SUMMARY Microtubules (MTs) serve important roles in cell trafficking, division and signaling. MTs polymerize via net addition of GTP-tubulin subunits to the MT plus end, which subsequently hydrolyze to GDP-tubulin after incorporation into the MT lattice. The relatively stable GTP-tubulin subunits create a “GTP cap” at the growing MT plus end that protects the MT from catastrophe. Therefore, to understand MT assembly regulation we need to understand GTP hydrolysis reaction kinetics and the size of the GTP cap. In vitro, the GTP cap has been estimated to be as small as one layer [1-3](13 subunits) or as large as 100-200 subunits [4]. GTP cap size estimates in vivo have not yet been reported. Using EB1-EGFP as a marker for GTP-tubulin in LLCPK1 epithelial cells, we find on average: (1) 270 EB1 dimers bound to growing MT plus ends, and (2) a GTP cap size of ~750 tubulin subunits. Thus, in vivo, the GTP cap size is far larger than previous estimates in vitro, and ~60-fold larger than a single layer cap. Consistent with these findings, we also find the tail region of a large GTP cap, 0.5-2.0 μm from the tip, promotes MT rescue and suppresses shortening. We speculate that a large GTP cap provides a locally concentrated (~100 μM) scaffold for tip-tracking proteins, and confers persistence to assembly in the face of physical barriers such as the cell cortex.

Human mesenchymal stem cells (hMSCs) receive differentiation cues from a number of stimuli, including extracellular matrix (ECM) stiffness. The pathways used to sense stiffness and other physical cues are just now being understood and include proteins within focal adhesions. To rapidly advance the pace of discovery for novel mechanosensitive proteins, we employed a combination of in silico and high throughput in vitro methods to analyze 47 different focal adhesion proteins for cryptic kinase binding sites. High content imaging of hMSCs treated with small interfering RNAs for the top 6 candidate proteins showed novel effects on both osteogenic and myogenic differentiation; Vinculin and SORBS1 were necessary for stiffness-mediated myogenic and osteogenic differentiation, respectively. Both of these proteins bound to MAPK1 (also known as ERK2), suggesting that it plays a context-specific role in mechanosensing for each lineage; validation for these sites was performed. This high throughput system, while specifically built to analyze stiffness-mediated stem cell differentiation, can be expanded to other physical cues to more broadly assess mechanical signaling and increase the pace of sensor discovery.