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First published online 31 October 2006
doi: 10.1242/jcs.03261

Journal of Cell Science 119, 4710-4718 (2006)
Published by The Company of Biologists 2006
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Pom1 kinase links division plane position to cell polarity by regulating Mid1p cortical distribution

Séverine Celton-Morizur*, Victor Racine, Jean-Baptiste Sibarita and Anne Paoletti{ddagger}

Institut Curie, Centre de Recherche and CNRS UMR144, Paris, 75248 cedex 05, France


Figure 1
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  		Fig. 1. Cortical mid1-4GFP expands towards the non-growing cell end in pom1{Delta} mutants. (A) GFP fluorescence in living wild-type (left panel-AP 1442) and pom1{Delta} cells (right panel-AP 1502) expressing mid1-4GFP, grown at 25°C. Bar, 5 µm. (B) GFP (upper panel) and fluostain I fluorescence (lower panel) in living pom1{Delta} cells expressing mid1-4GFP. Note that mid1-4GFP is present at the non-growing tip. Bar, 5 µm. (C) Distribution of mid1-4GFP in highly elongated pom1{Delta} cells obtained by treatment with 11 mM HU for 5 hours at 25°C. Note that HU treatment increases the pool of nuclear mid1-4GFP. mid1-4GFP spots are less abundant in the tip region compared with the medial or lateral cortex. Bar, 5 µm. (D) mid1-4GFP fluorescence profile along the cortex in wild-type (left panel) and pom1{Delta} cells (middle and right panels). Cortical intensity of mid1-4GFP was measured clockwise along the entire cell cortex of cells shown on the bottom, starting at the left cell tip. Dotted lines represent the position of the right cell tip. (E) Distribution of mid1-4GFP cortical spots in wild-type and pom1{Delta} cells. 223 wild-type and 268 pom1{Delta} cells from three independent experiments were analyzed. The presence of one or more mid1-4GFP spots at the medial (a), lateral (b) and tip (c) cortex (see scheme on the right) was scored in each cell, and correlated to cell length to monitor cell-cycle progression. Typical a-, abc- and ab-cells are shown in D (from left to right).

 

Figure 2
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  		Fig. 2. Contractile ring position is uncoupled from nuclear position in pom1{Delta} mutant. (A) Time-lapse video-microscopy of wild-type (a) or pom1{Delta} cells (b,c) that express mid1-4GFP and were grown at 25°C during entry into mitosis. Cells were imaged for GFP fluorescence every 4 minutes. Note that, in pom1{Delta} cells the contractile ring forms away from the position of the premitotic nucleus (star), closer to the non-growing end where mid1-4GFP spots were distributed before mitosis. Bar, 5 µm. (B) Localization of myo2p in the pom1{Delta} mutant: myo2-GFP fluorescence in pom1{Delta} cells grown at 25°C (left) and time-lapse video-microscopy of a similar cell in early mitosis (right). Note the asymmetrical distribution of myo2-GFP cortical spots in early mitosis (arrow). In the early mitotic cell on the right, asymmetrically distributed spots of myo2-GFP (t=0-6 minutes) compact into an offset tight ring (t=14 minutes) that contracts (t=34 and 50 minutes) to form two daughter cells of unequal size (t=78 minutes). Star, position of the premitotic nucleus. Bar, 5 µm.

 

Figure 3
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  		Fig. 3. Mid1p is located at the medial cortex before NETO. GFP fluorescence (a,c) and fluostain I staining of the cell wall (b,d) in wild-type cells expressing mid1-4GFP (AP1442) grown at 25°C, or wild-type cells grown for 3 hours in presence (c,d) or absence (a,b) of 11 mM HU to prevent DNA synthesis and to block cells before NETO. mid1-4GFP spots are medially located in monopolarly growing cells after HU treatment (arrowheads), indicating that Mid1p distribution to the medial cortex is established before NETO. Bar, 5 µm.

 

Figure 4
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  		Fig. 4. Distribution of mid1-4GFP in late mitosis in wild-type and pom1{Delta} cells. (A) Time-lapse video-microscopy of wild-type (a) or pom1{Delta} cells (b-c) expressing mid1-4GFP grown at 25°C in late mitosis. Bar, 5 µm. (B,C) Distribution of mid1-4GFP cortical spots in living wild-type (B) and pom1{Delta} cells (C) during late anaphase and septation. Note that cortical spots of mid1-4GFP are frequently observed at the old end in a pom1{Delta} background. Bar, 5 µm.

 

Figure 5
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  		Fig. 5. Distribution of mid1-4GFP in other polarity mutants. (A) GFP fluorescence in live bud6{Delta} (AP1758), tea3{Delta} (AP1717), tea1{Delta} (AP1727) and pom1{Delta}tea3{Delta} (AP1737) strains expressing mid1-4GFP, grown at 25°C. Bar, 5 µm. (B) Distribution of mid1-4GFP spots on the cortex of wild-type, bud6{Delta}, tea3{Delta}, tea1{Delta}, pom1{Delta} and pom1{Delta}tea3{Delta} cells. Presence of one or more spots of mid1-4GFP at the medial (a), lateral (b) and tip (c) cortex was recorded in 300 to 400 cells from three to four independent experiments. Error bars give standard deviation (s.d.). (C) Localization of pom1-GFP in wild-type (AP1329) and tea1{Delta} cells (AP1760) grown at 25°C. In tea1{Delta} cells, pom1-GFP is still consistently detected at the cell tips and in the region of septum formation. Bar, 5 µm. (D) Septum position defects in polarity mutants. Cells were grown at 25°C and stained for septa with fluostain I. The percentage of abnormally placed or oriented septa was recorded in 400 to 800 septated cells from two to four independent experiments. Error bars give s.d. 1, wild type; 2, bud6{Delta}; 3, tea3{Delta}; 4, tea1{Delta}; 5, pom1{Delta}; 6, pom1{Delta}tea3{Delta}. (E) mid1-4GFP expression level in polarity mutants. Western blot analysis of Mid1p [FC418 strain (Celton-Morizur et al., 2004Go), first lane and left arrow] and mid1-4GFP expression levels (right arrow) in wild type (1), bud6{Delta} (2), tea3{Delta} (3), tea1{Delta} (4), pom1{Delta} (5) and pom1{Delta}tea3{Delta} (6) strains grown at 25°C using affinity purified anti-Mid1p antibody and TAT1 monoclonal antibody against tubulin.

 

Figure 6
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  		Fig. 6. Automated analysis of the distribution of mid1-4GFP cortical spots. (A) After manual selection (a), and determination of cell medial and long axes by ellipse fitting (b), coordinates of cortical spots (c) and nuclei (d) on cell axes were automatically extracted. (B) Distribution of distances between cortical spots (left) or spots centroids (right) and the medial axis in wild-type (n=107), bud6{Delta} (n=96), tea3{Delta} (n=95), tea1{Delta} (n=103), pom1{Delta} (n=77) and pom1{Delta}tea3{Delta} (n=94) cells expressing mid1-4GFP, grown at 25°C. Spots centroids represent the mean distribution point of spots in individual cells. (C) Percentage of off-set spots (2 µm apart from the medial axis or more) or spots centroids (0.75 µm apart from the medial axis or more) in wild-type (1), bud6{Delta} (2), tea3{Delta} (3), tea1{Delta} (4), pom1{Delta} (5) and pom1{Delta}tea3{Delta} (6) cells expressing mid1-4GFP, grown at 25°C.

 

Figure 7
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  		Fig. 7. A model of negative regulation of Mid1p cortical distribution. In wild-type cells, Pom1 kinase (green) is delivered to the cell tips in a microtubule-dependent manner. Before NETO (top), Pom1p excludes Mid1p (red) from the non-growing tip while at the growing tip (blue arrow), other polarity factors (blue) may act in conjunction with Pom1p. These two mechanisms operate at the two growing tips after NETO (middle). This allows the formation of a medial cortical band of Mid1p in early phases of the cell cycle and yields to the formation of a centrally placed contractile ring during mitosis (bottom). In pom1-deficient cells, which have a monopolar pattern of growth, Mid1p is only excluded from the growing tips. Mid1p asymmetrical distribution is responsible for the displacement of the contractile ring towards the non-growing tip of the cell during mitosis. Microtubules (orange) are represented during interphase with their plus ends facing cell tips and their minus ends anchored on the nuclear envelope (black circle) and during mitosis (bottom) as they form an intranuclear mitotic spindle.

 

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© The Company of Biologists Ltd 2006