No abstract

It was recently demonstrated that strains homozygous for either of the mating type-like loci MTLa and MTL␣ 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 ␣ strains. This study provides the first description of the cell biology of the mating process of C. albicans.Candida albicans is a diploid organism that, until quite recently, was believed to have no sexual cycle. However, in 1999 Hull and Johnson (13) demonstrated that C. albicans strain CAI4 contained MTLa1 on one chromosome and MTL␣1 and MTL␣2 on the homolog. Soon after, fusion was genetically demonstrated to occur between MTLa and MTL␣ strains (a/Ϫ and ␣/Ϫ, respectively) in vivo (14) and in vitro (20). Recently, Miller and Johnson (21) demonstrated that the homozygous MTLa and MTL␣ strains engineered from strain CAI4 underwent white-opaque switching and that when both strains were in the opaque phase, mating occurred 10 6 times more efficiently than when one or both strains were in the white phase. Lockhart et al. (17) subsequently demonstrated that approximately 3% of 220 clinical isolates of C. albicans representing the major clades worldwide were homozygous for the mating type locus (i.e., either MTLa or MTL␣), that all tested clinical strains that underwent white-opaque switching were homozygous for MTL, that most clinical strains that were homozygous for MTL underwent the white-opaque transition, and that heterozygotes did not undergo white-opaque switching. Together, these recent results demonstrate for the first time that the mating type locus regulates switching and that switching facilitates mating.To visualize the mating process of C. albicans, we took advantage of the recent discovery that mating is facilitated by the opaque-phase phenotype (21). Using information obtained from videomicroscopy, computer-assisted dynami...

Myosin VII plays an important role in mediating the initial binding of cells to substrata, a novel role for an unconventional myosin.

We have investigated the effects of inhibiting the expression of cofilin to understand its role in protrusion dynamics in metastatic tumor cells, in particular. We show that the suppression of cofilin expression in MTLn3 cells (an apolar randomly moving amoeboid metastatic tumor cell) caused them to extend protrusions from only one pole, elongate, and move rectilinearly. This remarkable transformation was correlated with slower extension of fewer, more stable lamellipodia leading to a reduced turning frequency. Hence, the loss of cofilin caused an amoeboid tumor cell to assume a mesenchymal-type mode of movement. These phenotypes were correlated with the loss of uniform chemotactic sensitivity of the cell surface to EGF stimulation, demonstrating that to chemotax efficiently, a cell must be able to respond to chemotactic stimulation at any region on its surface. The changes in cell shape, directional migration, and turning frequency were related to the re-localization of Arp2/3 complex to one pole of the cell upon suppression of cofilin expression.

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.