Dictyostelium and Acanthamoeba myosin II assembly domains go to the cleavage furrow of Dictyostelium myosin II-null cells

Abstract

How myosin II localizes to the cleavage furrow of dividing cells is largely unknown. We show here that a 283-residue protein, assembly domain (AD)1, corresponding to the AD in the tail of Dictyostelium myosin II assembles into bundles of long tubules when expressed in myosin II-null cells and localizes to the cleavage furrow of dividing cells. AD1 mutants that do not polymerize in vitro do not go to the cleavage furrow in vivo. An assembly-competent polypeptide corresponding to the C-terminal 256 residues of Acanthamoeba myosin II also goes to the cleavage furrow of Dictyostelium myosin II-null cells. When overexpressed in wild-type cells, AD1 colocalizes with endogenous myosin II (possibly as a copolymer) in interphase, motile, and dividing cells and under caps of Con A receptors but has no effect on myosin II-dependent functions. These results suggest that neither a specific sequence, other than that required for polymerization, nor interaction with other proteins is required for localization of myosin II to the cleavage furrow.

Fig. 1.

AD constructs. Schematic representation of Dictyostelium myosin II heavy chain showing the coiled-coil helical tail (residues 819–2116) and the location of the AD (residues E1533–K1819). The AD mutants discussed in this article are AD1 (residues 1533–1815), AD2 (residues 1533–1804), AD3 (residues 1784–1804 of AD1 replaced by Dictyostelium myosin II tail residues 1833–1853), AD4 (residues 1694–1749, the homology region, of AD1 deleted), AD5 (residues 1694–1749 of AD1 replaced by the homology region of Acanthamoeba myosin II, residues 1303–1358), and AD6 (residues 1694–1749 of AD1 replaced by the homology region of smooth muscle myosin II, residues 1533–1588). All constructs had an N-terminal FLAG tag and are predicted to be 100% coiled-coil (21).

Fig. 2.

In vitro properties of AD1 and AD1 mutants. (A) Coomassie blue-stained SDS/PAGE of total mixture (T), supernatant (S), and pellet (P) after dialysis against 50 mM NaCl/10 mM MgCl₂ and centrifugation. (B) Negatively stained electron-microscopic images of AD1–AD6 after dialysis against 50 mM NaCl/10 mM MgCl₂.(C) Negatively stained images of AD1 after dialysis against 25 mM NaCl, 50 mM NaCl/2 mM MgCl₂, or 50 mM NaCl/10 mM MgCl₂.

Fig. 3.

Overexpression of AD1 and AD1 mutants in Dictyostelium myosin II-null cells. Shown are SDS/PAGE and Western blot with anti-FLAG antibody of total cell extracts of HS1 cells overexpressing FLAG-tagged AD1–AD6.

Fig. 4.

AD1 goes to the CF of Dictyostelium myosin II-null cells. (A) Immunostained interphase HS1 cells overexpressing FLAG-tagged AD1 showing predominantly cortical staining (wide arrow) and less frequent rods (thin arrow). (B and C) Thin-section electron micrographs showing longitudinal and cross-sectional views of bundles of tubules of AD1 in transfected interphase HS1 cells. (D–F) Immunostained dividing HS1 cells showing localization to the CF of overexpressed AD1.

Fig. 5.

Constructs AD2–AD6 do not assemble or go to the CF when expressed in Dictyostelium myosin II-null cells. Shown are immunostained interphase (A) and dividing (B) HS1 cells overexpressing AD2–AD6.

Fig. 6.

Assembly region of Acanthamoeba myosin II goes to the CF of Dictyostelium myosin II-null cells. (A) Schematic representation of Acanthamoeba myosin II showing the location of the homology region within the assembly region that spans the C-terminal half of the coiled-coil tail and the nonhelical tailpiece. (B) Negatively stained electron-microscopic image of filaments of the assembly region after dialysis against 50 mM NaCl/10 mM MgCl₂. (C) Micrograph of immunostained HS1 interphase cell overexpressing FLAG-tagged assembly region. (D–F) Micrographs of immunostained HS1 dividing cells overexpressing FLAG-tagged assembly region at progressive stages of division.

Fig. 7.

Expressed AD1 colocalizes with myosin II in Dictyostelium wild-type cells. (A) SDS/PAGE and Western blot of total cell proteins of AX3 cells and AX3 cells expressing AD1. (B) Thin-section electron micrograph showing longitudinal and cross-sectional (arrows) views of bundles of tubules of overexpressed AD1 in transfected interphase AX3 cells. (C) Colocalization of endogenous wild-type myosin II (Anti-Dd-wt) and AD1 (Anti-Flag) in interphase AX3 cells overexpressing AD1. (D) Colocalization of endogenous wild-type myosin II (Anti-Dd-wt) and AD1 (Anti-Flag) in dividing AX3 cells overexpressing AD1.

Fig. 8.

AD1 does not inhibit myosin II function in Dictyostelium wild-type cells. (A) Growth in suspension culture of HS1 (myosin-null) and AX3 (wild-type) cells and HS1 and AX3 cells overexpressing AD1, AD1-HS1, and AD1–AX3, respectively. (B) Development of cells after 24 h on starvation medium. (C) Colocalization of AD1 and myosin II at rear of a wild-type cell moving from right to left. (D) Co-capping of AD1 and myosin II under Con A caps at 5 min and redispersal of AD1 and myosin II by 25 min after capping in wild-type cells.