Ekkehard Leberer

CDC42 Is Required for Polarized Growth in Human Pathogen Candida albicans

ABSTRACT Cdc42p is a member of the RAS superfamily of GTPases and plays an essential role in polarized growth in many eukaryotic cells. We cloned the Candida albicans CaCDC42 by functional complementation in Saccharomyces cerevisiae and analyzed its function in C. albicans. A double deletion of CaCDC42 was made in a C. albicans strain containing CaCDC42 under the control of the PCK1 promoter. When expression of the heterologous copy of CaCDC42 was repressed in this strain, the cells ceased proliferation. These arrested cells were large, round, and unbudded and contained predominantly two nuclei. The PCK1-mediated overexpression of wild-type CaCdc42p had no effect on cells. However, in cells overexpressing CaCdc42p containing the dominant-negative D118A substitution, proliferation was blocked and the arrested cells were large, round, unbudded, and multinucleated, similar to the phenotype of the cdc42 double-deletion strain. Cells overexpressing CaCdc42p containing the hyperactive G12V substitution also ceased proliferation in yeast growth medium; in this case the arrested cells were multinucleated and multibudded. An intact CAAX box is essential for the phenotypes associated with either CaCdc42pG12V or CaCdc42pD118A ectopic expression, suggesting that membrane attachment is involved in CaCdc42p function. In addition, the lethality caused by ectopic expression of CaCdc42pG12V was suppressed by deletion of CST20 but not by deletion of CaCLA4. CaCdc42p function was also examined under hypha-inducing conditions. Cdc42p depletion prior to hyphal induction trapped cells in a round, unbudded state, while depletion triggered at the same time as hyphal induction permitted the initiation of germ tubes that failed to be extended. Ectopic expression of either the G12V or D118A substitution protein modified hyphal formation in a CAAX box-dependent manner. Thus, CaCdc42p function appears important for polarized growth of both the yeast and hyphal forms of C. albicans.

Myosin I Is Required for Hypha Formation in Candida albicans

ABSTRACT The pathogenic yeast Candidaalbicans can undergo a dramatic change in morphology from round yeast cells to long filamentous cells called hyphae. We have cloned the CaMYO5 gene encoding the only myosin I in C. albicans. A strain with a deletion of both copies of CaMYO5 is viable but cannot form hyphae under all hypha-inducing conditions tested. This mutant exhibits a higher frequency of random budding and a depolarized distribution of cortical actin patches relative to the wild-type strain. We found that polar budding, polarized localization of cortical actin patches, and hypha formation are dependent on a specific phosphorylation site on myosin I, called the “TEDS-rule” site. Mutation of this serine 366 to alanine gives rise to the null mutant phenotype, while a S366D mutation, the product of which mimics a phosphorylated serine, allows hypha formation. However, the S366D mutation still causes a depolarized distribution of cortical actin patches in budding cells, similar to that in the null mutant. The localization of CaMyo5-GFP together with cortical actin patches at the bud and hyphal tips is also dependent on serine 366. Intriguingly, the cortical actin patches in the majority of the hyphae of the mutant expressing Camyo5S366D were depolarized, suggesting that although their distribution is dependent on myosin I localization, polarized cortical actin patches may not be required for hypha formation.

Relationships between early alterations in parvalbumins, sarcoplasmic reticulum and metabolic enzymes in chronically stimulated fast twitch muscle

The present study compares the time courses of the early changes in parvalbumin content, in the properties of the sarcoplasmic reticulum (SR) and in activity and isozyme patterns of metabolic enzymes in chronically (12 h/day) stimulated fast twitch tibialis anterior (TA) muscle of the rabbit. Under the chosen conditions of stimulation, the first significant changes appeared after 6 days. Except for the delayed reduction in pyruvate kinase, the time course of the changes were the same. After 14 days of stimulation, parvalbumin decreased to 37% and Ca2+-ATPase activity of the SR to 29% of normal values. The transformation of the SR was also reflected by a 64% decrease of the 115000-Mr Ca2+-pumping peptide and a 5-fold increase in a 30000-Mr peptide. Following an identical time course, the mitochondrial activities of citrate synthase, 3-hydroxyacyl-CoA dehydrogenase and ketoacid-CoA transferase increased 2.9, 3.0 and 3.7-fold respectively. A similar time course was observed in the M to H-type transition of the lactate dehydrogenase isozyme. The cause of these changes is discussed at it relates to altered transcriptional and/or translational activities. It is suggested that an increase in free intracellular Ca2+ caused by increased contractile activity, which is then perpetuated by the decrease in Ca2+-binding and sequestering capacities, might be the signal for such altered synthetic activities.

Relationship between parvalbumin content and the speed of relaxation in chronically stimulated rabbit fast-twitch muscle

The time courses of changes in parvalbumin (PA) content, isometric twitch tension, and half-relaxation time (1/2RT) were studied in rabbit tibialis anterior muscle following chronic 10 Hz nerve stimulation of 1–21 days. Up to 5 days stimulation had no effect on PA content, but it induced a slight (10–15%) increase in the 1/2RT. This change occurred together with the previously observed 50% decrease in Ca2+-uptake by the SR (Leberer et al. 1987). While prolonged stimulation produced no further decrease in the Ca2+-uptake by the SR, PA content declined after 5 days of stimulation. The reduction in PA content was accompanied by a progressive lengthening of the 1/2RT. However, the increase in 1/2RT was particularly pronounced after PA had fallen below 50% of its normal value. A 90% reduction in PA coincided with a 60% increase in the 1/2RT. By this time the staircase phenomenon, normally observed in fast-twitch muscle, was completely abolished. Although the changes in PA content and 1/2RT were not linearly related, these results suggest that PA plays an important role in the relaxation process of mammalian fast-twitch muscle.

The fast-twitch muscle calsequestrin isoform predominates in rabbit slow-twitch soleus muscle

The major form of calsequestrin in rabbit slow‐twitch soleus muscle is shown to be identical to that isolated and cloned from rabbit fast‐twitch muscle on the following bases: identity of cDNAs cloned from mRNAs from the two muscle sources; equivalent hybridization of a fast‐twitch calsequestrin cDNA probe to mRNAs isolated from fast‐twitch and slow‐twitch muscles; identity of the 23 amino‐terminal amino acids; strong binding of 45Ca2+ in a gel overlay of slow muscle sarcoplasmic reticulum protein to a band at the level of the fast‐twitch calsequestrin isoform and only weak binding at the level of the cardiac isoform. No evidence was obtained for developmentally regulated alternative splicing of the calsequestrin transcript in mature slow or fast‐twitch muscle.

[56] Regulation of parvalbumin concentration in mammalian muscle

Publisher Summary Parvalbumin (PA), an acidic Ca 2+ - and Mg 2+ -binding protein of approximately 12,000 M r , is thought to act in skeletal muscle as a cytosolic Ca 2+ - and Mg 2+ -buffering compound. In mammalian muscle, it has been detected exclusively in fast-twitch fibers. Several studies have shown that the PA concentration in skeletal muscle is subject to regulation by exogenous factors and may be altered experimentally by changes in contractile and/or motoneuron activity. It has also been shown that PA is reduced in hereditary mammalian muscle diseases. The influence of neural activity upon PA expression is clearly shown by denervation, cross-reinnervation, and nerve-stimulation experiments. Denervation of neonatal and adult fast-twitch muscles suppresses PA synthesis. Its concentration remains low in denervated, presumptive fast-twitch muscles of the newborn rabbit. PA synthesis is suppressed in fast-twitch muscle after cross-reinnervation with a nerve that normally supplies a slow-twitch muscle. In view of the different activity patterns of fast and slow motoneurons, it seems likely that PA expression is under the positive control of phasic, high-frequency activity as delivered by fast motoneurons.