Molecular basis of the microtubule-regulating activity of microtubule crosslinking factor 1

<div><p>The variety of microtubule arrays observed across different cell types should require a diverse group of proteins that control microtubule organization. Nevertheless, mainly because of the intrinsic propensity of microtubules to easily form bundles upon stabilization, only a small number of microtubule crosslinking proteins have been identified, especially in postmitotic cells. Among them is microtubule crosslinking factor 1 (MTCL1) that not only interconnects microtubules via its N-terminal microtubule-binding domain (N-MTBD), but also stabilizes microtubules via its C-terminal microtubule-binding domain (C-MTBD). Here, we comprehensively analyzed the assembly structure of MTCL1 to elucidate the molecular basis of this dual activity in microtubule regulation. Our results indicate that MTCL1 forms a parallel dimer not only through multiple homo-interactions of the central coiled-coil motifs, but also the most C-terminal non-coiled-coil region immediately downstream of the C-MTBD. Among these homo-interaction regions, the first coiled-coil motif adjacent to N-MTBD is sufficient for the MTCL1 function to crosslink microtubules without affecting the dynamic property, and disruption of this motif drastically transformed MTCL1-induced microtubule assembly from tight to network-like bundles. Notably, suppression of the homo-interaction of this motif inhibited the endogenous MTCL1 function to stabilize Golgi-associated microtubules that are essential for Golgi-ribbon formation. Because the microtubule-stabilizing activity of MTCL1 is completely attributed to C-MTBD, the present study suggests possible interplay between N-MTBD and C-MTBD, in which normal crosslinking and accumulation of microtubules by N-MTBD is essential for microtubule stabilization by C-MTBD.</p></div

The coiled-coil interaction of CC1 plays an essential role in N1-mediated crosslinking of dynamic MTs.

<p>(A) Schematic representation of the N-terminal mutants used in the following experiments and the summary of results. Asterisks indicate previously reported results [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182641#pone.0182641.ref021" target="_blank">21</a>]. ND indicates not determined. (B) Immunostaining of V5 and α-tubulin in HeLa-K cells expressing V5-N1 wild-type, 5LP, or 5LA. The bottom panel is an enlarged view of the rectangle region of V5-N1 5LA-expressing cells. Scale bars, 10 μm. (C) Immunostaining of GFP, α-tubulin, and acetylated tubulin in HeLa-K cells expressing GFP, GFP-hCMTBD, and GFP-N1. Scale bar, 10 μm. (D) Immunostaining of GFP and α-tubulin in HeLa-K cells expressing GFP, GFP-hCMTBD, and GFP-N1 after cold treatment on ice for 1 h. Scale bar, 10 μm.</p

MTCL1 crosslinks MTs <i>in vitro</i>.

<p>(A) CBB staining of purified SBP-MTCL1 (lane 1; 2 μg, lane 2; 0.4 μg) separated by SDS-PAGE. (B) Visualization of taxol-stabilized MTs mixed with purified SBP-MTCL1 using an anti-tubulin antibody. Molar ratios of SBP-MTCL1 to the tubulin dimer (0, 0.07 or 0.2) are indicated. (C) Super-resolution image of MTs mixed with SBP-MTCL1 (molar ratio to tubulin dimer = 0.2) and double stained with anti-α-tubulin and anti-MTCL1 antibodies.</p

Identification of homo-interaction regions in the N-terminal half of MTCL1.

<p>(A) Schematic representation of the MTCL1 mutants used in the following experiments and the summary of results. Bidirectional black arrows indicate binding, and that with a cross mark indicates no binding. Brown bars with a blue arrow (top) indicate the identified homo-interaction regions. (B) Streptavidin pull-down assays performed with extracts of HEK293T cells co-expressing SBP-N or C and V5-tagged N. (C) Streptavidin pull-down assays performed with extracts of HEK293T cells co-expressing SBP-tagged CC2, N1 or N6 and V5-tagged N. CC2 was included to verify the specificity of the interactions. (D) Streptavidin pull-down assays performed with extracts of HEK293T cells co-expressing SBP-tagged N and individual N-terminal CCs with a GFP tag. (E) Streptavidin pull-down assays performed with extracts of HEK293T cells co-expressing SBP-tagged N6 and GFP-tagged CC3+CC4, CC5+CC6, N8, or N9.</p

Identification of homo-interaction regions in the C-terminal half of MTCL1.

<p>(A) Schematic representation of the MTCL1 mutants used in the following experiments and the summary of results. Bidirectional black arrows indicate binding, and those with a cross mark indicate no binding. Deep-blue bars with a yellow arrow (top) indicate the identified homo-interaction regions. (B) Streptavidin pull-down assays performed with extracts of HEK293T cells co-expressing SBP-CC2, C, C1 or C2 and V5-tagged C. CC2 was included to verify the specificity of the interactions. (C) Streptavidin pull-down assays performed with extracts of HEK293T cells co-expressing SBP-C1 or C2 and V5-tagged C1. (D) Streptavidin pull-down assays performed with extracts of HEK293T cells co-expressing GFP-C2, C3, C4, C5 or C2ΔC5 and SBP-tagged C2. (E) Streptavidin pull-down assays performed with extracts of HEK293T cells co-expressing SBP-C2 and GFP-tagged C6, C7, C8, or C9. (F) Immunoprecipitation assays performed using an anti-GFP antibody and extracts of HEK293T cells co-expressing GFP or GFP-C9 and RFP-C9.</p

The first coiled-coil motif is critical for oligomerization of the N-MTBD of MTCL1.

<p>(A) Domain organization of mouse MTCL1 (top) and the coiled-coil probability of each motif predicted by Coils (<a href="http://www.ch.embnet.org/software/COILS_form.html" target="_blank">http://www.ch.embnet.org/software/COILS_form.html</a>) (bottom). (B) Schematic representation of the N-terminal mutants used in the following experiments and the summary of results. Asterisks indicate previously reported results [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182641#pone.0182641.ref021" target="_blank">21</a>]. (C) Streptavidin pull-down assays performed with extracts of HEK293T cells co-expressing SBP-tagged MTCL1 mutants and V5-tagged N1. (D) Streptavidin pull-down assays performed with extracts of HEK293T cells co-expressing SBP-tagged N1 and GFP-tagged mutants. (E) GST pull-down assays performed with a mixture of the purified CC1 fragment and GST or GST-CC1. (F) Helical wheel presentation of the amino acid sequence of the first half of CC1. Blue dotted lines indicate predicted salt bridges enhancing the CC1-CC1 homo-interaction. Red asterisks indicate leucine residues mutated in 5LP and 5LA mutants. Blue asterisks indicate glutamate residues mutated in the 5EA mutant. (G) Immunoprecipitation assays performed using an anti-V5 antibody and extracts of HEK293T cells co-expressing V5-tagged mutants and HA-tagged N1. (H) Streptavidin pull-down assays performed with extracts of HEK293T cells co-expressing V5-tagged N1 and SBP-tagged mutants.</p

The coiled-coil interaction through CC1 is critically important for tight crosslinking of MTs.

<p>(A) Schematic representation of the MTCL1 mutants used in the following experiments. Red asterisks indicate the position of the 5LP mutation. (B) Immunostaining of V5 and α-tubulin in HeLa-K cells expressing V5-NCC4 wt or its 5LP mutant. Scale bar, 10 μm. (C) Immunostaining of V5 and α-tubulin in HeLa-K cells expressing V5-full ΔKR or its 5LP mutant. Scale bar, 10 μm. (D) Immunostaining of V5 (blue), GM130 (cis-Golgi marker) (green), and acetylated tubulin (red) in HeLa-K cells expressing V5-N3 wt or its 5LP mutant. Asterisks indicate cells expressing ectopic proteins. Scale bar, 50 mm. (E) Box plot presentation of the Golgi-ribbon index corresponding to the laterally expanding angle of the Golgi apparatus around nuclei (θ). The lines within each box represent medians. Data represent the results of the indicated number (n) of cells in three independent experiments. **P < 0.01, estimated by the two-tailed Mann-Whitney U-test.</p