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First published online 28 October 2008
doi: 10.1242/jcs.038497

Journal of Cell Science 121, 3834-3841 (2008)
Published by The Company of Biologists 2008
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Ncd motor binding and transport in the spindle

Mark A. Hallen, Zhang-Yi Liang and Sharyn A. Endow*

Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA


Figure 1
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  		Fig. 1. Ncd in the meiotic and mitotic spindle. (A) Ncd constructs. FLNcdVenus consists of an N-terminal basic, proline-rich tail, {alpha}-helical coiled-coil stalk (grey) and conserved motor domain or head, with the Venus fluorescent protein (green) at the C terminus. HLNcdVenus is deleted for the head and TLNcdVenus, for the tail. (B) FLncdVenus; cand oocyte meiosis I spindle (top), abnormal multi-polar and multiple small HLncdVenus; cand meiosis I spindles (middle) and absence of TLNcdVenus fluorescence in a TLncdVenus; ncdmRFP cand meiosis I spindle (bottom). Scale bars: 5 µm. (C) Prometaphase to telophase in a HLncdVenus cycle 10 (left) and TLncdVenus cycle 9 (right) mitotic division. Abnormal fused, spurred, bridged or bent spindles, or failure of midzone to form (arrows). Time, minutes:seconds. Scale bar: 10 µm.

 

Figure 2
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  		Fig. 2. Spindle length in FLncdVenus and HLncdVenus embryos. (A) Centrosome-to-centrosome spindle length in cycle 10 embryos from prometaphase to telophase. Data are the mean ± s.e.m. (B) Spindle images from time-lapse sequences. Note the difference in scale between the FLncdVenus and HLncdVenus images. ProM, prometaphase; M, metaphase; A, anaphase; T, telophase. Scale bars: 5 µm.

 

Figure 3
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  		Fig. 3. NcdVenus fluorescence recovery in the mitotic spindle. (A) FLncdVenus spindle FRAP assays at the equator in a small (radius, w=1.3 µm) (top) and large (w=2.66 µm) (bottom) ROI. (B) Mean fluorescence recovery curves (magenta; small ROI, n=5; large ROI, n=11) after normalization to the first prebleach image and correction for fluorescence loss during recovery imaging. Inset, curve fits to the new diffusion-binding-transport model (dark green). (C) HLncdVenus spindle FRAP assays at the equator in a small (w=1.3 µm) (top) and large (w=2.66 µm) (bottom) ROI. (D) Mean fluorescence recovery curves (purple; small ROI, n=8; large ROI, n=10) after normalization and correction for loss during imaging. Inset, curve fits to the new diffusion-binding-transport model (dark green). (E) TLncdVenus spindle FRAP assays at the equator in a small (w=1.3 µm) (top) and large (w=2.66 µm) (bottom) ROI. (F) Mean fluorescence recovery curves (violet; small ROI, n=8; large ROI, n=11) after normalization and correction for loss during imaging. Inset, curve fits to the diffusion-binding model (Sprague et al., 2004Go) (dark green). Scale bars: 3 µm.

 

Figure 4
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  		Fig. 4. Ncd transport in the mitotic spindle. (A) Median fluorescence position in FLncdVenus cycle 10 mitotic spindles versus time from early metaphase through anaphase. Distance from a point outside the pole in each half-spindle (n=14), normalized from zero to one, and averaged. Data are the mean ± s.e.m. The approximate time at which FRAP assays were performed is indicated. (B) Images from time-lapse sequences showing the equator (red line) and median fluorescence position in each half-spindle (blue lines) at the start, maximal position towards the equator, and end. Arrows (blue) in A indicate the times at which the images were acquired. Bar, 3 µm.

 

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© The Company of Biologists Ltd 2008