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First published online 14 April 2008
doi: 10.1242/jcs.023374

Journal of Cell Science 121, 1506-1513 (2008)
Published by The Company of Biologists 2008
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A new role for kinesin-directed transport of Bik1p (CLIP-170) in Saccharomyces cerevisiae

Fabrice Caudron, Annie Andrieux, Didier Job and Cécile Boscheron*

INSERM, U836, Groupe de Physiopathologie du Cytosquelette, Grenoble, F-38043, France; CEA, iRTSV, Grenoble, F-38054, France; Université Joseph Fourier, Institut des Neurosciences, Grenoble, F-38043, France


Figure 1
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  		Fig. 1. Bik1p localization is Bim1p-dependent in Glu tubulin strains. (A) Images of representative cells from different backgrounds expressing Bik1p-3GFP from the native locus. From left to right: Bik1p-3GFP fluorescence (green); Spc42p-RedStar fluorescence (red); GFP and RedStar merged imaged. Arrows indicate the plus end of astral MTs. Scale bar: 3 µm. (B) Shows the quantification of fluorescence intensity at the plus ends (+ ends) of astral MTs and at the SPB. 25-35 cells were scored for each strain. The bars indicate the s.e.m. (C) Left: western blotting of whole-cell extracts with anti-GFP against Bik1p-3GFP and with YoL1/34 against {alpha}-tubulin. Right: ratio between the anti-GFP signal and the anti-{alpha}-tubulin signal obtained for four independent western blotting experiments. The bars indicate the s.e.m. Strains are: (1) TUB1, (2) TUB1 bim1{Delta}, (3) tub1-Glu and (4) tub1-Glu bim1{Delta}.

 

Figure 2
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  		Fig. 2. Kip2p in Glu tubulin strains. (A) Merged images of representative cells of the indicated diploid strains, expressing Kip2p-3YFP (green) and Spc42p-RedStar (red). Arrows indicates the plus ends of astral MTs. (B) Shows the quantification of Kip2p-3YFP fluorescence intensity at the plus ends (+ ends) of astral MTs in the indicated strains. 50 cells were scored for each strain. The bars indicate the s.e.m. (C,D) Kip2p-3YFP localizes to plus ends of both growing (C) and shrinking (D) MTs in Glu tubulin cells. (E) Illustration of Kip2p-3YFP speckle movement on Glu MTs. (F) Illustration of Bik1p-3GFP speckle movement on Glu MTs. For the diploid cells in C-F, arrows indicate the plus end of astral MTs, arrowheads indicate the SPB and double arrowheads highlight speckles moving towards the plus ends. Time is in seconds. Scale bars: 3 µm.

 

Figure 3
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  		Fig. 3. Dynein in Glu tubulin strains. (A) Merged images of representative cells expressing Dyn1p-3GFP (green) and Spc42p-RedStar (red). The diploid strains are indicated. Arrows indicate the plus end of astral MTs. Scale bar: 3 µm. (B) Shows the quantification of Dyn1p-3GFP fluorescence intensity at the plus ends of astral MTs in the indicated strains. 50 cells were scored for each strain. No signal was detectable in Bik1-deleted cells. The bars indicate the s.e.m. N.D., not detectable. Fluorescence is in arbitrary units. (C,D) Dyn1p-3GFP localizes to the plus ends of both growing (C) and shrinking (D) MTs in Glu tubulin cells. (E) Illustration of Dyn1p-3GFP speckle movement on Glu MTs. For the diploid cells shown in C-E, arrows indicate the plus end of astral MTs, arrowheads indicate the SPB and double arrowheads highlight speckles moving towards the plus ends. Time is in seconds. Scale bars: 3 µm. (F) Shows the quantification of Bik1p-3GFP fluorescence intensity at the plus ends of astral MTs in the indicated strain. Fluorescence is in arbitrary units. 50 cells were scored for each strain. Bars are s.e.m. N.D., not detectable.

 

Figure 4
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  		Fig. 4. Mitosis in the tub1-Glu bim1{Delta} kip2{Delta} strain. We monitored mitosis in the tub1-Glu bim1{Delta} kip2{Delta} strain using GFP-tub1-Glu fusion integrated at the URA3 locus. (A) Viable cells from the tub1-Glu bim1{Delta} kip2{Delta} strain grew as well as the corresponding wild-type-tubulin TUB1 bim1{Delta} kip2{Delta} strain. (B) Illustration of MT behaviour. (Left) Contrast-phase image of cells; (right) fluorescent image of tub1-Glu-GFP cells. Astral MTs are visible at different cell stages (arrows). (C) Selected frames from a representative time-lapse series used to analyze mitosis. Astral MTs are not detectable. We imaged spindles that elongate in the mother cell. Finding the neck lumen was apparently problematic for the spindle and this delayed the penetration of the elongating spindle into the bud. (D) Selected frames from a representative time-lapse series of spindle bending in the mother cell. (B-D) The time is in hours:minutes. Scale bars: 3 µm. (E) Quantitative analysis of the percentage of spindles in contact with the cell cortex prior to passage of the spindle through the neck. 15 films were scored for each strain. Bars are s.e.m.

 

Figure 5
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  		Fig. 5. Schematic representation of the molecular interactions at MT plus ends. (A) Current models: the principal pathway for Bik1p localization involves Bik1p transport at plus ends as a cargo of Kip2p. Upon arrival at plus ends, Bik1p recruits dynein, which presumably also interacts with Pac1p. The Bik1p-dynein complex tracks plus ends as a cargo of Kip2p. (B) Present model: in wild-type (wt) strains, Bik1p is transported towards plus ends as a cargo of Kip2p, as in A. Additionally, dynein is recruited by Bik1p during end-ward transport. Upon arrival at plus ends, the Bik1p-Kip2p complex dissociates, and MT end tracking by Bik1p and Kip2p are distinct processes. Bik1p end tracks MTs based on the properties of its CAP-Gly domain, in a complex with dynein and, presumably, Pac1p. (C) Glu MTs: the Glu tubulin mutation disrupts the interaction between Bik1p and tubulin. As a result, Bik1p cannot track MT plus ends. Dynein, by contrast, still tracks plus ends, possibly in a complex with Pac1p. (D) KIP2-deleted strains: if Bik1p is present at plus ends either based on intrinsic interaction with tubulin (wild-type strains) or as a hitch-hiker of Bim1p (Glu tubulin strains), or through both mechanisms, then dynein can be directly recruited at plus ends by Bik1p. Note that Bim1p might localize Bik1p through mechanisms other than hitch-hiking, which is proposed here for the sake of simplicity. Note that D can co-exist with B and C.

 

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© The Company of Biologists Ltd 2008