1. Marking and Measuring Single Microtubules by PRC1 and Kinesin-4
Radhika Subramanian, Shih-Chieh Ti, Lei Tan, Seth A. Darst, Tarun M. Kapoor 
Cell 
Volume 154, Issue 2, Pages (July 2013)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1
The PRC1-Kinesin-4 Complex Tags the Ends of Single Microtubules
(A) Schematic of PRC1 and kinesin-4’s domain organization and the constructs used in binding and microscopy assays. PRC1: N-terminal domain (blue); spectrin domain (red); C-terminal domain (black). Kinesin-4: motor domain (blue); coiled coil domain (gray); C-terminal Cys-rich domain (yellow).
(B) Quantitative analysis of the PRC1-kinesin-4 binding interaction. Plot of the fraction kinesin-4ΔN (0.3 μM) bound to varying amounts of PRC1NSΔC (0.5–10 μM) (n = 3, mean ± SD). The data were fit to a hyperbola (see Experimental Procedures) to determine the dissociation constant (KD = 0.3 ± 0.03 μM).
(C) Schematic of the TIRF microscopy assay used for examining GFP-PRC1’s (purple) binding to a single microtubule (red). Microtubules were sparsely labeled with X-Rhodamine and biotin and immobilized on a glass surface (black line) via biotin-neutravidin linkages (black circles).
(D–F) Representative image shows microtubules (D), associated GFP-PRC1 (E) and overlay of the two images (red, microtubules; green, PRC1) (F).
(G) Linescan of GFP-PRC1-bound microtubule marked by an arrow in (F).
(H) Schematic of the assay used for examining kinesin-4-GFP’s (blue) binding to single microtubules.
(I–K) Representative image shows microtubules (I), associated kinesin-4-GFP (J), and overlay of the two images (red, microtubules; green, kinesin-4-GFP) (K).
(L) Linescan of kinesin-4-GFP-bound microtubule marked by an arrow in (J).
(M) Schematic of the assay used for examining GFP-PRC1’s (purple) binding to single microtubules in the presence of kinesin-4 (blue).
(N–P) Representative image shows microtubules (N), associated GFP-PRC1 in the presence of kinesin-4 (O) and overlay of the two images (red, microtubules; green, PRC1) (P).
(Q) Linescan of GFP-PRC1 and kinesin-4 bound microtubule marked by an arrow in (P).
Assay conditions: PRC1 (0.25 nM) and kinesin-4 (1.5 nM, 1 mM MgATP). Scale bars, 2.5 μm. See also Figure S1.
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4. Figure 2
Size of the PRC1-Kinesin-4 End-Tag Depends on Microtubule Length and Protein Concentration
(A) Schematic of the assay used for examining GFP-PRC1’s (purple) binding to single microtubules (red) in the presence of kinesin-4 (blue).
(B–D) Representative images of microtubules of different lengths (B), associated GFP-PRC1 (0.25 nM) (C), and overlay of the two images (red, microtubules; green: PRC1) (D). Kinesin-4 was at 1.5 nM.
(E) Plot of end-tag intensity as a function of microtubule length in assays with kinesin-4 (1.5 nM) and GFP-PRC1: 0.1 nM (blue; slope = 756 ± 56 a.u./μm, n = 195), 0.25 nM (red; slope = 1,960 ± 182 a.u./μm, n = 236), and 0.5 nM (black; slope = 3,561 ± 183 a.u./μm, n = 275).
(F) Plot of end-tag length as a function of microtubule length in assays with kinesin-4 (1.5 nM) and GFP-PRC1: 0.1 nM (blue; slope = 0.22 ± 0.02, n = 195), 0.25 nM (red; slope = 0.26 ± 0.02, n = 236), and 0.5 nM (black; slope = 0.42 ± 0.02, n = 275).
(G) Schematic of the assay used for examining kinesin-4-GFP’s binding to single microtubules.
(H–J) Representative image of a microtubule (H), associated kinesin-4-GFP (1.5 nM) (I) and overlay of the two images (red, microtubules; green, kinesin-4) (J).
(K) Schematic of the assay used for examining kinesin-4-GFP’s binding to single microtubules in the presence of PRC1.
(L–N) Representative image of a microtubule (L), associated kinesin-4-GFP (1.5 nM) in the presence PRC1 (0.4 nM) (M), and overlay of the two images (red, microtubules; green, kinesin-4) (N).
(O) Plot of end-tag intensity as a function of microtubule length in assays with kinesin-4-GFP (1.5 nM) and PRC1: 0 nM (blue; slope = 2,116 ± 171 a.u./μm, n = 116), 0.1 nM (red; slope = 3,085 ± 357 a.u./μm, n = 119), or 0.4 nM (black; slope = 5,337 ± 126 a.u./μm, n = 172).
(P) Plot of end-tag length as a function of microtubule length in assays with kinesin-4-GFP (1.5 nM) and PRC1: 0 nM (blue; slope = 0.09 ± 0.007, n = 116), 0.1 nM (red; slope = 0.12 ± 0.005, n = 119), or 0.4 nM (black; slope = 0.27 ± 0.03, n = 172).
All experiments include 1 mM MgATP. Error bars are SD. Scale bar, 2.5 μm. See also Figure S2.
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5. Figure 3
PRC1-Kinesin-4 Microtubule End-Tags Are Dynamic Steady-State Structures
(A) Schematic of the assay used for examining end-tag formation by GFP-PRC1 (purple) and kinesin-4 (blue) on single microtubules (red).
(B and C) Image of a microtubule (B) and associated GFP-PRC1 (C) from a time-lapse sequence acquired during end-tag formation. Assay conditions: PRC1 (0.1 nM) and kinesin-4 (1.5 nM).
(D) Kymograph corresponding to the time-lapse sequence in (C).
(E) Portion of the boxed region (yellow dashed rectangle) in (D). Image contrast (grayscale) is adjusted to highlight the GFP-PRC1 signal along the microtubule.
(F) Schematic of the “pulse-chase” type assay for examining GFP-PRC1 dynamics at the end-tag.
(G and H) Image of a microtubule (G) and associated GFP-PRC1 (H) from a time-lapse sequence acquired after addition of unlabeled proteins to microtubules end-tagged with GFP-PRC1 and kinesin-4. Assay conditions: PRC1 (0.15 nM) and kinesin-4 (0.5 nM).
(I) Kymograph corresponding to the time-lapse sequence in (H).
All assays include 1 mM MgATP. Scale bars, distance = 2.5 μm; time = 20 s. See also Figure S3.
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6. Figure 4 PRC1 Is an Elongated Rod-Shaped Molecule
(A) Schematic of PRC1’s domain organization and the construct used for X-ray crystallography (blue and orange, N-terminal domains; red, spectrin domain; black, C-terminal unstructured domain).
(B) Ribbon diagram shows the structure of a single PRC1 polypeptide within the homodimer. Dimerization domain (blue): helices H1–H2 and loop L1, rod domain (orange): helices H2–H7 and loops L2–L6, spectrin domain (red): helices H7–H9 and loops L7–L8.
(C) Secondary structure topology map corresponding to the ribbon diagram in (B).
(D) Examples of contacts between helices and loops mediated by conserved amino acid residues in the rod domain of PRC1. Side-chain atoms of key amino acid residues (labeled) in view are shown (N, blue; O, red; S, yellow; C, colored by percent conservation as in the scale bar). (i) Conserved contacts between helix H3 and helix H4. (ii) Conserved contacts between helix H4, helix H5, and loop L4.
See also Figure S4 and Table S1.
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7. Figure 5
PRC1 Dimerization Is Mediated by Bisecting N-Terminal Helix-Based Hairpins
(A) Ribbon diagram of the PRC1NSΔC dimer. The two monomers that form the homodimer are colored red and blue.
(B) Enlarged view of PRC1’s dimerization domain. Boxed sections are further enlarged in insets (i) and (ii). The views shown in the insets were generated by rotating the structure as indicated. Side-chain atoms of key amino-acid residues (labeled) mediating the interactions in PRC1’s dimerization domain are shown (N, blue; O, red; S, yellow; C, green).
(C) Schematic of the constructs generated to test PRC1’s dimerization.
(D) Elution profiles from size-exclusion chromatography of constructs PRC1NSΔC (gray), PRC1ΔN1SΔC (cyan), PRC1ΔN2SΔC (blue), PRC1ΔN3SΔC (black).
See also Figure S5.
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8. Figure 6
The Size of PRC1-Kinesin-4 End-Tag Depends on the Strength of the PRC1-Microtubule Interaction
(A) Schematic of PRC1 deletion constructs used in binding assays with the nonmotor domain at kinesin-4’s C terminus (kinesin-4ΔN: aa 733–1232).
(B) SDS-PAGE of the fraction of kinesin-4ΔN (1 μM) bound to PRC1 constructs (5 μM) shown in Figure 1A.
(C and D) Band intensities from gels were used to determine fraction kinesin-4ΔN bound to the PRC1 constructs in (A) and plotted against varying PRC1 concentration (n = 3, mean ± SD). The data were fit to a hyperbola to estimate the dissociation constant (KD). PRCΔN1SΔC: KD = 0.21 ± 0.01 μM; PRC1ΔN2SΔC: KD = 1.6 ± 0.11 μM; PRC1NSΔC5: KD = 0.3 ± 0.06 μM; PRC1NSΔC4: KD = 0.3 ± 0.04 μM; PRC1NSΔC3: KD = 1.2 ± 0.4 μM; PRC1NSΔC2: KD = 2.3 ± 0.6 μM.
(E) Ribbon diagram of the structure of the PRC1NSΔC dimer with the dimerization domain (blue) and the portion of the rod domain (orange) involved in kinesin-4ΔN binding highlighted. Estimated KD for constructs that terminate at different positions (black line) along PRC1’s rod domain are indicated.
(F) Schematic of the constructs generated to examine the effect of PRC1’s microtubule binding on plus-end-tagging (blue, dimerization domain; orange, rod domain; red, spectrin domain; black, unstructured domain).
(G–R) Representative images of a microtubule (G, J, M, P), associated (H) GFP-PRC1 (0.5 nM), (K) GFP-PRC1NSΔC (0.5 nM), (N) GFP-PRC1NSΔC4 (0.5 nM) and (Q) GFP-PRC1NSΔC4 (3 nM), and overlay of the two images (red, microtubules; green, PRC1) (I, L, O, R). Assay includes kinesin-4 (1.5 nM, 1 mM MgATP).
(S) Plot of end-tag intensity as a function of microtubule length in this assay: GFP-PRC1 (0.5 nM; black; slope = 1,864 ± 104 a.u./μm, n = 100), GFP-PRC1NSΔC (0.5 nM; red; slope = 1,051 ± 93 a.u./μm, n = 144), GFP-PRC1NSΔC4 (0.5 nM; blue; slope = 154 ± 16 a.u./μm, n = 148), or GFP-PRC1NSΔC4 (3.0 nM; green; slope = 465 ± 11 a.u./μm, n = 114).
(T) Plot of end-tag length as a function of microtubule length in this assay: GFP-PRC1 (0.5 nM; black; slope = 0.36 ± 0.004, n = 100), GFP-PRC1NSΔC (0.5 nM; red; slope = 0.21 ± a.u./μm, n = 144), GFP-PRC1NSΔC4 (0.5 nM; blue; slope = 0.06 ± 0.005, n = 148), or GFP-PRC1NSΔC4 (3.0 nM; green; slope = 0.06 ± a.u./μm, n = 114).
Scale bar, 2.5 μm. Error bars are SD. See also Figure S6.
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9. Figure 7 Microtubule End-Tagging in Dividing Cells during Anaphase
(A–C) Analysis of PRC1 localization in monopolar anaphase cells by immunofluorescence. Maximum intensity projections of DNA (blue), tubulin (green), PRC1 (red), and an overlay of the three images are shown. Insets: 2-fold enlargement of the outlined regions (white dashed rectangle) in the overlay image. Maximum intensity projections were generated from optical sections spanning the microtubule in the region.
(D) Plot of end-tag length as a function of microtubule length in monopolar cells undergoing anaphase (n = 48; slope = 0.4 ± 0.02).
(E–G) Analysis of PRC1 localization in bipolar anaphase cells by immunofluorescence. Maximum intensity projections of DNA (blue), tubulin (green), PRC1 (red), and an overlay of the three images are shown. Insets: 3-fold enlargement of the outlined regions (white dashed rectangle) in the overlay image. Maximum intensity projections were generated from optical sections spanning the microtubule in the region.
(H and I) Analysis of anaphase cells expressing GFP-PRC1 by live imaging (left: DIC; right: fluorescence). Insets: 2-fold enlargement of the outlined regions (white dashed rectangle) in the fluorescence image. Maximum intensity projections were generated from optical sections spanning the fluorescence signal in the region.
(J) Plot of end-tag length as a function of microtubule length in bipolar cells undergoing anaphase (n = 17; slope = 0.35 ± 0.06).
(K and L) A model for the formation of filament length-dependent microtubule end-tags by PRC1 and kinesin-4. PRC1 (purple) is transported to the filament plus end by kinesin-4 (blue) along a microtubule (α and β-tubulin are colored red and white, respectively; only one protofilament is shown for clarity). PRC1-kinesin-4 molecules persist at the filament end forming an end-tag. Additional molecules are transported to filament end and “line up” behind the previously occupied tubulin sites (i–iii). Eventually a steady state is reached when the number of PRC1-kinesin-4 molecules transported to microtubule ends equals the number of molecules lost due to unbinding (iv–vi) (K). Smaller end-tags form on shorter microtubules due to fewer PRC1-kinesin-4 binding sites on the lattice (L).
Scale bar, 2.5 μm. See also Figure S7.
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10. Figure S1
Characterization of Fluorescently Labeled and Unlabeled PRC1 and Kinesin-4 Constructs, Related to Figure 1
(A) SDS-PAGE analysis of kinesin-4-GFP, kinesin-4, PRC1, GFP-PRC1 and alexa546-SNAP-PRC1.
(B) Fluorescence intensity analysis of GFP tagged PRC1 and kinesin-4. GFP-PRC1 (aa: 1–620; intensity = 1426 ± 1053, N = 658) and kinesin-4-GFP (aa: 1–1232; intensity = 1356 ± 784, N = 650). Dimeric-kinesin-5-GFP (Intensity = 1251 ± 805, N = 481) and tetrameric-kinesin-5-GFP (intensity = 2638 ± 1578, N = 538) were used as references. Intensities are reported as mean ± SD.
(C) Kymograph from a time-lapse sequence of stochastic microtubule growth and shrinkage induced by addition of tubulin (20 μM, 1 mM MgGTP) to a GMP-CPP polymerized microtubule seed immobilized to a glass coverslip.
(D) Representative image shows immobilized X-rhodamine labeled microtubule seeds (bright red) in the presence of sparsely labeled X-rhodamine labeled tubulin (20 μM, 1 mM MgGTP; dim red) and kinesin-4-GFP (3 nM, 1mM MgATP; green).
(E and F) Kymographs generated from time-lapse sequence acquired under the same conditions as (D).
(G–I) Representative image shows Alexa546-SNAP-PRC1 (G), kinesin-4-GFP (H) and overlay of the two images (red: Alexa546-SNAP-PRC1; green: kinesin-4-GFP) (I). Assay condition: kinesin-4-GFP (1.5 nM), PRC1 (0.25 nM), alexa546-SNAP-PRC1 (0.05 nM).
Assay includes 1 mM MgATP. Scale bars, distance = 2.5 μm, time = 20 s. Error bars represent standard deviations.
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11. Figure S2
Additional Analyses of End-Tag Length and Density, Related to Figure 2
(A) Scatter plot of end-tag intensity as a function of microtubule length (5-22 μm) in assays with kinesin-4 (1.5 nM) and GFP-PRC1 (0.25 nM) (N = 89; number of microtubules > 14 μm = 12).
(B) Plot of end-tag density versus microtubule length in assays with kinesin-4-GFP (1.5 nM) and PRC1: 0 nM (blue, N = 116), 0.1 nM (red, N = 119) and 0.4 nM (black, N = 172).
(C) Mean end-tag density in this assay. 0 nM (mean = 7737 ± 2873 a.u./μm, N = 116), 0.1 nM (slope = 9009 ± 2533 a.u./μm, N = 119) or 0.4 nM (slope = ± 2407 a.u./μm, N = 172).
Error bars represent standard deviations.
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12. Figure S3
Pulse-Chase Assay to Examine Protein Dynamics at End-Tag, Related to Figure 3
(A) Schematic of the assay for examining PRC1 dynamics at end-tags (PRC1 (purple), kinesin-4 (blue), nonfluorescent microtubules (gray)).
(B–D) Pulse-chase was performed by addition of a mixture of PRC1 (0.5 nM), Alexa546-SNAP-PRC1 (0.05 nM) and kinesin-4 (1.5 nM) to nonfluorescent microtubules end-tagged with GFP-PRC1 (0.25 nM) and kinesin-4 (1.5 nM). Time-lapse sequence with near-simultaneous imaging of GFP and Alexa546 was acquired. Kymographs generated from the time-lapse imaging: GFP-PRC1 (B), Alexa546-SNAP-PRC1 (C), and overlay (D).
All assays include 1mM MgATP. Scale bars, distance = 2.5 μm; time = 20 s.
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13. Figure S4
Additional Analyses of PRC1’s Crystal Structure, Related to Figure 4
(A and B) Experimental electron density from a selenomethionine substituted PRC1 crystal (blue; contour = 2 σ) overlaid with the selenium electron density (red; contour = 5 σ). The Cα backbone from the refined model of the PRC1 homodimer is shown (the two monomers are colored pink and yellow) and the identified methionines are labeled. Electron density of PRC1’s (A) dimerization domain and (B) a section of the rod domain.
(C and D) Contacts between helices and loops in the PRC1 structure. (C) Secondary structure topology map of the dimerization-rod domain junction in PRC1. Inset shows contacts between helices H1, H2, H3 and loops L1 and L2. (D) Secondary structure topology map at a helical junction in PRC1. Inset shows contacts between helices H3, H4, H5 and loops L3 and L4. Sidechain atoms of key amino-acid residues (labeled) in view are shown (N: blue; O: red; S: yellow, C: colored by percent conservation as in the scale bar).
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14. Figure S5
Biochemical Characterization of Constructs Used to Test PRC1’s Dimerization, Related to Figure 5
(A) Amino-acid sequence alignment of residues in PRC1’s dimerization domain. Conserved residues are highlighted in orange.
(B) SDS-PAGE analysis of the constructs generated to test PRC1’s dimerization.
(C) HPLC Size Exclusion Chromatography/ Laser Light Scattering analysis of PRC1-N3SΔC (aa ). Plot shows elution profile from size exclusion chromatography and tandem light scattering analysis. The calculated average molecular mass (y axis of the plot) corresponding to the sample protein is indicated by the dotted line across the elution peak. This analysis shows that PRC1-N3SΔC is monomer (molecular weight = g/mol).
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15. Figure S6
Characterization of Constructs Used to Examine PRC1-Kinesin-4 Binding and Single-Molecule Analysis of Kinesin-4 Movement in the Presence of PRC1, Related to Figure 6
(A) Elution profiles from size-exclusion chromatography of PRC1NSΔC1 (aa: 1-66, black), PRC1NSΔC2 (aa: 1-117, red), PRC1NSΔC3 (aa: 1-168, purple), PRC1NSΔC4 (aa: 1-231, green), PRC1NSΔC5 (aa: 1-303, pink) and PRC1NSΔC (aa: 1-486, yellow).
(B and C) Kymographs generated from a time-lapse sequence acquired in assays examining the microtubule interaction of kinesin-4-GFP (20 pM) in the absence (B) or presence (C) of PRC1 (60 pM). Assays include 1mM MgATP.
(D–L) Representative images of a microtubule (D, G, J), associated (E) GFP-PRC1 (0.5 nM), (H) GFP-PRC1NSΔC (0.5 nM), (K) GFP-PRC1NSΔC4 (0.5 nM), and overlay of the two images (red: microtubules; green: PRC1) (F, I, L).
(M) Fluorescence intensity per unit filament length for GFP-PRC1 (mean = ± 6584 a.u./μm, N = 40) and GFP-PRC1NSΔC (mean = 1456 ± 386 a.u./μm, N = 34) bound to single immobilized microtubules. Errors are standard deviations.
(N) Table summarizing kinesin-4 binding affinity (Figure 6E), microtubule affinity (M) and fractional end-tag length (Figure 6T) for the PRC1 constructs in Figure 6F.
Scale bars, distance = 2.5 μm; time = 20 s. Error bars represent standard deviations.
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16. Figure S7
Additional Analyses of End-Tagging by GFP-PRC1 and Overlay of PRC1’s Spectrin Domains from Two Crystal Structures, Related to Figure 7 and Discussion
(A–C) Western blot analysis to assess depletion of endogenous PRC1 in human (A) RPE1 cells and (B-C) RPE1 cell line expressing PRC1-siRNA resistant GFP-PRC1. Lanes 1 and 2 correspond to mock-transfected and PRC1 siRNA-transfected cells respectively. Blots were probed with α-PRC1 or α-GFP antibody as indicated. Positions of endogenous and GFP-tagged PRC1 constructs on the blot are indicated by ‘∗’ and ‘∗∗’ respectively.
(D) Bargraph shows multinucleation index for mock-transfected RPE1 cells, PRC1 siRNA transfected RPE1 cells, and PRC1 siRNA transfected RPE1 cells expressing GFP-PRC1. N > 500 per sample. Error bars represent SD.
(E) Plot of experimental (black) and estimated (red; see Extended Experimental Procedures) slope of plots of end-tag length versus microtubule length at different PRC1 concentrations.
(F) Overlay of the two spectrin domains (red and blue) in the structure of the PRC1 dimer.
(G) Crystal structure of PRC1’s spectrin domain from a truncation construct of this domain alone (orange; PDB: 3NRY).
(H) Overlay of spectrin domains in (F) and (G). The bend in helix H7 is highlighted with a rectangle and an enlarged view is shown in the inset.
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