Characterization of Microtubule Lattice Heterogeneity by Segmented Subtomogram Averaging.

Area of Science:

• Structural biology
• Cell biology
• Biophysics

Background:

• Microtubule structure is typically studied using single-particle analysis (SPA) or subtomogram averaging (STA) to understand alpha-beta tubulin heterodimer organization and interactions.
• The conventional model depicts microtubules with regular head-to-tail alpha-beta tubulin heterodimer arrangement and specific lateral interactions, except at a single seam.
• Previous studies have suggested the presence of multiple seams in microtubules, contradicting the established structural model.

Purpose of the Study:

• To investigate the intrinsic structural heterogeneity of microtubules in greater detail.
• To develop and apply a novel segmented subtomogram averaging (SSTA) strategy for analyzing microtubule structure.
• To reveal variations in microtubule lattice organization, including the number and position of seams.

Main Methods:

• Development of a segmented subtomogram averaging (SSTA) strategy.
• Decoration of microtubules with kinesin motor-domains, which bind to every alpha-beta tubulin heterodimer.
• Modeling of individual protofilaments and microtubule centers, followed by sub-volume extraction and averaging for segmented analysis.

Main Results:

• The SSTA approach successfully revealed structural heterogeneity within individual microtubules.
• Analysis demonstrated variations in the number and location of seams along the microtubule shaft.
• These findings were observed in microtubules assembled both in vitro from purified tubulin and in Xenopus egg cytoplasmic extracts.

Conclusions:

• The study challenges the textbook description of a single, fixed seam in microtubules.
• Segmented subtomogram averaging (SSTA) provides a powerful tool for dissecting microtubule structural heterogeneity.
• Microtubules exhibit dynamic variations in their seam organization, impacting their overall lattice structure.