Quantifying BtubA/B Assembly in Living Cells

Microtubules are highly dynamic structures assembled from tubulin proteins. They play critical roles in cell division, transport and mobility. To study the assembly/disassembly of microtubules living cells, we selected BtubA/B as our model protein. two tubulin-like proteins bacterial genus Prosthecobacter, which show high sequence identity to mammalian alpha/beta tubulins but do not strongly interact with them. These features make good substrate. quantify interaction BtubA/B, pre-mixed FRET-labeled were microinjected into cells FRET responses measured using fluorescent microscopy. A new two-dimensional surface binding expriment was designed performed obtain in-cell dissociation constant Kd for first time. Comparing vitro, found has stronger i.e. cytoplasm matrix promotes assembly BtubA/B. The promotion is primarily due intracelluar crowding or ionic concentration other components under determination.