Dictyostelium Myosin Bipolar Thick Filament Formation: Importance of Charge and Specific Domains of the Myosin Rod

Myosin-II thick filament formation in Dictyostelium is an excellent system for investigating the phenomenon of self-assembly, as the myosin molecule itself contains all the information required to form a structure of defined size. Phosphorylation of only three threonine residues can dramatically change the assembly state of myosin-II. We show here that the C-terminal 68 kDa of the myosin-II tail (termed AD-Cterm) assembles in a regulated manner similar to full-length myosin-II and forms bipolar thick filament (BTF) structures when a green fluorescent protein (GFP) “head” is added to the N terminus. The localization of this GFP-AD-Cterm to the cleavage furrow of dividing Dictyostelium cells depends on assembly state, similar to full-length myosin-II. This tail fragment therefore represents a good model system for the regulated formation and localization of BTFs. By reducing regulated BTF assembly to a more manageable model system, we were able to explore determinants of myosin-II self-assembly. Our data support a model in which a globular head limits the size of a BTF, and the large-scale charge character of the AD-Cterm region is important for BTF formation. Truncation analysis of AD-Cterm tail fragments shows that assembly is delicately balanced, resulting in assembled myosin-II molecules that are poised to disassemble due to the phosphorylation of only three threonines

Localization of GFP-RLC-Tail Fragment Constructs in Live Dividing <i>Dictyostelium</i> Cells

<p>The localization of several GFP-RLC-myosin tail fragments during and just after cytokinesis in live <i>Dictyostelium</i> cells are shown. GFP-myosin (row 1) is clearly localized to the early and late cleavage furrow of the dividing cell and to the back end of the resulting daughter cells. By contrast, GFP-RLC-AD-Cterm (3xThr) (row 2) is localized correctly only at the late stages of cytokinesis and in the back end of one daughter cell. GFP-RLC-AD-Cterm (3xAla) (row 3) is localized to the furrow as well as to the back end of a daughter cell, while GFP-RLC-AD-Cterm (3xAsp) (row 4) shows diffuse localization throughout cytokinesis. The scale bar is 10 μm and the time is indicated in min:sec.</p

Characterization of “Headless” AD-Cterm Tail Fragments

<div><p>(A) Analysis of assembly by sedimentation. Fraction of soluble protein as a function of NaCl concentration is plotted for the constructs depicted adjacent to the graph. The solubility of “headless” AD-Cterm (3xThr) and AD-Cterm (3xAsp) are compared to the solubility of unphosphorylated and phosphorylated full-length myosin having the globular motor domain (full-length myosin data from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020356#pbio-0020356-Cote1" target="_blank">Cote and McCrea [1987]</a>).</p> <p>(B) Sedimentation equilibrium analysis of 52 μM “headless” AD-Cterm (3xThr) and AD-Cterm (3xAsp) in buffer containing 500 mM NaCl. The top graphs show the concentration distribution, fit, and residuals for AD-Cterm (3xThr), while the bottom graphs show the same data for AD-Cterm (3xAsp). The molecular weight obtained from the fit was 130 kDa for AD-Cterm (3xThr) and 120 kDa for AD-Cterm (3xAsp).</p> <p>(C) Thermal melts of “headless” AD-Cterm (3xThr) and AD-Cterm (3xAsp) in buffer containing 500 mM NaCl are shown as fraction of protein denatured as a function of temperature. The open circles are data for 50 μM “headless” AD-Cterm (3xThr), and the open squares are data for 50 μM “headless” AD-Cterm (3xAsp).</p></div

Tail Fragments Used for Truncation Analysis

<p>For tail fragments that include more than one domain, the name is determined by the first and last domain in the tail fragment. Phosphorylation sites are indicated in parentheses. 1xThr indicates that the fragment contains the threonine at a.a. 1,823; 2xThr indicates that the fragment contains the threonines at a.a. 1,823 and 1,833; and 3xThr indicates that the fragment contains the threonines at a.a. 1,823, 1,833, and 2,029. The same scheme is used to describe aspartic acid-containing constructs in the paper, except threonine is substituted with aspartic acid.</p

Design and Assembly Characteristics of AD-Cterm-GFP

<div><p>(A) The AD-Cterm charge distribution (top) is aligned with the reverse charge distribution (bottom), showing the overall symmetry of the 196-a.a. charge repeat in this region of the tail.</p> <p>(B) Analysis of assembly by EM. The AD-Cterm (3xThr) tail fragment has GFP on the C-terminus (AD-Cterm-GFP [3xThr]). The scale bar indicates a distance of 100 nm. Shown are three images of AD-Cterm GFP (3xThr), assembled 2–5 min.</p> <p>(C) Analysis of assembly by sedimentation. The solubility of AD-Cterm-GFP (3xThr) and AD-Cterm-GFP (3xAsp) tail fragments constructs are compared to GFP-AD-Cterm (3xThr) and “headless” AD-Cterm (3xThr) tail fragments.</p></div

Domains and Charge Distribution within the Myosin Tail

<div><p>(A) The myosin head (1–818) and light chains are shown at the N terminus. In the coiled-coil tail, Ala 1 is red (1,348–1,530), the AD is blue (1,531–1,824), Ala 2 is purple (1,825–1,966), the C-terminal domain is green (1,967–2,116), and the remainder is black (819–1,347). Phospho-threonines (at positions 1,823, 1,833, and 2,029) are indicated by the letter T.</p> <p>(B and C) Plots of the average charge of each tail domain color coded as in (A). The y-axis is average charge; the x-axis is tail position. Aspartic acid and glutamic acid are assigned –1, lysine and arginine are assigned +1, and all other a.a. are assigned 0. The average charge in (B) was determined with a window size of 14 a.a., and the average charge in (C) was determined with a window size of 28 a.a.. Arrows highlight the 28 a.a. charge repeat in (B) and the 196 a.a. charge repeat in (C).</p> <p>(D) “Headless” AD-Cterm (3xThr) (1,531–2,116).</p></div