<p>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.</p>
<p> </p>
<p>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).</p>
<p>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. </p>
<p> </p>
<p>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+. </p>
<p>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.</p>
Concentration Effects on the Dynamics of Liquid Crystalline Self-Assembly: Time-Resolved X-ray Scattering Studies