments from 3 independent experiments and expressed as a percentage of the control. The number of cells is indicated for each treatment on the graphs (n). The error bars represent the SEM.</p

C11ORF24 has a long luminal domain and a short cytosolic tail.

<p>(A) Cartoon of a cell expressing GFP-tagged GalT or C11ORF24 and stained with an anti-GFP antibody and an anti-GM130 antibody. When the cells are not permeabilized (left) the antibodies don’t bind their targets. After saponin permeabilization (middle) all antibodies are able to reach their targets since all membranes are permeabilized. After digitonin permeabilization (right) only the cytosolic epitopes (GM130) are labeled whereas the luminal epitopes (GFP of GalT or C11ORF24) are protected by the intact Golgi membranes. Protocol modified from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082223#B18" target="_blank">18</a>] (B) HeLa cells expressing GFP-GalT (a-c) or GFP-C11ORF24 (d-f) were stained with GFP and GM130 antibodies prior to fixation (a, d) or after fixation and permeabilization with saponin (b,e) or after fixation and permeabilization with digitonin (c,f). The GFP of C11ORF24 is accessible for the anti GFP antibody only after permeabilization of the Golgi membrane. (C) HeLa cells expressing GFP-GalT were stained with GFP and C11ORF24 antibodies prior to fixation (a) or after fixation and permeabilization with saponin (b) or after fixation and permeabilization with digitonin (c). The epitope of the C11ORF24 antibody is accessible only after permeabilization of the Golgi membrane. (D) HeLa cells were either transfected with GFP-RAB6 (line 1) or GFP-C11ORF24 (line 2) 18h prior to the incubation with the anti-GFP and internalization was performed at 37°C for 90 minutes. Cells were then fixed and stained with a secondary antibody and the localization of the anti-GFP antibody was compared with the GFP signal.</p

C11ORF24 is present on RAB6 positive transport carriers.

<p>(A) HeLa cells expressing GFP-C11ORF24 were imaged every second for 5 minutes by spinning disk microscopy. The red arrow indicates a small transport carrier whereas the green arrow point to a long tube connected to the Golgi apparatus. A magnified view of these structures is shown in line 2 and 3. (B) HeLa cells were fixed and co-stained with TGN46, C11ORF24 and GTP-RAB6. Colocalization is very strong between TGN46, C11ORF24 and GTP-RAB6. A line profile of the C11ORF24 (green), TGN46 (red) and GTP-RAB6 (blue) fluorescence intensities from the lines in the magnified views are shown in column 7. (C) HeLa cells expressing GFP-C11ORF24 and mCherry-RAB6 were imaged every second for 5 minutes by spinning disk microscopy. The arrow indicates a transport carrier positive for GFP-C11ORF24 (green) and for mCherry-RAB6 (red). A magnified view of this structure is shown lines 4-6. (D) Transport carriers were visualized using a kymograph that was made along the line drawn on the merge image. All the dynamic elements positive for GFP-C11ORF24 (left and merge green) were positive for mCherry-RAB6 (middle and merge red).</p

C11ORF24 sequence.

<p>(A) Putative domains of C11ORF24. SP: signal peptide, GFP: green fluorescent protein, TM: transmembrane domain, Tail: cytosolic tail. The numbers indicate the amino-acid position in the native protein. The putative signal peptide is underlined and colored in dark red and the putative transmembrane domain is underlined and colored in light blue. The tail domain is color-coded using Clustal W2 to highlight the similarities between different species. The position of the fragment used to raise the monoclonal antibody is indicated by the black bar. (B) The Homo sapiens (hs) C11ORF24 was aligned against its putative homologues in Mus musculus (mm, NP_082353.1) and <i>Dano </i><i>rerio</i> (<i>Dr</i>, XP_690904.4) using Clustal W2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082223#B13" target="_blank">13</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082223#B14" target="_blank">14</a>]. The cytosolic tail domain is well conserved in evolution.</p