Higher Order Assembly of Microtubules by Counter-ions: From Hexagonal Bundles to Living Necklaces

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Original entry: Tom Kodger, APPHY 226, Spring 2009

Overview

The authors explore the phase space of microtubule (MT) assembly in the presence of multi-valent cations using various experimental techniques. Analogies are made toward highly charged nanotube and nanowire assembly in technological endeavors. In cells, the presence of cations in the cytoplasm is functionally important and highly regulated. The controlled assembly and disassembly of highly charged polyelectrolytes including DNA, MTs, and filamentous actin is currently poorly understood. These processes play key roles in cellular functions such as cell division (MTs and actin (contractile ring)), intracellular transport (MTs), and cytoskeletal remodeling (MTs and filamentous actin). These processes are also proposed to affect gene regulation (heterochromatin-DNA).

Review of Microtubules

Microtubules are hollow cylindrical protein polymers with inner and outer diameters of 15nm and 25nm respectfully. They are composed of polymerized α and β tubulin subunits. MTs can be up to 10s of microns in length, with a bending radius on the order of millimeters to centimeters, but are typically short (<5um) in the cytoplasm due to active assembly and disassembly.

Naveen's comments: I though that many cells are on the order of microns in size. Is the typical length 
due to active reconstruction or just the dimensions of the cell?
Sung Hoon's comment: How are these microtubules formed? Is there any study about the mechanical properties of these
microtubules?

Experimental Techniques

SAXRD

Small angle x-ray diffraction is a common scattering technique that allows an experimental scattering vector (q) on the order of 10s of nm-1 to single A-1. For more information see Wikipedia-SAXS

<math> q\ =\ 2\ k\ \sin(\theta) = \frac{4 \pi\ \sin(\theta)}{\lambda}</math>


For SAXS, the θ range is typically defined as 0.01° to 20°. USAXS extends this range down to 0.001°.

Other

Transmission Electron Micrscopy (TEM) and Differential Interference Microscopy (DIC) are also used in this paper to visualize the MT assembly over different length scales.

Discussion

Direct Imaging

Fig.1, Cartoon of MT bundles

As seen in TEM, the valency of the cation is dropped from +5 to +2, the structral morphology of MT assembly is altered from a hexagonal to a flexible linear ordering, termed 'necklace-like'.

Fig.2, With +3,+4,+5 cations

Using multivalent cations (>+3) MT assemblies for a highly hexagonal cross-section (Fig.2B upper). Defects in ordered bundles are infrequent but still present (Fig.2B lower).

Fig.3, With divalent cations

At reasonable divalent concentrations (60mM BaCl2, 40mM CaCl2, 60mM SrCl2), linear MT assembly are seen. The authors note that thermal fluctuations allow these bundles to interconnect and generate a highly dynamic morphology, as seen with DIC microscopy. No theory can currently predict this phenomena which may be highly relevant in cellular activity.

Scattering

SAXS data is modeled assuming a hollow tube geometry with two free fitting parameters; aH (center to center distance) and w (peak width). The results are consistent with TEM images. Fits to the scattering curves resulted in the following findings:

  1. MT bundles vary from ~14MTs in cross-section for pentavalent, to ~8MTs for trivalent cations.
  2. MT-MT spacing decreases with cation physical size (oligoamine < oligolysines)
  3. Divalent samples have broad peaks, which is interpreted as a more polydispersed and dynamic bundle structure.

Importance

MT bundles are stabilized by a balance of hydration and long-range electrostatic repulsive forces, and attractive forces including van der Waals and ion correlations. Interestingly, the maximum MT-wall to MT-wall separation values are measured to be >5.5nm, which is beyond electrostatic and hydration repulsive distances. Overall, the experimental system studied here is highly complex, containing many repulsive and attractive forces including osmotic pressure of monovalent ions (not fully discussed). The result is a rich phase space which was not fully explored and should be highly relevant to the assembly of highly anisotropic nanomaterials, like nanowires.

Naveen's comments: This is an interesting study. Do you know how 
the ion concentrations studied here compare to those inside a living cell?

Tom's response: After some discussion with Prof. Needleman, while the ion concentration is comparable to cytoplasm, thermal affects are minimal due to rapid active rearrangements by motor proteins.

Could this structure act as an organic/sacrificial template for an inorganic skeleton, similar to surfactants?
--Lidiya 02:50, 18 February 2009 (UTC)