Deborah Wessels

A role for myosin VII in dynamic cell adhesion

BACKGROUND The initial stages of phagocytosis and cell motility resemble each other. The extension of a pseudopod at the leading edge of a migratory cell and the formation of a phagocytic cup are actin dependent, and each rely on the plasma membrane adhering to a surface during dynamic extension. RESULTS A myosin VII null mutant exhibited a drastic loss of adhesion to particles, consistent with the extent of an observed decrease in particle uptake. Additionally, cell-cell adhesion and the adhesion of the leading edge to the substratum during cell migration were defective in the myosin VII null cells. GFP-myosin VII rescued the phagocytosis defect of the null mutant and was distributed in the cytosol and recruited to the cortical cytoskeleton, where it appeared to be enriched at the tips of filopods. It was also localized to phagocytic cups, but only during the initial stages of particle engulfment. During migration, GFP-myosin VII is found at the leading edge of the cell. CONCLUSIONS Myosin VII plays an important role in mediating the initial binding of cells to substrata, a novel role for an unconventional myosin.

A novel cGMP signalling pathway mediating myosin phosphorylation and chemotaxis in Dictyostelium

Chemotactic stimulation of Dictyostelium cells results in a transient increase in cGMP levels, and transient phosphorylation of myosin II heavy and regulatory light chains. In Dictyostelium, two guanylyl cyclases and four candidate cGMP‐binding proteins (GbpA–GbpD) are implicated in cGMP signalling. GbpA and GbpB are homologous proteins with a Zn2+‐hydrolase domain. A double gbpA/gbpB gene disruption leads to a reduction of cGMP‐phosphodiesterase activity and a 10‐fold increase of basal and stimulated cGMP levels. Chemotaxis in gbpA−B− cells is associated with increased myosin II phosphorylation compared with wild‐type cells; formation of lateral pseudopodia is suppressed resulting in enhanced chemotaxis. GbpC is homologous to GbpD, and contains Ras, MAPKKK and Ras‐GEF domains. Inactivation of the gbp genes indicates that only GbpC harbours high affinity cGMP‐binding activity. Myosin phosphorylation, assembly of myosin in the cytoskeleton as well as chemotaxis are severely impaired in mutants lacking GbpC and GbpD, or mutants lacking both guanylyl cyclases. Thus, a novel cGMP signalling cascade is critical for chemotaxis in Dictyostelium, and plays a major role in myosin II regulation during this process.

Cell Biology of Mating in Candida albicans

ABSTRACT It was recently demonstrated that strains homozygous for either of the mating type-like loci MTLa and MTLα of Candida albicans undergo white-opaque switching and that expression of the opaque-phase phenotype greatly enhances mating between strains. Exploiting the latter property to obtain high-frequency mating, we have characterized the cell biology of the mating process of C. albicans. Employing continuous videomicroscopy, computer-assisted three-dimensional reconstruction of living cells, and fluorescence microscopy, we have monitored the mating-associated processes of conjugation, tube formation, fusion, budding, septum formation, and daughter cell development and the spatial and temporal dynamics of nuclear migration and division. From these observations, a model for the stages in C. albicans mating is formulated. The stages include shmooing, chemotropism of conjugation tubes, fusion of tubes and nuclear association, vacuole expansion and nuclear separation in the conjugation bridge, asynchronous nuclear division in the zygote, bud growth, nuclear migration into the daughter cell, septation, and daughter cell budding. Since there was no cytological indication of karyogamy, genetic experiments were performed to assess marker segregation. Recombination was not observed, suggesting that mating takes place in the absence of karyogamy between naturally occurring, homozygous a and α strains. This study provides the first description of the cell biology of the mating process of C. albicans.

A computer-assisted system for reconstructing and interpreting the dynamic three-dimensional relationships of the outer surface, nucleus and pseudopods of crawling cells

Newly developed software additions to the three-dimensional dynamic image analysis system, 3D-DIAS, are described for simultaneously reconstructing and motion analyzing in three dimensions the outer surface, nucleus and pseudopods of living, crawling cells. This new system is then used to describe for the first time a nuclear behavior cycle in translocating Dictyostelium discoideum amoebae and to investigate the role of pseudopod extension in this process. The nuclear behavior cycle is tuned to the two phases of the general cell behavior cycle [Wessels et al., 1994], and includes nuclear migration both in the z- and in the x,y-axes from the proximal border of the prior anterior pseudopod to the proximal border of a newly expanding anterior pseudopod. Nuclear migration is cued by pseudopod-substratum contact, achieves velocities in excess of 50 microm/min, and is accompanied by characteristic changes in nuclear shape. The rules and characteristics of nuclear behavior are demonstrated to be intact in two mutants affecting pseudopod formation, a myosin IB null mutant (myoB-) and a myosin II heavy chain phosphorylation mutant (3XALA). The rules and characteristics of nuclear migration, however, are disrupted upon dissolution of microtubules by colcemid. Together the above results demonstrate that the newly developed 3D-DIAS system can be used to gain new insights into the dynamic changes in the intracellular 3D architecture associated with cellular translocation.

Release of a Potent Polymorphonuclear Leukocyte Chemoattractant Is Regulated by White-Opaque Switching in Candida albicans

ABSTRACT Previous studies employing transmembrane assays suggested that Candida albicans and related species, as well as Saccharomyces cerevisiae, release chemoattractants for human polymorphonuclear leukocytes (PMNs). Because transmembrane assays do not definitively distinguish between chemokinesis and chemotaxis, single-cell chemotaxis assays were used to confirm these findings and test whether mating-type or white-opaque switching affects the release of attractant. Our results demonstrate that C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis, and C. glabrata release bona fide chemoattractants for PMNs. S. cerevisiae, however, releases a chemokinetic factor but not a chemoattractant. Characterization of the C. albicans chemoattractant revealed that it is a peptide of approximately 1 kDa. Whereas the mating type of C. albicans did not affect the release of chemoattractant, switching did. White-phase cells released chemoattractant, but opaque-phase cells did not. Since the opaque phase of C. albicans represents the mating-competent phenotype, it may be that opaque-phase cells selectively suppress the release of chemoattractant to facilitate mating.

A Dictyostelium myosin I plays a crucial role in regulating the frequency of pseudopods formed on the substratum

Analysis of the motile behavior of a strain of Dictyostelium lacking a myosin I, myoA, revealed that this mutant strain formed pseudopods and turned twice as frequently as wild type cells [Titus et al., 1993: Mol. Biol. Cell 4:233-246]. The basis for this aberrant behavior has been explored using three-dimensional reconstructions of translocating cells. Wild type cells form approximately 40% of pseudopods on the substratum and 60% off the substratum. The majority of pseudopods formed on the substratum initiate sharp turns while the majority of pseudopods formed off the substratum are retracted. Although myoA- cells form pseudopods at roughly twice the frequency of wild type cells, the increase in frequency is specific for only those pseudopods formed on the substratum. This increase is the basis for the aberrant increase in turning in myoA- cells. The selective increase in the frequency of pseudopods formed on the substratum correlates with a number of additional abnormalities in myoA- pseuodpod formation. First, myoA- cells can simultaneously extend more than one pseudopod, whereas wild type cells extend only one pseudopod at a time. Second, although wild type and myoA- pseudopods achieve the same final volumes, myoA- pseudopods grow at half the rate of wild type pseudopods and, therefore, take longer to achieve final volume. Third, while a wild type pseudopod grows in a continuous fashion, a myoA- pseudopod grows in a discontinuous fashion. Together, these results demonstrate that myoA plays a fundamental role in controlling the frequency of only those pseudopods formed on the substratum, and that maintenance of the normal frequency of pseudopod formation appears to be necessary for the normal velocity of cellular translocation, the normal frequency of turning, the normal rate of average pseudopod growth, and the high efficiency of chemotaxis. These results in turn indicate that pseudopod formation is precisely coordinated in space and time, and actin-associated proteins like myoA play key roles in coordination.

Phosphorylation of the Dictyostelium myosin II heavy chain is necessary for maintaining cellular polarity and suppressing turning during chemotaxis

Conversion of the three mapped threonine phosphorylation sites in the myosin II heavy chain tail to alanines results in a mutant (3XALA) in Dictyostelium discoideum, which displays constitutive myosin overassembly in the cytoskeleton and increased cortical tension. To assess the importance of myosin phosphorylation in cellular translocation and chemotaxis, 3XALA mutant cells have been analyzed by 2D and 3D computer-assisted methods in buffer, in a spatial gradient of cAMP, and after the rapid addition of cAMP. 3XALA cells crawling in buffer exhibit distinct abnormalities in cellular shape, the maintenance of polarity and the complexity of the pseudopod perimeter. 3XALA cells crawling in buffer also exhibit a decrease in directionality. In a spatial gradient of cAMP, the behavioral defects are accentuated. In a spatial gradient, 3XALA cells exhibit a repeating 1- to 2-min behavior cycle in which the shape of each cell changes abnormally from elongate to extremely wide with lateral, opposing pseudopods. At the end of each cycle, 3XALA cells turn 90 degrees into the left or right lateral pseudopod, resulting in a dramatic depression in chemotactic efficiency, even though 3XALA cells are chemotactically responsive to cAMP. These results demonstrate that the phosphorylation of myosin II heavy chain plays a critical role in the maintenance of cell shape and in persistent translocation in a spatial gradient of chemoattractant.

Asynchronous Cell Cycle and Asymmetric Vacuolar Inheritance in True Hyphae of Candida albicans

ABSTRACT Candida albicans forms unconstricted hyphae in serum-containing medium that are divided into discrete compartments. Time-lapse photomicroscopy, flow cytometry, and a novel three-dimensional imaging system were used to demonstrate that the kinetics and cell cycle events accompanying hyphal development were correlated with dynamic changes in vacuole morphology and the pattern of vacuole inheritance. Apical cells of hyphae underwent continuous extension before and after the first cytokinesis event. However, the resulting mother cell and sub-apical compartments did not immediately reenter the cell cycle and instead underwent cell cycle arrest before reentering the cycle. Vacuole was inherited asymmetrically at cytokinesis so that the distal, arrested compartments inherited most vacuole and the growing apical cell inherited most cytoplasm. Hydroxyurea release experiments demonstrated that the arrested, vacuolated hyphal compartments were in the G1 phase of the cycle. The period of cell cycle arrest was decreased by the provision of assimilatable forms of nitrogen, suggesting that the hyphal cell cycle is regulated by nitrogen limitation that results in sup-apical cell cycle arrest. This pattern of growth is distinct from that of the synchronous, symmetrical development of pseudohyphae of C. albicans and other yeast species. These observations suggest that the cellular vacuole space correlates with alterations in the cell cycles of different cell types and that the total organelle space may influence size-regulated functions and hence the timing of the eukaryotic cell cycle.

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.

RasGEF-containing proteins GbpC and GbpD have differential effects on cell polarity and chemotaxis in Dictyostelium

The regulation of cell polarity plays an important role in chemotaxis. Previously, two proteins termed GbpC and GbpD were identified in Dictyostelium, which contain RasGEF and cyclic nucleotide binding domains. Here we show that gbpC-null cells display strongly reduced chemotaxis, because they are unable to polarise effectively in a chemotactic gradient. However, gbpD-null mutants exhibit the opposite phenotype: cells display improved chemotaxis and appear hyperpolar, because cells make very few lateral pseudopodia, whereas the leading edge is continuously remodelled. Overexpression of GbpD protein results in severely reduced chemotaxis. Cells extend many bifurcated and lateral pseudopodia, resulting in the absence of a leading edge. Furthermore, cells are flat and adhesive owing to an increased number of substrate-attached pseudopodia. This GbpD phenotype is not dependent on intracellular cGMP or cAMP, like its mammalian homolog PDZ-GEF. Previously we showed that GbpC is a high-affinity cGMP-binding protein that acts via myosin II. We conclude that cGMP activates GbpC, mediating the chemoattractant-induced establishment of cell polarity through myosin. GbpD induces the formation of substrate-attached pseudopodia, resulting in increased attachment and suppression of polarity.