Lee H. Mitchell

Commitment to germ tube or bud formation during release from stationary phase in Candida albicans

Abstract Cells of the pathogenic yeast Candida albicans accumulate as unbudded singlets at stationary phase in defined medium at 25 °C. When released into fresh medium at 37 °C and pH 6.5, these cells will synchronously form elongate pseudomycelia, and when released into fresh medium at either 25 °C, pH 6.5, or 37 °C, pH 4.5, they will synchronously form buds. Using pH and temperature shift experiments, we have examined when cells become committed to pseudomycelium formation and bud formation under conditions conducive to each growth form respectively. It is demonstrated that in either case commitment occurs long after release from stationary phase, at approximately the same time the first evagination is visible on the cells surface. In addition, it is demonstrated that once a released cell has formed a bud, it and its progeny lose the capacity to form pseudomycelia until they re-enter stationary phase; on the other hand, elongating pseudomycelia retain the capacity to form buds. The possible relationships of the commitment events to septation and to the cell cycle are discussed.

Temporal and spatial differences in septation during synchronous mycelium and bud formation by Candida albicans

We have examined the time and location of the first septa formed by cells of Candida albicans when such cells were induced to grow synchronously as either mycelial or budding cultures. Synchronous growth was initiated by inoculating stationary-phase cells into an amino acid medium with a pH of either 6.5 or 4.5. Septa were visualized with the fluorescent stain Calcofluor. Evidence is presented that: (1) During both synchronous mycelium and bud formation, lightly staining septa first appear and 30 to 40 minutes later abruptly convert to darkly staining septa. (2) Although the times of evagination for mycelium and bud formation are the same, the times of septum formation differ. During bud formation the average septum forms very close to the time of initial evagination; during mycelium formation the average septum forms 30 minutes after evagination. (3) The location of septum formation differs. During bud formation, the septa invariably form at the junction of the mother cell and bud; during mycelium formation, the average septum forms in the tube approximately 2 μ m from the junction of mother cell and tube. (4) During mycelium formation, the average septum forms when the mycelium is approximately 7 μ m in length, and no tube elongation occurs between septum and mother cell once the initial septum is formed. A mutant strain, M11, is also described which begins to form a mycelium under conditions conducive to mycelium formation, but upon septation generates a bud distal to the septum.

An analysis of developmental timing in Dictyostelium discoideum

A new method has been developed to assess the minimum complexity and relationships of those pathways (developmental timers) which time the consecutive stages of a developing system (Soll, 1983). This method has been applied to the morphogenetic program of Dictyostelium discoideum and has resulted in (1) a minimum estimate of the number of components comprising the timers for the first seven stages of morphogenesis, (2) a characterization of the temperature sensitivities of these components including demonstration of a reversible timer component, (3) detained temporal definition of a number of transition points between rate-limiting components including a major branch point for the onset of several independent timer components coincident with the onset of aggregation, and (4) a temporal model for the relationships between the timers of the seven consecutive morphogenetic stages, including several examples of parallel timers.

Differentiation and dedifferentiation can function simultaneously and independently in the same cells in Dictyostelium discoideum

Abstract When developing cultures of Dictyostelium discoideum are disaggregated and morphogenesis is reinitiated, cells recapitulate the stages they had progressed through prior to disaggregation in a fraction of the original time. If developing cultures are disaggregated and the cells resuspended in nutrient medium, they retain this capacity for 1.5 hr and then synchronously and rapidly revert to the slow timing of log phase cells. Loss of the capacity to recapitulate morphogenesis rapidly is referred to as the “erasure event.” Following the erasure event, cells systematically lose developmentally acquired functions in a defined temporal sequence of dedifferentiation. Cells which have just passed through the erasure event can be stimulated to reenter the developmental program, even though they still possess several aggregation-associated functions acquired during the initial developmental program. In this report, we have tested whether cells stimulated to reenter the developmental program immediately after the erasure event progress along the same rate-limiting pathway leading to aggregation as they did during initial development and whether this rate-limiting pathway can run simultaneously with and independently of the sequence of dedifferentiation. Results are presented which demonstrate (1) that the erasure event resets the rate-limiting pathway for development back to zero and that erased cells reentering development progress along the same rate-limiting pathway as naive log phase cells, (2) that the loss of an aggregation-associated function late in the sequence of dedifferentiation is completely blocked by the addition of cycloheximide, but not cAMP, just prior to the expected time of loss, and (3) that differentiation and dedifferentiation can function simultaneously and independently in the same cells, even though the former leads to the acquisition and the latter to the loss of the same aggregation-associated functions (in this case EDTA-resistant adhesion and cAMP-stimulated motility).

Characterization of a timing mutant of Dictyostelium discoideum which exhibits “high frequency switching”

The preaggregative period of Dictyostelium discoideum is composed of two sequential rate-limiting components. The timing mutant FM-1 exhibits a decrease in the length of the preaggregative period and the interval between the maxifinger and early culminate II stage. In contrast, it is normal in all aspects of growth, in the sequence of morphogenetic stages, in spore formation, in the capacity to rapidly recapitulate morphogenesis, and in the erasure event and subsequent program of dedifferentiation. By the reciprocal shift experiment, it is demonstrated that FM-1 is completely missing the first of the two rate-limiting components comprising the preaggregative period. The FM-1 mutation is heritable and behaves as a single mutation mapping to linkage group II. However, the FM-1 variant switches at relatively high frequency to several other timing phenotypes with longer preaggregative periods which in turn switch at high frequency. The FM-1 phenotype is considered in terms of timing regulation, and the process of high frequency switching between timing phenotypes is compared to other newly discovered switching systems.