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, 16 (4), 1584-92

A Minus-End-Directed Kinesin With Plus-End Tracking Protein Activity Is Involved in Spindle Morphogenesis

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A Minus-End-Directed Kinesin With Plus-End Tracking Protein Activity Is Involved in Spindle Morphogenesis

J Christian Ambrose et al. Mol Biol Cell.

Abstract

Diverse kinesin motor proteins are involved in spindle function; however, the mechanisms by which they are targeted to specific sites within spindles are not well understood. Here, we show that a fusion between yellow fluorescent protein (YFP) and a minus-end-directed Kinesin-14 (C-terminal family) from Arabidopsis, ATK5, localizes to mitotic spindle midzones and regions rich in growing plus-ends within phragmoplasts. Notably, in Arabidopsis interphase cells, YFP::ATK5 localizes to microtubules with a preferential enrichment at growing plus-ends; indicating ATK5 is a plus-end tracking protein (+TIP). This +TIP activity is conferred by regions outside of the C-terminal motor domain, which reveals the presence of independent plus-end tracking and minus-end motor activities within ATK5. Furthermore, mitotic spindles of atk5 null mutant plants are abnormally broadened. Based on these data, we propose a model in which ATK5 uses plus-end tracking to reach spindle midzones, where it then organizes microtubules via minus-end-directed motor activity.

Figures

Figure 1.
Figure 1.
YFP::ATK5 localizes to microtubules in vivo and is concentrated at growing plus-ends. (A) Cartoon representation of the YFP::ATK5 construct used for transformation and stable expression in A. thaliana plants and cultured BY-2 cells. (B) YFP::ATK5 localization in Arabidopsis leaf guard cell. YFP::ATK5 localizes along the length of microtubules and is enriched at microtubule plus ends (arrowheads). (C) Slices from a time series showing plus-end enrichment of YFP::ATK5 fluorescence on a microtubule. Plus-end enrichment is lost upon microtubule catastrophe (asterisk). (D) Fluorescence profiles of YFP::ATK5 along a microtubule in growth state (diamonds) and shrinkage state (squares). Bars, 10 μm (B), 2.5 μm (C). Time scale, 35 s (C).
Figure 2.
Figure 2.
YFP::ATK5 localizes to mitotic spindles and phragmoplasts in BY-2 cells, with enrichment at spindle midzones and phragmoplast leading edges. During metaphase (top row), YFP::ATK5 localizes preferentially to microtubules in the spindle midzone (arrow). During anaphase (middle row), YFP::ATK5 remains localized on overlapping sectors of interpolar microtubules in the spindle midzone (arrow), whereas the kinetochore microtubules have moved to the poles. During cytokinesis (bottom row), YFP::ATK5 localizes to the phragmoplast advancing edge (dotted line), which contains numerous polymerizing microtubule plus-ends. Bar, 10 μm.
Figure 3.
Figure 3.
ATK5 is a minus-end–directed motor. Minus-end–labeled taxol-stabilized microtubules move with their plus-ends leading over a surface coated with cleared bacterial lysates from Escherichia coli expressing recombinant GST::ATK5, indicating the bound kinesin exhibits minus-end–directed motility. Bar, 1 μm.
Figure 4.
Figure 4.
YFP::TS localizes to microtubules in vivo and is concentrated at growing plus-ends. (A) Cartoon representation of YFP::TS construct used for transformation and stable expression in Arabidopsis plants and cultured BY-2 cells. (B) YFP::TS localization in an Arabidopsis hypocotyl epidermal cell. YFP::TS localizes to microtubules and is enriched at microtubule plus-ends (arrowheads). (C) Slices from a time series showing plus-end enrichment of YFP::TS fluorescence on a microtubule. Plus-end accumulation decreases upon microtubule catastrophe (asterisk) and is restored with rescue (arrow). (D) Slices from a time series showing localization of YFP::TS to dynamic microtubules. Arrows indicate rescue, asterisks indicate catastrophe. Bar, 10 μm (B), 2.5 μm (C), and 5 μm (D). Time scale (t), 25 s (C), 75 s (D).
Figure 5.
Figure 5.
YFP::TS localizes to mitotic spindles and phragmoplasts in BY-2 cells, with enrichment at spindle midzones and phragmoplast leading edges. All localization patterns are similar to that of full-length YFP::ATK5. During metaphase (top row), YFP::TS localizes preferentially to microtubules in the spindle midzone (arrow). During anaphase (middle row), YFP::TS remains localized on overlapping sectors of interpolar microtubules in the spindle midzone (arrow), whereas the kinetochore microtubules have moved to the poles. During cytokinesis (bottom row), YFP::TS localizes to the phragmoplast's advancing edge (dotted line). Bar, 10 μm.
Figure 6.
Figure 6.
atk5-1 and atk5-2 are insertional null alleles of ATK5. (A) Diagram showing locations of each T-DNA insertion. Exons are indicated by boxes, introns by lines. (B) RT-PCR showing loss of ATK5 transcript in atk5-1 and atk5-2 null mutants, relative to control (APT1).
Figure 7.
Figure 7.
Plants lacking ATK5 contain abnormally broadened mitotic spindles. (A) Metaphase spindle from a wild-type plant. (B) Metaphase spindle from an atk5-1 mutant. (C) Anaphase spindle from a wild-type plant. (D) Anaphase spindle from an atk5-1 mutant. Bar, 5 μm. Microtubules, green; chromatin, blue.
Figure 8.
Figure 8.
ATK5 mutant spindles are wider at both the midzone and the poles. (A) Mean spindle widths during metaphase and anaphase in wild-type and atk5-1 mutant plants. Dotted lines indicate measured parameters. Error bars indicate SE.
Figure 9.
Figure 9.
Hypothetical model for ATK5 function. The midzone region of the mitotic spindle (top) is projected into the underlying box. ATK5 is targeted to spindle midzones via plus-end tracking (either in a +TIP complex and/or by direct microtubule binding). At these sites of action, ATK5 functions by decreasing the lateral distance between neighboring microtubules via cross-linking and by promoting alignment of antiparallel microtubules via motor activity. ATK5 is shown here in association with antiparallel interpolar microtubules, although these functions may apply also to parallel microtubule interactions.

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