QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Stein, P. A. Articles by Rapoport, T. A. Search for Related Content PubMed PubMed Citation Articles by Stein, P. A. Articles by Rapoport, T. A.

A novel centrosome-associated protein with affinity for microtubules

¹ Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115-6091, USA
² Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115-6091, USA

View larger version (50K): [in a new window]   Fig. 1. Clone VLP27 encodes a novel microtubule-binding protein, MIR1. (A) The clone VLP27 (GFP-MIR1) was expressed at medium or low levels in BHK cells (first and third panel). The same cells were also stained with the tubulin antibody M1 (second and fourth panel). The magnification was 63x for the left two panels and 100x for the right ones. The arrows point to MIR1 staining of individual microtubules. (B) The diagram of the homology between MIR1 and its closest relative MID1/midin. The domain configuration is identical, except for the added P-stretch region and the missing N-terminal RING finger and B-Boxes in MIR1. (C) Northern blot of various human tissues probed against MIR1 RNA. (D) Immunoblot analysis of post-nuclear lysates from selected cells using the affinity-purified antibody Ab284 directed against MIR1. Lysate from BHK cells expressing untagged MIR1 was analyzed in parallel.

View larger version (39K): [in a new window]   Fig. 6. Microtubule association of overexpressed MIR1 point mutants. (A) The scheme shows point mutants made in the P-stretch of MIR1. MIR1A is a mutant in which all potential cdk phosphorylation sites were abolished. MIR1DE is a mutant that mimics the phosphorylated state. The scheme also shows the deletion mutants made. The N-terminal tag was either CFP, YFP, HA or 6xHis (full-length constructs only). (B) GFP-MIR1A was expressed in BHK cells during interphase or metaphase in mitosis. The cells were analyzed both for GFP fluorescence and for microtubules with a tubulin antibody. They were also stained for DNA. The arrows point to a mitotic cell showing the association of GFP-MIR1A with microtubules. (C) GFP-MIR1DE was expressed in interphase BHK cells. The cells were analyzed for GFP fluorescence and microtubule and DNA staining. The arrows point to a transfected cell showing the loss of association of GFP-MIR1DE with microtubules.

View larger version (32K): [in a new window]   Fig. 2. Endogenous MIR1 is localized at the centrosome. (A) To test the suitability of MIR1 antibodies in immunofluorescence experiments, GFP-MIR1 was expressed in BHK cells. Methanol-fixed cells were processed for immunofluorescence with either Ab284 (left column) or Ab284 that had previously been preadsorbed to recombinant MIR1 protein (right column). The arrow points to a cell that shows GFP fluorescence but no staining with the pre-saturated antibody. Exposure times and image processing parameters were kept constant for all four panels to allow direct comparison (63xfield). (B) Endogenous MIR1 in U2OS cells was visualized after fixation with methanol by immunofluorescence microscopy using Ab284. A low magnification view (upper right panel) shows prominent staining in the perinuclear region. A control shows staining with Ab284 that had previously been preadsorbed to recombinant MIR1 protein (lower right panel) (40x field). Bar 10 µm. A higher magnification picture is shown to the left. Co-staining with an antibody to acetylated tubulin reveals the location of the centrosome (arrows) (100x field). (C) MIR1 staining in different cell types was analyzed after fixation in methanol by immunofluorescence with Ab284 (rhodamine-conjugated secondary antibody). The cells were also stained with mAb GTU-88 to -tubulin (AlexaFluor-488-conjugated secondary antibody). Images, which were acquired at 100x on a Deltavision system, present a collapsed view of multiple processed sections. Bar, 5 µm. (D) MIR1 localization in U2OS cells persists at the centrosome in the absence of a microtubule network. MIR1 was stained with Ab284 and -tubulin with mAb GTU-88. (E) MIR1 localization with Ab520. Fixed U373 cells were stained for MIR1 with Ab520 and for centrosomes with an -centrin antibody (left panels). Samples stained with M1 and Ab520 reveal MIR1 localized along individual microtubules as distinct spots (arrows in right panels, enlarged view). Images, acquired at 100x on a Deltavision system, are of a particular Z-section and have been processed by with an iterative deconvolution algorithm. (F) MIR1 localization in U373 cells during different stages of the cell cycle. Fixed cells were stained for MIR1 with Ab284 anf for centrosomal antigens with human autoimmune serum 5051. The cells were also stained for DNA with Hoechst dye 33258. The arrow shows a cell going through the cell cycle. The arrowhead in the last panel shows another cell loss of MIR1 localization at the spindle poles in metaphase (100x field). (G) A higher magnification view of U373 cells in telophase. Staining was performed with Ab284 for MIR1 and with mAb GTU-88 for -tubulin. DNA was stained with Hoechst dye 33258. A Deltavision image was analyzed as in Fig. 1C. MIR1 staining is present at the centrosomes (arrows) and at the midbody (arrowhead) (100x field).

View larger version (91K): [in a new window]   Fig. 3. Overexpressed MIR1 localizes to newly polymerized microtubules. BHK cells transfected with GFP-MIR1 were treated with nocadozole to depolymerize the microtubules. The drug was washed out and the microtubules allowed to repolymerize for the indicated times. The cells were fixed and analyzed for MIR1 by GFP fluorescence (left column) and for tubulin using the M1 antibody (right column). Arrows highlight transfected cells with newly formed microtubules.

View larger version (52K): [in a new window]   Fig. 4. Microtubules are long-lived in interphase cells overexpressing MIR1. BHK cells, overexpressing GFP-MIR1, were analyzed both by GFP fluorescence and by immunofluorescence with antibody 6-11B-1, which recognizes acetylated tubulin. The arrow indicates a transfected cell containing long-lived, acetylated microtubules. The arrowhead points to an untransfected cell.

View larger version (100K): [in a new window]   Fig. 5. The association of overexpressed MIR1 with microtubules is dependent on the cell cycle stage. BHK cells expressing GFP-MIR1 were analyzed at different stages of the cell cycle by GFP fluorescence and by staining with tubulin antibody M1. The cells were also stained for DNA with Hoechst dye 33258.

View larger version (58K): [in a new window]   Fig. 7. Neuronal kinase cdk5 can modulate the affinity of MIR1 for microtubules. BHK cells were transfected with cdk5, its activator p35 and GFP-MIR1. Controls were performed with an inactive version of cdk5 (cdk5DN) and with GFP-MIR1A instead of the wild-type MIR1 fusion. Samples were processed for immunofluorescence microscopy 12 hours after transfection. The cells were analyzed for GFP fluorescence and for staining of microtubules. The arrow points to a cell expressing active cdk5 that shows diffuse MIR1 staining. The arrowheads point to cells expressing either inactive cdk5 or a non-phosphorylatable form of MIR1; these cells show colocalization of MIR1 and microtubules.

View larger version (66K): [in a new window]   Fig. 8. Effect of overexpression of MIR1 on mitotic spindles. (A) Mitotic BHK cells expressing GFP-MIR1 (middle panel) or GFP-MIR1A (bottom panel) were analyzed for GFP fluorescence (green) and stained for -tubulin with mAb GTU-88 (red) and for DNA with Hoechst dye 33258 (blue). Images were acquired with a Deltavision system and processed as in Fig. 2C. (B) Mitotic BHK cells expressing GFP-MIR1 were analyzed for GFP fluorescence and for stable microtubules using an antibody 6-11B-1 against acetylated tubulin. (C) As in B, but with cells expressing GFP-MIR1A. The arrows point to a transfected cell, the arrowhead to an untransfected cell.

View larger version (60K): [in a new window]   Fig. 9. Domain analysis of MIR1. (A) BHK cells were transfected with YFP fusions with different domains of MIR1 (Fig. 6A). The cells were analyzed for YFP fluorescence and for tubulin. The arrow indicates concentration of the C-terminal domains at the centrosome. (B) BHK cells were transfected with CFP fusions with N-terminal fragments of MIR1 (tN and tNP; Fig. 6A), as well as with YFP fusions with C-terminal fragments (tPC and tC). The cells were analyzed by CFP and YFP fluorescence and by staining for tubulin. The arrow points to cells in which the wild-type phenotype of microtubule bundling is restored by the simultaneous expression of N- and C-terminal domains that both contain the P-stretch. (C) BHK cells were transfected with a CFP fusion to a MIR1 fragment lacking the coiled-coil domain (delCC; Fig. 6A). The cells were analyzed for CFP fluorescence and for -tubulin staining with mAb GTU-88. CFP-delCC is visible in filaments and bundled structures (arrow heads), as well as at the centrosome (arrows). Colocolization with -tubulin is evident in the enlarged field. (D) MIR1 was expressed in a baculovirus system, purified, and subjected to sucrose gradient centrifugation. Fractions were analyzed by immunoblotting with MIR1 antibodies. Molecular weight standards were run in paralle. (E) Full-length MIR1 (wt) or a C-terminal truncation (tC) were synthesized in an in vitro translation system in the presence of 35S-methionine. The labeled proteins were combined with an excess of microtubules polymerized from purified tubulin (0.5 mg/ml), and binding was assessed in a microtubule co-sedimentation assay.