The role of calcium in the tropisms of Candida albicans hyphae

Lead Research Organisation: University of Aberdeen
Department Name: School of Medical Sciences


Many cells grow only at their ends / a mode of cell development that requires delivery of new membrane and assembly of new cell wall at a single point of the cell surface. This polarised form of development is common to nerve cells, pollen tubes and many other eukaryotic cell types, most notably fungal hyphae. Tip growing hyphae also have to be steered towards nutrients, oxygen, appropriate mating partners or other cells, and away from toxic compounds and around inpenetratable objects. Therefore tip growth has to be coupled to the ability to orient the tip. We know that the cytoskeleton and several important protein complexes located at the cell tip are required to organise the cell in such a way that secretory vesicles, which carry membrane and proteins for growth, are delivered to and fuse with the apical plasma membrane. We do not know how the cytoskeleton and these protein complexes are regulated to enable the growing hyphal tip to be steered and to respond to environmental cues. This research programme rests on the foundation of our recent observations that have shown that the ability of a hypha to turn requires a supply of calcium ions in the growth medium and several calcium channel proteins. These insights are embodied in our hypothesis that the molecular machinery required for hyphal orientation is regulated by local uptake of calcium ions at the hyphal apex. In our experiments we use hyphal orientation responses of the human pathogenic fungus Candida albicans as our model system. This is because we can genetically manipulate this organism to create mutations and protein-tagged strains that can be used to address our hypothesis, and because hyphal tropisms are likely to be important in the pathogenic life style of this fungus. We have also collected a wide range of mutants and strains for this project from colleagues who are investigating the tip growth process, but have not considered tip orientation as a separate phenomenon. We have developed two tractable assays that allow us to observe and quantify hyphal orientation responses. Firstly, we can measure how many hypha become deflected to a new axis of growth as the encounter a ridge underlying the cell (contact guidance or thigmotropism). Secondly we can override all endogenous and exogenous guidance cues by placing hyphae in an electrical field and observing them reorienting towards the cathodic pole (galvanotropism). So far our findings show that calcium ions are vital for both tropic responses suggesting that the steering machinery is the same, even for different tropisms. We will test the hypothesis that hyphal orientation is regulated by calcium ions in several ways. [i] We will localise the calcium channel complex proteins in the hyphae using fluorescently labelled YFP and CFP- fusions and observe the cellular localisation of proteins in this complex and other tip-growth related protein complexes (polarisome, exocyst, Cdc42 and Arp2/3 complexes) as hyphae undergo thigmotropic and galvanotropic alignments. [ii] We will determine whether calcium ions that originate from cellular stores are also important in supporting tropic orientations. [iii] We will characterise calcium-ion dependent processes that could translate calcium signals into tropic growth responses. [iv] Finally, we will determine the significance of tropic growth for the ability of C. albicans hyphae to invade host tissues.

Technical Summary

This proposal will investigate the relationship between polarized tip growth and the process of hyphal orientation. The mechanism of polarised cell growth has been shown to be related to activities of four protein complexes - the polarisome, exocyst, Arp2/3 and Cdc42-complexes. In previous studies we observed that exogenous Ca2+ and three Ca2+ channel proteins Cch1, Mid1 and Fig1 were all required for the ability of Candida albicans hyphae to respond thigmotropically to microfabricated ridges on quartz slides and galvanotropically to exogenous applied electrical fields. This suggests that Ca2+-signals mediated via these channels may influence the position and/or activity of proteins in these complexes. We propose to test this hypothesis by localising the calcium channel proteins in the cell membrane and by observing the position of the channels and marker proteins in the four protein complexes in cells that are responding to exogenous growth-directing signals. We will also determine the importance of Ca2+ stores in facilitating tropic growth and characterise the role of Cdc42 and other downstream components that may couple Ca2+signals to polarised cell growth. Finally, we will establish the role of hyphal orientation in the infiltration and invasion of host tissues using tropism-attenuated mutants and ex-vivo infection models. Because orthologues of many components of the cell orientation machinery are ubiquitous in eukaryotes we believe these studies will provide fundamental insights into the ways in which cells navigate during growth and development.
Description Fungal hyphae grow only at their ends so they must control the direction they grow in order to find nutrients or avoid unfavourable environments. Tip growth therefore has to be coupled to tip steering so that new cell wall material is not only delivered to the tip but is incorporated at the correct site. We tested the hypothesis that localised calcium uptake at the hyphal tip dictates the site at which tip growth occurs. Calcium signals are induced by various external features, such as obstacles or electric fields, and blocking or deleting the calcium channels in the fungus, Candida albicans, reduces the ability of its hyphae to steer their tips (Brand et al, 2007).

In this study, we accomplished all four objectives stated in the original proposal. We have strengthened the link between calcium influx and tip directionality by showing that the highly-regular, wavy growth by hyphae grown on semi-solid surfaces depends on calcium influx and homeostasis (Brand et al, 2009). We have also shown that deletion of the calcium channel, Fig1, severely restricts calcium uptake during mating, confirming a role for this protein as a regulator of calcium influx for the first time in C. albicans. We generated a yvc1 mutant that is defective in calcium export from the primary intracellular store in C. albicans, the vacuole. Using this we demonstrated that the release of calcium from the vacuole is not required for hyphae to respond to external cues. However, although we labelled all three plasma membrane channels with a green fluorescent protein (GFP), only Fig1-GFP has been localised. A manuscript on the biological relevance of Fig1 in C albicans mating is close to submission.The point of growth at the hyphal tip is controlled by proteins that form a complex called the polarisome. We made considerable progress in understanding how the different polarisome components contribute to tip steering by generating mutant strains where cell elongation is maintained but tips do not respond normally. The mutant tips display three different types of behaviour, which we term 'unanchored', 'locked' and 'unlocked'. When 'unanchored', the polarisome complex wanders erratically within the hyphal tip, which becomes blind to all directional cues. Even adding calcium does not reverse this behaviour, which suggests that calcium signalling acts on the polarisome anchor. Tip-steering was so reduced in this strain that it was less able to invade mammalian tissue, suggesting that control of hyphal tips could be important for the ability of C. albicans to cause disease (Brand et al, 2008). When the polarisome is 'locked' or 'unlocked', tips become less or more responsive, respectively, to directional cues than wild-type hyphae. Both the 'unanchored' and 'locked' phenotypes result from the inability of certain proteins to cycle between an 'on' and an 'off' phase, while the 'unlocked' phenotype is due to the reduced 'stickiness' of a protein that holds the other polarisome proteins together. Lastly, we found that, of two proteins that were thought to carry out very similar functions within the polarisome, only one is important for hyphae to re-orient themselves (Brand et al, in preparation). Together these results suggest that the polarisome complex lies centrally within the pathway that regulates the direction of hyphal growth.

This study has established some key features of tip regulation and provided a strong platform for the resulting successful awards to Alexandra Brand of a Royal Society University Research Fellowship and an MRC New Investigator Grant in order to develop this field. Seven peer reviewed papers have been published, one of which was designated as 'Must Read' by Faculty of 1000 Biology and another was selected as a Research Highlight by Nature Microbiology Reviews. These have generated considerable interest at an international research level, resulting in 7 invitations to speak at conferences, 3 offered talks and 4 poster presentations.
Exploitation Route Mutants strains have been supplied to other research groups.
Collaborative projects have been established.
Grant applications have been submitted.
Sectors Agriculture, Food and Drink,Healthcare

Description A number of output publications were generated. The named researcher went on to win two personal fellowships and to become a member of staff running her own laboratory at Aberdeen University. Contributed to the success of a new EU grant and the employment of a new ECR. Has provided strong preliminary data contributing to a PNAS paper and a further BBSRC project grant in 2014.
First Year Of Impact 2009
Sector Education,Healthcare
Impact Types Economic