Tubulin dimers are flexible entities serving as nanometric 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 the spermine. Using solution X-ray scattering (static and time-resolved) and cryo-TEM (static, time-resolved and tomography) measurements we studied the behavior of tubulin assembly in the presence of millimolar spermine concentrations. The results discovered novel nanostructural architectures of tubulin polymers and revealing fascinating hierarchical self-associations based on unique tubulin conical-spiral (TCS) subunits. The series of self-assembled tubulin structures are significantly different than the classical transition between twisted ribbons, helical, and tubular assemblies, and are not predicted by current theories of self-assemblies.

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 short tubulin concial spiral with about one helical turn nanostructures, resembling tubulin-rings. Above 1.5 mM spermine, tubulin assembled into tubulin conical-spirals with 3 helical turns. Tubulin conical-spiral is a unique tubulin assembly, serving as a new building block subunit. The tubulin conical-spirals assembled into different architectures. The predominant nanostructure was tubulin conical-spiral tubule (TCST) that further assembled in a remarkable antiparallel orientation, which formed bundles with 2D-cubic and a unique disordered 2D hexagonal lattices. Each tubulin conical-spiral tubule in the disordered hexagonal lattice was surrounded by four antiparallel tubulin conical-spiral tubules and two parallel tubulin conical-spiral tubules (ACS Nano 2021). An electron tomography of the TCST bundles is here.  At higher spermine concentrations, tubulin assembled into inverted tubulin tubules (ITTs), previously discovered by one of us (Nature Materials 2014). An electron tomography of the ITT bundles is here. Electron tomography was performed in the lab of Gabriel A. Frank, Ben Gurion University. 

We also showed the hierarchical assembly pathways from tubulin dimer to each of the above nanostructures, using time-resolved experiments with millisecond temporal resolution. We discovered that the structures that formed at low spermine concentrations were transient intermediates of the structures observed at higher spermine concentrations.

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