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. 2012 Jan 3;109(1):185-90.
doi: 10.1073/pnas.1118560109. Epub 2011 Dec 21.

Cellulose synthase interactive protein 1 (CSI1) links microtubules and cellulose synthase complexes

Shundai Li  1 , Lei Lei, Chris R Somerville, Ying Gu
Affiliations

Cellulose synthase interactive protein 1 (CSI1) links microtubules and cellulose synthase complexes

Shundai Li et al. Proc Natl Acad Sci U S A. .

Abstract

Cellulose synthase (CESA) complexes can be observed by live-cell imaging to move with trajectories that parallel the underlying cortical microtubules. Here we report that CESA interactive protein 1 (CSI1) is a microtubule-associated protein that bridges CESA complexes and cortical microtubules. Simultaneous in vivo imaging of CSI1, CESA complexes, and microtubules demonstrates that the association of CESA complexes and cortical microtubules is dependent on CSI1. CSI1 directly binds to microtubules as demonstrated by in vitro microtubule-binding assay.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
CSI1 colocalized with cortical microtubules. (A) Two-channel confocal imaging of epidermal cells in 3-d-old dark-grown hypocotyls expressing markers for cortical microtubules (YFP-TUA5) and CSI1 (RFP-CSI1). (Scale bar, 5 μm.) (B) Plot of a line scan showing a strong correlation between the spatial localization of YFP-TUA5 and RFP-CSI1. (C) Colocalization analysis of YFP-TUA5 and RFP-CSI1. White dots represent colocalized RFP-CSI1 with microtubules. RFP-CSI1 particles that did not colocalize with microtubules are green. Analysis was performed in six cells from six seedlings (Table 1). YFP-TUA5 is displayed in pseudocolor red and RFP-CSI1 is displayed in green for better visualization in colocalization analysis. (Scale bar, 10 μm.) (D) Time-average image showing that CSI1 moved along the underlying microtubules. Average of 31 frames (duration 150 s, 5-s interval) shows identical localization of YFP-TUA5 and RFP-CSI1. (Scale bar, 10 μm.)
Fig. 2.
Fig. 2.
CSI1 is a microtubule-binding protein. (A) Microtubule-binding assay. Coomassie-stained gels show supernatants and pellets after the microtubule-binding assay. S, supernatant fraction; P, pellet fraction; + or −, presence or absence of microtubules in the assay. The positions of positive control (MAP2), negative control (BSA), tubulin, and CSI1 are indicated by arrows. (B) Quantitative analysis of the binding properties between CSI1 and microtubules. The disassociation constant (Kd) for CSI1, determined by best fit to the data, is 1.07 ± 0.33 μM. The data were collected from three technical replicates.
Fig. 3.
Fig. 3.
Temporal distinction in localization changes upon oryzalin treatment. (A) 2-d-old dark-grown seedlings coexpressing GFP-CESA6 and RFP-CSI1 were incubated in Murashige and Skoog liquid solution containing 0.1% methanol (control) or 20 μM oryzalin. Single frame shows distribution of RFP-CSI1 and GFP-CESA6. Time average of 61 frames (5-min duration, 5-s interval) shows linear trajectories of RFP-CSI1 and GFP-CESA6. (Scale bar, 10 μm.) (B) Histogram of GFP-CESA6 particle velocities in mock control or oryzalin treatment for indicated time.
Fig. 4.
Fig. 4.
Mis-alignment of CESA complexes and cortical microtubules in csi1. Single optical section of epidermal cells in 3-d-old dark-grown hypocotyls expressing RFP-TUA5 and YFP-CESA6 in wild type (A–D) or csi1-3 (E–H). (A and E) RFP-TUA5. (B and F) YFP-CESA6. (C and G) Merge. (D) Representative image from five cells used for colocalization analysis (Table 1). In the wild-type cell shown here, observed coincidence is 71% and expected random coincidence is 42%. (H) Representative image from six cells used for colocalization analysis (Table 1). In the csi1-3 cell shown here, 60/150 (40%) of YFP-CESA6 particles were coaligned with microtubules in cells, which is not significantly different from the expected random coincidence of 69/178 (39%). White dots represent YFP-CESA6 particles that coincide with microtubules. YFP-CESA6 particles that did not colocalize with microtubules are green. (Scale bar, 5 μm.)
Fig. 5.
Fig. 5.
Colocalization of CESA complexes, CSI1, and cortical microtubules. Quantification of colocalization pattern in three-channel imaging of epidermal cells expressing CFP-TUA1, YFP-CESA6, and RFP-CSI1 (n = 3 cells from three seedlings). Shown is a representative image from three cells used for colocalization analysis. White dots represent colocalized CFP-TUA1, YFP-CESA6, and RFP-CSI1. Orange dots represent colocalized YFP-CESA6 and RFP-CSI1 but not CFP-TUA1. Pink dots represent colocalized RFP-CSI1 and CFP-TUA1 but not YFP-CESA6. Cyan dots represent colocalized CFP-TUA1 and YFP-CESA6 but not RFP-CSI1. Blue dots represent YFP-CESA6 that did not colocalize with others. Purple dots represent RFP-CSI1 that did not colocalize with others. (Scale bar, 10 μm.)
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