Steimle Lab Research

The function of a cell is dependent on its ability to undergo highly coordinated changes in shape and movement. In my lab, we use cellular, biochemical, and molecular approaches to study the processes driving changes in cell shape and how these processes are differentially regulated during diverse cellular motility events such as cell migration and cell division. To this end, we use the highly versatile eukaryotic model organism, Dictyostelium discoideum, Our studies provide an opportunity to identify how myosin II-mediated cellular activities (i.e. cytokinesis and cellular migration) can be regulated in a nonmuscle cell context, and by extension, how these processes can go awry in cancer cells exhibiting uncontrolled cell division and metastasis. Moreover, the studies proposed here will be important for understanding the basic mechanisms by which cellular migration is achieved in other contexts such as wound healing, chemotaxis, and metazoan development.

A major focus of the studies in my lab is to gain further insight into the the basic processes controlling myosin II filament assembly and how variations in such regulatory mechanisms can ultimately specify which of the diverse roles myosin II plays in the dynamic environment of the cell.

Numerous studies have established that the ability of Dictyostelium myosin II to assemble into filaments is inhibited dramatically upon  phosphorylation of three specific threonine residues (sites 1823, 1833, and 2029) located toward the carboxy-terminus of the MHC tail.  These MHC phosphorylation sites were initially recognized as target sites for the enzyme MHC kinase A (MHCK A). A schematic of MHCK A is provided below:

Domain Structure of MHCK A

MHCK A possesses a novel, modular organization containing an amino-terminal domain that is predicted to have significant a-helical coiled-coil structure and we have shown previously that the coiled-coild domain mediates the oligomerization, cellular localization, and actin-binding activities of the kinase (reference). We have recently demonstrated that F-actin binding by the coiled-coil domain also leads to a 40-fold increase in MHCK A activity (reference). The central catalytic region of MHCK A is the prototype for a novel family of protein kinases, referred to as "a-kinases", which have been identified in diverse groups of organisms, with several members present in Dictyostelium. At its carboxyl-terminus, MHCK A possesses a domain containing a 7-fold WD repeat motif that we have shown facilitates phosphorylation of myosin heavy chain by physically targeting MHCK A directly to myosin II filaments (reference).

Our studies of MHCK A cellular localization revealed that the kinase is recruited to actin-rich cortical sites and is preferentially enriched at sites of pseudopod formation, and thus MHCK A is proposed to play a role in regulating localized disassembly of myosin II filaments in the cell. The timelapse videos presented on this page show the localization characteristics of green fluorescent protein (GFP)-tagged MHCK A expressed from a plasmid in living cells; the images for these videos were collected at 10 min intervals.. The video below (to the left) is a good representation of the persistent enrichment of GFP-MHCK A at the leading edges of the cellular projections that is characteristic of this enzyme kinase (reference). In the context of chemoattractant stimulation, we have found that MHCK A undergoes a dramatic translocation from a diffuse cytoplasmic distribution to a highly enriched cortical localization (see video below and to the left) when exposed to a uniform, saturating concentration of cyclic-AMP. These localization properties are mediated by the coiled-coil domain of MHCK A, since a GFP-tagged truncation of MHCK A containing only the coiled-coil domain exhibits all of the localization properties of the full-length kinase (see video labeled "GFP-Coil") - reference. In the context of cytokinesis, MHCK A is enriched at the actin-rich polar regions of the dividing cell (Liang et al., 2002; Nagasaki et al., 2002).

The animation below depicts our working model of MHCK A function in the cell-- in which we have proposed that MHCK A is preferentially enriched at sites of pseudopod formation at the cell anterior where the kinase is expected to catalyze the disassembly of myosin II-based cross-linking of actin filaments which, in turn, may facilitate the reorganization of the actin cytoskeleton necessary for the formation of cellular extensions.

We are continuing our studies of MHCK A, by focusing on the actin-binding activity of the coiled-coil domain of the kinase. It is particularly noteworthy that the coiled-coil domain appears to be a novel actin-binding domain since it exhibits no sequence similarity to any known actin-binding proteins. We are also exploring similar structure-function studies of other myosin II heavy chain kinases in Dictyostelium with the aim of understanding how the activities of these enzymes are coordinated in the cell.