Daniel F. Lusche

PTEN plays a role in the suppression of lateral pseudopod formation during Dictyostelium motility and chemotaxis.

It has been suggested that the phosphatydylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] phosphatase and tensin homolog PTEN plays a fundamental role in Dictyostelium discoideum chemotaxis. To identify that role, the behavior of a pten– mutant was quantitatively analyzed using two-dimensional and three-dimensional computer-assisted methods. pten– cells were capable of polarizing and translocating in the absence of attractant, and sensing and responding to spatial gradients, temporal gradients and natural waves of attractant. However, all of these responses were compromised (i.e. less efficient) because of the fundamental incapacity of pten– cells to suppress lateral pseudopod formation and turning. This defect was equally manifested in the absence, as well as presence, of attractant. PTEN, which is constitutively localized in the cortex of polarized cells, was found essential for the attractant-stimulated increase in cortical myosin II and F-actin that is responsible for the increased suppression of pseudopods during chemotaxis. PTEN, therefore, plays a fundamental role in the suppression of lateral pseudopod formation, a process essential for the efficiency of locomotion and chemotaxis, but not in directional sensing.

Interferon regulatory factor 6 regulates keratinocyte migration

ABSTRACT Interferon regulatory factor 6 (Irf6) regulates keratinocyte proliferation and differentiation. In this study, we tested the hypothesis that Irf6 regulates cellular migration and adhesion. Irf6-deficient embryos at 10.5 days post-conception failed to close their wound compared with wild-type embryos. In vitro, Irf6-deficient murine embryonic keratinocytes were delayed in closing a scratch wound. Live imaging of the scratch showed deficient directional migration and reduced speed in cells lacking Irf6. To understand the underlying molecular mechanisms, cell–cell and cell–matrix adhesions were investigated. We show that wild-type and Irf6-deficient keratinocytes adhere similarly to all matrices after 60 min. However, Irf6-deficient keratinocytes were consistently larger and more spread, a phenotype that persisted during the scratch-healing process. Interestingly, Irf6-deficient keratinocytes exhibited an increased network of stress fibers and active RhoA compared with that observed in wild-type keratinocytes. Blocking ROCK, a downstream effector of RhoA, rescued the delay in closing scratch wounds. The expression of Arhgap29, a Rho GTPase-activating protein, was reduced in Irf6-deficient keratinocytes. Taken together, these data suggest that Irf6 functions through the RhoA pathway to regulate cellular migration.

The effects of extracellular calcium on motility, pseudopod and uropod formation, chemotaxis, and the cortical localization of myosin II in Dictyostelium discoideum.

Extracellular Ca(++), a ubiquitous cation in the soluble environment of cells both free living and within the human body, regulates most aspects of amoeboid cell motility, including shape, uropod formation, pseudopod formation, velocity, and turning in Dictyostelium discoideum. Hence it affects the efficiency of both basic motile behavior and chemotaxis. Extracellular Ca(++) is optimal at 10 mM. A gradient of the chemoattractant cAMP generated in the absence of added Ca(++) only affects turning, but in combination with extracellular Ca(++), enhances the effects of extracellular Ca(++). Potassium, at 40 mM, can partially substitute for Ca(++). Mg(++), Mn(++), Zn(++), and Na(+) cannot. Extracellular Ca(++), or K(+), also induce the cortical localization of myosin II in a polar fashion. The effects of Ca(++), K(+) or a cAMP gradient do not appear to be similarly mediated by an increase in the general pool of free cytosolic Ca(++). These results suggest a model, in which each agent functioning through different signaling systems, converge to affect the cortical localization of myosin II, which in turn effects the behavioral changes leading to efficient cell motility and chemotaxis. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.

How a cell crawls and the role of cortical myosin II.

ABSTRACT The movements of Dictyostelium discoideum amoebae translocating on a glass surface in the absence of chemoattractant have been reconstructed at 5-second intervals and motion analyzed by employing 3D-DIAS software. A morphometric analysis of pseudopods, the main cell body, and the uropod provides a comprehensive description of the basic motile behavior of a cell in four dimensions (4D), resulting in a list of 18 characteristics. A similar analysis of the myosin II phosphorylation mutant 3XASP reveals a role for the cortical localization of myosin II in the suppression of lateral pseudopods, formation of the uropod, cytoplasmic distribution of cytoplasm in the main cell body, and efficient motility. The results of the morphometric analysis suggest that pseudopods, the main cell body, and the uropod represent three motility compartments that are coordinated for efficient translocation. It provides a contextual framework for interpreting the effects of mutations, inhibitors, and chemoattractants on the basic motile behavior of D. discoideum. The generality of the characteristics of the basic motile behavior of D. discoideum must now be tested by similar 4D analyses of the motility of amoeboid cells of higher eukaryotic cells, in particular human polymorphonuclear leukocytes.

Ca2+ chemotaxis in Dictyostelium discoideum.

Using a newly developed microfluidic chamber, we have demonstrated in vitro that Ca2+ functions as a chemoattractant of aggregation-competent Dictyostelium discoideum amoebae, that parallel spatial gradients of cAMP and Ca2+ are more effective than either alone, and that cAMP functions as a stronger chemoattractant than Ca2+. Effective Ca2+ gradients are extremely steep compared with effective cAMP gradients. This presents a paradox because there is no indication to date that steep Ca2+ gradients are generated in aggregation territories. However, given that Ca2+ chemotaxis is co-acquired with cAMP chemotaxis during development, we speculate on the role that Ca2+ chemotaxis might have and the possibility that steep, transient Ca2+ gradients are generated during natural aggregation in the interstitial regions between cells.

The IplA Ca2+ channel of Dictyostelium discoideum is necessary for chemotaxis mediated through Ca2+, but not through cAMP, and has a fundamental role in natural aggregation.

During aggregation of Dictyostelium discoideum, nondissipating, symmetrical, outwardly moving waves of cAMP direct cells towards aggregation centers. It has been assumed that the spatial and temporal characteristics of the front and back of each cAMP wave regulate both chemokinesis and chemotaxis. However, during the period preceding aggregation, cells acquire not only the capacity to chemotax in a spatial gradient of cAMP, but also in a spatial gradient of Ca2+. The null mutant of the putative IplA Ca2+ channel gene, iplA−, undergoes normal chemotaxis in spatial gradients of cAMP and normal chemokinetic responses to increasing temporal gradients of cAMP, both generated in vitro. However, iplA− cells lose the capacity to undergo chemotaxis in response to a spatial gradient of Ca2+, suggesting that IplA is either the Ca2+ chemotaxis receptor or an essential component of the Ca2+ chemotaxis regulatory pathway. In response to natural chemotactic waves generated by wild-type cells, the chemokinetic response of iplA− cells to the temporal dynamics of the cAMP wave is intact, but the capacity to reorient in the direction of the aggregation center at the onset of each wave is lost. These results suggest that transient Ca2+ gradients formed between cells at the onset of each natural cAMP wave augment reorientation towards the aggregation center. If this hypothesis proves correct, it will provide a more complex contextual framework for interpreting D. discoideum chemotaxis.

Nhe1 Is Essential for Potassium but Not Calcium Facilitation of Cell Motility and the Monovalent Cation Requirement for Chemotactic Orientation in Dictyostelium discoideum

ABSTRACT In Dictyostelium discoideum, extracellular K+ or Ca2+ at a concentration of 40 or 20 mM, respectively, facilitates motility in the absence or presence of a spatial gradient of chemoattractant. Facilitation results in maximum velocity, cellular elongation, persistent translocation, suppression of lateral pseudopod formation, and myosin II localization in the posterior cortex. A lower threshold concentration of 15 mM K+ or Na or 5 mM Ca2+ is required for chemotactic orientation. Although the common buffer solutions used by D. discoideum researchers to study chemotaxis contain sufficient concentrations of cations for chemotactic orientation, the majority contain insufficient levels to facilitate motility. Here it has been demonstrated that Nhe1, a plasma membrane protein, is required for K+ but not Ca2+ facilitation of cell motility and for the lower K+ but not Ca2+ requirement for chemotactic orientation.

Arachidonic acid is a chemoattractant for Dictyostelium discoideum cells.

Cyclic AMP (cAMP) is a natural chemoattractant of the social amoeba Dictyostelium discoideum. It is detected by cell surface cAMP receptors. Besides a signalling cascade involving phosphatidylinositol 3,4,5-trisphosphate (PIP3), Ca2+ signalling has been shown to have a major role in chemotaxis. Previously, we have shown that arachidonic acid (AA) induces an increase in the cytosolic Ca2+ concentration by causing the release of Ca2+ from intracellular stores and activating influx of extracellular Ca2+. Here we report that AA is a chemoattractant for D. discoideum cells differentiated for 8–9 h. Motility towards a glass capillary filled with an AA solution was dose-dependent and qualitatively comparable to cAMP-induced chemotaxis. Ca2+ played an important role in AA chemotaxis of wild-type Ax2 as ethyleneglycolbis(b-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA) added to the extracellular buffer strongly inhibited motility. In the HM1049 mutant whose iplA gene encoding a putative Ins(1,4,5)P3-receptor had been knocked out, chemotaxis was only slightly affected by EGTA. Chemotaxis in the presence of extracellular Ca2+ was similar in both strains. Unlike cAMP, addition of AA to a cell suspension did not change cAMP or cGMP levels. A model for AA chemotaxis based on the findings in this and previous work is presented.

Huntingtin regulates Ca2+ chemotaxis and K+-facilitated cAMP chemotaxis, in conjunction with the monovalent cation/H+ exchanger Nhe1, in a model developmental system: Insights into its possible role in Huntington׳s disease

Huntington׳s disease is a neurodegenerative disorder, attributable to an expanded trinucleotide repeat in the coding region of the human HTT gene, which encodes the protein huntingtin. These mutations lead to huntingtin fragment inclusions in the striatum of the brain. However, the exact function of normal huntingtin and the defect causing the disease remain obscure. Because there are indications that huntingtin plays a role in Ca(2+) homeostasis, we studied the deletion mutant of the HTT ortholog in the model developmental system Dictyostelium discoideum, in which Ca(2+) plays a role in receptor-regulated behavior related to the aggregation process that leads to multicellular morphogenesis. The D. discoideum htt(-)-mutant failed to undergo both K(+)-facilitated chemotaxis in spatial gradients of the major chemoattractant cAMP, and chemotaxis up a spatial gradient of Ca(2+), but behaved normally in Ca(2+)-facilitated cAMP chemotaxis and Ca(2+)-dependent flow-directed motility. This was the same phenotypic profile of the null mutant of Nhel, a monovalent cation/H(+)exchanger. The htt(-)-mutant also failed to orient correctly during natural aggregation, as was the case for the Nhel mutant. Moreover, in a K(+)-based buffer the normal localization of actin was similarly defective in both htt(-) and nhe1(-) cells in a K(+)-based buffer, and the normal localization of Nhe1 was disrupted in the htt(-) mutant. These observations demonstrate that Htt and Nhel play roles in the same specific cation-facilitated behaviors and that Nhel localization is directly or indirectly regulated by Htt. Similar cation-dependent behaviors and a similar relationship between Htt and Nhe1 have not been reported for mammalian neurons and deserves investigation, especially as it may relate to Huntington׳s disease.

A Computer-Assisted 3D Model for Analyzing the Aggregation of Tumorigenic Cells Reveals Specialized Behaviors and Unique Cell Types that Facilitate Aggregate Coalescence

We have developed a 4D computer-assisted reconstruction and motion analysis system, J3D-DIAS 4.1, and applied it to the reconstruction and motion analysis of tumorigenic cells in a 3D matrix. The system is unique in that it is fast, high-resolution, acquires optical sections using DIC microscopy (hence there is no associated photoxicity), and is capable of long-term 4D reconstruction. Specifically, a z-series at 5 μm increments can be acquired in less than a minute on tissue samples embedded in a 1.5 mm thick 3D Matrigel matrix. Reconstruction can be repeated at intervals as short as every minute and continued for 30 days or longer. Images are converted to mathematical representations from which quantitative parameters can be derived. Application of this system to cancer cells from established lines and fresh tumor tissue has revealed unique behaviors and cell types not present in non-tumorigenic lines. We report here that cells from tumorigenic lines and tumors undergo rapid coalescence in 3D, mediated by specific cell types that we have named “facilitators” and “probes.” A third cell type, the “dervish”, is capable of rapid movement through the gel and does not adhere to it. These cell types have never before been described. Our data suggest that tumorigenesis in vitro is a developmental process involving coalescence facilitated by specialized cells that culminates in large hollow spheres with complex architecture. The unique effects of select monoclonal antibodies on these processes demonstrate the usefulness of the model for analyzing the mechanisms of anti-cancer drugs.