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. 2012 Oct 15;125(Pt 20):4934-44.
doi: 10.1242/jcs.112474. Epub 2012 Aug 16.

Myosin heavy chain kinases play essential roles in Ca2+, but not cAMP, chemotaxis and the natural aggregation of Dictyostelium discoideum

Deborah Wessels  1 , Daniel F Lusche, Paul A Steimle, Amanda Scherer, Spencer Kuhl, Kristen Wood, Brett Hanson, Thomas T Egelhoff, David R Soll
Affiliations

Myosin heavy chain kinases play essential roles in Ca2+, but not cAMP, chemotaxis and the natural aggregation of Dictyostelium discoideum

Deborah Wessels et al. J Cell Sci. .

Abstract

Behavioral analyses of the deletion mutants of the four known myosin II heavy chain (Mhc) kinases of Dictyostelium discoideum revealed that all play a minor role in the efficiency of basic cell motility, but none play a role in chemotaxis in a spatial gradient of cAMP generated in vitro. However, the two kinases MhckA and MhckC were essential for chemotaxis in a spatial gradient of Ca(2+), shear-induced directed movement, and reorientation in the front of waves of cAMP during natural aggregation. The phenotypes of the mutants mhckA(-) and mhckC(-) were highly similar to that of the Ca(2+) channel/receptor mutant iplA(-) and the myosin II phosphorylation mutant 3XALA, which produces constitutively unphosphorylated myosin II. These results demonstrate that IplA, MhckA and MhckC play a selective role in chemotaxis in a spatial gradient of Ca(2+), but not cAMP, and suggest that Ca(2+) chemotaxis plays a role in the orientation of cells in the front of cAMP waves during natural aggregation.

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Figures

Fig. 1.
Fig. 1.
Deleting the individual MHCK genes can have minor effects on the basic motile behavior of cells in the absence of chemoattractant. Cell behavior was assessed in a chamber perfused with buffer lacking attractant. (A–D) Representative perimeter tracks of cells of parental strain JH10 and mutants mhckA−, mhckB− and mhckC−, respectively. (E) Quantification of the motility paremeters instantaneous velocity (Inst. vel.), percentage of cells with velocities ≧9 µm per min (Percent cells ≧9 µm/min), positive flow (Posit. Flow) and persistence (Persist.). Arrows in A–D indicate net direction of a cell.
Fig. 2.
Fig. 2.
Deleting the individual MHCK genes does not affect the efficiency of chemotaxis in a spatial gradient of cAMP. Chemotaxis was assessed in a gradient chamber designed after that of Zigmond (Zigmond, 1977; Varnum and Soll, 1984). (A–D) Representative perimeter tracks of cells of parental strain JH10 and mutants mhckA−, mhckB− and mhckC−, respectively. (E) Quantification of motility and chemotaxis parameters. The motility parameters are defined in the legend in Fig. 1. The chemotaxis parameters were chemotactic index (Chem. Index, C.I.) and percentage of cells with a positive chemotactic index (Percent + C.I.). Large arrows in A–D represent direction of cAMP gradient.
Fig. 3.
Fig. 3.
Deleting the individual MHCK genes does not affect the chemokinetic response to temporal waves of cAMP generated in vitro that mimic the temporal dynamics of naturally relayed cAMP waves. Instantaneous velocity is plotted as a function of time. The cAMP waves are plotted above the velocity plots. “f” and blue shading denotes the front (increasing temporal gradient) of each wave.
Fig. 4.
Fig. 4.
The mutants mhckA−, mhckB− and mhckA−B−C− have lost the capacity to undergo chemotaxis in a spatial gradient of Ca2+. They also lose flow-induced directed motility. Chemotaxis and flow-induced directed motility were assessed in a microfluidic chamber (Scherer et al., 2010). (A) Quantification of motility parameters, flow-induced movement and chemotaxis. Motility and chemotaxis parameters are defined in the legends to Figs 1 and 2, respectively. Right. direct., rightward directionality. (B–E) Representative perimeter tracks of cells of parental strain Ax2 and mutants mhckA−, mhckB− and mhckC−, respectively. (F,G) Consecutive video frames at 2 minute intervals of Ax2 and mhckA− cells, respectively. In B–E, thick and thin arrows represent direction of Ca2+ gradient and cell movement, respectively. In F and G, the white dashed line denotes the middle of the chamber, and time is presented in minutes (M).
Fig. 5.
Fig. 5.
The mutants mhckA−,mhckC−, and mhckA−B−C− have lost the capacity to reorient in the front of each natural wave, in the direction of the aggregation center, the source of the waves. Parental and mutant cells were mixed at a ratio of 9∶1 and allowed to aggregate. (A,C,E,G) Representative velocity plots of cells responding to the temporal dynamics of successive natural waves (drawn above each velocity plot) for parent strain JH10 and mutants mhckA−, mhckB− and mhckC−, respectively (mhckD− and mhckA−B−C− not shown). In A, the data are for two representative JH10 cells in a homogeneous JH10 population (both black plots), and in C, E and G, the data are for a JH10 (black) and mutant (red) cell. (B,D,F,H) Representative centroid tracks at 20 sec. intervals in successive natural waves. In B, the data are for two representative JH10 cells (black). In D, F and H, data are for a representative control cell (black) and representative mutant (red) cells in 9∶1 mixtures. (I) Method for assessing the angle in the front of a wave. (J) Measured average angle in the front of successive waves.
Fig. 6.
Fig. 6.
Myosin II distribution in wild-type and mutant strains during cellular translocation. Cells were mixed and stained with anti-myosin II antibody. ps, pseudopod; u, uropod.
Fig. 7.
Fig. 7.
Varying Ca2+ concentration or adding calmodulin has little effect on MhckA activity in vitro. (A–C) The phosphorylation reaction was performed with the MH-1 peptide described elsewhere (Steimle et al., 2001a). Similar results were obtained with MhckC.
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