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, 30 (24), 4942-54

Directionality of Individual kinesin-5 Cin8 Motors Is Modulated by Loop 8, Ionic Strength and Microtubule Geometry

Affiliations

Directionality of Individual kinesin-5 Cin8 Motors Is Modulated by Loop 8, Ionic Strength and Microtubule Geometry

Adina Gerson-Gurwitz et al. EMBO J.

Abstract

Kinesin-5 motors fulfil essential roles in mitotic spindle morphogenesis and dynamics as slow, processive microtubule (MT) plus-end directed motors. The Saccharomyces cerevisiae kinesin-5 Cin8 was found, surprisingly, to switch directionality. Here, we have examined directionality using single-molecule fluorescence motility assays and live-cell microscopy. On spindles, Cin8 motors mostly moved slowly (∼25 nm/s) towards the midzone, but occasionally also faster (∼55 nm/s) towards the spindle poles. In vitro, individual Cin8 motors could be switched by ionic conditions from rapid (380 nm/s) and processive minus-end to slow plus-end motion on single MTs. At high ionic strength, Cin8 motors rapidly alternated directionalities between antiparallel MTs, while driving steady plus-end relative sliding. Between parallel MTs, plus-end motion was only occasionally observed. Deletion of the uniquely large insert in loop 8 of Cin8 induced bias towards minus-end motility and affected the ionic strength-dependent directional switching of Cin8 in vitro. The deletion mutant cells exhibited reduced midzone-directed motility and efficiency to support spindle elongation, indicating the importance of directionality control for the anaphase function of Cin8.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
In vivo, Cin8 occasionally switches directionality. (A) 2D-projection time-lapse confocal fluorescence images of Cin8–3GFP localization in WT cells. Time intervals: 1 min. (B) Model of Cin8 plus-end and minus-end directed movement in the anaphase spindle in S. cerevisiae cells. (C, D) Kymographs of Cin8–3GFP localization to the anaphase spindle. (A, C, D) Top arrows: spindle poles; asterisk: spindle midzone. Arrowheads: plus-end-directed movements towards the midzone; yellow arrows: minus-end directed movements towards the poles.
Figure 2
Figure 2
At high salt in vitro, Cin8 is a minus-end directed motor. (A) Time-lapse recording showing a single Cin8–3GFP molecule from whole-cell extract (green) moving directionally along the MT (red) towards the bright seed (yellow). Time intervals: 2 s. (BD) Kymographs of Cin8 movement on polarity-marked MTs. (B) Cin8–3GFP from whole-cell extract (C) Cin8–GFP purified from yeast cells and (D) Cin8–GFP purified from Sf9 cells. S: bright seed indicating the MT minus end. Scale bars in (D) also apply to (B) and (C). * indicates diffusive episotes. See also Supplementary Movies S2 and S3.
Figure 3
Figure 3
The effect of spindle proteins on Cin8 in-vitro motile properties and in-vivo localization to anaphase spindles. (A, B) Single-molecule fluorescence motility assay was carried out on polarity-marked MTs with kip1Δ or ase1Δ cell extracts expressing Cin8–3GFP. Extracts were diluted in high-salt (175 mM NaCl) buffer. (A) Representative kymographs of in-vitro runs of Cin8–3GFP in the different extracts. (B) Histograms of run lengths of Cin8–3GFP directional episodes in WT (olive), kip1Δ (light green) or ase1Δ (orange) extracts. Fit: Gaussian distribution. (CE) Representative 2D-projection time-lapse confocal fluorescence images of Cin8–3GFP localization to anaphase spindles in (C) WT (D) kip1Δ and (E) ase1Δ cells. Asterisk: spindle midzone; Arrows: spindle poles; Time interval between frames: 1 min; Scale bar: 2 μm. Genotypes and Cin8 variants are marked on top.
Figure 4
Figure 4
Decrease in ionic strength induces plus-end motility of single molecules of Cin8. (A, B) Histograms of velocities of Cin8 in whole-cell yeast extracts (A) or affinity purified (B) with saturating ATP. Salt and buffer conditions are indicated for each panel: MB—motility buffer; numbers adjacent to ‘MB' indicate the concentration (mM) of added NaCl. Ionic strength (M) is indicated in parentheses. A control experiment of motility with ADP was carried out using buffer with 30 mM NaCl (see Supplementary Figure S2C). Velocity histograms were assembled by dividing kymograph traces in 3 s segments and piecewise linear fitting. Lines—Gaussian distribution fit. (C) Representative kymographs of Cin8 motility along polarity-marked MTs. Expression/purification conditions are indicated on top. Plus (+) and minus (−) ends of MTs are indicated. See also Supplementary Movie S4. (D) ATP concentration dependence of plus-end (top) and minus-end directed (bottom) velocity of Cin8 (average±s.e.m.). NaCl concentration and ionic strengths (parenthesis) are indicated. Michaelis–Menten parameters Vmax and Km are indicated.
Figure 5
Figure 5
Decrease in ionic strength induces plus-end motility of single molecules of Cin8Δ99. (A, B) Histograms of velocities of Cin8Δ99 in whole-cell yeast extracts (A) or affinity purified (B) with saturating ATP. Salt and buffer conditions are indicated for each panel: MB—motility buffer; numbers adjacent to ‘MB' indicate the concentration (mM) of added NaCl. Ionic strength (M) is indicated in parentheses. Velocity histograms were assembled by dividing kymograph traces in 3 s segments and piecewise linear fitting. A control experiment of motility with ADP was carried out using buffer with 30 mM NaCl (see Supplementary Figure S2C). Lines—Gaussian distribution fit. (C) Representative kymographs of Cin8Δ99 motility along polarity-marked MTs. Expression/purification conditions are indicated on top. Plus (+) and minus (−) ends of MTs are indicated. See also Supplementary Movie S5. (D) ATP concentration dependence of minus-end velocity of Cin8Δ99 (average±s.e.m.) at high ionic strength. NaCl concentration and ionic strength, M (parenthesis) are indicated. Michaelis–Menten parameters Vmax and Km are indicated.
Figure 6
Figure 6
Deletion of the 99aa insert of Cin8 induces bias towards minus-end motility. (A, B) Histograms of velocities of Cin8 (green) and Cin8Δ99 (red) in whole-cell yeast extracts (A) or affinity purified (B) in buffer containing 30 mM NaCl, with saturating ATP. Velocity histograms were assembled by dividing kymograph traces in 3 s segments and piecewise linear fitting. Lines—Gaussian distribution fit. See also Supplementary Movies S4 and S5. (C) Ionic strength dependence of mean velocity of Cin8 (green) and Cin8Δ99 (red) in whole-cell extracts in saturating ATP conditions. Values represent mean s.e.m. of 50–200 velocity values.
Figure 7
Figure 7
Movements of Cin8–3GFP (A), Cin8Δ99–3GFP (B) and Cin8-2A–3GFP (C) in anaphase spindles. Kymographs of movements of Cin8–3GFP variants (indicated on the left) in intermediate to long anaphase spindles. White arrows: midzone-directed movements that span from the SPBs to the midzone; Yellow arrows: SPB-directed movements; White arrowheads: movements towards the SPB during spindle disassembly; Top arrows: spindle poles; Asterisks: midzone.
Figure 8
Figure 8
MT orientation changes the motile properties of single Cin8 molecules. Kymographs of movements of purified Cin8–GFP between antiparallel (A) and parallel (B) MTs in high-salt buffer (MB-175). For each event, a merged kymograph in colour (red—MT; green—Cin8 and minus ends of MTs) is shown on the left and a kymograph of the GFP-channel only is shown on the right. Overlapping region between MTs is marked by dashed lines. Cartoon depicting the orientation of overlapping MTs is shown at the bottom of the colour kymographs. Arrows: minus-end directed motility events; arrowheads: plus-end directed motility events. See also Supplementary Movie clips S6,S7,S8,S9,S10.

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