Dissecting the Nanoscale Distributions and Functions of Microtubule-End-Binding Proteins EB1 and ch-TOG in Interphase HeLa Cells

Abstract

Recently, the EB1 and XMAP215/TOG families of microtubule binding proteins have been demonstrated to bind autonomously to the growing plus ends of microtubules and regulate their behaviour in in vitro systems. However, their functional redundancy or difference in cells remains obscure. Here, we compared the nanoscale distributions of EB1 and ch-TOG along microtubules using high-resolution microscopy techniques, and also their roles in microtubule organisation in interphase HeLa cells. The ch-TOG accumulation sites protruded ∼100 nm from the EB1 comets. Overexpression experiments showed that ch-TOG and EB1 did not interfere with each other’s localisation, confirming that they recognise distinct regions at the ends of microtubules. While both EB1 and ch-TOG showed similar effects on microtubule plus end dynamics and additively increased microtubule dynamicity, only EB1 exhibited microtubule-cell cortex attachment activity. These observations indicate that EB1 and ch-TOG regulate microtubule organisation differently via distinct regions in the plus ends of microtubules.

Drosophila melanogaster Mini Spindles TOG3 Utilizes Unique Structural Elements to Promote Domain Stability and Maintain a TOG1- and TOG2-like Tubulin-binding Surface

“…EB1 fenestrates the MT lattice, binding between four tubulin heterodimers where it recognizes tubulin in a GTP hydrolysis transition/post-hydrolysis state mimicked by the GTP analog GTP␥S (58 -60). Thus, EB1 binds just distal to the polymerizing MT GTP-cap and via SLAIN2/Sentin localizes the XMAP215 C terminus to this zone (33,34,61,62). In contrast, the XMAP215 N-terminal TOG domain array binds and incorporates ␣␤-tubulin into the growing MT plus-end (28 -31, 38, 40, 41).…”

“…In contrast, the XMAP215 N-terminal TOG domain array binds and incorporates ␣␤-tubulin into the growing MT plus-end (28 -31, 38, 40, 41). The differential positioning of the XMAP215 N-and C-terminal domains at the MT plus-end likely results in a dynamic/polarized arrangement on the MT and requires an extended XMAP215 configuration (31,62,63). How each TOG domain in the pentameric array is positioned along the growing MT plus-end remains to be determined.…”

Biased Brownian motion as a mechanism to facilitate nanometer-scale exploration of the microtubule plus end by a kinesin-8

“…This distance corresponds to roughly 24 tubulin subunits on a 13-protofilament MT. Kif18B joins a growing list of proteins that localize to the MT plus-end terminus, the best characterized of which is ch-TOG (32,33). To determine whether ch-TOG and Kif18B occupy similar or distinct regions of the plus end, we compared the plusend distributions of the two proteins in fixed mitotic cells.…”

Low intensity pulse ultrasound stimulate chondrocytes growth in a 3-D alginate scaffold through improved porosity and permeability

“…HeLa (human cervical epithelial carcinoma) cells labeled with GFP [18] were provided by the medical school at Nanjing University.…”