Hierarchical Assembly Pathways of Spermine Induced Tubulin Conical-Spiral Architectures

Tubulin dimers are flexible entities serving as building blocks for construction of cellular polymers essential for the cytoskeleton. The conformational state of the dimer dictates the exact formation of assembly and can be regulated by cellular factors including spermine. Using solution X-ray scattering and cryo-TEM measurements we studied the behavior of tubulin assembly in the presence of millimolar spermine concentrations. The results discovered novel structural architectures of tubulin polymers and revealing fascinating hierarchical self-associations based on unique tubulin conical-spiral (TCS) subunits.

We followed the assembly pathways of tubulin dimers with different spermine concentrations, from milliseconds to days, and discovered multiple phase transitions with increasing spermine concentration. At 1 mM spermine, tubulin assembled into tubulin helical-pitch (THP) structures, resembling tubulin-rings. Above 1.5 mM spermine, tubulin assembled into TCS architectures. TCS is a unique tubulin assembly, serving as a new building block subunit. TCS assembled into different architectures . The predominant structure was TCS-tube (TCST) that further assembled in a remarkable antiparallel orientation which formed bundles with 2D-cubic and unique quasi-2D hexagonal lattices. Each TCST in the quasi-2D hexagonal lattice was surrounded by four antiparallel TCSTs and two parallel TCSTs. All the above assemblies have never been observed before. At higher spermine concentrations, tubulin assembled into twisted inverted tubulin tubules (ITTs).

Here we also show for the first time, the hierarchical assembly pathways from tubulin dimer to each of the above structures, using time-resolved experiments with millisecond temporal resolution. We discovered that the structures that formed at low spermine concentrations were transient precursors of the structures formed at higher spermine concentrations.

The results are based on high quality cryo-TEM images, cutting edge synchrotron solution X-ray scattering measurements and state-of-the-art data analysis, using our home developed groundbreaking analysis software, D+.

The findings can be relevant to a broad research fields including studies which explore different arrangements of the cytoskeletal network, or studies exploring the attraction forces between proteins that dictate their mode of assembly and molecular designed self-assembly of natural and/or synthetic analogous.