A novel PAR-1-binding protein, MTCL1, plays critical roles in organizing microtubules in polarizing epithelial cells

Abstract

SummaryThe establishment of epithelial polarity is tightly linked to the dramatic reorganization of microtubules (MTs) from a radial array to a vertical alignment of non-centrosomal MT bundles along the lateral membrane, and a meshwork under the apical and basal membranes. However, little is known about the underlying molecular mechanism of this polarity-dependent MT remodeling. The evolutionarily conserved cell polarity-regulating kinase PAR-1 (known as MARK in mammals), whose activity is essential for maintaining the dynamic state of MTs, has indispensable roles in promoting this process. Here, we identify a novel PAR-1-binding protein, which we call microtubule crosslinking factor 1 (MTCL1), that crosslinks MTs through its N-terminal MT-binding region and subsequent coiledcoil motifs. MTCL1 colocalized with the apicobasal MT bundles in epithelial cells, and its knockdown impaired the development of these MT bundles and the epithelial-cell-specific columnar shape. Rescue experiments revealed that the N-terminal MT-binding region was indispensable for restoring these defects of the knockdown cells. MT regrowth assays indicated that MTCL1 was not required for the initial radial growth of MTs from the apical centrosome but was essential for the accumulation of non-centrosomal MTs to the sublateral regions. Interestingly, MTCL1 recruited a subpopulation of PAR-1b (known as MARK2 in mammals) to the apicobasal MT bundles, and its interaction with PAR-1b was required for MTCL1-dependent development of the apicobasal MT bundles. These results suggest that MTCL1 mediates the epithelial-cell-specific reorganization of non-centrosomal MTs through its MT-crosslinking activity, and cooperates with PAR-1b to maintain the correct temporal balance between dynamic and stable MTs within the apicobasal MT bundles.

MTCL1 crosslinks and stabilizes non-centrosomal microtubules on the Golgi membrane

“…These results suggest that MTCL1 has a role in mediating the association of MTs with the Golgi membrane. Moreover, the MTCL1 localization on acetylated MTs, sometimes observed in the regions lacking GM130 signals (a bracket in Fig.1c), is consistent with its MT-crosslinking activity we have previously demonstrated 15 . Analysis by super-resolution microscopy (G-STED) resolved the perinuclear tracks of acetylated MTs into finer MT strands and revealed that many MTCL1 signals were localized between them or at their intersection points (Fig.…”

“…As we have reported previously 15 , MTCL1 has two MT-binding domains at the N-terminal and C-terminal regions, the latter termed the KR-rich region (KR; Fig. 6a).…”

“…It should be noted that probably because of the C-terminal KR-rich region, which can induce MT bundling by itself (see Fig. 8) 15 , expression of MTCL1DN induced MT bundle formation rich in acetylated tubulin, to which the mutant itself localized. Nevertheless, these MT bundles straggled randomly and did not exhibit complete accumulation around the nucleus.…”

“…We have recently identified a novel MT-regulating protein, MTCL1, which crosslinks MTs through its N-terminal MTbinding region and subsequent coiled-coil motifs, and revealed its indispensable roles in accumulating the non-centrosomal MTs along the lateral membrane of polarized epithelial cells 15 . In the current study, we demonstrate that MTCL1 co-localizes with the Golgi membrane through interactions with CLASPs and AKAP450/CG-NAP, and plays indispensable roles in the development of the Golgi-derived MTs.…”

“…We have previously shown that immunostaining of endogenous MTCL1 in sparsely seeded, less polarized Madin-Darby canine kidney (MDCK) cells revealed granular structures, which intermittently decorated the lateral sides of individual MTs and concentrated at MT bundles 15 . Similar staining was observed in HeLa-K cells (Fig.…”